Generation of Envelope-Modified Baculoviruses for Gene Delivery into Mammalian Cells.

PubMed

Hofmann, Christian

2016-01-01

Genetically modified baculoviruses can efficiently deliver and express genes in mammalian cells. The major prerequisite for the expression of a gene transferred by baculovirus is its control by a promoter that is active in mammalian cells. This chapter describes methods for producing second generation baculovirus vectors through modification of their envelope. Envelope modified baculoviruses offer additional new applications of the system, such as their use in in vivo gene delivery, targeting, and vaccination. Methods of generating a recombinant baculovirus vector with a modified envelope and its amplification and purification, including technical scale production, are discussed. A variety of notes give clues regarding specific technical procedures. Finally, methods to analyze the virus and transduction procedures are presented.

Overcoming chemotherapy drug resistance by targeting inhibitors of apoptosis proteins (IAPs).

PubMed

Rathore, Rama; McCallum, Jennifer E; Varghese, Elizabeth; Florea, Ana-Maria; Büsselberg, Dietrich

2017-07-01

Inhibitors of apoptosis (IAPs) are a family of proteins that play a significant role in the control of programmed cell death (PCD). PCD is essential to maintain healthy cell turnover within tissue but also to fight disease or infection. Uninhibited, IAPs can suppress apoptosis and promote cell cycle progression. Therefore, it is unsurprising that cancer cells demonstrate significantly elevated expression levels of IAPs, resulting in improved cell survival, enhanced tumor growth and subsequent metastasis. Therapies to target IAPs in cancer has garnered substantial scientific interest and as resistance to anti-cancer agents becomes more prevalent, targeting IAPs has become an increasingly attractive strategy to re-sensitize cancer cells to chemotherapies, antibody based-therapies and TRAIL therapy. Antagonism strategies to modulate the actions of XIAP, cIAP1/2 and survivin are the central focus of current research and this review highlights advances within this field with particular emphasis upon the development and specificity of second mitochondria-derived activator of caspase (SMAC) mimetics (synthetic analogs of endogenously expressed inhibitors of IAPs SMAC/DIABLO). While we highlight the potential of SMAC mimetics as effective single agent or combinatory therapies to treat cancer we also discuss the likely clinical implications of resistance to SMAC mimetic therapy, occasionally observed in cancer cell lines.

Baculovirus: an Insect-derived Vector for Diverse Gene Transfer Applications

PubMed Central

Airenne, Kari J; Hu, Yu-Chen; Kost, Thomas A; Smith, Richard H; Kotin, Robert M; Ono, Chikako; Matsuura, Yoshiharu; Wang, Shu; Ylä-Herttuala, Seppo

2013-01-01

Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered. PMID:23439502

Inducing death in tumor cells: roles of the inhibitor of apoptosis proteins.

PubMed

Finlay, Darren; Teriete, Peter; Vamos, Mitchell; Cosford, Nicholas D P; Vuori, Kristiina

2017-01-01

The heterogeneous group of diseases collectively termed cancer results not just from aberrant cellular proliferation but also from a lack of accompanying homeostatic cell death. Indeed, cancer cells regularly acquire resistance to programmed cell death, or apoptosis, which not only supports cancer progression but also leads to resistance to therapeutic agents. Thus, various approaches have been undertaken in order to induce apoptosis in tumor cells for therapeutic purposes. Here, we will focus our discussion on agents that directly affect the apoptotic machinery itself rather than on drugs that induce apoptosis in tumor cells indirectly, such as by DNA damage or kinase dependency inhibition. As the roles of the Bcl-2 family have been extensively studied and reviewed recently, we will focus in this review specifically on the inhibitor of apoptosis protein (IAP) family. IAPs are a disparate group of proteins that all contain a baculovirus IAP repeat domain, which is important for the inhibition of apoptosis in some, but not all, family members. We describe each of the family members with respect to their structural and functional similarities and differences and their respective roles in cancer. Finally, we also review the current state of IAPs as targets for anti-cancer therapeutics and discuss the current clinical state of IAP antagonists.

Inhibitors of apoptosis (IAPs) regulate intestinal immunity and inflammatory bowel disease (IBD) inflammation.

PubMed

Pedersen, Jannie; LaCasse, Eric C; Seidelin, Jakob B; Coskun, Mehmet; Nielsen, Ole H

2014-11-01

The inhibitor of apoptosis (IAP) family members, notably cIAP1, cIAP2, and XIAP, are critical and universal regulators of tumor necrosis factor (TNF) mediated survival, inflammatory, and death signaling pathways. Furthermore, IAPs mediate the signaling of nucleotide-binding oligomerization domain (NOD)1/NOD2 and other intracellular NOD-like receptors in response to bacterial pathogens. These pathways are important to the pathogenesis and treatment of inflammatory bowel disease (IBD). Inactivating mutations in the X-chromosome-linked IAP (XIAP) gene causes an immunodeficiency syndrome, X-linked lymphoproliferative disease type 2 (XLP2), in which 20% of patients develop severe intestinal inflammation. In addition, 4% of males with early-onset IBD also have inactivating mutations in XIAP. Therefore, the IAPs play a greater role in gut homeostasis, immunity and IBD development than previously suspected, and may have therapeutic potential. Copyright © 2014 Elsevier Ltd. All rights reserved.

Baculovirus infection induces disruption of the nuclear lamina.

PubMed

Zhang, Xiaomei; Xu, Kaiyan; Wei, Denghui; Wu, Wenbi; Yang, Kai; Yuan, Meijin

2017-08-10

Baculovirus nucleocapsids egress from the nucleus primarily via budding at the nuclear membrane. The nuclear lamina underlying the nuclear membrane represents a substantial barrier to nuclear egress. Whether the nuclear lamina undergoes disruption during baculovirus infection remains unknown. In this report, we generated a clonal cell line, Sf9-L, that stably expresses GFP-tagged Drosophila lamin B. GFP autofluorescence colocalized with immunofluorescent anti-lamin B at the nuclear rim of Sf9-L cells, indicating GFP-lamin B was incorporated into the nuclear lamina. Meanwhile, virus was able to replicate normally in Sf9-L cells. Next, we investigated alterations to the nuclear lamina during baculovirus infection in Sf9-L cells. A portion of GFP-lamin B localized diffusely at the nuclear rim, and some GFP-lamin B was redistributed within the nucleus during the late phase of infection, suggesting the nuclear lamina was partially disrupted. Immunoelectron microscopy revealed associations between GFP-lamin B and the edges of the electron-dense stromal mattes of the virogenic stroma, intranuclear microvesicles, and ODV envelopes and nucleocapsids within the nucleus, indicating the release of some GFP-lamin B from the nuclear lamina. Additionally, GFP-lamin B phosphorylation increased upon infection. Based on these data, baculovirus infection induced lamin B phosphorylation and disruption of the nuclear lamina.

Design Considerations for IAP Charts Approach Course Track and Communication Frequencies

DOT National Transportation Integrated Search

1991-08-01

This report describes two experiments evaluating format changes on instrument approach : plates (IAPs). The study used a simple chart reading task to assess information : transfer in IAPs. : The first experiment addressed different methods of display...

Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

PubMed Central

Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

2015-01-01

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses.

PubMed

Kroemer, Jeremy A; Bonning, Bryony C; Harrison, Robert L

2015-01-21

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification.

Evaluation of the Insecticidal Efficacy of Wild Type and Recombinant Baculoviruses.

PubMed

Popham, Holly J R; Ellersieck, Mark R; Li, Huarong; Bonning, Bryony C

2016-01-01

A considerable amount of work has been undertaken to genetically enhance the efficacy of baculovirus insecticides. Following construction of a genetically altered baculovirus, laboratory bioassays are used to quantify various parameters of insecticidal activity such as the median lethal concentration (or dose) required to kill 50 % of infected larvae (LC50 or LD50), median survival of larvae infected (ST50), and feeding damage incurred by infected larvae. In this chapter, protocols are described for a variety of bioassays and the corresponding data analyses for assessment of the insecticidal activity of baculovirus insecticides.

Transduction of cultured fish cells with recombinant baculoviruses.

PubMed

Leisy, Douglas J; Lewis, Teresa D; Leong, Jo-Ann C; Rohrmann, George F

2003-05-01

Five fish cell lines were tested for their ability to be transduced by Ac-CAlacZ, a recombinant baculovirus that is capable of expressing a beta-galactosidase reporter gene from the CAG promoter (consisting of a cytomegalovirus enhancer element, a chicken actin promoter and rabbit beta-globin termination sequences). TO (Tilapia ovary), EPC (carp), CHH-1 (Chum salmon heart fibroblast) and CHSE-214 (chinook salmon embryo) cells were transducible, as demonstrated by an in situ beta-galactosidase assay, whereas RTG-2 (rainbow trout gonad) cells were not. The EPC cell line was used for more detailed studies on baculovirus transduction. The transduction frequency was found to be higher at 28 degrees C than at 21 degrees C. Addition of the histone deacetylase inhibitor sodium butyrate increased the number of blue cells detected 5- to 7-fold. The m.o.i. was positively correlated with transduction frequency, although the relationship did not appear to be strictly linear, as has been observed with mammalian cells. The temperature at which baculoviruses were adsorbed to EPC cells did not affect levels of beta-galactosidase expression. We also examined expression levels of beta-galactosidase in EPC cells after infection with a baculovirus construct that overexpresses the vesicular stomatitis virus G protein and displays it on the virion surface. Expression levels with this virus were approximately 15-fold higher than were observed with Ac-CAlacZ.

[Expression and significance of immunosuppressive acidic protein (IAP) in human esophageal squamous cell carcinoma].

PubMed

Guo, S Z; Shen, Q; Zhang, H B

1994-03-01

The expression of IAP in the esophageal tissues of 74 patients with esophageal squamous cell carcinoma and 12 normal controls were determined by using MI2, and anti-IAP monoclonal antibody, and ABC immunohistochemical staining. The results showed that there was no expression of IAP in normal esophageal epithelium of all control subjects, and the positive rate in specimens of the esophageal carcinomas was 90.3% (P < 0.001). The staining intensity of IAP was increasing with the decrease in degrees of cell differentiation of the tumors (P < 0.05). The expression of IAP in long survivors without lymph node metastasis were lower than that in cases with metastasis (P < 0.005) and short survivors (P < 0.001). The results suggest that IAP may play an important role in tumor cell differentiation, clinical course and prognosis of esophageal carcinoma, and may be used as a tumor marker for the diagnosis of this malignancy.

Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication.

PubMed

Chen, Tingting; Dong, Zhanqi; Hu, Nan; Hu, Zhigang; Dong, Feifan; Jiang, Yaming; Li, Jun; Chen, Peng; Lu, Cheng; Pan, Minhui

2017-06-15

Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

Drosophila BRUCE inhibits apoptosis through non-lysine ubiquitination of the IAP-antagonist REAPER

PubMed Central

Domingues, C; Ryoo, H D

2012-01-01

Active caspases execute apoptosis to eliminate superfluous or harmful cells in animals. In Drosophila, living cells prevent uncontrolled caspase activation through an inhibitor of apoptosis protein (IAP) family member, dIAP1, and apoptosis is preceded by the expression of IAP-antagonists, such as Reaper, Hid and Grim. Strong genetic modifiers of this pathway include another IAP family gene encoding an E2 ubiquitin conjugating enzyme domain, dBruce. Although the genetic effects of dBruce mutants are well documented, molecular targets of its encoded protein have remained elusive. Here, we report that dBruce targets Reaper for ubiquitination through an unconventional mechanism. Specifically, we show that dBruce physically interacts with Reaper, dependent upon Reaper's IAP-binding (IBM) and GH3 motifs. Consistently, Reaper levels were elevated in a dBruce −/− background. Unexpectedly, we found that dBruce also affects the levels of a mutant form of Reaper without any internal lysine residues, which normally serve as conventional ubiquitin acceptor sites. Furthermore, we were able to biochemically detect ubiquitin conjugation on lysine-deficient Reaper proteins, and knockdown of dBruce significantly reduced the extent of this ubiquitination. Our results indicate that dBruce inhibits apoptosis by promoting IAP-antagonist ubiquitination on unconventional acceptor sites. PMID:21886178

Identification of a high-efficiency baculovirus DNA replication origin that functions in insect and mammalian cells.

PubMed

Wu, Yueh-Lung; Wu, Carol-P; Huang, Yu-Hui; Huang, Sheng-Ping; Lo, Huei-Ru; Chang, Hao-Shuo; Lin, Pi-Hsiu; Wu, Ming-Cheng; Chang, Chia-Jung; Chao, Yu-Chan

2014-11-01

a large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting with gene expression in mammalian cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Derivatization of inhibitor of apoptosis protein (IAP) ligands yields improved inducers of estrogen receptor α degradation.

PubMed

Ohoka, Nobumichi; Morita, Yoko; Nagai, Katsunori; Shimokawa, Kenichiro; Ujikawa, Osamu; Fujimori, Ikuo; Ito, Masahiro; Hayase, Youji; Okuhira, Keiichiro; Shibata, Norihito; Hattori, Takayuki; Sameshima, Tomoya; Sano, Osamu; Koyama, Ryokichi; Imaeda, Yasuhiro; Nara, Hiroshi; Cho, Nobuo; Naito, Mikihiko

2018-05-04

Aberrant expression of proteins often underlies many diseases, including cancer. A recently developed approach in drug development is small molecule-mediated, selective degradation of dysregulated proteins. We have devised a protein-knockdown system that utilizes chimeric molecules termed specific and nongenetic IAP-dependent protein erasers (SNIPERs) to induce ubiquitylation and proteasomal degradation of various target proteins. SNIPER(ER)-87 consists of an inhibitor of apoptosis protein (IAP) ligand LCL161 derivative that is conjugated to the estrogen receptor α (ERα) ligand 4-hydroxytamoxifen by a PEG linker, and we have previously reported that this SNIPER efficiently degrades the ERα protein. Here, we report that derivatization of the IAP ligand module yields SNIPER(ER)s with superior protein-knockdown activity. These improved SNIPER(ER)s exhibited higher binding affinities to IAPs and induced more potent degradation of ERα than does SNIPER(ER)-87. Further, they induced simultaneous degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) and delayed degradation of X-linked IAP (XIAP). Notably, these reengineered SNIPER(ER)s efficiently induced apoptosis in MCF-7 human breast cancer cells that require IAPs for continued cellular survival. We found that one of these molecules, SNIPER(ER)-110, inhibits the growth of MCF-7 tumor xenografts in mice more potently than the previously characterized SNIPER(ER)-87. Mechanistic analysis revealed that our novel SNIPER(ER)s preferentially recruit XIAP, rather than cIAP1, to degrade ERα. Our results suggest that derivatized IAP ligands could facilitate further development of SNIPERs with potent protein-knockdown and cytocidal activities against cancer cells requiring IAPs for survival. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Perinatal lead (Pb) exposure results in sex and tissue-dependent adult DNA methylation alterations in murine IAP transposons.

PubMed

Montrose, L; Faulk, C; Francis, J; Dolinoy, D C

2017-10-01

Epidemiological and animal data suggest that adult chronic disease is influenced by early-life exposure-induced changes to the epigenome. Previously, we observed that perinatal lead (Pb) exposure results in persistent murine metabolic- and activity-related effects. Using phylogenetic and DNA methylation analysis, we have also identified novel intracisternal A particle (IAP) retrotransposons exhibiting regions of variable methylation as candidate loci for environmental effects on the epigenome. Here, we now evaluate brain and kidney DNA methylation profiles of four representative IAPs in adult mice exposed to human physiologically relevant levels of Pb two weeks prior to mating through lactation. When IAPs across the genome were evaluated globally, average (sd) methylation levels were 92.84% (3.74) differing by tissue (P < 0.001), but not sex or dose. By contrast, the four individual IAPs displayed tissue-specific Pb and sex effects. Medium Pb-exposed mice had 3.86% less brain methylation at IAP 110 (P < 0.01), while high Pb-exposed mice had 2.83% less brain methylation at IAP 236 (P = 0.01) and 1.77% less at IAP 506 (P = 0.05). Individual IAP DNA methylation differed by sex for IAP 110 in the brain and kidney, IAP 236 in the kidney, and IAP 1259 in the kidney. Using Tomtom, we identified three binding motifs that matched to each of our novel IAPs impacted by Pb, one of which (HMGA2) has been linked to metabolic-related conditions in both mice and humans. Thus, these recently identified IAPs display tissue-specific environmental lability as well as sex-specific differences supporting an epigenetic link between early exposure to Pb and later-in-life health outcomes. Environ. Mol. Mutagen. 58:540-550, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method

PubMed Central

Niarchos, Athanasios; Lagoumintzis, George; Poulas, Konstantinos

2015-01-01

Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions. PMID:26490731

Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

PubMed Central

2010-01-01

Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs. PMID:20587066

Loss of cIAP1 attenuates soleus muscle pathology and improves diaphragm function in mdx mice

PubMed Central

Enwere, Emeka K.; Boudreault, Louise; Holbrook, Janelle; Timusk, Kristen; Earl, Nathalie; LaCasse, Eric; Renaud, Jean-Marc; Korneluk, Robert G.

2013-01-01

The cellular inhibitor of apoptosis 1 (cIAP1) protein is an essential regulator of canonical and noncanonical nuclear factor κB (NF-κB) signaling pathways. NF-κB signaling is known to play important roles in myogenesis and degenerative muscle disorders such as Duchenne muscular dystrophy (DMD), but the involvement of cIAP1 in muscle disease has not been studied directly. Here, we asked whether the loss of cIAP1 would influence the pathology of skeletal muscle in the mdx mouse model of DMD. Double-mutant cIAP1−/−;mdx mice exhibited reduced muscle damage and decreased fiber centronucleation in the soleus, compared with single-mutant cIAP1+/+;mdx mice. This improvement in pathology was associated with a reduction in muscle infiltration by macrophages and diminished expression of inflammatory cytokines such as IL-6 and tumor necrosis factor-α. Furthermore, the cIAP1−/−;mdx mice exhibited reduced serum creatine kinase, and improved exercise endurance associated with improved exercise resilience by the diaphragm. Mechanistically, the loss of cIAP1 was sufficient to drive constitutive activation of the noncanonical NF-κB pathway, which led to increased myoblast fusion in vitro and in vivo. Collectively, these results show that the loss of cIAP1 protects skeletal muscle from the degenerative pathology resulting from systemic loss of dystrophin. PMID:23184147

IAP/APA evidence-based guidelines for the management of acute pancreatitis.

PubMed

2013-01-01

There have been substantial improvements in the management of acute pancreatitis since the publication of the International Association of Pancreatology (IAP) treatment guidelines in 2002. A collaboration of the IAP and the American Pancreatic Association (APA) was undertaken to revise these guidelines using an evidence-based approach. Twelve multidisciplinary review groups performed systematic literature reviews to answer 38 predefined clinical questions. Recommendations were graded using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system. The review groups presented their recommendations during the 2012 joint IAP/APA meeting. At this one-day, interactive conference, relevant remarks were voiced and overall agreement on each recommendation was quantified using plenary voting. The 38 recommendations covered 12 topics related to the clinical management of acute pancreatitis: A) diagnosis of acute pancreatitis and etiology, B) prognostication/predicting severity, C) imaging, D) fluid therapy, E) intensive care management, F) preventing infectious complications, G) nutritional support, H) biliary tract management, I) indications for intervention in necrotizing pancreatitis, J) timing of intervention in necrotizing pancreatitis, K) intervention strategies in necrotizing pancreatitis, and L) timing of cholecystectomy. Using the GRADE system, 21 of the 38 (55%) recommendations, were rated as 'strong' and plenary voting revealed 'strong agreement' for 34 (89%) recommendations. The 2012 IAP/APA guidelines provide recommendations concerning key aspects of medical and surgical management of acute pancreatitis based on the currently available evidence. These recommendations should serve as a reference standard for current management and guide future clinical research on acute pancreatitis. Copyright © 2013 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Bioengineered baculoviruses as new class of therapeutics using micro and nanotechnologies: principles, prospects and challenges.

PubMed

Paul, Arghya; Hasan, Anwarul; Rodes, Laetitia; Sangaralingam, Mugundhine; Prakash, Satya

2014-05-01

Designing a safe and efficient gene delivery system is required for success of gene therapy trials. Although a wide variety of viral, non-viral and polymeric nanoparticle based careers have been widely studied, the current gene delivery vehicles are limited by their suboptimal, non-specific therapeutic efficacy and acute immunological reactions, leading to unwanted side effects. Recently, there has been a growing interest in insect-cell-originated baculoviruses as gene delivery vehicles for diverse biomedical applications. Specifically, the emergence of diverse types of surface functionalized and bioengineered baculoviruses is posed to edge over currently available gene delivery vehicles. This is primarily because baculoviruses are comparatively non-pathogenic and non-toxic as they cannot replicate in mammalian cells and do not invoke any cytopathic effect. Moreover, emerging advanced studies in this direction have demonstrated that hybridizing the baculovirus surface with different kinds of bioactive therapeutic molecules, cell-specific targeting moieties, protective polymeric grafts and nanomaterials can significantly improve the preclinical efficacy of baculoviruses. This review presents a comprehensive overview of the recent advancements in the field of bioengineering and biotherapeutics to engineer baculovirus hybrids for tailored gene therapy, and articulates in detail the potential and challenges of these strategies for clinical realization. In addition, the article illustrates the rapid evolvement of microfluidic devices as a high throughput platform for optimizing baculovirus production and treatment conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

African swine fever virus IAP-like protein induces the activation of nuclear factor kappa B.

PubMed

Rodríguez, Clara I; Nogal, María L; Carrascosa, Angel L; Salas, María L; Fresno, Manuel; Revilla, Yolanda

2002-04-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-kappaB. Thus, transient transfection of the viral IAP increases the activity of an NF-kappaB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-kappaB-dependent gene. NF-kappaB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-kappaB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-kappaB activity seems to be the consequence of higher IkappaB kinase (IKK) basal activity in these cells. The NF-kappaB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2.

African Swine Fever Virus IAP-Like Protein Induces the Activation of Nuclear Factor Kappa B

PubMed Central

Rodríguez, Clara I.; Nogal, María L.; Carrascosa, Angel L.; Salas, María L.; Fresno, Manuel; Revilla, Yolanda

2002-01-01

African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-κB. Thus, transient transfection of the viral IAP increases the activity of an NF-κB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-κB-dependent gene. NF-κB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-κB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-κB activity seems to be the consequence of higher IκB kinase (IKK) basal activity in these cells. The NF-κB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2. PMID:11907233

Characterization of viral proteins of Oryctes baculovirus and comparison between two geographical isolates.

PubMed

Mohan, K S; Gopinathan, K P

1989-01-01

Bacilliform Oryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infected Oryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reported Oryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

Global Screening of Antiviral Genes that Suppress Baculovirus Transgene Expression in Mammalian Cells.

PubMed

Wang, Chia-Hung; Naik, Nenavath Gopal; Liao, Lin-Li; Wei, Sung-Chan; Chao, Yu-Chan

2017-09-15

Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.

BACULOVIRUS REPLICATION ALTERS HORMONE-REGULATED HOST DEVELOPMENT.

EPA Science Inventory

The baculovirus Lymantria dispar nuclear polyhedrosis virus interferes with insect larval development by altering the host's hormonal system. The level of haemolymph ecdysteroids, the insect moulting hormone, was found to be higher in virus-infected larvae than in uninfected cont...

Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives

PubMed Central

Haase, Santiago; Sciocco-Cap, Alicia; Romanowski, Víctor

2015-01-01

Baculoviruses are known to regulate many insect populations in nature. Their host-specificity is very high, usually restricted to a single or a few closely related insect species. They are amongst the safest pesticides, with no or negligible effects on non-target organisms, including beneficial insects, vertebrates and plants. Baculovirus-based pesticides are compatible with integrated pest management strategies and the expansion of their application will significantly reduce the risks associated with the use of synthetic chemical insecticides. Several successful baculovirus-based pest control programs have taken place in Latin American countries. Sustainable agriculture (a trend promoted by state authorities in most Latin American countries) will benefit from the wider use of registered viral pesticides and new viral products that are in the process of registration and others in the applied research pipeline. The success of baculovirus-based control programs depends upon collaborative efforts among government and research institutions, growers associations, and private companies, which realize the importance of using strategies that protect human health and the environment at large. Initiatives to develop new regulations that promote the use of this type of ecological alternatives tailored to different local conditions and farming systems are underway. PMID:25941826

Baculovirus replication induces the expression of heat shock proteins in vivo and in vitro

USDA-ARS?s Scientific Manuscript database

A recent handful of studies have linked baculovirus infection with the induction of heat shock proteins, a highly conserved family of cytoprotective proteins. Here, we demonstrate baculovirus-stimulated upregulation of hsp70 transcription in the natural host, Helicoverpa zea. Larvae lethally infec...

Reaching the Melting Point: Degradative Enzymes and Protease Inhibitors Involved in Baculovirus Infection and Dissemination

PubMed Central

Ishimwe, Egide; Hodgson, Jeffrey J.; Clem, Rollie J.; Passarelli, A. Lorena

2015-01-01

Baculovirus infection of a host insect involves several steps, beginning with initiation of virus infection in the midgut, followed by dissemination of infection from the midgut to other tissues in the insect, and finally culminating in “melting” or liquefaction of the host, which allows for horizontal spread of infection to other insects. While all of the viral gene products are involved in ultimately reaching this dramatic infection endpoint, this review focuses on two particular types of baculovirus-encoded proteins: degradative enzymes and protease inhibitors. Neither of these types of proteins is commonly found in other virus families, but they both play important roles in baculovirus infection. The types of degradative enzymes and protease inhibitors encoded by baculoviruses are discussed, as are the roles of these proteins in the infection process. PMID:25724418

Covert Infection of Insects by Baculoviruses.

PubMed

Williams, Trevor; Virto, Cristina; Murillo, Rosa; Caballero, Primitivo

2017-01-01

Baculoviruses ( Baculoviridae ) are occluded DNA viruses that are lethal pathogens of the larval stages of some lepidopterans, mosquitoes, and sawflies (phytophagous Hymenoptera). These viruses have been developed as biological insecticides for control of insect pests and as expression vectors in biotechnological applications. Natural and laboratory populations frequently harbor covert infections by baculoviruses, often at a prevalence exceeding 50%. Covert infection can comprise either non-productive latency or sublethal infection involving low level production of virus progeny. Latency in cell culture systems involves the expression of a small subset of viral genes. In contrast, covert infection in lepidopterans is associated with differential infection of cell types, modulation of virus gene expression and avoidance of immune system clearance. The molecular basis for covert infection may reside in the regulation of host-virus interactions through the action of microRNAs (miRNA). Initial findings suggest that insect nudiviruses and vertebrate herpesviruses may provide useful analogous models for exploring the mechanisms of covert infection by baculoviruses. These pathogens adopt mixed-mode transmission strategies that depend on the relative fitness gains that accrue through vertical and horizontal transmission. This facilitates virus persistence when opportunities for horizontal transmission are limited and ensures virus dispersal in migratory host species. However, when host survival is threatened by environmental or physiological stressors, latent or persistent infections can be activated to produce lethal disease, followed by horizontal transmission. Covert infection has also been implicated in population level effects on host-pathogen dynamics due to the reduced reproductive capacity of infected females. We conclude that covert infections provide many opportunities to examine the complexity of insect-virus pathosystems at the organismal level and to explore

Covert Infection of Insects by Baculoviruses

PubMed Central

Williams, Trevor; Virto, Cristina; Murillo, Rosa; Caballero, Primitivo

2017-01-01

Baculoviruses (Baculoviridae) are occluded DNA viruses that are lethal pathogens of the larval stages of some lepidopterans, mosquitoes, and sawflies (phytophagous Hymenoptera). These viruses have been developed as biological insecticides for control of insect pests and as expression vectors in biotechnological applications. Natural and laboratory populations frequently harbor covert infections by baculoviruses, often at a prevalence exceeding 50%. Covert infection can comprise either non-productive latency or sublethal infection involving low level production of virus progeny. Latency in cell culture systems involves the expression of a small subset of viral genes. In contrast, covert infection in lepidopterans is associated with differential infection of cell types, modulation of virus gene expression and avoidance of immune system clearance. The molecular basis for covert infection may reside in the regulation of host–virus interactions through the action of microRNAs (miRNA). Initial findings suggest that insect nudiviruses and vertebrate herpesviruses may provide useful analogous models for exploring the mechanisms of covert infection by baculoviruses. These pathogens adopt mixed-mode transmission strategies that depend on the relative fitness gains that accrue through vertical and horizontal transmission. This facilitates virus persistence when opportunities for horizontal transmission are limited and ensures virus dispersal in migratory host species. However, when host survival is threatened by environmental or physiological stressors, latent or persistent infections can be activated to produce lethal disease, followed by horizontal transmission. Covert infection has also been implicated in population level effects on host–pathogen dynamics due to the reduced reproductive capacity of infected females. We conclude that covert infections provide many opportunities to examine the complexity of insect–virus pathosystems at the organismal level and to

Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

PubMed

Xuan, X; Maeda, K; Mikami, T; Otsuka, H

1996-12-01

The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells

PubMed Central

Nogal, María L.; González de Buitrago, Gonzalo; Rodríguez, Clara; Cubelos, Beatriz; Carrascosa, Angel L.; Salas, María L.; Revilla, Yolanda

2001-01-01

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis. PMID:11222676

African swine fever virus IAP homologue inhibits caspase activation and promotes cell survival in mammalian cells.

PubMed

Nogal, M L; González de Buitrago, G; Rodríguez, C; Cubelos, B; Carrascosa, A L; Salas, M L; Revilla, Y

2001-03-01

African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

PubMed

López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

PubMed Central

Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

Baculovirus expression system and method for high throughput expression of genetic material

DOEpatents

Clark, Robin; Davies, Anthony

2001-01-01

The present invention provides novel recombinant baculovirus expression systems for expressing foreign genetic material in a host cell. Such expression systems are readily adapted to an automated method for expression foreign genetic material in a high throughput manner. In other aspects, the present invention features a novel automated method for determining the function of foreign genetic material by transfecting the same into a host by way of the recombinant baculovirus expression systems according to the present invention.

Pellino-1 confers chemoresistance in lung cancer cells by upregulating cIAP2 through Lys63-mediated polyubiquitination

PubMed Central

Koh, Jaemoon; Chung, Doo Hyun

2016-01-01

Pellino-1 is an E3 ubiquitin ligase that mediates immune receptor signaling pathways. The role of Pellino-1 in oncogenesis of lung cancer was investigated in this study. Pellino-1 expression was increased in human lung cancer cell lines compared with non-neoplastic lung cell lines. Pellino-1 overexpression in human lung cancer cells, A549 and H1299 cells, increased the survival and colony forming ability. Pellino-1 overexpression in these cells also conferred resistance to cisplatin- or paclitaxel-induced apoptosis. In contrast, depletion of Pellino-1 decreased the survival of A549 and H1299 cells and sensitized these cells to cisplatin- and paclitaxel-induced apoptosis. Pellino-1 overexpression in A549 and H1299 cells upregulated the expression of inhibitor of apoptosis (IAP) proteins, including cIAP1 and cIAP2, while Pellino-1 depletion downregulated these molecules. Notably, Pellino-1 directly interacted with cIAP2 and stabilized cIAP2 through lysine63-mediated polyubiquitination via its E3 ligase activity. Pellino-1-mediated chemoresistance in lung cancer cells was dependent on the induction of cIAP2. Moreover, a strong positive correlation between Pellino-1 and the cIAP2 expression was observed in human lung adenocarcinoma tissues. Taken together, these results demonstrate that Pellino-1 contributes to lung oncogenesis through the overexpression of cIAP2 and promotion of cell survival and chemoresistance. Pellino-1 might be a novel oncogene and potential therapeutic target in lung cancer. PMID:27248820

Construction of a highly efficient display system for baculovirus and its application on multigene co-display.

PubMed

Zheng, Hao; Wang, Xiong; Ren, Feifei; Zou, Shenglong; Feng, Min; Xu, Liangliang; Yao, Lunguang; Sun, Jingchen

2018-06-19

The classical baculovirus display system (BDS) has often recruited fields including gene delivery, gene therapy, and the genetic engineering of vaccines, as it is capable of presenting foreign polypeptides on the membranes of recombinant baculovirus through a transmembrane protein. However, classical BDS's high cost, complicated operation, low display efficiency and its inability to simultaneously display multiple gene products impede its practicality. In this study, we present a novel and highly efficient display system based on ires-dependent gp64 for rescuing gp64-null Bacmid of baculovirus construction without affecting the viral replication cycle, which we name the baculovirus multigene display system (BMDS). Laser scanning confocal microscopy demonstrated that eGFP, eYFP, and mCherry were translocated on the membrane of Spodoptera frugiperda 9 cell successfully as expected. Western blot analysis further confirmed the presence of the fluorescent proteins on the budded, mature viral particles. The results showed the display efficiency of target gene on cell surface is fourfold that of classical BDS. In addition, a recombinant baculovirus displaying three kinds of fluorescent proteins simultaneously was constructed, thereby demonstrating the effectiveness of BMDS as a co-display system.

Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes

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

Ono, M.

1986-06-01

By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identifiedmore » as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent.« less

Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology.

PubMed

Palomares, Laura A; Srivastava, Indresh K; Ramírez, Octavio T; Cox, Manon M J

2018-06-10

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

PubMed

Turunen, Tytteli Anni Kaarina; Laakkonen, Johanna Päivikki; Alasaarela, Laura; Airenne, Kari Juhani; Ylä-Herttuala, Seppo

2014-01-01

A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

PubMed

Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

2017-10-10

Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP + mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP + cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP + mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

Effect of spray drying processing parameters on the insecticidal activity of two encapsulated formulations of baculovirus

USDA-ARS?s Scientific Manuscript database

The aim of this work was to evaluate the effect of spray dryer processing parameters on the process yield and insecticidal activity of baculovirus to support the development of this beneficial group of microbes as biopesticides. For each of two baculoviruses [granulovirus (GV) from Pieris rapae (L....

Improved replication of the baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) in vitro using proteins from Lonomia obliqua hemolymph.

PubMed

Sousa, Álvaro P B; Moraes, Roberto H P; Mendonça, Ronaldo Z

2015-03-01

The baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV), a member of the family Baculoviridae, has been widely applied as a biopesticide for the control of the velvetbean caterpillar, a pest of soybean crop field. Baculoviruses are considered safe and efficient agents for this purpose, because they do not infect vertebrates, being safe for the health of humans and animals, as well as to the environment. The objective of this work was to identify proteins obtained from Lonomia obliqua hemolymph with potential application in the optimization of baculovirus AgMNPV replication in Sf9 insect cell culture. In this work the improvement of the cell culture and viral replication of the AgMNPV baculovirus was observed when Grace medium was supplemented with 10 % (v/v) Fetal Bovine Serum (FBS), 1 % (v/v) hemolymph extract, or 3 % (v/v) of hemolymph fractions or hemolymph sub-fractions obtained by purifying hemolymph through High Performance Liquid Chromatography. Hemolymph presented a positive effect on the synthesis of polyhedra and enhanced baculovirus replication in Spodoptera frugiperda (Sf9) cells (TCID50/mL), and led to Sf9 cell culture improvement. Grace medium supplemented with 10 % (v/v) FBS and 1 % (v/v) hemolymph provided an increase of baculovirus replication, when the cells were infected with multiplicity of infection of 1. In this case, the baculovirus replication was 6,443.91 times greater than that obtained with the control: Grace medium supplemented with 10 % (v/v) FBS. In addition, this work suggests that hemolymph from L. obliqua could have an interesting application in biotechnology, due to an increase in the viability of the cells and virus replication.

Baculovirus GP64-mediated entry into mammalian cells.

PubMed

Kataoka, Chikako; Kaname, Yuuki; Taguwa, Shuhei; Abe, Takayuki; Fukuhara, Takasuke; Tani, Hideki; Moriishi, Kohji; Matsuura, Yoshiharu

2012-03-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

Genetically-engineered baculovirus pesticides and their environmental safety

Treesearch

H. Alan Wood; Yu Zailin

1991-01-01

Baculoviruses such as the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) are ecologically attractive alternatives to chemical insect pesticides but have a slow rate of control. To overcome this we have developed and are field testing an environmentally acceptable strategy which can be used for the introduction and expression of pesticide-...

Tumors acquire inhibitor of apoptosis protein (IAP)-mediated apoptosis resistance through altered specificity of cytosolic proteolysis.

PubMed

Hong, Xu; Lei, Lu; Glas, Rickard

2003-06-16

Many tumors overexpress members of the inhibitor of apoptosis protein (IAP) family. IAPs contribute to tumor cell apoptosis resistance by the inhibition of caspases, and are degraded by the proteasome to allow further progression of apoptosis. Here we show that tumor cells can alter the specificity of cytosolic proteolysis in order to acquire apoptosis resistance, which promotes formation of rapidly growing tumors. Survival of tumor cells with low proteasomal activity can occur in the presence of high expression of Tri-peptidyl-peptidase II (TPP II), a large subtilisin-like peptidase that complements proteasomal activity. We find that this state leaves tumor cells unable of effectively degrading IAPs, and that cells in this state form rapidly growing tumors in vivo. We also find, in studies of apoptosis resistant cells derived from large in vivo tumors, that these have acquired an altered peptidase activity, with up-regulation of TPP II activity and decreased proteasomal activity. Importantly, we find that growth of subcutaneous tumors is limited by maintenance of the apoptosis resistant phenotype. The apoptosis resistant phenotype was reversed by increased expression of Smac/DIABLO, an antagonist of IAP molecules. Our data suggest a reversible mechanism in regulation of apoptosis resistance that drives tumor progression in vivo. These data are relevant in relation to the multitude of therapy-resistant clinical tumors that have increased levels of IAP molecules.

Revised IAP growth charts for height, weight and body mass index for 5- to 18-year-old Indian children.

PubMed

Khadilkar, Vaman; Yadav, Sangeeta; Agrawal, K K; Tamboli, Suchit; Banerjee, Monidipa; Cherian, Alice; Goyal, Jagdish P; Khadilkar, Anuradha; Kumaravel, V; Mohan, V; Narayanappa, D; Ray, I; Yewale, Vijay

2015-01-01

The need to revise Indian Academy of Pediatrics (IAP) growth charts for 5- to 18-year-old Indian children and adolescents was felt as India is in nutrition transition and previous IAP charts are based on data which are over two decades old. The Growth Chart Committee was formed by IAP in January 2014 to design revised growth charts. Consultative meeting was held in November 2014 in Mumbai. Studies performed on Indian children's growth, nutritional assessment and anthropometry from upper and middle socioeconomic classes in last decade were identified. Committee contacted 13 study groups; total number of children in the age group of 5 to 18 years were 87022 (54086 boys). Data from fourteen cities (Agartala, Ahmadabad, Chandigarh, Chennai, Delhi, Hyderabad, Kochi, Kolkata, Madurai, Mumbai, Mysore, Pune, Raipur and Surat) in India were collated. Data of children with weight for height Z scores >2 SD were removed from analyses. Data on 33148 children (18170 males, 14978 females) were used to construct growth charts using Cole's LMS method. To construct revised IAP growth charts for 5-18 year old Indian children based on collated national data from published studies performed on apparently healthy children and adolescents in the last 10 years. The IAP growth chart committee recommends these revised growth charts for height, weight and body mass index (BMI) for assessment of growth of 5-18 year old Indian children to replace the previous IAP charts; rest of the recommendations for monitoring height and weight remain as per the IAP guidelines published in 2007. To define overweight and obesity in children from 5-18 years of age, adult equivalent of 23 and 27 cut-offs presented in BMI charts may be used. IAP recommends use of WHO standards for growth assessment of children below 5 years of age.

Intrasystem Analysis Program (IAP) Structural Design Study.

DTIC Science & Technology

1981-06-01

accuracy constraints, and user competence . This report is designed to serve as a guide in con- structing procedures and identifying those aspects of the...parameters. 3.3.3 Userability The term "Userability" refers here to the level of competence assumed for an IAP analyst in need of a procedure. There...media the wires pass through is homogeneous along the length of the wires. Under these assumptions the wave propagation is predominantly tranverse

Inhibition of melanization by serpin-5 and serpin-9 promotes baculovirus infection in cotton bollworm Helicoverpa armigera

PubMed Central

Wang, Manli; Wang, Xi; Yin, Mengyi; Wang, Qianran; Hu, Zhihong

2017-01-01

Melanization, an important insect defense mechanism, is mediated by clip-domain serine protease (cSP) cascades and is regulated by serpins. Here we show that proteolytic activation of prophenoloxidase (PPO) and PO-catalyzed melanization kill the baculovirus in vitro. Our quantitative proteomics and biochemical experiments revealed that baculovirus infection of the cotton bollworm, Helicoverpa armigera, reduced levels of most cascade members in the host hemolymph and PO activity. By contrast, serpin-9 and serpin-5 were sequentially upregulated after the viral infection. The H. armigera serpin-5 and serpin-9 regulate melanization by directly inhibiting their target proteases cSP4 and cSP6, respectively and cSP6 activates PPO purified from hemolymph. Furthermore, serpin-5/9-depleted insects exhibited high PO activities and showed resistance to baculovirus infection. Together, our results characterize a part of the melanization cascade in H. armigera, and suggest that natural insect virus baculovirus has evolved a distinct strategy to suppress the host immune system. PMID:28953952

A cluster-based approach to selecting representative stimuli from the International Affective Picture System (IAPS) database.

PubMed

Constantinescu, Alexandra C; Wolters, Maria; Moore, Adam; MacPherson, Sarah E

2017-06-01

The International Affective Picture System (IAPS; Lang, Bradley, & Cuthbert, 2008) is a stimulus database that is frequently used to investigate various aspects of emotional processing. Despite its extensive use, selecting IAPS stimuli for a research project is not usually done according to an established strategy, but rather is tailored to individual studies. Here we propose a standard, replicable method for stimulus selection based on cluster analysis, which re-creates the group structure that is most likely to have produced the valence arousal, and dominance norms associated with the IAPS images. Our method includes screening the database for outliers, identifying a suitable clustering solution, and then extracting the desired number of stimuli on the basis of their level of certainty of belonging to the cluster they were assigned to. Our method preserves statistical power in studies by maximizing the likelihood that the stimuli belong to the cluster structure fitted to them, and by filtering stimuli according to their certainty of cluster membership. In addition, although our cluster-based method is illustrated using the IAPS, it can be extended to other stimulus databases.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

PubMed

Ardisson-Araújo, Daniel Mendes Pereira; Rocha, Juliana Ribeiro; da Costa, Márcio Hedil Oliveira; Bocca, Anamélia Lorenzetti; Dusi, André Nepomuceno; de Oliveira Resende, Renato; Ribeiro, Bergmann Morais

2013-08-15

Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in

IAP Antagonists Enhance Apoptotic Response to Enzalutamide in Castration-Resistant Prostate Cancer Cells via Autocrine TNF-α Signaling.

PubMed

Pilling, Amanda B; Hwang, Ok; Boudreault, Alain; Laurent, Alain; Hwang, Clara

2017-06-01

Castration-resistant prostate cancer (CRPC) remains incurable and identifying effective treatments continues to present a clinical challenge. Although treatment with enzalutamide, a second generation androgen receptor (AR) antagonist, prolongs survival in prostate cancer patients, responses can be limited by intrinsic resistance or acquired resistance. A potential mechanism of resistance to androgen axis inhibition is evasion of apoptosis. Inhibitor of apoptosis proteins (IAPs) are found to be overexpressed in prostate cancer and function to block apoptosis and promote survival signaling. Novel, small-molecule IAP antagonists, such as AEG40995, are emerging as a strategy to induce apoptosis and increase therapeutic response in cancer. Human prostate cancer cell lines LNCaP and C4-2 were treated with enzalutamide with or without addition of IAP antagonist AEG40995 and proliferation and survival were determined by MTS and clonogenic assay. Western blot was used to evaluate IAP protein expression changes and PARP-1 cleavage was assessed as indication of apoptosis. Flow cytometry was performed to analyze apoptosis in treated cells. Caspase activity was determined by luminescence assay. Quantitative real-time PCR and immunometric ELISA was used to assess TNF-α (transcript and protein levels, respectively) in response to treatment. In this study, we demonstrate that IAP antagonist AEG40995 exhibits minimal effects on prostate cancer cell proliferation or survival, but rapidly degrades cIAP1 protein. Combination treatment with enzalutamide demonstrates that AEG40995 increases apoptosis and reduces proliferation and clonogenic survival in cell line models of prostate cancer. Mechanistically, we demonstrate that apoptosis in response to enzalutamide and IAP antagonist requires activation of caspase-8, suggesting extrinsic/death receptor apoptosis signaling. Assessment of TNF-α in response to combination treatment with enzalutamide and AEG40995 reveals increased m

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

PubMed Central

Iacono-Connors, L C; Schmaljohn, C S; Dalrymple, J M

1990-01-01

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response. Images PMID:2105271

IKKε-mediated tumorigenesis requires K63-linked polyubiquitination by a cIAP1/cIAP2/TRAF2 E3 ubiquitin ligase complex

PubMed Central

Zhou, Alicia Y.; Shen, Rhine R.; Kim, Eejung; Lock, Ying J.; Xu, Ming; Chen, Zhijian J.; Hahn, William C.

2014-01-01

SUMMARY IκB kinase ε (IKKε, IKBKE) is a key regulator of innate immunity and a breast cancer oncogene, amplified in ~30% of breast cancers, that promotes malignant transformation through NF-κB activation. Here we show that IKKε is modified and regulated by K63-linked polyubiquitination at Lysine 30 and Lysine 401. TNFα and IL-1β stimulation induces IKKε K63-linked polyubiquitination over baseline levels in both macrophages and breast cancer cell lines, and this modification is essential for IKKε kinase activity, IKKε-mediated NF-κB activation and IKKε-induced malignant transformation. Disruption of K63-linked ubiquitination of IKKε does not affect its overall structure but impairs the recruitment of canonical NF-κB proteins. A cIAP1/cIAP2/TRAF2 E3 ligase complex binds to and ubiquitinates IKKε. Together, these observations demonstrate that K63-linked polyubiquitination regulates IKKε activity in both inflammatory and oncogenic contexts and suggests an alterative approach to target this breast cancer oncogene. PMID:23453969

Racial differences in the expression of inhibitors of apoptosis (IAP) proteins in extracellular vesicles (EV) from prostate cancer patients.

PubMed

Khan, Salma; Simpson, Jennifer; Lynch, James C; Turay, David; Mirshahidi, Saied; Gonda, Amber; Sanchez, Tino W; Casiano, Carlos A; Wall, Nathan R

2017-01-01

African-American men with prostate cancer typically develop more aggressive tumors than men from other racial/ethnic groups, resulting in a disproportionately high mortality from this malignancy. This study evaluated differences in the expression of inhibitors of apoptosis proteins (IAPs), a known family of oncoproteins, in blood-derived exosomal vesicles (EV) between African-American and European-American men with prostate cancer. The ExoQuick™ method was used to isolate EV from both plasma and sera of African-American (n = 41) and European-American (n = 31) men with prostate cancer, as well as from controls with no cancer diagnosis (n = 10). EV preparations were quantified by acetylcholinesterase activity assays, and assessed for their IAP content by Western blotting and densitometric analysis. Circulating levels of the IAP Survivin were evaluated by ELISA. We detected a significant increase in the levels of circulating Survivin in prostate cancer patients compared to controls (P<0.01), with the highest levels in African-American patients (P<0.01). African-American patients with prostate cancer also contained significantly higher amounts of EVs in their plasma (P<0.01) and sera (P<0.05) than European-American patients. In addition, EVs from African-American patients with prostate cancer contained significantly higher amounts of the IAPs Survivin (P<0.05), XIAP (P<0.001), and cIAP-2 (P<0.01) than EVs from European-American patients. There was no significant correlation between expression of IAPs and clinicopathological parameters in the two patient groups. Increased expression of IAPs in EVs from African-American patients with prostate cancer may influence tumor aggressiveness and contribute to the mortality disparity observed in this patient population. EVs could serve as reservoirs of novel biomarkers and therapeutic targets that may have clinical utility in reducing prostate cancer health disparities.

Display of a maize cDNA library on baculovirus infected insect cells.

PubMed

Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

2008-08-12

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

Localization of VP28 on the baculovirus envelope and its immunogenicity against white spot syndrome virus in Penaeus monodon

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

Syed Musthaq, S.; Madhan, Selvaraj; Sahul Hameed, A.S.

2009-09-01

White spot syndrome virus (WSSV) is a large dsDNA virus responsible for white spot disease in shrimp and other crustaceans. VP28 is one of the major envelope proteins of WSSV and plays a crucial role in viral infection. In an effort to develop a vaccine against WSSV, we have constructed a recombinant baculovirus with an immediate early promoter 1 which expresses VP28 at an early stage of infection in insect cells. Baculovirus expressed rVP28 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed thatmore » rVP28 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired rVP28 from the insect cell membrane via the budding process. Using this baculovirus displaying VP28 as a vaccine against WSSV, we observed a significantly higher survival rate of 86.3% and 73.5% of WSSV-infected shrimp at 3 and 15 days post vaccination respectively. Quantitative real-time PCR also indicated that the WSSV viral load in vaccinated shrimp was significantly reduced at 7 days post challenge. Furthermore, our RT-PCR and immunohistochemistry results demonstrated that the recombinant baculovirus was able to express VP28 in vivo in shrimp tissues. This study will be of considerable significance in elucidating the morphogenesis of WSSV and will pave the way for new generation vaccines against WSSV.« less

The small-molecule IAP antagonist AT406 inhibits pancreatic cancer cells in vitro and in vivo

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

Jiang, Yongsheng; Meng, Qinghua; Chen, Bo

In the present study, we tested the anti-pancreatic cancer activity by AT406, a small-molecule antagonist of IAP (inhibitor of apoptosis proteins). In established (Panc-1 and Mia-PaCa-2 lines) and primary human pancreatic cancer cells, treatment of AT406 significantly inhibited cell survival and proliferation. Yet, same AT406 treatment was non-cytotoxic to pancreatic epithelial HPDE6c7 cells. AT406 increased caspase-3/-9 activity and provoked apoptosis in the pancreatic cancer cells. Reversely, AT406′ cytotoxicity in these cells was largely attenuated with pre-treatment of caspase inhibitors. AT406 treatment caused degradation of IAP family proteins (cIAP1 and XIAP) and release of cytochrome C, leaving Bcl-2 unaffected in pancreaticmore » cancer cells. Bcl-2 inhibition (by ABT-737) or shRNA knockdown dramatically sensitized Panc-1 cells to AT406. In vivo, oral administration of AT406 at well-tolerated doses downregulated IAPs (cIAP1/XIAP) and inhibited Panc-1 xenograft tumor growth in severe combined immunodeficient (SCID) nude mice. Together, our preclinical results suggest that AT406 could be further evaluated as a promising anti-pancreatic cancer agent. - Highlights: • AT406 is cytotoxic to established/primary human pancreatic cancer cells. • AT406 provokes caspase-dependent apoptosis in pancreatic cancer cells. • AT406 causes degradation of key IAPs and promotes cytochrome C release. • Bcl-2 inhibition or knockdown dramatically sensitizes Panc-1 cells to AT406. • Oral administration of AT406 inhibits Panc-1 tumor growth in SCID nude mice.« less

Critical role of endogenous Akt/IAPs and MEK1/ERK pathways in counteracting endoplasmic reticulum stress-induced cell death.

PubMed

Hu, Ping; Han, Zhang; Couvillon, Anthony D; Exton, John H

2004-11-19

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of many diseases and in cancer therapy. Although the unfolded protein response is known to alleviate ER stress by reducing the accumulation of misfolded proteins, the exact survival elements and their downstream signaling pathways that directly counteract ER stress-stimulated apoptotic signaling remain elusive. Here, we have shown that endogenous Akt and ERK are rapidly activated and act as downstream effectors of phosphatidylinositol 3-kinase in thapsigargin- or tunicamycin-induced ER stress. Introduction of either dominant-negative Akt or MEK1 or the inhibitors LY294002 and U0126 sensitized cells to ER stress-induced cell death in different cell types. Reverse transcription-PCR analysis of gene expression during ER stress revealed that cIAP-2 and XIAP, members of the IAP family of potent caspase suppressors, were strongly induced. Transcription of cIAP-2 and XIAP was up-regulated by the phosphatidylinositol 3-kinase/Akt pathway as shown by its reversal by dominant-negative Akt or LY294002. Ablation of these IAPs by RNA interference sensitized cells to ER stress-induced death, which was reversed by the caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone. The protective role of IAPs in ER stress coincided with Smac release from mitochondria to the cytosol. Furthermore, it was shown that mTOR was not required for Akt-mediated survival. These results represent the first demonstration that activation of endogenous Akt/IAPs and MEK/ERK plays a critical role in controlling cell survival by resisting ER stress-induced cell death signaling.

Identification of mTOR as a primary resistance factor of the IAP antagonist AT406 in hepatocellular carcinoma cells

PubMed Central

Wu, Shao-Feng; Zhao, Yi-Lin; Liu, Ping-Guo; Yin, Zhen-Yu

2017-01-01

Dysregulation of inhibitor of apoptosis (IAP) proteins (IAPs) in hepatocellular carcinoma (HCC) is often associated with poor prognosis. Here we showed that AT406, an IAP antagonist, was cytotoxic and pro-apoptotic to both established (HepG2, SMMC-7721 lines) and primary HCC cells. Activation of mTOR could be a key resistance factor of AT406 in HCC cells. mTOR inhibition (by OSI-027), kinase-dead mutation or knockdown remarkably enhanced AT406-induced lethality in HCC cells. Reversely, forced-activation of mTOR by adding SC79 or exogenous expressing a constitutively active S6K1 (T389E) attenuated AT406-induced cytotoxicity against HCC cells. We showed that AT406 induced degradation of IAPs (cIAP-1 and XIAP), but didn't affect another anti-apoptosis protein Mcl-1. Co-treatment of OSI-027 caused simultaneous Mcl-1 downregulation to overcome AT406's resistance. Significantly, shRNA knockdown of Mcl-1 remarkably facilitated AT406-induced apoptosis in HCC cells. In vivo, AT406 oral administration suppressed HepG2 tumor growth in nude mice. Its activity was potentiated with co-administration of OSI-027. We conclude that mTOR could be a key resistance factor of AT406 in HCC cells. PMID:28036295

Identification of mTOR as a primary resistance factor of the IAP antagonist AT406 in hepatocellular carcinoma cells.

PubMed

Zhen, Mao-Chuan; Wang, Fu-Qiang; Wu, Shao-Feng; Zhao, Yi-Lin; Liu, Ping-Guo; Yin, Zhen-Yu

2017-02-07

Dysregulation of inhibitor of apoptosis (IAP) proteins (IAPs) in hepatocellular carcinoma (HCC) is often associated with poor prognosis. Here we showed that AT406, an IAP antagonist, was cytotoxic and pro-apoptotic to both established (HepG2, SMMC-7721 lines) and primary HCC cells. Activation of mTOR could be a key resistance factor of AT406 in HCC cells. mTOR inhibition (by OSI-027), kinase-dead mutation or knockdown remarkably enhanced AT406-induced lethality in HCC cells. Reversely, forced-activation of mTOR by adding SC79 or exogenous expressing a constitutively active S6K1 (T389E) attenuated AT406-induced cytotoxicity against HCC cells. We showed that AT406 induced degradation of IAPs (cIAP-1 and XIAP), but didn't affect another anti-apoptosis protein Mcl-1. Co-treatment of OSI-027 caused simultaneous Mcl-1 downregulation to overcome AT406's resistance. Significantly, shRNA knockdown of Mcl-1 remarkably facilitated AT406-induced apoptosis in HCC cells. In vivo, AT406 oral administration suppressed HepG2 tumor growth in nude mice. Its activity was potentiated with co-administration of OSI-027. We conclude that mTOR could be a key resistance factor of AT406 in HCC cells.

Baculovirus-based genome editing in primary cells.

PubMed

Mansouri, Maysam; Ehsaei, Zahra; Taylor, Verdon; Berger, Philipp

2017-03-01

Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template. Recombinant Lentivirus or Adenovirus genomes have enough capacity for a nuclease coding sequence and the gRNA but are usually too small to also carry large targeting constructs. We recently showed that a baculovirus-based multigene expression system (MultiPrime) can be used for genome editing in primary cells since it possesses the necessary capacity to carry the nuclease and gRNA expression constructs and the HDR targeting sequences. Here we present new Acceptor plasmids for MultiPrime that allow simplified cloning of baculoviruses for genome editing and we show their functionality in primary cells with limited life span and induced pluripotent stem cells (iPS). Copyright © 2017 Elsevier Inc. All rights reserved.

Construction of a recombinant baculovirus expressing swine hepatitis E Virus ORF2 and preliminary research on its immune effect.

PubMed

Yang, Z; Hu, Y; Yuan, P; Yang, Y; Wang, K; Xie, L Y; Huang, S L; Liu, J; Ran, L; Song, Z H

2018-03-01

In the swine hepatitis E virus (HEV), open reading frame 2 (ORF2) is rich in antigenic determinants and neutralizing epitopes that could induce immune protection. We chose the Bac-to-Bac® Baculovirus Expression System to express fragments containing the critical neutralizing antigenic sites within the HEV ORF2 protein of pigs to obtain a recombinant baculovirus. The fragment of swine HEV ORF2 region (1198-1881bp) was cloned into vector pFastBacTM. A recombinant baculovirus, rBacmid-ORF2, was obtained after transposition and transfection. The molecular mass of the recombinant protein was 26 kDa. Mice were immunized by the intraperitoneal and oral routes with cell lysates of recombinant baculovirus rBacmid-ORF2. Serum and feces of the mice were collected separately at 0, 14, 28, and 42 d after immunization and the antibody levels of IgG and secretory IgA against swine HEV were determined using an enzyme-linked immunosorbent assay. The results suggested that rBacmid-ORF2 induced antibodies of the humoral and mucosal immune responses in mice and that the oral route was significantly superior to the intraperitoneal route. This is the first study to demonstrate that that recombinant baculovirus swine HEV ORF2 could induce humoral and mucosal immune responses in mice. Copyright© by the Polish Academy of Sciences.

cIAPs promote the proteasomal degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis.

PubMed

Choi, Jin Sun; Kim, Kidae; Lee, Do Hee; Cho, Sayeon; Ha, Jae Du; Park, Byoung Chul; Kim, Sunhong; Park, Sung Goo; Kim, Jeong-Hoon

2016-11-18

Although the ubiquitin-proteasome system is believed to play an important role in the pathogenesis of familial amyotrophic lateral sclerosis (FALS), caused by mutations in Cu/Zn-superoxide dismutase 1 (SOD1), the mechanism of how mutant SOD1 protein is regulated in cells is still poorly understood. Here we have demonstrated that cellular inhibitor of apoptosis proteins (cIAPs) are specifically associated with FALS-linked mutant SOD1 (mSOD1) and that this interaction promotes the ubiquitin-dependent proteasomal degradation of mutant SOD1. By utilizing cumate inducible SOD1 cells, we also showed that knock-down or pharmacologic depletion of cIAPs leads to H 2 O 2 induced cytotoxicity in mSOD1 expressing cells. Altogether, our results reveal a novel role of cIAPs in FALS-associated mutant SOD1 regulation. Copyright © 2016 Elsevier Inc. All rights reserved.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

PubMed Central

Armstrong, Chris W.D.; Maxwell, Pamela J.; Ong, Chee Wee; Redmond, Kelly M.; McCann, Christopher; Neisen, Jessica; Ward, George A.; Chessari, Gianni; Johnson, Christopher; Crawford, Nyree T.; LaBonte, Melissa J.; Prise, Kevin M.; Robson, Tracy; Salto-Tellez, Manuel; Longley, Daniel B.; Waugh, David J.J.

2016-01-01

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy. PMID:26799286

Expression of the hemagglutinin HA1 subunit of the equine influenza virus using a baculovirus expression system.

PubMed

Sguazza, Guillermo H; Fuentealba, Nadia A; Tizzano, Marco A; Galosi, Cecilia M; Pecoraro, Marcelo R

2013-01-01

Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens' eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests. Copyright © 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

Insect cells-baculovirus system for the production of difficult to express proteins.

PubMed

Osz-Papai, Judit; Radu, Laura; Abdulrahman, Wassim; Kolb-Cheynel, Isabelle; Troffer-Charlier, Nathalie; Birck, Catherine; Poterszman, Arnaud

2015-01-01

The production of sufficient quantities of homogenous protein not only is an essential prelude for structural investigations but also represents a rate-limiting step for many human functional studies. Although technologies for expression of recombinant proteins and complexes have been improved tremendously, in many cases, protein production remains a challenge and can be associated with considerable investment. This chapter describes simple and efficient protocols for expression screening and optimization of protein production in insect cells using the baculovirus expression system. We describe the procedure, starting from the cloning of a gene of interest into an expression transfer baculovirus vector, followed by generation of the recombinant virus by homologous recombination, evaluation of protein expression, and scale-up. Handling of insect cell cultures and preparation of bacmid for co-transfection are also detailed.

Ground correlation investigation of thruster spacecraft interactions to be measured on the IAPS flight test

NASA Technical Reports Server (NTRS)

Power, J. L.

1984-01-01

Preliminary ground correlation testing has been conducted with an 8 cm mercury ion thruster and diagnostic instrumentation replicating to a large extent the IAPS flight test hardware, configuration, and electrical grounding/isolation. Thruster efflux deposition retained at 25 C was measured and characterized. Thruster ion efflux was characterized with retarding potential analyzers. Thruster-generated plasma currents, the spacecraft common (SCC) potential, and ambient plasma properties were evaluated with a spacecraft potential probe (SPP). All the measured thruster/spacecraft interactions or their IAPS measurements depend critically on the SCC potential, which can be controlled by a neutralizer ground switch and by the SPP operation.

Modularity and evolutionary constraints in a baculovirus gene regulatory network

PubMed Central

2013-01-01

Background The structure of regulatory networks remains an open question in our understanding of complex biological systems. Interactions during complete viral life cycles present unique opportunities to understand how host-parasite network take shape and behave. The Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is a large double-stranded DNA virus, whose genome may encode for 152 open reading frames (ORFs). Here we present the analysis of the ordered cascade of the AgMNPV gene expression. Results We observed an earlier onset of the expression than previously reported for other baculoviruses, especially for genes involved in DNA replication. Most ORFs were expressed at higher levels in a more permissive host cell line. Genes with more than one copy in the genome had distinct expression profiles, which could indicate the acquisition of new functionalities. The transcription gene regulatory network (GRN) for 149 ORFs had a modular topology comprising five communities of highly interconnected nodes that separated key genes that are functionally related on different communities, possibly maximizing redundancy and GRN robustness by compartmentalization of important functions. Core conserved functions showed expression synchronicity, distinct GRN features and significantly less genetic diversity, consistent with evolutionary constraints imposed in key elements of biological systems. This reduced genetic diversity also had a positive correlation with the importance of the gene in our estimated GRN, supporting a relationship between phylogenetic data of baculovirus genes and network features inferred from expression data. We also observed that gene arrangement in overlapping transcripts was conserved among related baculoviruses, suggesting a principle of genome organization. Conclusions Albeit with a reduced number of nodes (149), the AgMNPV GRN had a topology and key characteristics similar to those observed in complex cellular organisms, which indicates

Iron levels change in larval Heliothis virescens tissues following baculovirus infection

USDA-ARS?s Scientific Manuscript database

Inductively-coupled plasma mass spectrometry (ICP-MS) and 59Fe radiotracers were used to investigate changes in levels of iron (Fe) in the tissues of Heliothis virescens following baculovirus infection. Fe concentrations were determined by ICP-MS in hemolymph collected from 4th instar larvae infect...

Expression of the lef5 gene from Spodoptera exigua multiple nucleopolyhedrovirus contributes to the baculovirus stability in cell culture.

PubMed

Martínez-Solís, María; Jakubowska, Agata K; Herrero, Salvador

2017-10-01

Baculoviruses are a broad group of viruses infecting insects, predominately of the order Lepidoptera. They are used worldwide as biological insecticides and as expression vectors to produce recombinant proteins. Baculoviruses replicate in their host, although several cell lines have been developed for in vitro replication. Nevertheless, replication of baculoviruses in cell culture involves the generation of defective viruses with a decrease in productivity and virulence. Transcriptional studies of the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infective process revealed differences in the expression patterns when the virus replicated under in vitro (Se301 cells) or in vivo (S. exigua larvae) conditions. The late expression factor 5 (lef5) gene was found to be highly overexpressed when the virus replicates in larvae. To test the possible role of lef5 expression in viral stability, recombinant AcMNPV expressing the lef5 gene from SeMNPV (Se-lef5) was generated and its stability was monitored during successive infection passages in Sf21 cells by evaluating the loss of several essential and non-essential genes. The gfp transgene was more stable in those viruses expressing the Se-LEF5 protein and the GFP-defective viruses were accumulated at a lower level when compared to its control viruses, confirming the positive influence of lef5 in viral stability during the multiplication process. This work describes for the first time a viral factor involved in transgene stability when baculoviruses replicate in cell culture, opening new ways to facilitate the in vitro production of recombinant proteins using baculovirus.

EnOI-IAU Initialization Scheme Designed for Decadal Climate Prediction System IAP-DecPreS

NASA Astrophysics Data System (ADS)

Wu, Bo; Zhou, Tianjun; Zheng, Fei

2018-02-01

A decadal climate prediction system named as IAP-DecPreS was constructed in the Institute of Atmospheric Physics (IAP), Chinese Academy of Sciences, based on a fully coupled model FGOALS-s2 and a newly developed initialization scheme, referred to as EnOI-IAU. In this paper, we introduce the design of the EnOI-IAU scheme, assess the accuracies of initialization integrations using the EnOI-IAU and preliminarily evaluate hindcast skill of the IAP-DecPreS. The EnOI-IAU scheme integrates two conventional assimilation approaches, ensemble optimal interpolation (EnOI) and incremental analysis update (IAU). The EnOI and IAU were applied to calculate analysis increments and incorporate them into the model, respectively. Three continuous initialization (INIT) runs were conducted for the period of 1950-2015, in which observational sea surface temperature (SST) from the HadISST1.1 and subsurface ocean temperature profiles from the EN4.1.1 data set were assimilated. Then nine-member 10 year long hindcast runs initiated from the INIT runs were conducted for each year in the period of 1960-2005. The accuracies of the INIT runs are evaluated from the following three aspects: upper 700 m ocean temperature, temporal evolution of SST anomalies, and dominant interdecadal variability modes, Pacific Decadal Oscillation (PDO) and Atlantic Multidecadal Oscillation (AMO). Finally, preliminary evaluation of the ensemble mean of the hindcast runs suggests that the IAP-DecPreS has skill in the prediction of the PDO-related SST anomalies in the midlatitude North Pacific and AMO-related SST anomalies in the tropical North Atlantic.

Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody.

PubMed

Dhungel, Bidur; Ohno, Yoshikazu; Matayoshi, Rie; Otaki, Joji M

2013-03-25

Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally

Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody

PubMed Central

2013-01-01

Background Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. Results A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Conclusions Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer

[Use of a novel baculovirus vector to express nucleoprotein gene of Crimean-Congo hemorrhagic fever virus in both insect and mammalian cells].

PubMed

Ma, Benjiang; Hang, Changshou; Zhao, Yun; Wang, Shiwen; Xie, Yanxiang

2002-09-01

To construct a novel baculovirus vector which is capable of promoting the high-yield expression of foreign gene in mammalian cells and to express by this vector the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV) Chinese isolate (Xinjiang hemorrhagic fever virus, XHFV) BA88166 in insect and Vero cells. Human cytomegalovirus (CMV) immediate early (IE) promoter was ligated to the baculovirus vector pFastBac1 downstream of the polyhedrin promoter to give rise to the novel vector pCB1. XHFV NP gene was cloned to this vector and was well expressed in COS-7 cells and Vero cells by means of recombinant plasmid transfection and baculovirus infection. The XHFV NP gene in vector pCB1 could be well expressed in mammalian cells. Vero cells infected with recombinant baculovirus harboring NP gene could be employed as antigens to detect XHF serum specimens whose results were in good correlation with those of ELISA and in parallel with clinical diagnoses. This novel baculovirus vector is able to express the foreign gene efficiently in both insect and mammalian cells, which provides not only the convenient diagnostic antigens but also the potential for developing recombinant virus vaccines and gene therapies.

Description and Evaluation of IAP-AACM: A Global-regional Aerosol Chemistry Model for the Earth System Model CAS-ESM

NASA Astrophysics Data System (ADS)

Wei, Y.; Chen, X.

2017-12-01

We present a first description and evaluation of the IAP Atmospheric Aerosol Chemistry Model (IAP-AACM) which has been integrated into the earth system model CAS-ESM. In this way it is possible to research into interaction of clouds and aerosol by its two-way coupling with the IAP Atmospheric General Circulation Model (IAP-AGCM). The model has a nested global-regional grid based on the Global Environmental Atmospheric Transport Model (GEATM) and the Nested Air Quality Prediction Modeling System (NAQPMS). The AACM provides two optional gas chemistry schemes, the CBM-Z gas chemistry as well as a sulfur oxidize box designed specifically for the CAS-ESM. Now the model driven by AGCM has been applied to a 1-year simulation of tropospheric chemistry both on global and regional scales for 2014, and been evaluated against various observation datasets, including aerosol precursor gas concentration, aerosol mass and number concentrations. Furthermore, global budgets in AACM are compared with other global aerosol models. Generally, the AACM simulations are within the range of other global aerosol model predictions, and the model has a reasonable agreement with observations of gases and particles concentration both on global and regional scales.

The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

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

Long, C.M.; Rohrmann, G.F.; Merrill, G.F., E-mail: merrillg@onid.orst.ed

2009-06-05

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involvedmore » in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.« less

The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

PubMed

Long, C M; Rohrmann, G F; Merrill, G F

2009-06-05

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

MEMBRANOUS LABYRINTH IN BACULOVIRUS-INFECTED CRUSTRACEAN CELLS: POSSIBLE ROLES IN VIRAL REPRODUCTION

EPA Science Inventory

The origins and morphogenesis of the membranous labyrinth (ML) in Baculovirus penaei (BP) infected cells of penaeid shrimps (Crustacea:Decapoda) are described. t is hypothesized that, because of the close parallel and concurrent development of the ML and virus reproduction, and o...

In Vivo Knockdown of Pathogenic Proteins via Specific and Nongenetic Inhibitor of Apoptosis Protein (IAP)-dependent Protein Erasers (SNIPERs).

PubMed

Ohoka, Nobumichi; Okuhira, Keiichiro; Ito, Masahiro; Nagai, Katsunori; Shibata, Norihito; Hattori, Takayuki; Ujikawa, Osamu; Shimokawa, Kenichiro; Sano, Osamu; Koyama, Ryokichi; Fujita, Hisashi; Teratani, Mika; Matsumoto, Hirokazu; Imaeda, Yasuhiro; Nara, Hiroshi; Cho, Nobuo; Naito, Mikihiko

2017-03-17

Many diseases, especially cancers, result from aberrant or overexpression of pathogenic proteins. Specific inhibitors against these proteins have shown remarkable therapeutic effects, but these are limited mainly to enzymes. An alternative approach that may have utility in drug development relies on selective degradation of pathogenic proteins via small chimeric molecules linking an E3 ubiquitin ligase to the targeted protein for proteasomal degradation. To this end, we recently developed a protein knockdown system based on hybrid small molecule SNIPERs ( S pecific and N ongenetic I AP-dependent P rotein Er asers) that recruit inhibitor of the apoptosis protein (IAP) ubiquitin ligases to specifically degrade targeted proteins. Here, we extend our previous study to show a proof of concept of the SNIPER technology in vivo By incorporating a high affinity IAP ligand, we developed a novel SNIPER against estrogen receptor α (ERα), SNIPER(ER)-87, that has a potent protein knockdown activity. The SNIPER(ER) reduced ERα levels in tumor xenografts and suppressed the growth of ERα-positive breast tumors in mice. Mechanistically, it preferentially recruits X-linked IAP (XIAP) rather than cellular IAP1, to degrade ERα via the ubiquitin-proteasome pathway. With this IAP ligand, potent SNIPERs against other pathogenic proteins, BCR-ABL, bromodomain-containing protein 4 (BRD4), and phosphodiesterase-4 (PDE4) could also be developed. These results indicate that forced ubiquitylation by SNIPERs is a useful method to achieve efficient protein knockdown with potential therapeutic activities and could also be applied to study the role of ubiquitylation in many cellular processes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Autophagy-mediated degradation of IAPs and c-FLIP L potentiates apoptosis induced by combination of TRAIL and Chal-24

DOE PAGES

Xu, Jennings; Xu, Xiuling; Shi, Shaoqing; ...

2015-11-02

Combination chemotherapy is an effective strategy for increasing anticancer efficacy, reducing side effects and alleviating drug resistance. In this paper, we report that combination of the recently identified novel chalcone derivative, chalcone-24 (Chal-24), and TNF-related apoptosis-inducing ligand (TRAIL) significantly increases cytotoxicity in lung cancer cells. Chal-24 treatment significantly enhanced TRAIL-induced activation of caspase-8 and caspase-3, and the cytotoxicity induced by combination of these agents was effectively suppressed by the pan-caspase inhibitor z-VAD-fmk. Chal-24 and TRAIL combination suppressed expression of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (c-FLIPL) and cellular inhibitor of apoptosis proteins (c-IAPs), and ectopic expression of c-FLIPL andmore » c-IAPs inhibited the potentiated cytotoxicity. In addition, TRAIL and Chal-24 cooperatively activated autophagy. Suppression of autophagy effectively attenuated cytotoxicity induced by Chal-24 and TRAIL combination, which was associated with attenuation of c-FLIPL and c-IAPs degradation. In conclusion, these results suggest that Chal-24 potentiates the anticancer activity of TRAIL through autophagy-mediated degradation of c-FLIPL and c-IAPs, and that combination of Chal-24 and TRAIL could be an effective approach in improving chemotherapy efficacy.« less

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

PubMed

Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

2018-05-02

The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

Baculovirus-mediated vascular endothelial growth factor-D(ΔNΔC) gene transfer induces angiogenesis in rabbit skeletal muscle.

PubMed

Heikura, Tommi; Nieminen, Tiina; Roschier, Miia M; Karvinen, Henna; Kaikkonen, Minna U; Mähönen, Anssi J; Lesch, Hanna P; Rissanen, Tuomas T; Laitinen, Olli H; Airenne, Kari J; Ylä-Herttuala, Seppo

2012-01-01

Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding β-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene. Copyright © 2012 John Wiley & Sons, Ltd.

DEVELOPMENT OF AN IN SITU TOXICITY ASSAY SYSTEM USING RECOMBINANT BACULOVIRUSES. (R825433)

EPA Science Inventory

A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or m...

Development of protein degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL kinase inhibitors and IAP ligands.

PubMed

Shibata, Norihito; Miyamoto, Naoki; Nagai, Katsunori; Shimokawa, Kenichiro; Sameshima, Tomoya; Ohoka, Nobumichi; Hattori, Takayuki; Imaeda, Yasuhiro; Nara, Hiroshi; Cho, Nobuo; Naito, Mikihiko

2017-08-01

Chromosomal translocation occurs in some cancer cells, which results in the expression of aberrant oncogenic fusion proteins that include BCR-ABL in chronic myelogenous leukemia (CML). Inhibitors of ABL tyrosine kinase, such as imatinib and dasatinib, exhibit remarkable therapeutic effects, although emergence of drug resistance hampers the therapy during long-term treatment. An alternative approach to treat CML is to downregulate the BCR-ABL protein. We have devised a protein knockdown system by hybrid molecules named Specific and Non-genetic inhibitor of apoptosis protein [IAP]-dependent Protein Erasers (SNIPER), which is designed to induce IAP-mediated ubiquitylation and proteasomal degradation of target proteins, and a couple of SNIPER(ABL) against BCR-ABL protein have been developed recently. In this study, we tested various combinations of ABL inhibitors and IAP ligands, and the linker was optimized for protein knockdown activity of SNIPER(ABL). The resulting SNIPER(ABL)-39, in which dasatinib is conjugated to an IAP ligand LCL161 derivative by polyethylene glycol (PEG) × 3 linker, shows a potent activity to degrade the BCR-ABL protein. Mechanistic analysis suggested that both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) play a role in the degradation of BCR-ABL protein. Consistent with the degradation of BCR-ABL protein, the SNIPER(ABL)-39 inhibited the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and Crk like proto-oncogene (CrkL), and suppressed the growth of BCR-ABL-positive CML cells. These results suggest that SNIPER(ABL)-39 could be a candidate for a degradation-based novel anti-cancer drug against BCR-ABL-positive CML. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

[Expression of goat IL-18 mature protein in insect/baculovirus and determination of bioactivity of the recombinant protein].

PubMed

Wang, Ting-Ting; Wang, Xi-Hui; Fan, Zhong-Ling; Chen, Jin-Long; Cao, Bing-Lei; Kong, Na; Hu, Jing-Dong; Zhao, Hong-Kun

2011-02-01

To express goat IL-18 in insect/baculovirus and detect the bioactivity of the recombinant protein. The mature goat interleukin-18(gIL-18) gene was cloned into the baculovirus transfer vector pFastBac Dual, and then the resulting eukaryotic expression plasmid pFastBac Dual-gIL18 was transformed into DH10Bac, followed by the identification of Bacmid-gIL18 recombinat plosmid by three antibiotics and blue-white patch. Finally, the recombinant bacmid was transfected into sf9 insect cells by Cellfectin and the transfected cells were harvested at different times. Then the expressed protein was identified by SDS-PAGE, Western blot and bioactivity assay. The recombinant protein recognized and bound to its specific antibody. Bioactivity assay showed that the recombinant protein stimulated the proliferation of lymphocytes and induced IFN-γproduction in spleen lymphocytes. The mature gIL-18 protein has been expressed successfully in insect/baculovirus expression system, and have good immunogenicity and bioactivity. The study paves a way for application of gIL-18 as an immunomodulator or immune adjuvant.

Novel baculovirus-derived p67 subunit vaccines efficacious against East Coast fever in cattle.

PubMed

Kaba, Stephen A; Musoke, Anthony J; Schaap, Dick; Schetters, Theo; Rowlands, John; Vermeulen, Arno N; Nene, Vishvanath; Vlak, Just M; van Oers, Monique M

2005-04-15

Two novel baculovirus-derived recombinant Theileria parva p67 constructs were tested for their vaccine potential against East Coast fever. Boran calves were immunized with a his-GFP-p67 fusion protein (GFP:p67deltaSS) or with GP64:p67C, a protein fusion between a C-terminal domain of p67 and the baculovirus envelope protein GP64. Both GFP:p67deltaSS and GP64:p67C induced antibodies with high ELISA titers that neutralized T. parva sporozoites with high efficiency. Upon challenge, a correlation was observed between the in vitro neutralizing capacity and the reduction in severe ECF for individual animals. A protection level upto 85% was obtained. This level of protection was achieved with only two inoculations of 100 microg per dose, which is a major improvement over previous recombinant p67 products.

Cloning and baculovirus expression of a desiccation stress gene from the beetle, Tenebrio molitor.

PubMed

Graham, L A; Bendena, W G; Walker, V K

1996-02-01

The cDNA sequence encoding a novel desiccation stress protein (dsp28) found in the hemolymph of the common yellow mealworm beetle, Tenebrio molitor, has been determined. The sequence encodes a 225 amino acid protein containing a 20 amino acid signal peptide. Dsp28 shows no significant similarity to any known nucleic acid or protein sequence. Levels of dsp28 mRNA were found to increase approx 5-fold following desiccation. Dsp28 cDNA has been cloned into a baculovirus expression vector and the expressed protein was compared to native dsp28. Both dsp28 expressed by recombinant baculovirus and native dsp28 are glycosylated and N-terminally processed. Although dsp28 is induced by cold in addition to desiccation stress, it does not contribute to the freezing point depression (thermal hysteresis) observed in Tenebrio hemolymph.

Development of Impregnated Agglomerate Pelletization (IAP) process for fabrication of (Th,U)O 2 mixed oxide pellets

NASA Astrophysics Data System (ADS)

Khot, P. M.; Nehete, Y. G.; Fulzele, A. K.; Baghra, Chetan; Mishra, A. K.; Afzal, Mohd.; Panakkal, J. P.; Kamath, H. S.

2012-01-01

Impregnated Agglomerate Pelletization (IAP) technique has been developed at Advanced Fuel Fabrication Facility (AFFF), BARC, Tarapur, for manufacturing (Th, 233U)O 2 mixed oxide fuel pellets, which are remotely fabricated in hot cell or shielded glove box facilities to reduce man-rem problem associated with 232U daughter radionuclides. This technique is being investigated to fabricate the fuel for Indian Advanced Heavy Water Reactor (AHWR). In the IAP process, ThO 2 is converted to free flowing spheroids by powder extrusion route in an unshielded facility which are then coated with uranyl nitrate solution in a shielded facility. The dried coated agglomerate is finally compacted and then sintered in oxidizing/reducing atmosphere to obtain high density (Th,U)O 2 pellets. In this study, fabrication of (Th,U)O 2 mixed oxide pellets containing 3-5 wt.% UO 2 was carried out by IAP process. The pellets obtained were characterized using optical microscopy, XRD and alpha autoradiography. The results obtained were compared with the results for the pellets fabricated by other routes such as Coated Agglomerate Pelletization (CAP) and Powder Oxide Pelletization (POP) route.

Effect of different baculovirus inactivation procedures on the integrity and immunogenicity of porcine parvovirus-like particles.

PubMed

Rueda, P; Fominaya, J; Langeveld, J P; Bruschke, C; Vela, C; Casal, J I

2000-11-22

We have demonstrated earlier the usefulness of recombinant porcine parvovirus (PPV) virus-like particles (VLPs) as an efficient recombinant vaccine for PPV. Here, we have demonstrated that preparations of PPV VLPs could be contaminated by recombinant baculoviruses. Since these baculoviruses can be a problem for the registration and safety requirements of the recombinant vaccine, we have tested different baculovirus inactivation strategies, studying simultaneously the integrity and immunogenicity of the VLPs. These methods were pasteurization, treatment with detergents and alkylation with binary ethylenimine (BEI). The structural and functional integrity of the PPV VLPs after the inactivation treatments were analyzed by electron microscopy, hemagglutination, double antibody sandwich (DAS)-ELISA and immunogenicity studies. Binary ethylenimine and Triton X-100 inactivated particles maintained all the original structural and antigenic properties. In addition, PPV VLPs were subjected to size-exclusion chromatography to analyze the presence of VP2 monomers or any other contaminant. The resulting highly purified material was used as the standard of reference to quantify PPV VLPs in order to determine the dose of vaccine by DAS-ELISA. After immunization experiments in guinea pigs, the antibody titers obtained with all the inactivation procedures were very similar. Triton X-100 treatment was selected for further testing in animals because of the speed, simplicity and safety of the overall procedure.

Reduction of the infectivity of baculovirus stocks frozen at ultra-low temperature in serum-free media: The role of lipid emulsions.

PubMed

Eberhardt, Ignacio; Gioria, Verónica Viviana; Micheloud, Gabriela Analía; Claus, Juan Daniel

2016-11-01

The infectivity of stocks of baculoviruses produced in serum-free media is sensitive to freezing at ultra-low temperatures. The objective of this work was to elucidate the causes of such sensitivity, using as a model the freezing of stocks of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), a baculovirus widely employed as biological insecticide. Titers of supernatants of cell cultures infected with AgMNPV in four different serum-free media supplemented with lipid emulsions were reduced by 50 to 90% after six months freezing. By using a full factorial experiment, freezing and lipid emulsion, as well as the interaction between them, were identified as the main factors reducing the viral titer. The virucidal effect of the lipid emulsion was reproduced by one of their components, the surfactant Polysorbate 80. Damaged viral envelopes were observed by transmission electron microscopy in most particles frozen in a medium supplemented with lipid emulsion or Polysorbate 80. Additionally, Polysorbate 80 also affected the infectivity of AgMNPV stocks that were incubated at 27°C. The identification of the roles played by the lipid emulsion and Polysorbate 80 is not only a contribution to the understanding of the mechanisms underlying the inactivation of baculovirus stocks produced in serum-free media during storage at ultra-low temperature, but is also an input for the rational development of new procedures aimed at improving both the preservation of baculovirus stocks and the composition of culture media for the production of baculovirus-based bioproducts in insect cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1559-1569, 2016. © 2016 American Institute of Chemical Engineers.

Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

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

Mohareer, Krishnaveni; Institute of Life Sciences, University of Hyderabad Campus, Prof. C.R. Rao Road, Gachibowli, Hyderabad 500046; Sahdev, Sudhir

2011-11-04

Highlights: Black-Right-Pointing-Pointer Baculovirus p35 is regulated by both viral and host factors. Black-Right-Pointing-Pointer Baculovirus p35 is negatively regulated by SfP53-like factor. Black-Right-Pointing-Pointer Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors,more » which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.« less

THE EFFECT OF BACULOVIRUS INFECTION ON ECDYSTEROID TITER IN GYPSY MOTH LARVAE (LYMANTRIA DISPAR).

EPA Science Inventory

Insect baculovirus carries a gene refered to as egt. This gene encodes an enzyme known as ecdysteroid UDP-glucosyl transferase which catalyzes the sugar conjugation of ecdysteroids. Using a gypsy moth embryonic cell line EGT activity of Lymantria dispar nuclear polyhedrosis virus...

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

Structural Organization of Baculovirus Occlusion Bodies and Protective Role of Multilayered Polyhedron Envelope Protein.

PubMed

Sajjan, Dayanand B; Hinchigeri, Shivayogeppa B

2016-03-01

Baculoviruses are the ingenious insect pathogens. Outside the host, baculovirus occlusion bodies (OB) provide stability to occlusion-derived viruses (ODV) embedded within. The OB is an organized structure, chiefly composed of proteins namely polyhedrin, polyhedron envelope protein (PEP) and P10. Currently, the structural organization of OB is poorly understood and the role of OB proteins in conferring the stability to ODV is unknown. Here we have shown that the assembly of polyhedrin unit cells into an OB is a rapid process; the PEP forms in multiple layers; the PEP layers predominantly contribute to ODV viability. Full-grown OBs (n = 36) were found to be 4.0 ± 1.0 µm in diameter and possessed a peculiar geometry of a truncated rhombic dodecahedron. The atomic force microscopy (AFM) study on the structure of OBs at different stages of growth in insect cells revealed polyhedrin assembly and thickness of PEP layers. The thickness of PEP layers at 53 h post-transfection (hpt) ranged from 56 to 80 nm. Mature PEP layers filled up approximately one third of the OB volume. The size of ODV nucleocapsid was found to be 433 ± 10 nm in length. The zeta potential and particle size distribution study of viruses revealed the protective role of PEP layers. The presence of a multilayered PEP confers a viable advantage to the baculoviruses compared to single-layered PEP. Thus, these findings may help in developing PEP layer-based biopolymers for protein-based nanodevices, nanoelectrodes and more stable biopesticides.

Connective tissue growth factor confers drug resistance in breast cancer through concomitant up-regulation of Bcl-xL and cIAP1.

PubMed

Wang, Ming-Yang; Chen, Pai-Sheng; Prakash, Ekambaranellore; Hsu, Hsing-Chih; Huang, Hsin-Yi; Lin, Ming-Tsan; Chang, King-Jen; Kuo, Min-Liang

2009-04-15

Connective tissue growth factor (CTGF) expression is elevated in advanced breast cancer and promotes metastasis. Chemotherapy response is only transient in most metastatic diseases. In the present study, we examined whether CTGF expression could confer drug resistance in human breast cancer. In breast cancer patients who received neoadjuvant chemotherapy, CTGF expression was inversely associated with chemotherapy response. Overexpression of CTGF in MCF7 cells (MCF7/CTGF) enhanced clonogenic ability, cell viability, and resistance to apoptosis on exposure to doxorubicin and paclitaxel. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) mitigated this drug resistance capacity. CTGF overexpression resulted in resistance to doxorubicin- and paclitaxel-induced apoptosis by up-regulation of Bcl-xL and cellular inhibitor of apoptosis protein 1 (cIAP1). Knockdown of Bcl-xL or cIAP1 with specific small interfering RNAs abolished the CTGF-mediated resistance to apoptosis induced by the chemotherapeutic agents in MCF7/CTGF cells. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 effectively reversed the resistance to apoptosis as well as the up-regulation of Bcl-xL and cIAP1 in MCF7/CTGF cells. A neutralizing antibody against integrin alpha(v)beta(3) significantly attenuated CTGF-mediated ERK1/2 activation and up-regulation of Bcl-xL and cIAP1, indicating that the integrin alpha(v)beta(3)/ERK1/2 signaling pathway is essential for CTGF functions. The Bcl-xL level also correlated with the CTGF level in breast cancer patients. We also found that a COOH-terminal domain peptide from CTGF could exert activities similar to full-length CTGF, in activation of ERK1/2, up-regulation of Bcl-xL/cIAP1, and resistance to apoptosis. We conclude that CTGF expression could confer resistance to chemotherapeutic agents through augmenting a survival pathway through ERK1/2-dependent Bcl-xL/cIAP1 up-regulation.

Soluble forms of the cell adhesion molecule L1 produced by insect and baculovirus-transduced mammalian cells enhance Schwann cell motility.

PubMed

Lavdas, Alexandros A; Efrose, Rodica; Douris, Vassilis; Gaitanou, Maria; Papastefanaki, Florentia; Swevers, Luc; Thomaidou, Dimitra; Iatrou, Kostas; Matsas, Rebecca

2010-12-01

For biotechnological applications, insect cell lines are primarily known as hosts for the baculovirus expression system that is capable to direct synthesis of high levels of recombinant proteins through use of powerful viral promoters. Here, we demonstrate the implementation of two alternative approaches based on the baculovirus system for production of a mammalian recombinant glycoprotein, comprising the extracellular part of the cell adhesion molecule L1, with potential important therapeutic applications in nervous system repair. In the first approach, the extracellular part of L1 bearing a myc tag is produced in permanently transformed insect cell lines and purified by affinity chromatography. In the second approach, recombinant baculoviruses that express L1-Fc chimeric protein, derived from fusion of the extracellular part of L1 with the Fc part of human IgG1, under the control of a mammalian promoter are used to infect mammalian HEK293 and primary Schwann cells. Both the extracellular part of L1 bearing a myc tag accumulating in the supernatants of insect cultures as well as L1-Fc secreted by transduced HEK293 or Schwann cells are capable of increasing the motility of Schwann cells with similar efficiency in a gap bridging bioassay. In addition, baculovirus-transduced Schwann cells show enhanced motility when grafted on organotypic cultures of neonatal brain slices while they retain their ability to myelinate CNS axons. This proof-of-concept that the migratory properties of myelin-forming cells can be modulated by recombinant protein produced in insect culture as well as by means of baculovirus-mediated adhesion molecule expression in mammalian cells may have beneficial applications in the field of CNS therapies. ©2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry.

Recommendations of 2nd National Consultative Meeting of Indian Academy of Pediatrics (IAP) on polio eradication and improvement of routine immunization.

PubMed

Vashishtha, Vipin M; Kalra, Ajay; John, T Jacob; Thacker, Naveen; Agarwal, R K

2008-05-01

Persistence of intense wild poliovirus (WPV) transmission, particularly type 3 in northern India necessitated the Indian Academy of Pediatrics (IAP) to convene a National Consultative Meeting to review its earlier recommendations on polio eradication and improvement of routine immunization. More than thirty experts were invited and intense deliberations were held over two days to draw consensus statements on various issues related with polio eradication. To review the ongoing strategy, identify the existing challenges, and suggest modifications to the current strategy for eradication of poliomyelitis in India. IAP reiterates its support to ongoing efforts on polio eradication but demand some flexibility in the strategy. The immediate challenges identified include persistent WPV type 1 transmission in Uttar Pradesh (UP) and Bihar, intense type 3 transmission also in UP and Bihar, and maintaining polio-free status of all other states. Circulating vaccine derived poliovirus (cVDPV), particularly type 2, was identified as a great future threat. Neglect of routine immunization (RI), poor efficacy of oral polio vaccine (OPV), operational issues, and inadequate uptake of OPV in the 2 endemic states are the main reasons of failure to interrupt transmission of WPV 1 and 3. However, for the first time in history the intensity of WPV 1 circulation is very low in western UP. IAP suggests that high-quality, uniform and consistent performance of supplementary immunization activities (SIAs) in all districts of western UP, particularly using mOPV1(monovalent OPV1) should be maintained to avoid reestablishment of circulation of type 1 poliovirus. A judicious mix of mOPV1 and mOPV3, given sequentially or even simultaneously (after validating the efficacies) will be necessary to address the upsurge of WPV3. Re-establishing routine immunization should be the foremost priority. IAP strongly recommends to Government of India (GOI) to take urgent measures to attain coverage of a minimum

Purification of proteins from baculovirus-infected insect cells.

PubMed

O'Shaughnessy, Luke; Doyle, Sean

2011-01-01

Expression of recombinant proteins in the baculovirus/insect cell expression system is employed because it enables post-translational protein modification and high yields of recombinant protein. The system is capable of facilitating the functional expression of many proteins - either secreted or intracellularly located within infected insect cells. Strategies for the isolation and extraction of soluble proteins are presented in this chapter and involve selective cell lysis, precipitation and chromatography. Protein insolubility, following recombinant expression in insect cells, can occur. However, using the methods described herein, it is possible to extract and purify insoluble protein using affinity, ion-exchange and gel filtration chromatography. Indeed, protein insolubility often aids protein purification.

Physical mapping of the genomic DNA of the Oryctes rhinoceros baculovirus, KI.

PubMed

Mohan, K S; Gopinathan, K P

1991-11-15

A non-occluded baculovirus, OBV-KI has been isolated from the insect pest, Oryctes rhinoceros. The viral genome is estimated to be 123 kb, with a G + C content of 43 mol% and no detectible methylated bases. A restriction map of the OBV-KI genome for BamHI, EcoRI, HindIII, PstI, SalI and XbaI has been constructed.

Contributions of immune responses to developmental resistance in Lymantria dispar challenged with baculovirus

Treesearch

James McNeil; Diana Cox-Foster; James Slavicek; Kelli Hoover

2010-01-01

How the innate immune system functions to defend insects from viruses is an emerging field of study. We examined the impact of melanized encapsulation, a component of innate immunity that integrates both cellular and humoral immune responses, on the success of the baculovirus Lymantria dispar multiple nucleocapsid nucleopolyhedrovirus (LdMNPV) in its...

The xeroderma pigmentosum group B protein ERCC3 produced in the baculovirus system exhibits DNA helicase activity.

PubMed Central

Ma, L; Siemssen, E D; Noteborn, H M; van der Eb, A J

1994-01-01

The XPB/ERCC3 gene corrects the nucleotide excision-repair defect in the human hereditary disease xeroderma pigmentosum group B and encodes the largest subunit of the basal transcription factor BTF2/TFIIH. The primary sequence of the XPB/ERCC3 protein features the hallmarks of seven helicase motifs found in many known and putative helicases or helicase-related proteins. Recently, the multiprotein BTF2/TFIIH complex has been found to be associated with DNA helicase activity. To explore the properties and functions of XPB/ERCC3, we have used the baculovirus/insect-cell expression system to produce recombinant protein. We report here the construction and analysis of recombinant baculovirus expressing XPB/ERCC3. The XPB/ERCC3 protein is synthesized at a relatively high level in baculovirus-infected insect cells. While the majority of XPB/ERCC3 end up in the insoluble fraction of insect cell lysates, a minor fraction of recombinant protein is present in soluble form which can be purified under native conditions. We have found that a DNA helicase activity is associated with the purified XPB/ERCC3 protein, suggesting that XPB/ERCC3 may function as a DNA helicase in local unwinding of DNA template both in the context of transcription and nucleotide excision repair. Images PMID:7937133

Protein Expression Profiles of Permissive, Semi-Permissive and Non-Permissive Cells Infected by Baculovirus

USDA-ARS?s Scientific Manuscript database

Amassing information on the in vitro protein expression of an insect host challenged by an entomopathogenic agent, such as a baculovirus, is paramount to an enhanced understanding of how host-pathogen interactions determine the success or failure of a pathogen. In this study, 2D-gel electrophoresis...

Baculovirus IE2 Stimulates the Expression of Heat Shock Proteins in Insect and Mammalian Cells to Facilitate Its Proper Functioning.

PubMed

Tung, Hsuan; Wei, Sung-Chan; Lo, Huei-Ru; Chao, Yu-Chan

2016-01-01

Baculoviruses have gained popularity as pest control agents and for protein production in insect systems. These viruses are also becoming popular for gene expression, tissue engineering and gene therapy in mammalian systems. Baculovirus infection triggers a heat shock response, and this response is crucial for its successful infection of host insect cells. However, the viral protein(s) or factor(s) that trigger this response are not yet clear. Previously, we revealed that IE2-an early gene product of the baculovirus-could form unique nuclear bodies for the strong trans-activation of various promoters in mammalian cells. Here, we purified IE2 nuclear bodies from Vero E6 cells and investigated the associated proteins by using mass spectrometry. Heat shock proteins (HSPs) were found to be one of the major IE2-associated proteins. Our experiments show that HSPs are greatly induced by IE2 and are crucial for the trans-activation function of IE2. Interestingly, blocking both heat shock protein expression and the proteasome pathway preserved the IE2 protein and its nuclear body structure, and revived its function. These observations reveal that HSPs do not function directly to assist the formation of the nuclear body structure, but may rather protect IE2 from proteasome degradation. Aside from functional studies in mammalian cells, we also show that HSPs were stimulated and required to determine IE2 protein levels, in insect cells infected with baculovirus. Upon inhibiting the expression of heat shock proteins, baculovirus IE2 was substantially suppressed, resulting in a significantly suppressed viral titer. Thus, we demonstrate a unique feature in that IE2 can function in both insect and non-host mammalian cells to stimulate HSPs, which may be associated with IE2 stabilization and lead to the protection of the its strong gene activation function in mammalian cells. On the other hand, during viral infection in insect cells, IE2 could also strongly stimulate HSPs and

A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

PubMed Central

Ardisson-Araújo, Daniel Mendes Pereira; Morgado, Fabrício Da Silva; Schwartz, Elisabeth Ferroni; Corzo, Gerardo; Ribeiro, Bergmann Morais

2013-01-01

Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells. PMID

How baculovirus polyhedra fit square pegs into round holes to robustly package viruses.

PubMed

Ji, Xiaoyun; Sutton, Geoff; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Stuart, David I

2010-01-20

Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.

Design, synthesis, and biological activities of novel hexahydropyrazino[1,2-a]indole derivatives as potent inhibitors of apoptosis (IAP) proteins antagonists with improved membrane permeability across MDR1 expressing cells.

PubMed

Shiokawa, Zenyu; Hashimoto, Kentaro; Saito, Bunnai; Oguro, Yuya; Sumi, Hiroyuki; Yabuki, Masato; Yoshimatsu, Mie; Kosugi, Yohei; Debori, Yasuyuki; Morishita, Nao; Dougan, Douglas R; Snell, Gyorgy P; Yoshida, Sei; Ishikawa, Tomoyasu

2013-12-15

We previously reported octahydropyrrolo[1,2-a]pyrazine derivative 2 (T-3256336) as a potent antagonist for inhibitors of apoptosis (IAP) proteins. Because compound 2 was susceptible to MDR1 mediated efflux, we developed another scaffold, hexahydropyrazino[1,2-a]indole, using structure-based drug design. The fused benzene ring of this scaffold was aimed at increasing the lipophilicity and decreasing the basicity of the scaffold to improve the membrane permeability across MDR1 expressing cells. We established a chiral pool synthetic route to yield the desired tricyclic chiral isomers. Chemical modification of the core scaffold led to a representative compound 50, which showed strong inhibition of IAP binding (X chromosome-linked IAP [XIAP]: IC50 23 nM and cellular IAP [cIAP]: IC50 1.1 nM) and cell growth inhibition (MDA-MB-231 cells: GI50 2.8 nM) with high permeability and low potential of MDR1 substrate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Role of Microphysical Parameterizations with Droplet Relative Dispersion in IAP AGCM 4.1

DOE PAGES

Xie, Xiaoning; Zhang, He; Liu, Xiaodong; ...

2018-01-10

In previous studies we see that accurate descriptions of the cloud droplet effective radius (Re) and the autoconversion process of cloud droplets to raindrops (Au) can effectively improve simulated clouds and surface precipitation, and reduce the uncertainty of aerosol indirect effects in global climate models (GCMs). In this paper, we implement cloud microphysical schemes including two-moment Au and R e considering relative dispersion of the cloud droplet size distribution into version 4.1 of the Institute of Atmospheric Physics atmospheric GCM (IAP AGCM 4.1), which is the atmospheric component of the Chinese Academy of Sciences-Earth System model (CAS-ESM 1.0). An analysismore » of the effects of different schemes shows that the newly implemented schemes can improve both the simulated shortwave (SWCF) and longwave cloud radiative forcings (LWCF) as compared to the standard scheme in IAP AGCM 4.1. The new schemes also effectively enhance the large-scale precipitation, especially over low latitudes, although the influences of total precipitation are insignificant for different schemes. Further studies show that similar results can be found with the Community Atmosphere Model 5.1 (CAM5.1).« less

Role of Microphysical Parameterizations with Droplet Relative Dispersion in IAP AGCM 4.1

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

Xie, Xiaoning; Zhang, He; Liu, Xiaodong

In previous studies we see that accurate descriptions of the cloud droplet effective radius (Re) and the autoconversion process of cloud droplets to raindrops (Au) can effectively improve simulated clouds and surface precipitation, and reduce the uncertainty of aerosol indirect effects in global climate models (GCMs). In this paper, we implement cloud microphysical schemes including two-moment Au and R e considering relative dispersion of the cloud droplet size distribution into version 4.1 of the Institute of Atmospheric Physics atmospheric GCM (IAP AGCM 4.1), which is the atmospheric component of the Chinese Academy of Sciences-Earth System model (CAS-ESM 1.0). An analysismore » of the effects of different schemes shows that the newly implemented schemes can improve both the simulated shortwave (SWCF) and longwave cloud radiative forcings (LWCF) as compared to the standard scheme in IAP AGCM 4.1. The new schemes also effectively enhance the large-scale precipitation, especially over low latitudes, although the influences of total precipitation are insignificant for different schemes. Further studies show that similar results can be found with the Community Atmosphere Model 5.1 (CAM5.1).« less

Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding

PubMed Central

2011-01-01

Background Baculovirus, which has a width of 40 nm and a length of 250-300 nm, can display functional peptides, receptors and antigens on its surface by their fusion with a baculovirus envelop protein, GP64. In addition, some transmembrane proteins can be displayed without GP64 fusion, using the native transmembrane domains of the baculovirus. We used this functionality to display human prorenin receptor fused with GFPuv (GFPuv-hPRR) on the surface of silkworm Bombyx mori nucleopolyhedrovirus (BmNPV) and then tested whether these baculovirus particles could be used to detect protein-protein interactions. Results BmNPV displaying GFPuv-hPRR (BmNPV-GFPuv-hPRR) was purified from hemolymph by using Sephacryl S-1000 column chromatography in the presence of 0.01% Triton X-100. Its recovery was 86% and the final baculovirus particles number was 4.98 × 108 pfu. Based on the results of enzyme-linked immunosorbent assay (ELISA), 3.1% of the total proteins in BmNPV-GFPuv-hPRR were GFPuv-hPRR. This value was similar to that calculated from the result of western blot by a densitometry (2.7%). To determine whether BmNPV-GFPuv-hPRR particles were bound to human prorenin, ELISA results were compared with those from ELISAs using protease negative BmNPV displaying β1,3-N-acetylglucosaminyltransferase 2 fused with the gene encoding GFPuv (GGT2) (BmNPV-CP--GGT2) particles, which do not display hPRR on their surfaces. Conclusion The display of on the surface of the BmNPV particles will be useful for the detection of protein-protein interactions and the screening of inhibitors and drugs in their roles as nanobioparticles. PMID:21635720

The phosphotransferase system-dependent sucrose utilization regulon in enteropathogenic Escherichia coli strains is located in a variable chromosomal region containing iap sequences.

PubMed

Treviño-Quintanilla, Luis Gerardo; Escalante, Adelfo; Caro, Alma Delia; Martínez, Alfredo; González, Ricardo; Puente, José Luis; Bolívar, Francisco; Gosset, Guillermo

2007-01-01

The capacity to utilize sucrose as a carbon and energy source (Scr(+) phenotype) is a highly variable trait among Escherichia coli strains. In this study, seven enteropathogenic E. coli (EPEC) strains from different sources were studied for their capacity to grow using sucrose. Liquid media cultures showed that all analyzed strains have the Scr(+) phenotype and two distinct groups were defined: one of five and another of two strains displaying doubling times of 67 and 125 min, respectively. The genes conferring the Scr(+) phenotype in one of the fast-growing strains (T19) were cloned and sequenced. Comparative sequence analysis revealed that this strain possesses the scr regulon genes scrKYABR, encoding phosphoenolpyruvate:phosphotransferase system-dependent sucrose transport and utilization activities. Transcript level quantification revealed sucrose-dependent induction of scrK and scrR genes in fast-growing strains, whereas no transcripts were detected in slow-growing strains. Sequence comparison analysis revealed that the scr genes in strain T19 are almost identical to those present in the scr regulon of prototype EPEC E2348/69 and in both strains, the scr genes are inserted in the chromosomal intergenic region of hypothetical genes ygcE and ygcF. Comparison of the ygcE-ygcF intergenic region sequence of strains MG1655, enterohemorrhagic EDL933, uropathogenic ECFT073 and EPEC T19-E2348/69 revealed that the number of extragenic highly repeated iap sequences corresponded to nine, four, two and none, respectively. These results show that the iap sequence-containing chromosomal ygcE-ygcF intergenic region is highly variable in E. coli. Copyright (c) 2007 S. Karger AG, Basel.

MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection.

PubMed

Mehrabadi, Mohammad; Hussain, Mazhar; Asgari, Sassan

2013-06-01

MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signalling and immune response. Studies also suggest that miRNAs are important in host-virus interactions where the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we identified conserved and new miRNAs from Spodoptera frugiperda cells (Sf9) using a combination of deep sequencing and bioinformatics as well as experimental approaches. S. frugiperda miRNAs share common features of miRNAs in other organisms, such as uracil (U) at the 5' end of miRNA. The 5' ends of the miRNAs were more conserved than the 3' ends, revealing evolutionary protection of the seed region in miRNAs. The predominant miRNAs were found to be conserved among arthropods. The majority of homologous miRNAs were found in Bombyx mori, with 76 of the 90 identified miRNAs. We found that seed shifting and arm switching have happened in this insect's miRNAs. Expression levels of the majority of miRNAs changed following baculovirus infection. Results revealed that baculovirus infection mainly led to an overall suppression of cellular miRNAs. We found four different genes being regulated by sfr-miR-184 at the post-transcriptional level. The data presented here further support conservation of miRNAs in insects and other organisms. In addition, the results reveal a differential expression of host miRNAs upon baculovirus infection, suggesting their potential roles in host-virus interactions. Seed shifting and arm switching happened during evolution of miRNAs in different insects and caused miRNA diversification, which led to changes in the target repository of miRNAs.

How baculovirus polyhedra fit square pegs into round holes to robustly package viruses

PubMed Central

Ji, Xiaoyun; Sutton, Geoff; Evans, Gwyndaf; Axford, Danny; Owen, Robin; Stuart, David I

2010-01-01

Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles. PMID:19959989

Laboratory and field evaluations for efficacy of a fast-killing baculovirus isolate from Spodoptera frugiperda

USDA-ARS?s Scientific Manuscript database

Three biopesticide parameters were evaluated for a fast-killing isolate (3AP2) Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) and a wild-type isolate (Sf3) of the same baculovirus. Both isolates were evaluated for virus production using in vivo methods, for speed of kill based on bioas...

Optimizing working space in laparoscopy: CT measurement of the effect of pre-stretching of the abdominal wall in a porcine model.

PubMed

Vlot, John; Wijnen, René; Stolker, Robert Jan; Bax, Klaas N

2014-03-01

Determinants of working space in minimal access surgery have not been well studied. Using computed tomography (CT) to measure volumes and linear dimensions, we are studying the effect of a number of determinants of CO2 working space in a porcine laparoscopy model. Here we report the effects of pre-stretching of the abdominal wall. Earlier we had noted an increase in CO2 pneumoperitoneum volume at repeat insufflation with an intra-abdominal pressure (IAP) of 5 mmHg after previous stepwise insufflation up to an IAP of 15 mmHg. We reviewed the data of this serendipity group; data of 16 pigs were available. In a new group of eight pigs, we also explored this effect at repeat IAPs of 10 and 15 mmHg. Volumes and linear dimensions of the CO2 pneumoperitoneum were measured on reconstructed CT images and compared between the initial and repeat insufflation runs. Previous stepwise insufflation of the abdomen with CO2 up to 15 mmHg significantly (p < 0.01) increased subsequent working-space volume at a repeat IAP of 5 mmHg by 21 %, 7 % at a repeat IAP of 10 mmHg and 3 % at a repeat IAP of 15 mmHg. The external anteroposterior diameter significantly (p < 0.01) increased by 0.5 cm (14 %) at repeat 5 mmHg. Other linear dimensions showed a much smaller change. There was no statistically significant correlation between the duration of the insufflation run and the volume increase after pre-stretching at all IAP levels. Pre-stretching of the abdominal wall allows for the same surgical-field exposure at lower IAPs, reducing the negative effects of prolonged high-pressure CO2 pneumoperitoneum on the cardiorespiratory system and microcirculation. Pre-stretching has important scientific consequences in studies addressing ways of increasing working space in that its effect may confound the possible effects of other interventions aimed at increasing working space.

Status of indoor air pollution (IAP) through particulate matter (PM) emissions and associated health concerns in South Asia.

PubMed

Junaid, Muhammad; Syed, Jabir Hussain; Abbasi, Naeem Akhtar; Hashmi, Muhammad Zaffar; Malik, Riffat Naseem; Pei, De-Sheng

2018-01-01

Exposure to particulate emissions poses a variety of public health concerns worldwide, specifically in developing countries. This review summarized the documented studies on indoor particulate matter (PM) emissions and their major health concerns in South Asia. Reviewed literature illustrated the alarming levels of indoor air pollution (IAP) in India, Pakistan, Nepal, and Bangladesh, while Sri Lanka and Bhutan are confronted with relatively lower levels, albeit not safe. To our knowledge, data on this issue are absent from Afghanistan and Maldives. We found that the reported levels of PM 10 and PM 2.5 in Nepal, Pakistan, Bangladesh, and India were 2-65, 3-30, 4-22, 2-28 and 1-139, 2-180, 3-77, 1-40 fold higher than WHO standards for indoor PM 10 (50 μg/m 3 ) and PM 2.5 (25 μg/m 3 ), respectively. Regarding IAP-mediated health concerns, mortality rates and incidences of respiratory and non-respiratory diseases were increasing with alarming rates, specifically in India, Pakistan, Nepal, and Bangladesh. The major cause might be the reliance of approximately 80% population on conventional biomass burning in the region. Current review also highlighted the prospects of IAP reduction strategies, which in future can help to improve the status of indoor air quality and public health in South Asia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Finite-volume Atmospheric Model of the IAP/LASG (FAMIL)

NASA Astrophysics Data System (ADS)

Bao, Q.

2015-12-01

The Finite-volume Atmospheric Model of the IAP/LASG (FAMIL) is introduced in this work. FAMIL have the flexible horizontal and vertical resolutions up to 25km and 1Pa respectively, which currently running on the "Tianhe 1A&2" supercomputers. FAMIL is the atmospheric component of the third-generation Flexible Global Ocean-Atmosphere-Land climate System model (FGOALS3) which will participate in the Coupled Model Intercomparison Project Phase 6 (CMIP6). In addition to describing the dynamical core and physical parameterizations of FAMIL, this talk describes the simulated characteristics of energy and water balances, precipitation, Asian Summer Monsoon and stratospheric circulation, and compares them with observational/reanalysis data. Finally, the model biases as well as possible solutions are discussed.

The Env-like open reading frame of the baculovirus-integrated retrotransposon TED encodes a retrovirus-like envelope protein.

PubMed

Ozers, M S; Friesen, P D

1996-12-15

TED is a 7.5-kbp member of the gypsy family of retrotransposons that was first identified by its integration within the baculovirus DNA genome. This lepidopteran (moth) transposon contains three retrovirus-like genes, including functional gag and pol that yield reverse transcriptase-containing virus-like particles. To identify and characterize the product(s) of the third env-like open reading frame, TED ORF3 was expressed in homologous lepidopteran cells by using a baculovirus vector, vENV. Immunoblots and immunoprecipitations with antiserum raised against a bacterial ORF3-fusion protein detected two ORF3-encoded proteins, p68env and gp75env. On the basis of selective incorporation of [3H]mannose and inhibition of modification by tunicamycin which blocks N-linked glycosylation, gp75env is a glycoprotein derived from core precursor p68env. As predicted by the presence of a transmembrane domain near the carboxyl terminus, both p68env and gp75env were associated with heavy membranes of vENV-infected cells. Thus, TED ORF3 encodes a membrane glycoprotein with properties characteristic of retroviral env proteins. These data are consistent with the hypothesis that TED is an invertebrate retrovirus. Moreover, TED integration within the baculovirus genome provides an example of retroelement-mediated acquisition of host genes that may contribute to virus evolution.

Electron Tomography and Simulation of Baculovirus Actin Comet Tails Support a Tethered Filament Model of Pathogen Propulsion

PubMed Central

Mueller, Jan; Pfanzelter, Julia; Winkler, Christoph; Narita, Akihiro; Le Clainche, Christophe; Nemethova, Maria; Carlier, Marie-France; Maeda, Yuichiro; Welch, Matthew D.; Ohkawa, Taro; Schmeiser, Christian; Resch, Guenter P.; Small, J. Victor

2014-01-01

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion. PMID:24453943

Electron tomography and simulation of baculovirus actin comet tails support a tethered filament model of pathogen propulsion.

PubMed

Mueller, Jan; Pfanzelter, Julia; Winkler, Christoph; Narita, Akihiro; Le Clainche, Christophe; Nemethova, Maria; Carlier, Marie-France; Maeda, Yuichiro; Welch, Matthew D; Ohkawa, Taro; Schmeiser, Christian; Resch, Guenter P; Small, J Victor

2014-01-01

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.

CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus-insect cell system.

PubMed

Mabashi-Asazuma, Hideaki; Jarvis, Donald L

2017-08-22

The baculovirus-insect cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of insect cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors-which has simplified the system-and transform insect cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two insect cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous insect cell gene and alter protein glycosylation in the BICS.

Proteasome inhibitor PS-341 limits macrophage necroptosis by promoting cIAPs-mediated inhibition of RIP1 and RIP3 activation.

PubMed

Zhang, Yuchen; Cheng, Junjun; Zhang, Junmeng; Wu, Xiaofan; Chen, Fang; Ren, Xuejun; Wang, Yunlong; Li, Quan; Li, Yu

2016-09-02

Apoptotic and necrotic macrophages have long been known for their existence in atherosclerotic lesions. However, the mechanisms underlying the choice of their death pattern have not been fully elucidated. Here, we report the effects of PS-341, a potent and specific proteasome inhibitor, on the cell death of primary bone marrow-derived macrophages (BMDMs) in vitro. The results showed that PS-341 could not induce macrophage apoptosis or promote TNF-induced macrophage apoptosis, on the other hand, PS-341 could significantly inhibit macrophage necroptosis induced by TNF and pan-caspase inhibitor z-VAD treatment. Remarkably, high-dose of PS-341 showed similar inhibitory effects on macrophage necroptosis comparable to that of kinase inhibition of RIP1 through specific inhibitor Nec-1 or inhibition of RIP3 via specific genetical ablation. Furthermore, the degradation of cellular inhibitor of apoptosis proteins (cIAPs) was suppressed by PS-341, which could antagonize the activation of RIP1 kinase via post-translational mechanism. Further evidences demonstrated reduced levels of both RIP1 and RIP 3 upon PS-341 treatment, concomitantly, a more strong association of RIP1 with cIAPs and less with RIP3 was found following PS-341 treatment, these findings suggested that PS-341 may disrupt the formation of RIP1-RIP3 complex (necrosome) through stabilizing cIAPs. Collectively, our results indicated that the proteasome-mediated degradation of cIAPs could be inhibited by PS-341 and followed by limited RIP1 and RIP3 kinase activities, which were indispensable for necroptosis, thus eliciting a significant necroptosis rescue in BMDMs in vitro. Overall, our study has identified a new role of PS-341 in the cell death of BMDMs and provided a novel insight into the atherosclerotic inflammation caused by proteasome-mediated macrophage necroptosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

PubMed

Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

2004-10-01

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice.

PubMed

Dai, Shiyu; Zhang, Tao; Zhang, Yanfang; Wang, Hualin; Deng, Fei

2018-06-01

The newly emerged mosquito-borne Zika virus (ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles (VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane (prM) and envelope (E) proteins were co-expressed in insect cells and self-assembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.

Antigenic characterization of bovine ephemeral fever rhabdovirus G and GNS glycoproteins expressed from recombinant baculoviruses.

PubMed

Johal, Jasjit; Gresty, Karryn; Kongsuwan, Kritaya; Walker, Peter J

2008-01-01

Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein G(NS) were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed G(NS) protein was also located on the cell surface but did not exhibit fusogenic activity. The G(NS) protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant G(NS) but did not react with G protein antibodies. A His(6)-tagged, soluble form of the G protein was expressed and purified by Ni(2+)-NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.

CHARACTERIZATION OF THE GLYCOSYLATED ECDYSTEROIDS IN THE HEMOLYMPH OF BACULOVIRUS-INFECTED GYPSY MOTH LARVAE AND CELLS IN CULTURE

EPA Science Inventory

Fourth-instar gypsy moth (Lymantria dispar; Lepidoptera: Lymantriidae) larvae, infected with the gypsy moth baculovirus (LdNPV), show an elevated and prolonged extension of the hemolymph ecdysteroid titer peak associated with molting. The ecdysteroid immunoreactivity associated w...

The genome sequence of Condylorrhiza vestigialis NPV, a novel baculovirus for the control of the Alamo moth on Populus spp. in Brazil.

PubMed

Castro, Maria Elita B; Melo, Fernando L; Tagliari, Marina; Inglis, Peter W; Craveiro, Saluana R; Ribeiro, Zilda Maria A; Ribeiro, Bergmann M; Báo, Sônia N

2017-09-01

Condylorrhiza vestigialis (Lepidoptera: Cambridae), commonly known as the Brazilian poplar moth or Alamo moth, is a serious defoliating pest of poplar, a crop of great economic importance for the production of wood, fiber, biofuel and other biomaterials as well as its significant ecological and environmental value. The complete genome sequence of a new alphabaculovirus isolated from C. vestigialis was determined and analyzed. Condylorrhiza vestigialis nucleopolyhedrovirus (CoveNPV) has a circular double-stranded DNA genome of 125,767bp with a GC content of 42.9%. One hundred and thirty-eight putative open reading frames were identified and annotated in the CoveNPV genome, including 38 core genes and 9 bros. Four homologous regions (hrs), a feature common to most baculoviruses, and 19 perfect and imperfect direct repeats (drs) were found. Phylogenetic analysis confirmed that CoveNPV is a Group I Alphabaculovirus and is most closely related to Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) and Choristoneura fumiferana DEF multiple nucleopolyhedrovirus CfDEFMNPV. The gp37 gene was not detected in the CoveNPV genome, although this gene is found in many NPVs. Two other common NPV genes, chitinase (v-chiA) and cathepsin (v-cath), that are responsible for host insect liquefaction and melanization, were also absent, where phylogenetic analysis suggests that the loss these genes occurred in the common ancestor of AgMNPV, CfDEFMNPV and CoveNPV, with subsequent reacquisition of these genes by CfDEFMNPV. The molecular biology and genetics of CoveNPV was formerly very little known and our expectation is that the findings presented here should accelerate research on this baculovirus, which will facilitate the use of CoveNPV in integrated pest management programs in Poplar crops. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of an Sf-rhabdovirus-negative Spodoptera frugiperda cell line as an alternative host for recombinant protein production in the baculovirus-insect cell system.

PubMed

Maghodia, Ajay B; Geisler, Christoph; Jarvis, Donald L

2016-06-01

Cell lines derived from the fall armyworm, Spodoptera frugiperda (Sf), are widely used as hosts for recombinant protein production in the baculovirus-insect cell system (BICS). However, it was recently discovered that these cell lines are contaminated with a virus, now known as Sf-rhabdovirus [1]. The detection of this adventitious agent raised a potential safety issue that could adversely impact the BICS as a commercial recombinant protein production platform. Thus, we examined the properties of Sf-RVN, an Sf-rhabdovirus-negative Sf cell line, as a potential alternative host. Nested RT-PCR assays showed Sf-RVN cells had no detectable Sf-rhabdovirus over the course of 60 passages in continuous culture. The general properties of Sf-RVN cells, including their average growth rates, diameters, morphologies, and viabilities after baculovirus infection, were virtually identical to those of Sf9 cells. Baculovirus-infected Sf-RVN and Sf9 cells produced equivalent levels of three recombinant proteins, including an intracellular prokaryotic protein and two secreted eukaryotic glycoproteins, and provided similar N-glycosylation patterns. In fact, except for the absence of Sf-rhabdovirus, the only difference between Sf-RVN and Sf9 cells was SF-RVN produced higher levels of infectious baculovirus progeny. These results show Sf-RVN cells can be used as improved, alternative hosts to circumvent the potential safety hazard associated with the use of Sf-rhabdovirus-contaminated Sf cells for recombinant protein manufacturing with the BICS. Copyright © 2016 Elsevier Inc. All rights reserved.

Evolution in Oryctes baculovirus: rate and types of genomic change.

PubMed

Crawford, A M; Zelazny, B

1990-01-01

Three cloned strains of Oryctes baculovirus were released into a previously unexposed population of the host insect, the coconut palm rhinoceros beetle, Oryctes rhinoceros. The experiment was conducted on Meemu Atoll in the Maldive Islands. Viruses were isolated from the beetle population at 1 year, 1.75 years, and 4 years after release. No changes in genotype were observed in viruses isolated after 1 and 1.75 years. After 4 years, however, three types of genomic change had occurred. A recombinant derived from two of the released strains, an isolate containing a 100-bp insert, and one example of a point mutation were found in the 22 isolates examined.

Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays.

PubMed

Freystetter, Andrea; Paar, Christian; Stekel, Herbert; Berg, Jörg

2017-10-01

The widespread use of molecular PCR-based assays in analytical and clinical laboratories brings about the need for test-specific, stable, and reliable external controls (EC) as well as standards and internal amplification controls (IC), in order to arrive at consistent test results. In addition, there is also a growing need to produce and provide stable, well-characterized molecular controls for quality assurance programs. In this study, we describe a novel approach to generate armored double-stranded DNA controls, which are encapsulated in baculovirus (BV) particles of the species Autographa californica multiple nucleopolyhedrovirus. We used the well-known BacPAK™ Baculovirus Expression System (Takara-Clontech), removed the polyhedrin promoter used for protein expression, and generated recombinant BV-armored DNAs. The obtained BV-armored DNAs were readily extracted by standard clinical DNA extraction methods, showed favorable linearity and performance in our clinical PCR assays, were resistant to DNase I digestion, and exhibited marked stability in human plasma and serum. BV-armored DNA ought to be used as ECs, quantification standards, and ICs in molecular assays, with the latter application allowing for the entire monitoring of clinical molecular assays for sample adequacy. BV-armored DNA may also be used to produce double-stranded DNA reference materials for, e.g., quality assurance programs. The ease to produce BV-armored DNA should make this approach feasible for a broad spectrum of molecular applications. Finally, as BV-armored DNAs are non-infectious to mammals, they may be even more conveniently shipped than clinical specimen.

[Demonstration, stabilization and purification of an intracapsid nucleoprotein structure of Baculovirus of Oryctes rhinoceros L].

PubMed

Monsarrat, P; Revet, B; Gourevitch, I

1975-11-10

The presence of a structurally organized nucleoproteic structure in the capsid of the Baculovirus of Oryctes rhinoceros L. is shown. This structure is stabilized under definite conditions described in detail in the paper. It possesses a rope-like structure of about 280 nm in length on 15 nm in diameter containing the DNA molecule. A basic protein is found in the virus.

Characterization of 5-HT₁A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.

PubMed

Tõntson, Lauri; Kopanchuk, Sergei; Rinken, Ago

2014-02-01

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+). Copyright © 2014 Elsevier Ltd. All rights reserved.

Isolation of a baculovirus variant that exhibits enhanced polyhedra production stability during serial passage in cell culture

Treesearch

James M. Slavicek; Melissa J. Mercer; Mary Ellen Kelly; Nancy Hayes-Plazolles

1996-01-01

The formation of few polyhedra mutants during serial propagation of baculoviruses in cell culture encumbers commercial scale production in this system. A Lymantria dispar nuclear polyhedrosis virus (LdMNPV) variant (isolate A21-MPV) has been isolated and the traits of budded virus (BV) production, synthesis of polyhedra, the...

Enhanced effect of fluorescent whitening agent on peroral infection for recombinant baculovirus in the host Bombyx mori L.

PubMed

Wang, Bing; Shang, Jinyan; Liu, Xunli; Cui, Weizheng; Wu, Xiaofeng; Zhao, Na

2007-01-01

The low efficiency of the oral infectivity of recombinant polyhedrin-negative baculovirus is a major bottleneck in the application of the baculovirus expression system in the silkworm (Bombyx mori L). In this study, the effects of a fluorescent whitening agent on improving the oral infection for the recombinant Bombyx mori nuclear polyhedrosis virus in silkworm larva and their possible mechanism were investigated. The results showed that the peroral infection can be remarkably enhanced by adding VBL into the larval artificial diet. The maximum infection rate reached as high as 90% with the concentration of VBL (1%), which was then considered as optimal. The total protease activity and pH value of the larval intestinal juice were found to be lower when compared to the control, indicating an abnormal physiological change of the larval digestive system by VBL, which, in turn, resulted in improved peroral infection of recombinant virus.

Effect of baculovirus P35 protein on apoptosis in brain tissue of rats with acute cerebral infarction.

PubMed

Ji, J F; Ma, X H

2015-08-10

We explored the effect of baculovirus P35 protein on apoptosis in the brain tissue of rats with acute cerebral infarction (ACI). A rat model of middle cerebral artery infarction was created. The rats were randomly divided into sham, model, and treatment groups. Baculovirus P35 protein was injected into the intracranial arteries of the treatment group rats. The rats in the model group were given an equal volume of phosphate-buffered saline. The rats were sacrificed after 72 h and the brain tissue was separated. The levels of caspase-3, Bcl-2, and Bax mRNA, the brain cell apoptosis index, and the infarct size were determined. After 72 h, the levels of caspase-3 and Bax mRNA in the model and treatment groups were significantly greater than in the sham group, and the levels of Bcl-2 mRNA were significantly smaller (P < 0.05). The levels of caspase-3 and Bax mRNA were significantly lower in the treatment group than in the model group, and the level of Bcl-2 mRNA was significantly greater (P < 0.05). Compared with the sham group, the brain tissue apoptosis index and the cerebral infarction area increased significantly in the model and treatment groups (P < 0.05). The brain tissue apoptosis index and cerebral infarction area in the treatment group were significantly lower than in the model group (P < 0.05). Baculovirus P35 protein can effectively inhibit brain cell apoptosis in rats with ACI. It delayed apoptosis and necrosis in subjects with ACI tissue and had a protective effect on brain tissue.

N-TERMINALLY ELONGATED SpliInx2 AND SpliInx3 REDUCE BACULOVIRUS-TRIGGERED APOPTOSIS VIA HEMICHANNEL CLOSURE.

PubMed

Chen, Ya-Bin; Xiao, Wei; Li, Ming; Zhang, Yan; Yang, Yang; Hu, Jian-Sheng; Luo, Kai-Jun

2016-05-01

The hemichannel and gap junction channel are major portals for the release of factors responsible for the effects of apoptotic cells on the spread of apoptosis to neighboring cells and apoptotic corpse clearance, typically by phagocytes. The N-terminal cytoplasmic domain in the connexins, gap junction proteins in vertebrate, has been implicated in regulating channel closure. However, little is known about how the hemichannel close responds to apoptotic signaling transduction leading to the reduction of neighboring cellular apoptosis in an invertebrate. An insect Bac-to-Bac expression system, pFastBac(TM) HT A, allows us to construct an N-terminally elongated SpliInx2 (Nte-Inx2) and SpliInx3 (Nte-Inx3). Here, we demonstrated that recombinant baculovirus Bac-Nte-Inx2 (reBac-Net-Inx2) and Bac-Nte-Inx3 (reBac-Nte-Inx3) closed the endogenous hemichannel on the Sf9 cell surface. Importantly, primary baculovirus infections significantly caused early apoptosis, and this apoptosis was reduced by hemichannel-closed Sf9 cells at 24-h post-infection (PI). Although N-terminal-elongated residue led to the increase in the phosphorylated sites in both Nte-Inx2 and Nte-Inx3 and an additional transmembrane domain in Nte-Inx3, both the proteins localized on the cell surface, suggesting Nte-Inxs proteins could mediate hemichannel closure. Further supporting evidence showed that hemichannel closure was dependent on N-Inxs expressed by baculovirus polyhedrin promoter, which began to express at 18-24 h PI. These results identify an unconventional function of N-terminal-elongated innexins that could act as a plug to manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction from intracellular to extracellular compartment. © 2016 Wiley Periodicals, Inc.

Chaperokine function of recombinant Hsp72 produced in insect cells using a baculovirus expression system is retained.

PubMed

Zheng, Hongying; Nagaraja, Ganachari M; Kaur, Punit; Asea, Edwina E; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72(bv) (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72(bv) enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72(bv) in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72(bv) can now be used to unlock the important role Hsp72 plays in modulating immune function.

Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained*

PubMed Central

Zheng, Hongying; Nagaraja, Ganachari M.; Kaur, Punit; Asea, Edwina E.; Asea, Alexzander

2010-01-01

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72bv (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72bv enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72bv in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72bv can now be used to unlock the important role Hsp72 plays in modulating immune function. PMID:19861412

A Novel Ideal Radionuclide Imaging System for Non-invasively Cell Monitoring built on Baculovirus Backbone by Introducing Sleeping Beauty Transposon

PubMed Central

Lv, Jing; Pan, Yu; Ju, Huijun; Zhou, Jinxin; Cheng, Dengfeng; Shi, Hongcheng; Zhang, Yifan

2017-01-01

Sleeping Beauty (SB) transposon is an attractive tool in stable transgene integration both in vitro and in vivo; and we introduced SB transposon into recombinant sodium-iodide symporter baculovirus system (Bac-NIS system) to facilitate long-term expression of recombinant sodium-iodide symporter. In our study, two hybrid baculovirus systems (Bac-eGFP-SB-NeoR and Bac-NIS-SB-NeoR) were successfully constructed and used to infect U87 glioma cells. After G418 selection screening, the Bac-eGFP-SB-NeoR-U87 cells remained eGFP positive, at the 18th and 196th day post transfection (96.03 ± 0.21% and 97.43 ± 0.81%), while eGFP positive population declined significantly at 18 days in cells transfected with unmodified baculovirus construct. NIS gene expression by Bac-NIS-SB-NeoR-U87 cells was also maintained for 28 weeks as determined by radioiodine uptake assay, reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot (WB) assay. When transplanted in mice, Bac-NIS-SB-NeoR-U87 cells also expressed NIS gene stably as monitored by SPECT imaging for 43 days until the tumor-bearing mice were sacrificed. Herein, we showed that incorporation of SB in Bac-NIS system (hybrid Bac-NIS-SB-NeoR) can achieve a long-term transgene expression and can improve radionuclide imaging in cell tracking and monitoring in vivo. PMID:28262785

Baculovirus-mediated expression of GPCRs in insect cells.

PubMed

Saarenpää, Tuulia; Jaakola, Veli-Pekka; Goldman, Adrian

2015-01-01

G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations. © 2015 Elsevier Inc. All rights reserved.

Meet OLAF, a Good Friend of the IAPS! The Open Library of Affective Foods: A Tool to Investigate the Emotional Impact of Food in Adolescents

PubMed Central

Miccoli, Laura; Delgado, Rafael; Rodríguez-Ruiz, Sonia; Guerra, Pedro; García-Mármol, Eduardo; Fernández-Santaella, M. Carmen

2014-01-01

In the last decades, food pictures have been repeatedly employed to investigate the emotional impact of food on healthy participants as well as individuals who suffer from eating disorders and obesity. However, despite their widespread use, food pictures are typically selected according to each researcher's personal criteria, which make it difficult to reliably select food images and to compare results across different studies and laboratories. Therefore, to study affective reactions to food, it becomes pivotal to identify the emotional impact of specific food images based on wider samples of individuals. In the present paper we introduce the Open Library of Affective Foods (OLAF), which is a set of original food pictures created to reliably select food pictures based on the emotions they prompt, as indicated by affective ratings of valence, arousal, and dominance and by an additional food craving scale. OLAF images were designed to allow simultaneous use with affective images from the International Affective Picture System (IAPS), which is a well-known instrument to investigate emotional reactions in the laboratory. The ultimate goal of the OLAF is to contribute to understanding how food is emotionally processed in healthy individuals and in patients who suffer from eating and weight-related disorders. The present normative data, which was based on a large sample of an adolescent population, indicate that when viewing affective non-food IAPS images, valence, arousal, and dominance ratings were in line with expected patterns based on previous emotion research. Moreover, when viewing food pictures, affective and food craving ratings were consistent with research on food cue processing. As a whole, the data supported the methodological and theoretical reliability of the OLAF ratings, therefore providing researchers with a standardized tool to reliably investigate the emotional and motivational significance of food. The OLAF database is publicly available at zenodo

Meet OLAF, a good friend of the IAPS! The Open Library of Affective Foods: a tool to investigate the emotional impact of food in adolescents.

PubMed

Miccoli, Laura; Delgado, Rafael; Rodríguez-Ruiz, Sonia; Guerra, Pedro; García-Mármol, Eduardo; Fernández-Santaella, M Carmen

2014-01-01

In the last decades, food pictures have been repeatedly employed to investigate the emotional impact of food on healthy participants as well as individuals who suffer from eating disorders and obesity. However, despite their widespread use, food pictures are typically selected according to each researcher's personal criteria, which make it difficult to reliably select food images and to compare results across different studies and laboratories. Therefore, to study affective reactions to food, it becomes pivotal to identify the emotional impact of specific food images based on wider samples of individuals. In the present paper we introduce the Open Library of Affective Foods (OLAF), which is a set of original food pictures created to reliably select food pictures based on the emotions they prompt, as indicated by affective ratings of valence, arousal, and dominance and by an additional food craving scale. OLAF images were designed to allow simultaneous use with affective images from the International Affective Picture System (IAPS), which is a well-known instrument to investigate emotional reactions in the laboratory. The ultimate goal of the OLAF is to contribute to understanding how food is emotionally processed in healthy individuals and in patients who suffer from eating and weight-related disorders. The present normative data, which was based on a large sample of an adolescent population, indicate that when viewing affective non-food IAPS images, valence, arousal, and dominance ratings were in line with expected patterns based on previous emotion research. Moreover, when viewing food pictures, affective and food craving ratings were consistent with research on food cue processing. As a whole, the data supported the methodological and theoretical reliability of the OLAF ratings, therefore providing researchers with a standardized tool to reliably investigate the emotional and motivational significance of food. The OLAF database is publicly available at zenodo.org.

A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells.

PubMed

Wu, Chunxiao; Wang, Shu

2012-01-01

Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.

Trypsin cleavage of the baculovirus occlusion-derived virus attachment protein P74 is prerequisite in per os infection.

PubMed

Slack, Jeffrey M; Lawrence, Susan D; Krell, Peter J; Arif, Basil M

2008-10-01

Baculovirus occlusion-derived virions (ODVs) contain a number of infectivity factors essential for the initiation of infection in larval midgut cells. Deletion of any of these factors neutralizes infectivity by the per os route. We have observed that P74 of the group I alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is N-terminally cleaved when a soluble form of the protein was incubated with insect midgut tissues under alkaline conditions and that cleavage was prevented by soybean trypsin inhibitor (SBTI). Presently, biological assays were carried out that suggest SBTI inhibits and trypsin enhances baculovirus per os infectivity. We developed a method to rescue per os infectivity of a P74 null virus involving co-transfection of viral DNA with a plasmid that transiently expresses p74. We used this plasmid rescue method to functionally characterize P74. A series of site-directed mutants were generated at the N terminus to evaluate if trypsin cleavage sites were necessary for function. Mutagenesis of R195, R196 and R199 compromised per os infectivity and rendered P74 resistant to midgut trypsin.

Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.

PubMed

Sequeira, Daniela P; Correia, Ricardo; Carrondo, Manuel J T; Roldão, António; Teixeira, Ana P; Alves, Paula M

2018-05-24

Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains and promising approaches based on the expression of multiple hemagglutinins (HA) in a virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can lead to its instability due to tandem repetition of similar sequences. By combining stable with transient expression systems we can rationally distribute the number of genes to be expressed per platform and thus mitigate this risk. In this work, we developed a modular system comprising stable and baculovirus-mediated expression in insect cells for production of multi-HA influenza enveloped VLPs. First, a stable insect High Five cell population expressing two different HA proteins from subtype H3 was established. Infection of this cell population with a baculovirus vector encoding three other HA proteins from H3 subtype proved to be as competitive as traditional co-infection approaches in producing a pentavalent H3 VLP. Aiming at increasing HA expression, the stable insect cell population was infected at increasingly higher cell concentrations (CCI). However, cultures infected at CCI of 3×10 6 cells/mL showed lower HA titers per cell in comparison to standard CCI of 2×10 6 cells/mL, a phenomenon named "cell density effect". To lessen the negative impact of this phenomenon, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth. Noteworthy, cultures supplemented and infected at a CCI of 4×10 6 cells/mL showed comparable HA titers per cell to those of CCI of 2×10 6 cells/mL, thus leading to an increase of up to 4-fold in HA titers per mL. Scalability of the modular strategy herein proposed was successfully demonstrated in 2L stirred tank bioreactors with comparable HA protein levels observed between bioreactor and shake flasks cultures. Overall, this work demonstrates the suitability of combining stable

Restriction by APOBEC3 proteins of endogenous retroviruses with an extracellular life cycle: ex vivo effects and in vivo "traces" on the murine IAPE and human HERV-K elements

PubMed Central

Esnault, Cécile; Priet, Stéphane; Ribet, David; Heidmann, Odile; Heidmann, Thierry

2008-01-01

Background APOBEC3 cytosine deaminases have been demonstrated to restrict infectivity of a series of retroviruses, with different efficiencies depending on the retrovirus. In addition, APOBEC3 proteins can severely restrict the intracellular transposition of a series of retroelements with a strictly intracellular life cycle, including the murine IAP and MusD LTR-retrotransposons. Results Here we show that the IAPE element, which is the infectious progenitor of the strictly intracellular IAP elements, and the infectious human endogenous retrovirus HERV-K are restricted by both murine and human APOBEC3 proteins in an ex vivo assay for infectivity, with evidence in most cases of strand-specific G-to-A editing of the proviruses, with the expected signatures. In silico analysis of the naturally occurring genomic copies of the corresponding endogenous elements performed on the mouse and human genomes discloses "traces" of APOBEC3-editing, with the specific signature of the murine APOBEC3 and human APOBEC3G enzymes, respectively, and to a variable extent depending on the family member. Conclusion These results indicate that the IAPE and HERV-K elements, which can only replicate via an extracellular infection cycle, have been restricted at the time of their entry, amplification and integration into their target host genomes by definite APOBEC3 proteins, most probably acting in evolution to limit the mutagenic effect of these endogenized extracellular parasites. PMID:18702815

MacoNPV baculovirus midgut-specific gene expression during infection of the bertha armyworm, Mamestra configurata

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

Donly, B. Cameron, E-mail: Cam.Donly@agr.gc.ca

Baculoviruses have two forms, occlusion derived virus (ODV) which is responsible for primary infection in host midgut tissue and budded virus (BV), which infects all other host tissues during secondary infection. This study examined the primary infection by ODV of midgut cells of bertha armyworm Mamestra configurata fourth instar larvae and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those produced by AcMNPV BV infection of in vitro cell cultures. This included having unique collections of genes expressed early, asmore » well as much greater late gene expression of p6.9 and much reduced expression of polh and p10. These differences likely reflect characteristics unique to the critical step of in vivo midgut cell infection, and provide insights into the processes that regulate viral gene expression in different host tissues. -- Highlights: •The transcriptome of MacoNPV ODV in larval midgut was measured by RNA-seq and digital PCR. •The earliest genes expressed included fusion protein, hoar, and me53. •p6.9 was highly expressed late but polH and p10 were less so. •These patterns are unique from BV of other baculoviruses in tissue culture cells.« less

Protective Efficacy of a Single Dose of Baculovirus Hemagglutinin-Based Vaccine in Chickens and Ducks Against Homologous and Heterologous H5N1 Virus Infections

PubMed Central

Park, Eun Hye; Song, Byung Min; Yum, Jung; Kim, Ji An; Oh, Seung Kyoo; Kim, Hyun Soo; Cho, Gil Jae

2014-01-01

Abstract Outbreaks of the highly pathogenic H5N1 virus in poultry and humans are ongoing. Vaccination is an efficient method for prevention of H5N1 infection. Using chickens and ducks, we assessed the efficacy of a vaccine comprising H5N1 hemagglutinin (HA) protein produced in a baculovirus expression system. The immunized chickens and ducks were protected against lethal infection by H5N1 in an antigen dose-dependent manner. Complete protection against homologous challenge and partial protection against heterologous challenge were achieved in chickens immunized with 5 μg HA protein and in ducks immunized with 10 μg HA protein. The IgG antibody subtype was mainly detected in the sera and tissues, including the lungs. The neuraminidase (NA) inhibition assay was negative in immunized chickens and ducks. Our results indicated that the expressed HA protein by baculovirus was immunogenic to both chickens and ducks, and the immunized chickens and ducks were protected from the lethal infections of highly pathogenic H5N1 influenza virus, though ducks required more HA protein than chickens to be protected. Also, baculovirus HA-vaccinated poultry can be differentiated from infected poultry by NA inhibition assay. PMID:25211640

Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression

PubMed Central

Li, Ao; Zhao, Haizhou; Lai, Qingying; Huang, Zhihong; Yuan, Meijin

2015-01-01

ABSTRACT Many viruses utilize viral or cellular chromatin machinery for efficient infection. Baculoviruses encode a conserved protamine-like protein, P6.9. This protein plays essential roles in various viral physiological processes during infection. However, the mechanism by which P6.9 regulates transcription remains unknown. In this study, 7 phosphorylated species of P6.9 were resolved in Sf9 cells infected with the baculovirus type species Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Mass spectrometry identified 22 phosphorylation and 10 methylation sites but no acetylation sites in P6.9. Immunofluorescence demonstrated that the P6.9 and virus-encoded serine/threonine kinase PK1 exhibited similar distribution patterns in infected cells, and coimmunoprecipitation confirmed the interaction between them. Upon pk1 deletion, nucleocapsid assembly and polyhedron formation were interrupted and the transcription of viral very late genes was downregulated. Interestingly, we found that the 3 most phosphorylated P6.9 species vanished from Sf9 cells transfected with the pk1 deletion mutant, suggesting that PK1 is involved in the hyperphosphorylation of P6.9. Mass spectrometry suggested that the phosphorylation of the 7 Ser/Thr and 5 Arg residues in P6.9 was PK1 dependent. Replacement of the 7 Ser/Thr residues with Ala resulted in a P6.9 phosphorylation pattern similar to that of the pk1 deletion mutant. Importantly, the decreases in the transcription level of viral very late genes and viral infectivity were consistent. Our findings reveal that P6.9 hyperphosphorylation is a precondition for the maximal hyperexpression of baculovirus very late genes and provide the first experimental insights into the function of the baculovirus protamine-like protein and the related protein kinase in epigenetics. IMPORTANCE Diverse posttranslational modifications (PTMs) of histones constitute a code that creates binding platforms that recruit transcription factors to

Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

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

Vanarsdall, Adam L.; Mikhailov, Victor S.; N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808

2007-08-01

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbpmore » knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp

Generation of PCV2 in PK15 cells transfected with recombinant baculovirus containing a 1.1 copy of the PCV2 genome.

PubMed

Cai, Jie; Xie, Xiaohong; Hu, Yi; Zhan, Yang; Yu, Wanting; Wang, Aibing; Wang, Naidong

2017-06-01

Porcine circovirus associated diseases (PCVAD) caused by PCV2 are responsible for severe economic losses in the swine industry. The mechanism of PCV2 replication has not been fully elucidated yet. PCV2 may be successfully rescued by means of either an infectious DNA clone containing the full length of the viral genomic DNA, or from PCV2-infected clinical tissues in PK15 cell culture. However, viruses harvested by both methods have low titres. In this study, PCV2 was prepared with a higher titre from PK15 cells infected by recombinant baculoviruses containing 1PCV2 (one stem-loop structure) or 1.1PCV2 (two stem-loop structure) genomic DNA copy. In addition, infectious DNA clones containing two stem-loop structures in either plasmid or baculovirus backbones are capable of generating a higher virus titre than the DNA clones with only one copy of stem-loop structure.

The production of multiprotein complexes in insect cells using the baculovirus expression system.

PubMed

Abdulrahman, Wassim; Radu, Laura; Garzoni, Frederic; Kolesnikova, Olga; Gupta, Kapil; Osz-Papai, Judit; Berger, Imre; Poterszman, Arnaud

2015-01-01

The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of multiprotein complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.

Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems.

PubMed

Vettath, Sunitha Kodengil; Shivashankar, Gaganashree; Menon, Krishnakumar N; Vijayachandran, Lakshmi S

2018-04-15

Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved. Copyright © 2018 Elsevier B.V. All rights reserved.

Immune responses to baculovirus-displayed enterovirus 71 VP1 antigen.

PubMed

Kiener, Tanja K; Premanand, Balraj; Kwang, Jimmy

2013-04-01

The increased distribution and neurovirulence of enterovirus 71 is an important health threat for young children in Asia Pacific. Vaccine design has concentrated on inactivated virus with the most advanced undergoing Phase III clinical trials. By using a subunit vaccine approach, production costs could be reduced by lowering the need for biocontainment. In addition, novel mutations could be rapidly incorporated to reflect the emergence of new enterovirus 71 subgenogroups. To circumvent the problems associated with conventional subunit vaccines, the antigen can be displayed on a viral vector that conveys stability and facilitates purification. Additional advantages of viral-vectored subunit vaccines are their ability to stimulate the innate immune system by transducing cells and the possibility of oral or nasal delivery, which dispenses with the need for syringes and medical personnel. Baculovirus-displayed VP1 combines all these benefits with protection that is as efficient as inactivated virus.

Recombinant ELISA using baculovirus-expressed VP2 for detection of antibodies against canine parvovirus.

PubMed

Elia, Gabriella; Desario, Costantina; Pezzoni, Giulia; Camero, Michele; Brocchi, Emiliana; Decaro, Nicola; Martella, Vito; Buonavoglia, Canio

2012-09-01

The gene encoding the VP2 protein of canine parvovirus type 2 was expressed in an insect-baculovirus system. The recombinant (r) VP2 was similar antigenically/functionally to the native capsid protein as demonstrated by hemagglutination, Western blotting and hemagglutination inhibition test, using Canine parvovirus type-2 (CPV-2) positive sera. An enzyme-linked immunosorbent assay (ELISA) using the rVP2 was used for testing CPV-2 positive and negative sera from dogs and for determining the threshold of maternally derived antibodies interfering with successful vaccination of pups against CPV-2. Copyright © 2012 Elsevier B.V. All rights reserved.

Friendship IAP, Maryland. Revised Uniform Summary of Surface Weather Observations (RUSSWO). Parts A-F.

DTIC Science & Technology

1982-05-04

USAFETAC PSYCHROMETRIC SUMMARY’k A1 dEATHER SERVXCE/MAC STATIO STATION 041A vt LS MONT. PAGE I1 - i(Fl WI~III~i~i1IIi ET SUL& TESIPEXATUnE DEPRESION (F...WEATHER SERVICE/MAC L2AD0. FRIENDOSHIP IAP MD 74-S1orf STATION STATION *464 VtAAS M. PAGE 2 -nn-nn Th.~.WIT SUL$ TEMPERATURE DEPRESION (F) ITOTAL TOTAL () 0...Alte WEAT.4ER SERVICE/IAC 776ff F7hnSHTP Ti A mn-t ER STAION NWA YA PAGE 1 WE? BULB TEMPERATURE DEPRESION (F) TOTAL 1 TOTAL (I 0 1.2 j3-43. 7. 9. . 1o 1

Development of a subunit vaccine for infectious pancreatic necrosis virus using a baculovirus insect/larvae system

USGS Publications Warehouse

Shivappa, R.B.; McAllister, P.E.; Edwards, G.H.; Santi, N.; Evensen, O.; Vakharia, V.N.; ,

2005-01-01

Various attempts to develop a vaccine against infectious pancreatic necrosis virus (IPNV) have not yielded consistent results. Thus, at present, no commercial vaccine is available that can be used with confidence to immunize fry of salmon and trout. We generated a cDNA clone of the large genome segment A of an IPNV Sp strain and expressed all structural protein genes in insect cells and larvae using a baculovirus expression system. Green fluorescent protein was also co-expressed as a reporter molecule. High yields of IPNV proteins were obtained and the structural proteins self assembled to form virus-like particles (VLPs). We tested the immunogenicity of the putative VLP antigen in immersion vaccine experiments (two concentrations) in rainbow trout (Oncorhynchus mykiss) fry, and by intraperitoneal immunisation of Atlantic salmon (Salmo salar) pre-smolts using an oil adjuvant formulation. Rainbow trout were challenged by immersion using either the Sp or the VR-299 strain of IPNV two or three weeks post-vaccination, while Atlantic salmon were bath challenged with Sp strain after two months, after parr-smolt transformation. In the rainbow trout fry challenged two weeks post-immunization, cumulative mortality rates three weeks post challenge were 14 % in the fry that had received the highest dose versus 8 % in the control groups. No indication of protection was seen in repeated trials using a lower dose of antigen and challenge three weeks post-immunisation. The cumulative mortality rate of intraperitoneally immunised Atlantic salmon post-smolts four weeks post challenge was lower (56 %) than in the control fish (77 %), showing a dose-response pattern.

High-yield production of canine parvovirus virus-like particles in a baculovirus expression system.

PubMed

Jin, Hongli; Xia, Xiaohong; Liu, Bing; Fu, Yu; Chen, Xianping; Wang, Huihui; Xia, Zhenqiang

2016-03-01

An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.

The silencing suppressor (NSs) protein of the plant virus Tomato spotted wilt virus enhances heterologous protein expression and baculovirus pathogenicity in cells and lepidopteran insects.

PubMed

de Oliveira, Virgínia Carla; da Silva Morgado, Fabricio; Ardisson-Araújo, Daniel Mendes Pereira; Resende, Renato Oliveira; Ribeiro, Bergmann Morais

2015-11-01

In this work, we showed that cell death induced by a recombinant (vAcNSs) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expressing the silencing suppressor (NSs) protein of Tomato spotted wilt virus (TSWV) was enhanced on permissive and semipermissive cell lines. The expression of a heterologous gene (firefly luciferase) during co-infection of insect cells with vAcNSs and a second recombinant baculovirus (vAgppolhfluc) was shown to increase when compared to single vAgppolhfluc infections. Furthermore, the vAcNSs mean time-to-death values were significantly lower than those for wild-type AcMNPV on larvae of Spodoptera frugiperda and Anticarsia gemmatalis. These results showed that the TSWV-NSs protein could efficiently increase heterologous protein expression in insect cells as well as baculovirus pathogenicity and virulence, probably by suppressing the gene-silencing machinery in insects.

Physiologically based pharmacokinetic and pharmacodynamic modeling of an antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in a mouse xenograft model of human breast cancer.

PubMed

Zhang, Tao; Li, Yanyan; Zou, Peng; Yu, Jing-yu; McEachern, Donna; Wang, Shaomeng; Sun, Duxin

2013-09-01

The inhibitors of apoptosis proteins (IAPs) are a class of key apoptosis regulators overexpressed or dysregulated in cancer. SM-406/AT-406 is a potent and selective small molecule mimetic of Smac that antagonizes the inhibitor of apoptosis proteins (IAPs). A physiologically based pharmacokinetic and pharmacodynamic (PBPK-PD) model was developed to predict the tissue concentration-time profiles of SM-406, the related onco-protein levels in tumor, and the tumor growth inhibition in a mouse model bearing human breast cancer xenograft. In the whole body physiologically based pharmacokinetic (PBPK) model for pharmacokinetics characterization, a well stirred (perfusion rate-limited) model was used to describe SM-406 pharmacokinetics in the lung, heart, kidney, intestine, liver and spleen, and a diffusion rate-limited (permeability limited) model was used for tumor. Pharmacodynamic (PD) models were developed to correlate the SM-406 concentration in tumor to the cIAP1 degradation, pro-caspase 8 decrease, CL-PARP accumulation and tumor growth inhibition. The PBPK-PD model well described the experimental pharmacokinetic data, the pharmacodynamic biomarker responses and tumor growth. This model may be helpful to predict tumor and plasma SM-406 concentrations in the clinic. Copyright © 2013 John Wiley & Sons, Ltd.

A novel SMAC mimetic APG-1387 exhibits dual antitumor effect on HBV-positive hepatocellular carcinoma with high expression of cIAP2 by inducing apoptosis and enhancing innate anti-tumor immunity.

PubMed

Pan, Wentao; Luo, Qiuyun; Yan, Xianglei; Yuan, Luping; Yi, Hanjie; Zhang, Lin; Li, Baoxia; Zhang, Yuxin; Sun, Jian; Qiu, Miao-Zhen; Yang, Da-Jun

2018-04-18

Check point inhibitor anti-PD1 antibody produced some efficacy in Hepatocellular Carcinoma (HCC) patients previously treated with sorafenib. Unfortunately, HCC patients with hepatitis B virus (HBV) infection did not respond as well as uninfected patients. Previously, Second mitochondria-derived activator of caspases (SMAC) mimetics-the antagonist for inhibitor of apoptosis proteins (IAPs) can rapidly reduce serum hepatitis B virus DNA in animal model. APG-1387 is a novel SMAC-mimetic, small molecule inhibitor targeting inhibitor of apoptosis proteins (IAPs). In our study, firstly, we found that HCC patients with copy number alteration of cIAP1, cIAP2, and XIAP had a dismal prognosis. Then, we discovered that APG-1387 alone could induce apoptosis of PLC/PRF/5 which was HBV positive both in-vitro and in-vivo. Furthermore, we found that APG-1387 significantly up-regulated the expression of calreticulin and HLA-DR in PLC/PRF/5 via activating non-classic NF-κB pathway. Also, compared to vehicle group, APG-1387 increased NK cell counts by 5 folds in PLC/PRF/5 xenograft model. In-vitro, APG-1387 positively regulated T cells by reducing Treg differentiation and down-regulating PD1 expression in CD4 T cell. Moreover, APG-1387 had no impact on memory T cells. Consequently, our results suggest that APG1387 could be a good candidate to combine with anti-PD1 antibody treatment to overcome low responds of check point inhibitors in HBV positive HCC. Copyright © 2018 Elsevier Inc. All rights reserved.

DEPENDENCE OF ECDYSTEROID METABOLISM AND DEVELOPMENT IN HOST LARVAE ON THE TIME OF BACULOVIRUS INFECTION AND THE ACTIVITY OF THE UDP-GLUCOSYL TRANSFERASE GENE.

EPA Science Inventory

Infection of fourth-instar gypsy moth (Lymantria dispar, Lepidoptera: Lymantriidae) larvae with the wild-type (Wt) gypsy moth baculovirus, LdNPV on the first day post-molt, or infection of fifth instars on the fifth day post-molt, results in elevated ecdysteroid levels in both he...

The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, Mythimna unipuncta, contains two copies of the lef-7 gene

USDA-ARS?s Scientific Manuscript database

A baculovirus isolate from a USDA Forest Service collection was examined by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species My...

The activity of phenoloxidase in haemolymph plasma is not a predictor of Lymantria dispar resistance to its baculovirus

PubMed Central

Kasianov, Nikita S.; Belousova, Irina A.; Pavlushin, Sergey V.; Dubovskiy, Ivan M.; Podgwaite, John D.; Bakhvalov, Stanislav A.

2017-01-01

Host innate immunity is one of the factors that determines the resistance of insects to their entomopathogens. In the research reported here we studied whether or not phenoloxidase (PO), a key enzyme in the melanogenesis component of humoral immunity of insects, plays a role in the protection of Lymantria dispar larvae from infection by L. dispar multiple nucleopolyhedrovirus. We studied two types of viral infection: overt and covert. The following lines of investigation were tested: i) the intravital individual estimation of baseline PO activity in haemolymph plasma followed by virus challenging; ii) the specific inhibition of PO activity in vivo by peroral treatment of infected larvae with phenylthiourea (PTU), a competitive inhibitor of PO; iii) the evaluation of PO activity in the haemolymph plasma after larval starvation. Starvation is a stress that activates the covert infection to an overt form. All of these experiments did not show a relationship between PO activity in haemolymph plasma of L. dispar larvae and larval susceptibility to baculovirus. Moreover, starvation-induced activation of covert viral infection to an overt form occurred in 70 percent of virus-carrying larvae against the background of a dramatic increase of PO activity in haemolymph plasma in the insects studied. Our conclusion is that in L. dispar larvae PO activity is not a predictor of host resistance to baculovirus. PMID:28854240

Baculovirus directly activates murine NK cells via TLR9.

PubMed

Moriyama, T; Suzuki, T; Chang, M O; Kitajima, M; Takaku, H

2017-04-01

The importance of natural killer (NK) cells in innate immune responses against tumors or viral infections enhances the appeal of NK cell-based immunotherapeutic approaches. We have recently reported that baculovirus (BV)-infected dendritic cells (DCs; BV-DCs) induce antitumor immunity against established tumors in mice. These antitumor effects were CD8 + T-cell and NK cell dependent; however, they were found to be CD4 + T-cell independent. In this study, we investigated the involvement of Toll-like receptor 9 (TLR9) in the process of BV recognition by NK cells. We found that BV directly stimulated NK cells, induced the expression of the activation marker CD69 and promoted interferon-gamma (IFN-γ) production and cytotoxicity. Moreover, TLR9 knockout in mice (tlr9-/- NK cells) inhibited NK cell responses to BV, indicating that TLR9 may have a relevant role in the BV-induced upregulation of NK cell functions. Our data demonstrated for the first time that NK cells directly recognize BV via TLR9, which provides opportunities for the use of this technique as an effective tool for BV-based immunotherapies against malignancies.

Nudiviruses and other large, double-stranded circular DNA viruses of invertebrates: new insights on an old topic.

PubMed

Wang, Yongjie; Jehle, Johannes A

2009-07-01

Nudiviruses (NVs) are a highly diverse group of large, circular dsDNA viruses pathogenic for invertebrates. They have rod-shaped and enveloped nucleocapsids, replicate in the nucleus of infected host cells, and possess interesting biological and molecular properties. The unassigned viral genus Nudivirus has been proposed for classification of nudiviruses. Currently, the nudiviruses comprise five different viruses: the palm rhinoceros beetle virus (Oryctes rhinoceros NV, OrNV), the Hz-1 virus (Heliothis zea NV-1, HzNV-1), the cricket virus (Gryllus bimaculatus NV, GbNV), the corn earworm moth Hz-2 virus (HzNV-2), and the occluded shrimp Monodon Baculovirus reassigned as Penaeus monodon NV (PmNV). Thus far, the genomes of OrNV, GbNV, HzNV-1 and HzNV-2 have been completely sequenced. They vary between 97 and 230kbp in size and encode between 98 and 160 open reading frames (ORFs). All sequenced nudiviruses have 33 ORFs in common. Strikingly, 20 of them are homologous to baculovirus core genes involved in RNA transcription, DNA replication, virion structural components and other functions. Another nine conserved ORFs are likely associated with DNA replication, repair and recombination, and nucleotide metabolism; one is homologous to baculovirus iap-3 gene; two are nudivirus-specific ORFs of unknown function. Interestingly, one nudivirus ORF is similar to polh/gran gene, encoding occlusion body protein matrix and being conserved in Alpha- Beta- and Gammabaculoviruses. Members of nudiviruses are closely related and form a monophyletic group consisting of two sister clades of OrNV/GbNV and HzNVs/PmNV. It is proposed that nudiviruses and baculoviruses derived from a common ancestor and are evolutionarily related to other large DNA viruses such as the insect-specific salivary gland hypertrophy virus (SGHV) and the marine white spot syndrome virus (WSSV).

IAP Guidelines on Rickettsial Diseases in Children.

PubMed

Rathi, Narendra; Kulkarni, Atul; Yewale, Vijay

2017-03-15

To formulate practice guidelines on rickettsial diseases in children for pediatricians across India. Rickettsial diseases are increasingly being reported from various parts of India. Due to low index of suspicion, nonspecific clinical features in early course of disease, and absence of easily available, sensitive and specific diagnostic tests, these infections are difficult to diagnose. With timely diagnosis, therapy is easy, affordable and often successful. On the other hand, in endemic areas, where healthcare workers have high index of suspicion for these infections, there is rampant and irrational use of doxycycline as a therapeutic trial in patients of undifferentiated fevers. Thus, there is a need to formulate practice guidelines regarding rickettsial diseases in children in Indian context. A committee was formed for preparing guidelines on rickettsial diseases in children in June 2016. A meeting of consultative committee was held in IAP office, Mumbai and scientific content was discussed. Methodology and results were scrutinized by all members and consensus was reached. Textbook references and published guidelines were also used in few instances to make recommendations. Various Indian and international publications pertinent to present study were collated and guidelines were approved by all committee members. Future updates in these guidelines will be dictated by new scientific data in the field of rickettsial diseases in children. Indian tick typhus and scrub typhus are commonly seen rickettsial diseases in India. It is recommended that practicing pediatricians should be well conversant with compatible clinical scenario, suggestive epidemiological features, differential diagnoses and suggestive laboratory features to make diagnosis and avoid over diagnosis of these infections, as suggested in these guidelines. Doxycycline is the drug of choice and treatment should begin promptly without waiting for confirmatory laboratory results.

Initial preclinical safety of non-replicating human endogenous retrovirus envelope protein-coated baculovirus vector-based vaccines against human papillomavirus.

PubMed

Han, Su-Eun; Kim, Mi-Gyeong; Lee, Soondong; Cho, Hee-Jeong; Byun, Youngro; Kim, Sujeong; Kim, Young Bong; Choi, Yongseok; Oh, Yu-Kyoung

2013-12-01

Human endogenous retrovirus (HERV) envelope protein-coated, baculovirus vector-based HPV 16 L1 (AcHERV-HPV16L1) is a non-replicating recombinant baculoviral vaccine. Here, we report an initial evaluation of the preclinical safety of AcHERV-HPV16L1 vaccine. In an acute toxicity study, a single administration of AcHERV-HPV16L1 DNA vaccine given intramuscularly (i.m.) to mice at a dose of 1 × 10(8) plaque-forming units (PFU) did not cause significant changes in body weight compared with vehicle-treated controls. It did cause a brief increase in the weights of some organs on day 15 post-treatment, but by day 30, all organ weights were not significantly different from those in the vehicle-treated control group. No hematological changes were observed on day 30 post-treatment. In a range-finding toxicity study with three doses of 1 × 10(7) , 2 × 10(7) and 5 × 10(7) PFU once daily for 5 days, the group treated with 5 × 10(7) PFU showed a transient decrease in the body weights from day 5 to day 15 post-treatment, but recovery to the levels similar to those in the vehicle-treated control group by post-treatment day 20. Organ weights were slightly higher for lymph nodes, spleen, thymus and liver after repeated dosing with 5 × 10(7) PFU on day 15, but had normalized by day 30. Moreover, repeated administration of AcHERV-HPV16L1 did not induce myosin-specific autoantibody in serum, and did not cause immune complex deposition or tissue damage at injection sites. Taken together, these results provide preliminary evidence of the preclinical safety of AcHERV-based HPV16L1 DNA vaccines in mice. Copyright © 2012 John Wiley & Sons, Ltd.

Comparison of two eukaryotic systems for the expression of VP6 protein of rotavirus specie A: transient gene expression in HEK293-T cells and insect cell-baculovirus system.

PubMed

da Silva Junior, Haroldo Cid; da Silva E Mouta Junior, Sérgio; de Mendonça, Marcos César Lima; de Souza Pereira, Mirian Claudia; da Rocha Nogueira, Alanderson; de Azevedo, Maria Luiza Borges; Leite, José Paulo Gagliardi; de Moraes, Márcia Terezinha Baroni

2012-09-01

The VP6 protein of rotavirus A (RVA) is a target antigen used for diagnostic assays and also for the development of new RVA vaccines. We have compared the expression of VP6 protein in human embryonic kidney (HEK293-T) cells with results obtained using a well-established insect cell-baculovirus system. The recombinant VP6 (rVP6) expressed in HEK293-T cells did not present degradation and also retained the ability to form trimers. In the insect cell-baculovirus system, rVP6 was expressed at higher levels and with protein degradation as well as partial loss of ability to form trimers was observed. Therefore, HEK293-T cells represent a less laborious alternative system than insect cells for expression of rVP6 from human RVA.

The baculovirus-integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase.

PubMed Central

Lerch, R A; Friesen, P D

1992-01-01

TED is a lepidopteran retrotransposon found inserted within the DNA genome of the Autographa californica nuclear polyhedrosis virus mutant, FP-D. To examine the proteins and functions encoded by this representative of the gypsy family of retrotransposons, the gag- and pol-like open reading frames (ORFs 1 and 2) were expressed in homologous lepidopteran cells by using recombinant baculovirus vectors. Expression of ORF 1 resulted in synthesis of an abundant TED-specific protein (Pr55gag) that assembled into viruslike particles with a diameter of 55 to 60 nm. Expression of ORF 2, requiring a -1 translational frameshift, resulted in synthesis of a protease that mediated cleavage of Pr55gag to generate p37, the major protein component of the resulting particles. Expression of ORF 2 also produced reverse transcriptase that associated with these particles. Both protease and reverse transcriptase activities mapped to domains within ORF 2 that contain sequence similarities with the corresponding functional domains of the pol gene of the vertebrate retroviruses. These results indicated that TED ORFs 1 and 2 functionally resemble the retrovirus gag and pol genes and demonstrated for the first time that an invertebrate member of the gypsy family of elements encodes active forms of the structural and enzymatic functions necessary for transposition via an RNA intermediate. TED integration within the baculovirus genome thus represents one of the first examples of transposon-mediated transfer of host-derived genes to an eukaryotic virus. Images PMID:1371168

Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis.

PubMed

Hepp, Susan E; Borgo, Gina M; Ticau, Simina; Ohkawa, Taro; Welch, Matthew D

2018-06-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex. IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which Ac

Seroprevalence of sapovirus in dogs using baculovirus-expressed virus-like particles.

PubMed

Melegari, Irene; Marsilio, Fulvio; Di Profio, Federica; Sarchese, Vittorio; Massirio, Ivano; Palombieri, Andrea; D'Angelo, Anna Rita; Lanave, Gianvito; Diakoudi, Georgia; Cavalli, Alessandra; Martella, Vito; Di Martino, Barbara

2018-06-02

Caliciviruses of the Sapovirus genus have been recently detected in dogs. Canine sapoviruses (SaVs) have been identified in the stools of young or juvenile animals with gastro-enteric disease at low prevalence (2.0-2.2%), but whether they may have a role as enteric pathogens and to which extent dogs are exposed to SaVs remains unclear. Here, we report the expression in a baculovirus system of virus like-particles (VLPs) of a canine SaV strain, the prototype virus Bari/4076/2007/ITA. The recombinant antigen was used to develop an enzyme-linked immunosorbent assay (ELISA). By screening an age-stratified collection of serum samples from 516 dogs in Italy, IgG antibodies specific for the canine SaV VLPs were detected in 40.3% (208/516) of the sera. Also, as observed for SaV infection in humans, we observed a positive association between seropositivity and age, with the highest prevalence rates in dogs older than 4 years of age. Copyright © 2018 Elsevier B.V. All rights reserved.

Temporal regulation of Drosophila IAP1 determines caspase functions in sensory organ development

PubMed Central

Koto, Akiko; Kuranaga, Erina

2009-01-01

The caspases comprise a family of cysteine proteases that function in various cellular processes, including apoptosis. However, how the balance is struck between the caspases’ role in cell death and their nonapoptotic functions is unclear. To address this issue, we monitored the protein turnover of an endogenous caspase inhibitor, Drosophila IAP1 (DIAP1). DIAP1 is an E3 ubiquitin ligase that promotes the ubiquitination of caspases and thereby prevents caspase activation. For this study, we developed a fluorescent probe to monitor DIAP1 turnover in the external sensory organ precursor (SOP) lineage of living Drosophila. The SOP divides asymmetrically to make the shaft, socket, and sheath cells, and the neuron that comprise each sensory organ. We found that the quantity of DIAP1 changed dramatically depending on the cell type and maturity, and that the temporal regulation of DIAP1 turnover determines whether caspases function nonapoptotically in cellular morphogenesis or cause cell death. PMID:19822670

A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

PubMed

Oliveira, Virgínia Carla; Bartasson, Lorrainy; de Castro, Maria Elita Batista; Corrêa, José Raimundo; Ribeiro, Bergmann Morais; Resende, Renato Oliveira

2011-01-01

The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector. Copyright © 2010 Elsevier B.V. All rights reserved.

Dengue-1 Virus Envelope Glycoprotein Gene Expressed in Recombinant Baculovirus Elicits Virus-Neutralizing Antibody in Mice and Protects them from Virus Challenge

DTIC Science & Technology

1991-01-01

8217 terminus of E. When the recombinant virus was grown in Spodoptera frugiperda cells. about I mg of E antigen was made per 10’ cells. Recombinant E antigen...assay with DEN-I virus coprotein gene and its expression in Spodoptera hyperimmune mouse ascitic fluid. This heat-in- frugiperda cells activated...immunization, S. frugiperda cells infected with tion with BstNI (cuts at nucleotides 801 and recombinant baculovirus were pelleted. lysed by 2150). The

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

PubMed

Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

2017-12-01

Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a

Phenotypic Variation in Overwinter Environmental Transmission of a Baculovirus and the Cost of Virulence.

PubMed

Fleming-Davies, Arietta E; Dwyer, Greg

2015-12-01

A pathogen's ability to persist in the environment is an ecologically important trait, and variation in this trait may promote coexistence of different pathogen strains. We asked whether naturally occurring isolates of the baculovirus that infects gypsy moth larvae varied in their overwinter environmental transmission and whether this variation was consistent with a trade-off or an upper limit to virulence that might promote pathogen diversity. We used experimental manipulations to replicate the natural overwinter infection process, using 16 field-collected isolates. Virus isolates varied substantially in the fraction of larvae infected, leading to differences in overwinter transmission rates. Furthermore, isolates that killed more larvae also had higher rates of early larval death in which no infectious particles were produced, consistent with a cost of high virulence. Our results thus support the existence of a cost that could impose an upper limit to virulence even in a highly virulent pathogen.

Is heart rate variability (HRV) an adequate tool for evaluating human emotions? - A focus on the use of the International Affective Picture System (IAPS).

PubMed

Choi, Kwang-Ho; Kim, Junbeom; Kwon, O Sang; Kim, Min Ji; Ryu, Yeon Hee; Park, Ji-Eun

2017-05-01

Because human emotion varies greatly among individuals and is a qualitative factor, measuring it with any degree of accuracy is very difficult. Heart rate variability (HRV), which is used in evaluations of the autonomic nervous system (ANS), is used to evaluate human emotions. This study examines the validity of HRV as a tool to evaluate emotions using the International Affective Picture System (IAPS). For experimentation, five photos were selected for each of the categories of "happy," "unhappy", and "neutral" from among the images provided by the IAPS. The subjects were required to complete the Self-Assessment Manikin (SAM) after being shown each picture. We extracted the R-R interval (RRI) value of each photo from the PPG, as well as the valence, arousal, and dominance value of each photo from the SAM to analyze their correlation. As results, there was significant positive correlation with valence and significant negative correlation with dominance in the photo simulation associated with the "unhappy" emotion, only when the arousal value exceeded a critical value. Therefore, the findings of this study suggest that it is possible to use an HRV-based evaluation only when a high level of emotion is induced by visual stimulation. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration.

PubMed

Oliveira, Catarina R; Lemaitre, Regis; Murawala, Prayag; Tazaki, Akira; Drechsel, David N; Tanaka, Elly M

2018-01-15

Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Prevalence, diversity and co-occurrence of the white spot syndrome virus, monodon baculovirus and Penaeus stylirostris densovirus in wild populations of Penaeus monodon in the Philippines.

PubMed

Orosco, Fredmoore L; Lluisma, Arturo O

2017-08-09

The farming of the black tiger shrimp Penaeus monodon in the Philippines relies on wild broodstock. PCR was thus used to determine the prevalence of white spot syndrome virus (WSSV), monodon baculovirus (MBV) and Penaeus stylirostris densovirus (PstDV) in a total of 178 shrimp from 6 geographically disparate locations where broodstock are captured for use in hatcheries. PCR amplicons were also sequenced to identify phylogenetic relationships of the virus haplotypes detected. Shrimp from southeastern Luzon (Camarines Norte) had the highest prevalence of each of the 3 viruses and were frequently co-infected with 2 or more viruses. No viruses were detected in shrimp from northwestern Luzon (Pangasinan). MBV was most prevalent and PstDV strains displayed the most genetic diversity. WSSV was detected at 3 sites, and a VP28 gene sequence examined was invariant and consistent with strains found in many countries, including Thailand, China, Japan, Korea, Indonesia, Iran, Brazil and Mexico. WSSV open reading frame 94 gene sequence analysis identified location-specific repeat types. MBV sequences were dissimilar to haplotypes detected in India. PstDV sequences were diverse and included 2 lineages detected either in Australia or in the United States, Ecuador, Taiwan, China and Vietnam. The PCR data confirmed that WSSV, MBV and PstDV are endemic in P. monodon in the Philippines but that populations at some locations might remain free of infection.

Comprehensive analysis of single molecule sequencing-derived complete genome and whole transcriptome of Hyposidra talaca nuclear polyhedrosis virus.

PubMed

Nguyen, Thong T; Suryamohan, Kushal; Kuriakose, Boney; Janakiraman, Vasantharajan; Reichelt, Mike; Chaudhuri, Subhra; Guillory, Joseph; Divakaran, Neethu; Rabins, P E; Goel, Ridhi; Deka, Bhabesh; Sarkar, Suman; Ekka, Preety; Tsai, Yu-Chih; Vargas, Derek; Santhosh, Sam; Mohan, Sangeetha; Chin, Chen-Shan; Korlach, Jonas; Thomas, George; Babu, Azariah; Seshagiri, Somasekar

2018-06-12

We sequenced the Hyposidra talaca NPV (HytaNPV) double stranded circular DNA genome using PacBio single molecule sequencing technology. We found that the HytaNPV genome is 139,089 bp long with a GC content of 39.6%. It encodes 141 open reading frames (ORFs) including the 37 baculovirus core genes, 25 genes conserved among lepidopteran baculoviruses, 72 genes known in baculovirus, and 7 genes unique to the HytaNPV genome. It is a group II alphabaculovirus that codes for the F protein and lacks the gp64 gene found in group I alphabaculovirus viruses. Using RNA-seq, we confirmed the expression of the ORFs identified in the HytaNPV genome. Phylogenetic analysis showed HytaNPV to be closest to BusuNPV, SujuNPV and EcobNPV that infect other tea pests, Buzura suppressaria, Sucra jujuba, and Ectropis oblique, respectively. We identified repeat elements and a conserved non-coding baculovirus element in the genome. Analysis of the putative promoter sequences identified motif consistent with the temporal expression of the genes observed in the RNA-seq data.

Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice

PubMed Central

Jin, Ling; Carpenter, Dale; Moerdyk-Schauwecker, Megan; Vanarsdall, Adam L; Osorio, Nelson; Hsiang, Chinhui; Jones, Clinton; Wechsler, Steven L

2010-01-01

Latency-associated transcript (LAT) deletion mutants of herpes simplex virus type 1 (HSV-1) have reduced reactivation phenotypes. Thus, LAT plays an essential role in the latency-reactivation cycle of HSV-1. We have shown that LAT has antiapoptosis activity and demonstrated that the chimeric virus, dLAT-cpIAP, resulting from replacing LAT with the baculovirus antiapoptosis gene cpIAP, has a wild-type HSV-1 reactivation phenotype in mice and rabbits. Thus, LAT can be replaced by an alternative antiapoptosis gene, confirming that LAT’s antiapoptosis activity plays an important role in the mechanism by which LAT enhances the virus’ reactivation phenotype. However, because cpIAP interferes with both of the major apoptosis pathways, these studies did not address whether LAT’s proreactivation phenotype function was due to blocking the extrinsic (Fas-ligand–, caspase-8–, or caspase-10–dependent pathway) or the intrinsic (mitochondria-, caspase-9–dependent pathway) pathway, or whether both pathways must be blocked. Here we constructed an HSV-1 LAT(−) mutant that expresses cellular FLIP (cellular FLICE-like inhibitory protein) under control of the LAT promoter and in place of LAT nucleotides 76 to 1667. Mice were ocularly infected with this mutant, designated dLAT-FLIP, and the reactivation phenotype was determined using the trigeminal ganglia explant model. dLAT-FLIP had a reactivation phenotype similar to wild-type virus and significantly higher than the LAT(−) mutant dLAT2903. Thus, the LAT function responsible for enhancing the reactivation phenotype could be replaced with an antiapoptosis gene that primarily blocks the extrinsic signaling apoptosis pathway. PMID:18989818

Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection.

PubMed

Zhang, Yuan; Qiao, Lei; Hu, Xiao; Zhao, Kang; Zhang, Yanwen; Chai, Feng; Pan, Zishu

2016-01-04

Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV. Copyright © 2015 Elsevier Ltd. All rights reserved.

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

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

Ju, Huanyu; Wei, Na; Wang, Qian

Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particlesmore » (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.« less

Proteomics of the Autographa californica Nucleopolyhedrovirus Budded Virions ▿

PubMed Central

Wang, RanRan; Deng, Fei; Hou, Dianhai; Zhao, Yong; Guo, Lin; Wang, Hualin; Hu, Zhihong

2010-01-01

Baculoviruses produce two progeny phenotypes during their replication cycles. The occlusion-derived virus (ODV) is responsible for initiating primary infection in the larval midgut, and the budded virus (BV) phenotype is responsible for the secondary infection. The proteomics of several baculovirus ODVs have been revealed, but so far, no extensive analysis of BV-associated proteins has been conducted. In this study, the protein composition of the BV of Autographa californica nucleopolyhedrovirus (AcMNPV), the type species of baculoviruses, was analyzed by various mass spectrometry (MS) techniques, including liquid chromatography-triple quadrupole linear ion trap (LC-Qtrap), liquid chromatography-quadrupole time of flight (LC-Q-TOF), and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). SDS-PAGE and MALDI-TOF analyses showed that the three most abundant proteins of the AcMNPV BV were GP64, VP39, and P6.9. A total of 34 viral proteins associated with the AcMNPV BV were identified by the indicated methods. Thirteen of these proteins, PP31, AC58/59, AC66, IAP-2, AC73, AC74, AC114, AC124, chitinase, polyhedron envelope protein (PEP), AC132, ODV-E18, and ODV-E56, were identified for the first time to be BV-associated proteins. Western blot analyses showed that ODV-E18 and ODV-E25, which were previously thought to be ODV-specific proteins, were also present in the envelop fraction of BV. In addition, 11 cellular proteins were found to be associated with the AcMNPV BV by both LC-Qtrap and LC-Q-TOF analyses. Interestingly, seven of these proteins were also identified in other enveloped viruses, suggesting that many enveloped viruses may commonly utilize certain conserved cellular pathways. PMID:20444894

The heptad repeats region is essential for AcMNPV P10 filament formation and not the proline-rich or the C-terminus basic regions.

PubMed

Dong, Chunsheng; Deng, Fei; Li, Dan; Wang, Hualin; Hu, Zhihong

2007-09-01

Baculovirus P10 protein is a small conserved protein and is expressed as bundles of filaments in the host cell during the late phase of virus infection. So far the published results on the domain responsible for filament structural formation have been contradictory. Electron microscopy revealed that the C-terminus basic region was involved in filament structural formation in the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) [van Oers, M.M., Flipsen, J.T., Reusken, C.B., Sliwinsky, E.L., Vlak, J.M., 1993. Functional domains of the p10 protein of Autographa californica nuclear polyhedorsis virus. J. Gen. Virol. 74, 563-574.]. While in the Helicoverpa armigera nucleopolyhedrovirus (HearNPV), the heptad repeats region but not the C-terminus domain was proven to be responsible for filament formation [Dong, C., Li, D., Long, G., Deng, F., Wang, H., Hu, Z., 2005. Identification of functional domains required for HearNPV P10 filament formation. Virology 338, 112-120.]. In this manuscript, fluorescence confocal microscopy was applied to study AcMNPV P10 filament formation. A set of plasmids containing different P10 structural domains fused with a fluorescent protein were constructed and transfected into Sf-9 cells. The data indicated that the heptad repeats region, but not the proline-rich region or the C-terminus basic region, is essential for AcMNPV P10 filament formation. Co-transfection of P10s tagged with different fluorescent revealed that P10s with defective heptad repeats region could not interact with intact heptad repeats region or even full-length P10s to form filament structure. Within the heptad repeats region, deletion of the three amino acids spacing of AcMNPV P10 appeared to have no significant impact on the formation of filament structures, but the content of the heptad repeats region appeared to play a role in the morphology of the filaments.

Baculovirus expression of the avian paramyxovirus 2 HN gene for diagnostic applications.

PubMed

Choi, Kang-Seuk; Kye, Soo-Jeong; Kim, Ji-Ye; Seul, Hee-Jeong; Lee, Hee-Soo; Kwon, Hyuk-Moo; Sung, Haan-Woo

2014-03-01

Avian paramyxovirus 2 (APMV-2) infections are associated with respiratory diseases in poultry worldwide. The hemagglutination inhibition (HI) test is a useful tool for surveillance and monitoring of this virus. In this study, full-length hemagglutinin (HN) gene of APMV-2 was chemically synthesized based on its published sequence, cloned and expressed in Spodoptera frugiperda insect cells using recombinant baculoviruses. The biological, antigenic and immunogenic properties of the expressed protein were evaluated to assess its ability to produce diagnostic reagents for HI testing. Recombinant APMV-2 HN protein showed two distinct bands with molecular masses of 64 and 75kDa, which showed hemagglutination (HA) and neuraminidase activities, respectively. The recombinant HN (rHN) protein extracted from infected cells produced high HA titers (2(13) per 25μL). HA activity of the protein was inhibited by APMV-2 antiserum, although there were weak cross reactions with other APMV serotype antisera. The rHN protein induced high titers of APMV-2-specific antibodies in immunized chickens based on the HI test. These results indicated that recombinant APMV-2 HN protein is a useful alternative to the APMV-2 antigen in HI assays. Copyright © 2013 Elsevier B.V. All rights reserved.

A nanobiohybrid complex of recombinant baculovirus and Tat/DNA nanoparticles for delivery of Ang-1 transgene in myocardial infarction therapy.

PubMed

Paul, Arghya; Binsalamah, Zyad M; Khan, Afshan A; Abbasia, Sana; Elias, Cynthia B; Shum-Tim, Dominique; Prakash, Satya

2011-11-01

The study aims to design a new gene delivery method utilizing the complementary strengths of baculovirus, such as relatively high transduction efficiency and easy scale-up, and non-viral nanodelivery systems, such as low immunogenicity. This formulation was developed by generating a self assembled binary complex of negatively charged baculovirus (Bac) and positively charged endosomolytic histidine rich Tat peptide/DNA nanoparticles (NP). The synergistic effect of this hybrid (Bac-NP) system to induce myocardial angiogenesis in acute myocardial infarction (AMI) model has been explored in this study, using Angiopoietin-1 (Ang-1) as the transgene carried by both vector components. Under optimal transduction conditions, Bac-NP(Ang1) showed 1.75 times higher and sustained Ang-1 expression in cardiomyocytes than Bac(Ang1), with significantly high angiogenic potential as confirmed by functional assays. For in vivo analysis, we intramyocardially delivered Bac-NP(Ang1) to AMI rat model. 3 weeks post AMI, data showed increase in capillary density (p < 0.01) and reduction in infarct sizes (p < 0.05) in Bac-NP(Ang1) compared to Bac(Ang1), NP(Ang1) and control groups due to enhanced myocardial Ang-1 expression at peri-infarct regions (1.65 times higher than Bac(Ang1)). Furthermore, the Bac-NP(Ang1) group showed significantly higher cardiac performance in echocardiography than Bac(Ang1) (44.2 ± 4.77% vs 37.46 ± 5.2%, p < 0.01), NP(Ang1) and the control group (32.26 ± 2.49% and 31.58 ± 2.26%). Collectively, this data demonstrates hybrid Bac-NP as a new and improved gene delivery system for therapeutic applications. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Expression and Self-Assembly in Baculovirus of Porcine Enteric Calicivirus Capsids into Virus-Like Particles and Their Use in an Enzyme-Linked Immunosorbent Assay for Antibody Detection in Swine

PubMed Central

Guo, Mingzhang; Qian, Yuan; Chang, Kyeong-Ok; Saif, Linda J.

2001-01-01

Porcine enteric calicivirus (PEC) causes diarrhea and intestinal lesions in pigs. PEC strain Cowden grows to low to moderate titers in cell culture but only with the addition of intestinal contents from uninfected gnotobiotic pigs (W. T. Flynn and L. J. Saif, J. Clin. Microbiol. 26:206–212, 1988; A. V. Parwani, W. T. Flynn, K. L. Gadfield, and L. J. Saif, Arch. Virol. 120:115–122, 1991). Cloning and sequence analysis of the PEC Cowden full-length genome revealed that it is most closely related genetically to the human Sapporo-like viruses. In this study, the complete PEC capsid gene was subcloned into the plasmid pBlueBac4.5 and the recombinant baculoviruses were identified by plaque assay and PCR. The PEC capsid protein was expressed in insect (Sf9) cells inoculated with the recombinant baculoviruses, and the recombinant capsid proteins self- assembled into virus-like particles (VLPs) that were released into the cell supernatant and purified by CsCl gradient centrifugation. The PEC VLPs had the same molecular mass (58 kDa) as the native virus capsid and reacted with pig hyperimmune and convalescent-phase sera to PEC Cowden in enzyme-linked immunosorbent assay (ELISA) and Western blotting. The PEC capsid VLPs were morphologically and antigenically similar to the native virus by immune electron microscopy. High titers (1:102,400 to 204,800) of PEC-specific antibodies were induced in guinea pigs inoculated with PEC VLPs, suggesting that the VLPs could be useful for future candidate PEC vaccines. A fixed-cell ELISA and VLP ELISA were developed to detect PEC serum antibodies in pigs. For the fixed-cell ELISA, Sf9 cells were infected with recombinant baculoviruses expressing PEC capsids, followed by cell fixation with formalin. For the VLP ELISA, the VLPs were used for the coating antigen. Our data indicate that both tests were rapid, specific, and reproducible and might be used for large-scale serological investigations of PEC antibodies in swine. PMID:11283075

Prevention and Treatment of Vitamin D and Calcium Deficiency in Children and Adolescents: Indian Academy of Pediatrics (IAP) Guidelines.

PubMed

Khadilkar, Anuradha; Khadilkar, Vaman; Chinnappa, Jagdish; Rathi, Narendra; Khadgawat, Rajesh; Balasubramanian, S; Parekh, Bakul; Jog, Pramod

2017-07-15

Vitamin D deficiency (VDD) is being increasingly reported from India from all age-groups. Reports suggest that VDD affects all age groups, from neonates to adolescents. Further, habitually low calcium intakes are also reported in Indian children. Given the multiple guidelines, peculiarities of Indian circumstances, changing lifestyles, and lack of fortification, the Indian Academy of Pediatrics (IAP) felt the need for a Practice Guideline for Pediatricians for the prevention and treatment of vitamin D and calcium deficiency in children and adolescents. The 'Guideline for Vitamin D and Calcium in Children' committee was formed by the IAP in September 2016. A consultative committee meeting was held in November 2016 in Mumbai. Evidence from Indian and international studies and other previous published recommendations, which were pertinent to the Indian circumstances, were collated for the preparation of these guidelines. To present a practice guideline for pediatricians for the prevention and treatment of deficiency of vitamin D and calcium in the Indian context. For the prevention of rickets in premature infants, 400 IU of vitamin D and 150-220 mg/kg of calcium, and in neonates, 400 IU of vitamin D and 200 mg of calcium are recommended daily. For prevention of rickets and hypocalcemia in infants (after neonatal period) upto 1 year of age, and from 1-18 years, 400 IU and 600 IU vitamin D/day and 250-500 mg/day and 600-800 mg/day of calcium, respectively, are recommended. For treatment of rickets in premature neonates, infants upto 1 year and from 1-18 years, 1000 IU, 2000 IU and 3000-6000 IU of vitamin D daily, respectively, and elemental calcium of 70-80 mg/kg/day in premature neonates and 500-800 mg daily for all children over that age are recommended. Larger doses of vitamin D may be given from 3 months to 18 years of age as 60,000 IU/week for 6 weeks.

Comparative studies of lepidopteran baculovirus-specific protein FP25K: development of a novel Bombyx mori nucleopolyhedrovirus-based vector with a modified fp25K gene.

PubMed

Nakanishi, Tadashi; Goto, Chie; Kobayashi, Michihiro; Kang, Wonkyung; Suzuki, Takehiro; Dohmae, Naoshi; Matsumoto, Shogo; Shimada, Toru; Katsuma, Susumu

2010-05-01

Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.

Intracellular Trafficking of Baculovirus Particles: A Quantitative Study of the HearNPV/HzAM1 Cell and AcMNPV/Sf9 Cell Systems.

PubMed

Matindoost, Leila; Nielsen, Lars K; Reid, Steve

2015-05-05

To replace the in vivo production of baculovirus-based biopesticides with a more convenient in vitro produced product, the limitations imposed by in vitro production have to be solved. One of the main problems is the low titer of HearNPV budded virions (BV) in vitro as the use of low BV titer stocks can result in non-homogenous infections resulting in multiple virus replication cycles during scale up that leads to low Occlusion Body yields. Here we investigate the baculovirus traffic in subcellular fractions of host cells throughout infection with an emphasis on AcMNPV/Sf9 and HearNPV/HzAM1 systems distinguished as "good" and "bad" BV producers, respectively. qPCR quantification of viral DNA in the nucleus, cytoplasm and extracellular fractions demonstrated that although the HearNPV/HzAM1 system produces twice the amount of vDNA as the AcMNPV/Sf9 system, its percentage of BV to total progeny vDNA was lower. vDNA egress from the nucleus to the cytoplasm is sufficient in both systems, however, a higher percentage of vDNA in the HearNPV/HzAM1 system remain in the cytoplasm and do not bud out of the cells compared to the AcMNPV/Sf9 system. In both systems more than 75% of the vDNA produced in the nuclear fraction go unused, without budding or being encapsulated in OBs showing the capacity for improvements that could result from the engineering of the virus/cell line systems to achieve better productivities for both BV and OB yields.

Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

PubMed

Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

2015-11-01

Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

Structural Insight into Inhibitor of Apoptosis Proteins Recognition by a Potent Divalent Smac-Mimetic

PubMed Central

Vachette, Patrice; Malvezzi, Francesca; Grassi, Serena; Lecis, Daniele; Delia, Domenico; Drago, Carmelo; Seneci, Pierfausto; Bolognesi, Martino; Mastrangelo, Eloise

2012-01-01

Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy. PMID:23166698

Deuteration and selective labeling of alanine methyl groups of β2-adrenergic receptor expressed in a baculovirus-insect cell expression system.

PubMed

Kofuku, Yutaka; Yokomizo, Tomoki; Imai, Shunsuke; Shiraishi, Yutaro; Natsume, Mei; Itoh, Hiroaki; Inoue, Masayuki; Nakata, Kunio; Igarashi, Shunsuke; Yamaguchi, Hideyuki; Mizukoshi, Toshimi; Suzuki, Ei-Ichiro; Ueda, Takumi; Shimada, Ichio

2018-03-08

G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl- 13 C 1 H 3 -labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β 2 -adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.

Use of baculovirus expression system for generation of virus-like particles: successes and challenges.

PubMed

Liu, Fuxiao; Wu, Xiaodong; Li, Lin; Liu, Zengshan; Wang, Zhiliang

2013-08-01

The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs. Copyright © 2013 Elsevier Inc. All rights reserved.

Construction of baculovirus expression vector of miRNAs and its expression in insect cells.

PubMed

Huang, Yong; Zou, Quan; Shen, Xing Jia; Yu, Xue Li; Wang, Zhan Bin; Cheng, Xiang Chao

2012-01-01

MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. The miR-9a is very conservative in animals from flies to humans. Studies indicated that miR-9a is involved in the regulation of neurogenesis in animals. In our study, the baculovirus expression system was used to transcribe a recombinant vector containing miR-9a for further analysis the function ofmiR-9a. The sequence ofpre-miR-9a from silkworm DNA was first cloned into the donor pFastBac. The enhanced green fluorescent protein (EGFP) was used as reporter gene. The recombinant donor plasmid pFastBac-miR-9a was transformed into E.coli DH10Bac/AcNPV forming Bacmid-9a which was transfected into insect cells with cational lipofectin. The transcription of mature miR-9a was detected by Real-time PCR. The results show the recombinant Bacmid-9a was successfully constructed and effectively transcribed miR-9a in infected Sf21 insect cells.

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.

PubMed

Mori, Takaaki; Kamiya, Koki; Tomita, Masahiro; Yoshimura, Tetsuro; Tsumoto, Kanta

2014-06-01

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system.

PubMed

Masoomi Dezfooli, Seyedehsara; Tan, Wen Siang; Tey, Beng Ti; Ooi, Chien Wei; Hussain, Siti Aslina

2016-01-01

Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig. © 2015 American Institute of Chemical Engineers.

Genetic and pharmacological screens converge in identifying FLIP, BCL2 and IAP proteins as key regulators of sensitivity to the TRAIL-inducing anti-cancer agent ONC201/TIC10

PubMed Central

Allen, Joshua E.; Prabhu, Varun V.; Talekar, Mala; van den Heuvel, AP; Lim, Bora; Dicker, David T.; Fritz, Jennifer L.; Beck, Adam; El-Deiry, Wafik S.

2015-01-01

ONC201/TIC10 is a small molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the anti-apoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes including the multi-kinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death. In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib co-treatment to enhance anticancer responses. PMID:25681273

Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties

PubMed Central

2013-01-01

Background Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. Methods A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3’-5’-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman’s assay in microplates. Results A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for

Genetic and Pharmacological Screens Converge in Identifying FLIP, BCL2, and IAP Proteins as Key Regulators of Sensitivity to the TRAIL-Inducing Anticancer Agent ONC201/TIC10.

PubMed

Allen, Joshua E; Prabhu, Varun V; Talekar, Mala; van den Heuvel, A Pieter J; Lim, Bora; Dicker, David T; Fritz, Jennifer L; Beck, Adam; El-Deiry, Wafik S

2015-04-15

ONC201/TIC10 is a small-molecule inducer of the TRAIL gene under current investigation as a novel anticancer agent. In this study, we identify critical molecular determinants of ONC201 sensitivity offering potential utility as pharmacodynamic or predictive response markers. By screening a library of kinase siRNAs in combination with a subcytotoxic dose of ONC201, we identified several kinases that ablated tumor cell sensitivity, including the MAPK pathway-inducer KSR1. Unexpectedly, KSR1 silencing did not affect MAPK signaling in the presence or absence of ONC201, but instead reduced expression of the antiapoptotic proteins FLIP, Mcl-1, Bcl-2, cIAP1, cIAP2, and survivin. In parallel to this work, we also conducted a synergy screen in which ONC201 was combined with approved small-molecule anticancer drugs. In multiple cancer cell populations, ONC201 synergized with diverse drug classes, including the multikinase inhibitor sorafenib. Notably, combining ONC201 and sorafenib led to synergistic induction of TRAIL and its receptor DR5 along with a potent induction of cell death. In a mouse xenograft model of hepatocellular carcinoma, we demonstrated that ONC201 and sorafenib cooperatively and safely triggered tumor regressions. Overall, our results established a set of determinants for ONC201 sensitivity that may predict therapeutic response, particularly in settings of sorafenib cotreatment to enhance anticancer responses. ©2015 American Association for Cancer Research.

Sequence analysis of the complete genome of Trichoplusia ni single nucleopolyhedrovirus and the identification of a baculoviral photolyase gene

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

Willis, Leslie G.; Siepp, Robyn; Stewart, Taryn M.

2005-08-01

The genome of the Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), a group II NPV which infects the cabbage looper (T. ni), has been completely sequenced and analyzed. The TnSNPV DNA genome consists of 134,394 bp and has an overall G + C content of 39%. Gene analysis predicted 144 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. Comparisons with previously sequenced baculoviruses indicate that 119 TnSNPV ORFs were homologues of previously reported viral gene sequences. Ninety-four TnSNPV ORFs returned an Autographa californica multiple NPV (AcMNPV) homologue while 25 ORFs returned poor or no sequence matches withmore » the current databases. A putative photolyase gene was also identified that had highest amino acid identity to the photolyase genes of Chrysodeixis chalcites NPV (ChchNPV) (47%) and Danio rerio (zebrafish) (40%). In addition unlike all other baculoviruses no obvious homologous repeat (hr) sequences were identified. Comparison of the TnSNPV and AcMNPV genomes provides a unique opportunity to examine two baculoviruses that are highly virulent for a common insect host (T. ni) yet belong to diverse baculovirus taxonomic groups and possess distinct biological features. In vitro fusion assays demonstrated that the TnSNPV F protein induces membrane fusion and syncytia formation and were compared to syncytia formed by AcMNPV GP64.« less

A Repeat Look at Repeating Patterns

ERIC Educational Resources Information Center

Markworth, Kimberly A.

2016-01-01

A "repeating pattern" is a cyclical repetition of an identifiable core. Children in the primary grades usually begin pattern work with fairly simple patterns, such as AB, ABC, or ABB patterns. The unique letters represent unique elements, whereas the sequence of letters represents the core that is repeated. Based on color, shape,…

Genomic Sequences of Five Helicoverpa armigera Nucleopolyhedrovirus Genotypes from Spain That Differ in Their Insecticidal Properties

PubMed Central

Arrizubieta, Maite; Williams, Trevor; Caballero, Primitivo

2015-01-01

Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has proved effective as the basis for various biological insecticides. Complete genome sequences of five Spanish HearNPV genotypes differed principally in the homologous regions (hrs) and the baculovirus repeat open reading frame (bro) genes, suggesting that they may be involved in the phenotypic differences observed among genotypes. PMID:26067949

Reward modulation of contextual cueing: Repeated context overshadows repeated target location.

PubMed

Sharifian, Fariba; Contier, Oliver; Preuschhof, Claudia; Pollmann, Stefan

2017-10-01

Contextual cueing can be enhanced by reward. However, there is a debate if reward is associated with the repeated target-distractor configurations or with the repeated target locations that occur in both repeated and new displays. Based on neuroimaging evidence, we hypothesized that reward becomes associated with the target location only in new displays, but not in repeated displays, where the repeated target location is overshadowed by the more salient repeated target-distractor configuration. To test this hypothesis, we varied the reward value associated with the same target location in repeated and new displays. The results confirmed the overshadowing hypothesis in that search facilitation in repeated target-distractor configurations was modulated by the variable value associated with the target location. This effect was observed mainly in early learning.

The components of shear stress affecting insect cells used with the baculovirus expression vector system.

PubMed

Weidner, Tobias; Druzinec, Damir; Mühlmann, Martina; Buchholz, Rainer; Czermak, Peter

2017-09-26

Insect-based expression platforms such as the baculovirus expression vector system (BEVS) are widely used for the laboratory- and industrial-scale production of recombinant proteins. Thereby, major drawbacks to gain high-quality proteins are the lytic infection cycle and the shear sensitivity of infected insect cells due to turbulence and aeration. Smaller bubbles were formerly assumed to be more harmful than larger ones, but we found that cell damage is also dependent on the concentration of protective agents such as Pluronic®. At the appropriate concentration, Pluronic forms a layer around air bubbles and hinders the attachment of cells, thus limiting the damage. In this context, we used microaeration to vary bubble sizes and confirmed that size is not the most important factor, but the total gas surface area in the reactor is. If the surface area exceeds a certain threshold, the concentration of Pluronic is no longer sufficient for cell protection. To investigate the significance of shear forces, a second study was carried out in which infected insect cells were cultivated in a hollow fiber module to protect them from shear forces. Both model studies revealed important aspects of the design and scale-up of BEVS processes for the production of recombinant proteins.

Protein Expression in Insect and Mammalian Cells Using Baculoviruses in Wave Bioreactors.

PubMed

Kadwell, Sue H; Overton, Laurie K

2016-01-01

Many types of disposable bioreactors for protein expression in insect and mammalian cells are now available. They differ in design, capacity, and sensor options, with many selections available for either rocking platform, orbitally shaken, pneumatically mixed, or stirred-tank bioreactors lined with an integral disposable bag (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). WAVE Bioreactors™ were among the first disposable systems to be developed (Singh, Cytotechnology 30:149-158, 1999). Since their commercialization in 1999, Wave Bioreactors have become routinely used in many laboratories due to their ease of operation, limited utility requirements, and protein expression levels comparability to traditional stirred-tank bioreactors. Wave Bioreactors are designed to use a presterilized Cellbag™, which is attached to a rocking platform and inflated with filtered air provided by the bioreactor unit. The Cellbag can be filled with medium and cells and maintained at a set temperature. The rocking motion, which is adjusted through angle and rock speed settings, provides mixing of oxygen (and CO2, which is used to control pH in mammalian cell cultures) from the headspace created in the inflated Cellbag with the cell culture medium and cells. This rocking motion can be adjusted to prevent cell shear damage. Dissolved oxygen and pH can be monitored during scale-up, and samples can be easily removed to monitor other parameters. Insect and mammalian cells grow very well in Wave Bioreactors (Shukla and Gottschalk, Trends Biotechnol 31(3):147-154, 2013). Combining Wave Bioreactor cell growth capabilities with recombinant baculoviruses engineered for insect or mammalian cell expression has proven to be a powerful tool for rapid production of a wide range of proteins.

Genome sequence of an enhancin gene-rich nucleopolyhedrovirus (NPV) from Agrotis segetum: collinearity with Spodoptera exigua multiple NPV.

PubMed

Jakubowska, Agata K; Peters, Sander A; Ziemnicka, Jadwiga; Vlak, Just M; van Oers, Monique M

2006-03-01

The genome sequence of a Polish isolate of Agrotis segetum nucleopolyhedrovirus (AgseNPV-A) was determined and analysed. The circular genome is composed of 147,544 bp and has a G+C content of 45.7 mol%. It contains 153 putative, non-overlapping open reading frames (ORFs) encoding predicted proteins of more than 50 aa, together making up 89.8 % of the genome. The remaining 10.2 % of the DNA constitutes non-coding regions and homologous-repeat regions. One hundred and forty-three AgseNPV-A ORFs are homologues of previously reported baculovirus gene sequences. There are ten unique ORFs and they account for 3 % of the genome in total. All 62 lepidopteran baculovirus genes, including the 29 core baculovirus genes, were found in the AgseNPV-A genome. The gene content and gene order of AgseNPV-A are most similar to those of Spodoptera exigua (Se) multiple NPV and their shared homologous genes are 100 % collinear. Three putative enhancin genes were identified in the AgseNPV-A genome. In phylogenetic analysis, the AgseNPV-A enhancins form a cluster separated from enhancins of the Mamestra species NPVs.

The genomic underpinnings of apoptosis in the silkworm, Bombyx mori

PubMed Central

2010-01-01

Background Apoptosis is regulated in an orderly fashion by a series of genes, and has a crucial role in important physiological processes such as growth development, immunological response and so on. Recently, substantial studies have been undertaken on apoptosis in model animals including humans, fruit flies, and the nematode. However, the lack of genomic data for silkworms limits their usefulness in apoptosis studies, despite the advantages of silkworm as a representative of Lepidoptera and an effective model system. Herein we have identified apoptosis-related genes in the silkworm Bombyx mori and compared them to those from insects, mammals, and nematodes. Results From the newly assembled genome databases, a genome-wide analysis of apoptosis-related genes in Bombyx mori was performed using both nucleotide and protein Blast searches. Fifty-two apoptosis-related candidate genes were identified, including five caspase family members, two tumor necrosis factor (TNF) superfamily members, one Bcl-2 family member, four baculovirus IAP (inhibitor of apoptosis) repeat (BIR) domain family members and 1 RHG (Reaper, Hid, Grim, and Sickle; Drosophila cell death activators) family member. Moreover, we identified a new caspase family member, BmCaspase-New, two splice variants of BmDronc, and Bm3585, a mammalian TNF superfamily member homolog. Twenty-three of these apoptosis-related genes were cloned and sequenced using cDNA templates isolated from BmE-SWU1 cells. Sequence analyses revealed that these genes could have key roles in apoptosis. Conclusions Bombyx mori possesses potential apoptosis-related genes. We hypothesized that the classic intrinsic and extrinsic apoptotic pathways potentially are active in Bombyx mori. These results lay the foundation for further apoptosis-related study in Bombyx mori. PMID:21040523

Screening and Characterization of Hydrate Forms of T-3256336, a Novel Inhibitor of Apoptosis (IAP) Protein Antagonist.

PubMed

Takeuchi, Shoko; Kojima, Takashi; Hashimoto, Kentaro; Saito, Bunnai; Sumi, Hiroyuki; Ishikawa, Tomoyasu; Ikeda, Yukihiro

2015-01-01

Different crystal packing of hydrates from anhydrate crystals leads to different physical properties, such as solubility and stability. Investigation of the potential of varied hydrate formation, and understanding the stability in an anhydrous/hydrate system, are crucial to prevent an undesired transition during the manufacturing process and storage. Only one anhydrous form of T-3256336, a novel inhibitor of apoptosis (IAP) protein antagonist, was discovered during synthesis, and no hydrate form has been identified. In this study, we conducted hydrate screening such as dynamic water vapor sorption/desorption (DVS), and the slurry experiment, and characterized the solid-state properties of anhydrous/hydrate forms to determine the most desirable crystalline form for development. New hydrate forms, both mono-hydrate and hemi-hydrate forms, were discovered as a result of this hydrate screening. The characterization of two new hydrate forms was conducted, and the anhydrous form was determined to be the most desirable development form of T-3256336 in terms of solid-state stability. In addition, the stability of the anhydrous form was investigated using the water content and temperature controlled slurry experiment to obtain the desirable crystal form in the crystallization process. The water content regions of the stable phase of the desired form, the anhydrous form, were identified for the cooling crystallization process.

The effect of intermittent intraabdominal pressure elevations and low cardiac output on the femoral to carotid arterial blood pressure difference in piglets.

PubMed

Aksakal, Devrim; Hückstädt, Thomas; Richter, Steffen; Klitscher, Daniela; Wowra, Tobias; Schier, Felix; Wessel, Lucas M; Kubiak, Rainer

2016-11-01

Our previous work in a laparoscopic setting in piglets revealed that the systolic femoral artery pressure was approximately 5 % higher than its carotid counterpart, whereas the mean and diastolic values showed no significant difference. This remained idem when the intraabdominal pressure (IAP) was gradually increased. In this study, we aimed to investigate the effect of (1) intermittent IAP elevations and (2) a low cardiac output (CO) on the blood pressure (BP) difference cranially (carotid artery) and caudally (femoral artery) of a capnoperitoneum (ΔP = P a fem -P a carot ). A total of twenty-two piglets (mean body weight 11.0 kg; range 8.9-13.3 kg) were studied. Of these, 14 underwent intermittent IAP elevations at 8 and 16 mmHg, and ΔP was measured. In another 8 piglets, a model of reduced CO was created by introducing an air embolism (2 ml/kg over 30 s) in the inferior caval vein (VCI) at 12 mmHg IAP to further assess the influence of this variable on ΔP. Systolic ΔP remained at a mean of 5.6 mmHg and was not significantly affected by insufflation or exsufflation up to an IAP of 16 mmHg. Diastolic and mean values showed no differences between P a carot and P a fem . P a fem, systol remained higher than its carotid counterpart as long as the cardiac index (CI) was above 1.5 l/min/m 2 , but fell significantly below P a carot, systol at a low CI. There was no CO-dependent effect on diastolic and mean ΔP. Repeated IAP elevations do not significantly influence ΔP. Intermittent IAP elevations do not significantly influence ΔP. Despite of a CO-dependent inversion of systolic ΔP, mean BP measurements at the leg during laparoscopy remain representative even at low CO values.

Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

PubMed

Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

2001-06-01

1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

To Repeat or Not to Repeat a Course

ERIC Educational Resources Information Center

Armstrong, Michael J.; Biktimirov, Ernest N.

2013-01-01

The difficult transition from high school to university means that many students need to repeat (retake) 1 or more of their university courses. The authors examine the performance of students repeating first-year core courses in an undergraduate business program. They used data from university records for 116 students who took a total of 232…

Repeating and non-repeating fast radio bursts from binary neutron star mergers

NASA Astrophysics Data System (ADS)

Yamasaki, Shotaro; Totani, Tomonori; Kiuchi, Kenta

2018-04-01

Most fast radio bursts (FRB) do not show evidence of repetition, and such non-repeating FRBs may be produced at the time of a merger of binary neutron stars (BNS), provided that the BNS merger rate is close to the high end of the currently possible range. However, the merger environment is polluted by dynamical ejecta, which may prohibit the radio signal from propagating. We examine this by using a general-relativistic simulation of a BNS merger, and show that the ejecta appears about 1 ms after the rotation speed of the merged star becomes the maximum. Therefore there is a time window in which an FRB signal can reach outside, and the short duration of non-repeating FRBs can be explained by screening after ejecta formation. A fraction of BNS mergers may leave a rapidly rotating and stable neutron star, and such objects may be the origin of repeating FRBs like FRB 121102. We show that a merger remnant would appear as a repeating FRB on a time scale of ˜1-10 yr, and expected properties are consistent with the observations of FRB 121102. We construct an FRB rate evolution model that includes these two populations of repeating and non-repeating FRBs from BNS mergers, and show that the detection rate of repeating FRBs relative to non-repeating ones rapidly increases with improving search sensitivity. This may explain why only the repeating FRB 121102 was discovered by the most sensitive FRB search with Arecibo. Several predictions are made, including the appearance of a repeating FRB 1-10 yr after a BNS merger that is localized by gravitational waves and subsequent electromagnetic radiation.

Repeating and non-repeating fast radio bursts from binary neutron star mergers

NASA Astrophysics Data System (ADS)

Yamasaki, Shotaro; Totani, Tomonori; Kiuchi, Kenta

2018-06-01

Most fast radio bursts (FRB) do not show evidence of repetition, and such non-repeating FRBs may be produced at the time of a merger of binary neutron stars (BNS), provided that the BNS merger rate is close to the high end of the currently possible range. However, the merger environment is polluted by dynamical ejecta, which may prohibit the radio signal from propagating. We examine this by using a general-relativistic simulation of a BNS merger, and show that the ejecta appears about 1 ms after the rotation speed of the merged star becomes the maximum. Therefore there is a time window in which an FRB signal can reach outside, and the short duration of non-repeating FRBs can be explained by screening after ejecta formation. A fraction of BNS mergers may leave a rapidly rotating and stable neutron star, and such objects may be the origin of repeating FRBs like FRB 121102. We show that a merger remnant would appear as a repeating FRB on a time scale of ˜1-10 yr, and expected properties are consistent with the observations of FRB 121102. We construct an FRB rate evolution model that includes these two populations of repeating and non-repeating FRBs from BNS mergers, and show that the detection rate of repeating FRBs relative to non-repeating ones rapidly increases with improving search sensitivity. This may explain why only the repeating FRB 121102 was discovered by the most sensitive FRB search with Arecibo. Several predictions are made, including the appearance of a repeating FRB 1-10 yr after a BNS merger that is localized by gravitational waves and subsequent electromagnetic radiation.

Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus-silkworm expression system.

PubMed

Muraki, Michiro; Honda, Shinya

2011-11-01

To achieve an efficient isolation of human Fas receptor extracellular domain (hFasRECD), a fusion protein of hFasRECD with human IgG1 heavy chain Fc domain containing thrombin cleavage sequence at the junction site was overexpressed using baculovirus-silkworm larvae expression system. The hFasRECD part was separated from the fusion protein by the effective cleavage of the recognition site with bovine thrombin. Protein G column treatment of the reaction mixture and the subsequent cation-exchange chromatography provided purified hFasRECD with a final yield of 13.5mg from 25.0 ml silkworm hemolymph. The functional activity of the product was examined by size-exclusion chromatography analysis. The isolated hFasRECD less strongly interacted with human Fas ligand extracellular domain (hFasLECD) than the Fc domain-bridged counterpart, showing the contribution of antibody-like avidity in the latter case. The purified glycosylated hFasRECD presented several discrete bands in the disulphide-bridge non-reducing SDS-PAGE analysis, and virtually all of the components were considered to participate in the binding to hFasLECD. The attached glycans were susceptible to PNGase F digestion, but mostly resistant to Endo Hf digestion under denaturing conditions. One of the components exhibited a higher susceptibility to PNGase F digestion under non-denaturing conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

The Apis mellifera Filamentous Virus Genome

PubMed Central

Gauthier, Laurent; Cornman, Scott; Hartmann, Ulrike; Cousserans, François; Evans, Jay D.; de Miranda, Joachim R.; Neumann, Peter

2015-01-01

A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family. PMID:26184284

The Apis mellifera Filamentous Virus Genome.

PubMed

Gauthier, Laurent; Cornman, Scott; Hartmann, Ulrike; Cousserans, François; Evans, Jay D; de Miranda, Joachim R; Neumann, Peter

2015-07-09

A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family.

Study of different operational modes of the IAP 2-port-DOAS instrument for atmospheric trace gases investigation during CINDI-2 campaign basing on residual noise analysis

NASA Astrophysics Data System (ADS)

Borovski, A.; Postylyakov, O.; Elokhov, A.; Bruchkovski, I.

2017-11-01

An instrument for measuring atmospheric trace gases by DOAS method using scattered solar radiation was developed in A.M.Obukhov IAP RAS. The instrument layout is based on the lab Shamrock 303i spectrograph supplemented by 2-port radiation input system employing optical fiber. Optical ports may be used with a telescope with fixed field of view or with a scanning MAX-DOAS unit. MAX-DOAS unit port will be used for investigation of gas contents and profiles in the low troposphere. In September 2016 the IAP instrument participated in the CINDI-2 campaign, held in the Netherlands. CINDI 2 (2nd Cabauw Intercomparison of Nitrogen Dioxide Measuring Instruments) involves about 40 instruments quasi-synchronously performing DOAS measurements of NO2 and other trace gases. During the campaign the instrument ports had telescopes A and B with similar field of view of about 0.3°. Telescope A was always directed to the zenith. Telescope B was directed at 5° elevation angle. Two gratings were installed in the spectrometer. They provide different spectral resolution (FWHM 0.4 and 0.8 nm respectively) and spectral window width ( 70 and 140 nm respectively). During CINDI-2 campaign we performed test measurements in UV and visible wavelength ranges to investigate instrument stability and retrieval errors of NO2 and HCHO contents. We perform the preliminary error analysis of retrieval of the NO2 and HCHO differential slant column densities using spectra measured in four modes of the instrument basing on residual noise analysis in this paper. It was found that rotation of grating turret does not significantly affected on quality of NO2 DSCD retrieval from spectra which measured in visible spectral region. Influence of grating turret rotation is much more significant for gas DSCD retrieval from spectra which measured in UV spectral region. Standard deviation of retrieval error points to presence of some systematic error.

A Unified Model for Repeating and Non-repeating Fast Radio Bursts

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

Bagchi, Manjari, E-mail: manjari@imsc.res.in

The model that fast radio bursts (FRBs) are caused by plunges of asteroids onto neutron stars can explain both repeating and non-repeating bursts. If a neutron star passes through an asteroid belt around another star, there would be a series of bursts caused by a series of asteroid impacts. Moreover, the neutron star would cross the same belt repetitively if it were in a binary with the star hosting the asteroid belt, leading to a repeated series of bursts. I explore the properties of neutron star binaries that could lead to the only known repeating FRB so far (FRB121102). Inmore » this model, the next two epochs of bursts are expected around 2017 February 27 and 2017 December 18. On the other hand, if the asteroid belt is located around the neutron star itself, then a chance fall of an asteroid from that belt onto the neutron star would lead to a non-repeating burst. Even a neutron star grazing an asteroid belt can lead to a non-repeating burst caused by just one asteroid plunge during the grazing. This is possible even when the neutron star is in a binary with the asteroid-hosting star, if the belt and the neutron star orbit are non-coplanar.« less

Repeatability of Cryogenic Multilayer Insulation

NASA Technical Reports Server (NTRS)

Johnson, W. L.; Vanderlaan, M.; Wood, J. J.; Rhys, N. O.; Guo, W.; Van Sciver, S.; Chato, D. J.

2017-01-01

Due to the variety of requirements across aerospace platforms, and one off projects, the repeatability of cryogenic multilayer insulation has never been fully established. The objective of this test program is to provide a more basic understanding of the thermal performance repeatability of MLI systems that are applicable to large scale tanks. There are several different types of repeatability that can be accounted for: these include repeatability between multiple identical blankets, repeatability of installation of the same blanket, and repeatability of a test apparatus. The focus of the work in this report is on the first two types of repeatability. Statistically, repeatability can mean many different things. In simplest form, it refers to the range of performance that a population exhibits and the average of the population. However, as more and more identical components are made (i.e. the population of concern grows), the simple range morphs into a standard deviation from an average performance. Initial repeatability testing on MLI blankets has been completed at Florida State University. Repeatability of five GRC provided coupons with 25 layers was shown to be +/- 8.4 whereas repeatability of repeatedly installing a single coupon was shown to be +/- 8.0. A second group of 10 coupons have been fabricated by Yetispace and tested by Florida State University, through the first 4 tests, the repeatability has been shown to be +/- 16. Based on detailed statistical analysis, the data has been shown to be statistically significant.

Repeatability of Cryogenic Multilayer Insulation

NASA Technical Reports Server (NTRS)

Johnson, W. L.; Vanderlaan, M.; Wood, J. J.; Rhys, N. O.; Guo, W.; Van Sciver, S.; Chato, D. J.

2017-01-01

Due to the variety of requirements across aerospace platforms, and one off projects, the repeatability of cryogenic multilayer insulation has never been fully established. The objective of this test program is to provide a more basic understanding of the thermal performance repeatability of MLI systems that are applicable to large scale tanks. There are several different types of repeatability that can be accounted for: these include repeatability between multiple identical blankets, repeatability of installation of the same blanket, and repeatability of a test apparatus. The focus of the work in this report is on the first two types of repeatability. Statistically, repeatability can mean many different things. In simplest form, it refers to the range of performance that a population exhibits and the average of the population. However, as more and more identical components are made (i.e. the population of concern grows), the simple range morphs into a standard deviation from an average performance. Initial repeatability testing on MLI blankets has been completed at Florida State University. Repeatability of five GRC provided coupons with 25 layers was shown to be +/- 8.4% whereas repeatability of repeatedly installing a single coupon was shown to be +/- 8.0%. A second group of 10 coupons have been fabricated by Yetispace and tested by Florida State University, through the first 4 tests, the repeatability has been shown to be +/- 15-25%. Based on detailed statistical analysis, the data has been shown to be statistically significant.

Repeatability of Cryogenic Multilayer Insulation

NASA Astrophysics Data System (ADS)

Johnson, W. L.; Vanderlaan, M.; Wood, J. J.; Rhys, N. O.; Guo, W.; Van Sciver, S.; Chato, D. J.

2017-12-01

Due to the variety of requirements across aerospace platforms, and one off projects, the repeatability of cryogenic multilayer insulation (MLI) has never been fully established. The objective of this test program is to provide a more basic understanding of the thermal performance repeatability of MLI systems that are applicable to large scale tanks. There are several different types of repeatability that can be accounted for: these include repeatability between identical blankets, repeatability of installation of the same blanket, and repeatability of a test apparatus. The focus of the work in this report is on the first two types of repeatability. Statistically, repeatability can mean many different things. In simplest form, it refers to the range of performance that a population exhibits and the average of the population. However, as more and more identical components are made (i.e. the population of concern grows), the simple range morphs into a standard deviation from an average performance. Initial repeatability testing on MLI blankets has been completed at Florida State University. Repeatability of five Glenn Research Center (GRC) provided coupons with 25 layers was shown to be +/- 8.4% whereas repeatability of repeatedly installing a single coupon was shown to be +/- 8.0%. A second group of 10 coupons has been fabricated by Yetispace and tested by Florida State University, the repeatability between coupons has been shown to be +/- 15-25%. Based on detailed statistical analysis, the data has been shown to be statistically significant.

Reconfigurable multiport EPON repeater

NASA Astrophysics Data System (ADS)

Oishi, Masayuki; Inohara, Ryo; Agata, Akira; Horiuchi, Yukio

2009-11-01

An extended reach EPON repeater is one of the solutions to effectively expand FTTH service areas. In this paper, we propose a reconfigurable multi-port EPON repeater for effective accommodation of multiple ODNs with a single OLT line card. The proposed repeater, which has multi-ports in both OLT and ODN sides, consists of TRs, BTRs with the CDR function and a reconfigurable electrical matrix switch, can accommodate multiple ODNs to a single OLT line card by controlling the connection of the matrix switch. Although conventional EPON repeaters require full OLT line cards to accommodate subscribers from the initial installation stage, the proposed repeater can dramatically reduce the number of required line cards especially when the number of subscribers is less than a half of the maximum registerable users per OLT. Numerical calculation results show that the extended reach EPON system with the proposed EPON repeater can save 17.5% of the initial installation cost compared with a conventional repeater, and can be less expensive than conventional systems up to the maximum subscribers especially when the percentage of ODNs in lightly-populated areas is higher.

PAMAM dendrimer-baculovirus nanocomplex for microencapsulated adipose stem cell-gene therapy: in vitro and in vivo functional assessment.

PubMed

Paul, Arghya; Shao, Wei; Abbasi, Sana; Shum-Tim, Dominique; Prakash, Satya

2012-09-04

The present study aims to develop a new stem cell based gene delivery system consisting of human adipose tissue derived stem cells (hASCs) genetically modified with self-assembled nanocomplex of recombinant baculovirus and PAMAM dendrimer (Bac-PAMAM) to overexpress the vascular endothelial growth factor (VEGF). Cells were enveloped into branched PEG surface functionalized polymeric microcapsules for efficient transplantation. In vitro analysis confirmed efficient transduction of hASCs expressing 7.65 ± 0.86 ng functionally active VEGF per 10(6) microencapsulated hASCs (ASC-VEGF). To determine the potential of the developed system, chronically infarcted rat hearts were treated with either empty microcapsules (MC), microencapsulated hASCs expressing MGFP reporter protein (MC+ASC-MGFP), or MC+ASC-VEGF, and analyzed for 10 weeks. Post-transplantation data confirmed higher myocardial VEGF expressions with significantly enhanced neovasculature in the MC+ASC-VEGF group. In addition, the cardiac performance, as measured by percentage ejection fraction, also improved significantly in the MC+ASC-VEGF group (48.6 ± 6.1%) compared to that in MC+ASC-MGFP (38.8 ± 5.3%) and MC groups (31.5 ± 3.3%). Collectively, these data demonstrate the feasibility of this system for improved stem cell therapy applications.

Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells.

PubMed Central

Manneberg, M.; Friedlein, A.; Kurth, H.; Lahm, H. W.; Fountoulakis, M.

1994-01-01

A soluble form of the human interferon gamma receptor that is required for the identification of interferon gamma antagonists was expressed in baculovirus-infected insect cells. The protein carried N-linked carbohydrate and showed a heterogeneity on denaturing polyacrylamide gels. We investigated the utilization of the potential sites for N-linked glycosylation and the structure of the carbohydrate moieties of this soluble receptor. Amino acid sequence analysis and ion spray mass spectrometry revealed that of the five potential sites for N-linked glycosylation, Asn17 and Asn69 were always utilized, whereas Asn62 and Asn162 were utilized in approximately one-third of the protein population. Asn223 was never found to be glycosylated. The soluble receptor was treated with N-glycosidase F and the oligosaccharides released were analyzed by matrix-assisted laser desorption mass spectrometry, which showed that the protein carried six types of short carbohydrate chains. The predominant species was a hexasaccharide of molecular mass 1,039, containing a fucose subunit linked to the proximal N-acetylglucosamine residue: [formula: see text] PMID:8142896

PolyQ repeat expansions in ATXN2 associated with ALS are CAA interrupted repeats.

PubMed

Yu, Zhenming; Zhu, Yongqing; Chen-Plotkin, Alice S; Clay-Falcone, Dana; McCluskey, Leo; Elman, Lauren; Kalb, Robert G; Trojanowski, John Q; Lee, Virginia M-Y; Van Deerlin, Vivianna M; Gitler, Aaron D; Bonini, Nancy M

2011-03-29

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27-33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥ 34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1-3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2.

The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors.

PubMed

Felberbaum, Rachael S

2015-05-01

The baculovirus expression vector system (BEVS) platform has become an established manufacturing platform for the production of viral vaccines and gene therapy vectors. Nine BEVS-derived products have been approved - four for human use (Cervarix(®), Provenge(®), Glybera(®) and Flublok(®)) and five for veterinary use (Porcilis(®) Pesti, BAYOVAC CSF E2(®), Circumvent(®) PCV, Ingelvac CircoFLEX(®) and Porcilis(®) PCV). The BEVS platform offers many advantages, including manufacturing speed, flexible product design, inherent safety and scalability. This combination of features and product approvals has previously attracted interest from academic researchers, and more recently from industry leaders, to utilize BEVS to develop next generation vaccines, vectors for gene therapy, and other biopharmaceutical complex proteins. In this review, we explore the BEVS platform, detailing how it works, platform features and limitations and important considerations for manufacturing and regulatory approval. To underscore the growth in opportunities for BEVS-derived products, we discuss the latest product developments in the gene therapy and influenza vaccine fields that follow in the wake of the recent product approvals of Glybera(®) and Flublok(®), respectively. We anticipate that the utility of the platform will expand even further as new BEVS-derived products attain licensure. Finally, we touch on some of the areas where new BEVS-derived products are likely to emerge. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fullerene-like organization of HIV gag-protein shell in virus-like particles produced by recombinant baculovirus.

PubMed

Nermut, M V; Hockley, D J; Jowett, J B; Jones, I M; Garreau, M; Thomas, D

1994-01-01

Virus-like particles produced by a recombinant baculovirus containing the HIV gag gene were examined by negative staining after delipidization. This technique demonstrated that the gag-protein shell consisted of radially arranged short rods which formed a network of ring-like structures. Similar structures were observed at the plasma membrane of infected cells which had been opened by wet-cleaving. Occasionally five or six subunits were observed forming a ring. These findings suggest that the gag-encoded precursor (pr55) is a rod-like molecule about 34 A in diameter and 85 A in length. A protein cylinder of such dimensions would have a molecular weight of 56K. The center-to-center distance of two neighboring rings formed by the rods was 66 +/- 8 A (N = 200) by direct measurements and 65 A as obtained from averaged images. This morphology and these dimensions indicate that the virus-like particles contain the gag precursor in the form of a near-spherical "fullerene-like" icosahedral shell. Our data indicate that the triangulation number of the rings equals 63. However, since one rod of pr55 is shared by two rings, the number of copies of the precursor will be 1890 as opposed to 2522 if the molecules were closely packed. The particle diameter of 102 nm deduced from the proposed model was close to the diameter obtained from thin sections of low-temperature-embedded specimens (103-108 nm).

Risks of repeated miscarriage.

PubMed

George, Lena; Granath, Fredrik; Johansson, Anna L V; Olander, Bodil; Cnattingius, Sven

2006-03-01

There is a lack of well-designed epidemiological studies of possible risk factors for repeated miscarriage. In this Swedish population-based case-control study, we investigated the association between sociodemographic and anthropometric factors, obstetric history and life-style factors, with respect to the risks of first-trimester repeated miscarriage. Information on maternal characteristics was collected through in-person interviews. Plasma blood samples were analysed for cotinine and folate concentrations. Adjusted odds ratios (OR) with 95% confidence interval [CI] were used to estimate the relative risk of repeated miscarriage. The risks of repeated miscarriage were increased for women aged > or = 35 years (adjusted OR 2.9 [95% CI 1.4, 5.8]), as well as for women aged < or = 24 years (OR 2.8 [95% CI 1.1, 6.8]). Women with a history of at least one preceding miscarriage prior to the two index pregnancies, women reporting prolonged time to conceive, and women with a history of myoma, faced a more than fourfold increased risk of repeated miscarriage. Smokers were at an increased risk of repeated miscarriage (OR 2.1 [95% CI 1.1, 4.1]). Among non-smoking women with high caffeine intake, there was an increased risk of repeated miscarriage, whereas there was no such association among smokers. Low plasma folate levels were not associated with increased risks.

Revisiting the TALE repeat.

PubMed

Deng, Dong; Yan, Chuangye; Wu, Jianping; Pan, Xiaojing; Yan, Nieng

2014-04-01

Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33-35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity.

All-photonic quantum repeaters

PubMed Central

Azuma, Koji; Tamaki, Kiyoshi; Lo, Hoi-Kwong

2015-01-01

Quantum communication holds promise for unconditionally secure transmission of secret messages and faithful transfer of unknown quantum states. Photons appear to be the medium of choice for quantum communication. Owing to photon losses, robust quantum communication over long lossy channels requires quantum repeaters. It is widely believed that a necessary and highly demanding requirement for quantum repeaters is the existence of matter quantum memories. Here we show that such a requirement is, in fact, unnecessary by introducing the concept of all-photonic quantum repeaters based on flying qubits. In particular, we present a protocol based on photonic cluster-state machine guns and a loss-tolerant measurement equipped with local high-speed active feedforwards. We show that, with such all-photonic quantum repeaters, the communication efficiency scales polynomially with the channel distance. Our result paves a new route towards quantum repeaters with efficient single-photon sources rather than matter quantum memories. PMID:25873153

RepeatsDB-lite: a web server for unit annotation of tandem repeat proteins.

PubMed

Hirsh, Layla; Paladin, Lisanna; Piovesan, Damiano; Tosatto, Silvio C E

2018-05-09

RepeatsDB-lite (http://protein.bio.unipd.it/repeatsdb-lite) is a web server for the prediction of repetitive structural elements and units in tandem repeat (TR) proteins. TRs are a widespread but poorly annotated class of non-globular proteins carrying heterogeneous functions. RepeatsDB-lite extends the prediction to all TR types and strongly improves the performance both in terms of computational time and accuracy over previous methods, with precision above 95% for solenoid structures. The algorithm exploits an improved TR unit library derived from the RepeatsDB database to perform an iterative structural search and assignment. The web interface provides tools for analyzing the evolutionary relationships between units and manually refine the prediction by changing unit positions and protein classification. An all-against-all structure-based sequence similarity matrix is calculated and visualized in real-time for every user edit. Reviewed predictions can be submitted to RepeatsDB for review and inclusion.

Baculovirus-vectored multistage Plasmodium vivax vaccine induces both protective and transmission-blocking immunities against transgenic rodent malaria parasites.

PubMed

Mizutani, Masanori; Iyori, Mitsuhiro; Blagborough, Andrew M; Fukumoto, Shinya; Funatsu, Tomohiro; Sinden, Robert E; Yoshida, Shigeto

2014-10-01

A multistage malaria vaccine targeting the pre-erythrocytic and sexual stages of Plasmodium could effectively protect individuals against infection from mosquito bites and provide transmission-blocking (TB) activity against the sexual stages of the parasite, respectively. This strategy could help prevent malaria infections in individuals and, on a larger scale, prevent malaria transmission in communities of endemicity. Here, we describe the development of a multistage Plasmodium vivax vaccine which simultaneously expresses P. vivax circumsporozoite protein (PvCSP) and P25 (Pvs25) protein of this species as a fusion protein, thereby acting as a pre-erythrocytic vaccine and a TB vaccine, respectively. A new-concept vaccine platform based on the baculovirus dual-expression system (BDES) was evaluated. The BDES-Pvs25-PvCSP vaccine displayed correct folding of the Pvs25-PvCSP fusion protein on the viral envelope and was highly expressed upon transduction of mammalian cells in vitro. This vaccine induced high levels of antibodies to Pvs25 and PvCSP and elicited protective (43%) and TB (82%) efficacies against transgenic P. berghei parasites expressing the corresponding P. vivax antigens in mice. Our data indicate that our BDES, which functions as both a subunit and DNA vaccine, can offer a promising multistage vaccine capable of delivering a potent antimalarial pre-erythrocytic and TB response via a single immunization regimen. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Characterization of self-assembled virus-like particles of dromedary camel hepatitis e virus generated by recombinant baculoviruses.

PubMed

Zhou, Xianfeng; Kataoka, Michiyo; Liu, Zheng; Takeda, Naokazu; Wakita, Takaji; Li, Tian-Cheng

2015-12-02

Dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus, has been identified in dromedary camels in Dubai, United Arab Emirates. The antigenicity, pathogenicity and epidemiology of this virus have been unclear. Here we first used a recombinant baculovirus expression system to express the 13 and 111 N-terminus amino-acid-truncated DcHEV ORF2 protein in insect Tn5 cells, and we obtained two types of virus-like particles (VLPs) with densities of 1.300 g/cm(3) and 1.285 g/cm(3), respectively. The small VLPs (Dc4sVLPs) were estimated to be 24 nm in diameter, and were assembled by a protein with the molecular mass 53 kDa. The large VLPs (Dc3nVLPs and Dc4nVLPs) were 35 nm in diameter, and were assembled by a 64-kDa protein. An antigenic analysis demonstrated that DcHEV was cross-reactive with G1, G3-G6, ferret and rat HEVs, and DcHEV showed a stronger cross-reactivity to G1 G3-G6 HEV than it did to rat and ferret HEV. In addition, the antibody against DcHEV-LPs neutralized G1 and G3 HEV in a cell culture system, suggesting that the serotypes of these HEVs are identical. We also found that the amino acid residue Met-358 affects the small DcHEV-LPs assembly. Copyright © 2015 Elsevier B.V. All rights reserved.

The Baculovirus-Expressed Binding Region of Plasmodium falciparum EBA-140 Ligand and Its Glycophorin C Binding Specificity

PubMed Central

Rydzak, Joanna; Kaczmarek, Radoslaw; Czerwinski, Marcin; Lukasiewicz, Jolanta; Tyborowska, Jolanta; Szewczyk, Boguslaw; Jaskiewicz, Ewa

2015-01-01

The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36–63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands. PMID:25588042

Repeatability of paired counts.

PubMed

Alexander, Neal; Bethony, Jeff; Corrêa-Oliveira, Rodrigo; Rodrigues, Laura C; Hotez, Peter; Brooker, Simon

2007-08-30

The Bland and Altman technique is widely used to assess the variation between replicates of a method of clinical measurement. It yields the repeatability, i.e. the value within which 95 per cent of repeat measurements lie. The valid use of the technique requires that the variance is constant over the data range. This is not usually the case for counts of items such as CD4 cells or parasites, nor is the log transformation applicable to zero counts. We investigate the properties of generalized differences based on Box-Cox transformations. For an example, in a data set of hookworm eggs counted by the Kato-Katz method, the square root transformation is found to stabilize the variance. We show how to back-transform the repeatability on the square root scale to the repeatability of the counts themselves, as an increasing function of the square mean root egg count, i.e. the square of the average of square roots. As well as being more easily interpretable, the back-transformed results highlight the dependence of the repeatability on the sample volume used.

Genome of Epinotia aporema granulovirus (EpapGV), a polyorganotropic fast killing betabaculovirus with a novel thymidylate kinase gene

PubMed Central

2012-01-01

Background Epinotia aporema (Lepidoptera: Tortricidae) is an important pest of legume crops in South America. Epinotia aporema granulovirus (EpapGV) is a baculovirus that causes a polyorganotropic infection in the host larva. Its high pathogenicity and host specificity make EpapGV an excellent candidate to be used as a biological control agent. Results The genome of Epinotia aporema granulovirus (EpapGV) was sequenced and analyzed. Its circular double-stranded DNA genome is 119,082 bp in length and codes for 133 putative genes. It contains the 31 baculovirus core genes and a set of 19 genes that are GV exclusive. Seventeen ORFs were unique to EpapGV in comparison with other baculoviruses. Of these, 16 found no homologues in GenBank, and one encoded a thymidylate kinase. Analysis of nucleotide sequence repeats revealed the presence of 16 homologous regions (hrs) interspersed throughout the genome. Each hr was characterized by the presence of 1 to 3 clustered imperfect palindromes which are similar to previously described palindromes of tortricid-specific GVs. Also, one of the hrs (hr4) has flanking sequences suggestive of a putative non-hr ori. Interestingly, two more complex hrs were found in opposite loci, dividing the circular dsDNA genome in two halves. Gene synteny maps showed the great colinearity of sequenced GVs, being EpapGV the most dissimilar as it has a 20 kb-long gene block inversion. Phylogenetic study performed with 31 core genes of 58 baculoviral genomes suggests that EpapGV is the baculovirus isolate closest to the putative common ancestor of tortricid specific betabaculoviruses. Conclusions This study, along with previous characterization of EpapGV infection, is useful for the better understanding of the pathology caused by this virus and its potential utilization as a bioinsecticide. PMID:23051685

Sequence repeats and protein structure

NASA Astrophysics Data System (ADS)

Hoang, Trinh X.; Trovato, Antonio; Seno, Flavio; Banavar, Jayanth R.; Maritan, Amos

2012-11-01

Repeats are frequently found in known protein sequences. The level of sequence conservation in tandem repeats correlates with their propensities to be intrinsically disordered. We employ a coarse-grained model of a protein with a two-letter amino acid alphabet, hydrophobic (H) and polar (P), to examine the sequence-structure relationship in the realm of repeated sequences. A fraction of repeated sequences comprises a distinct class of bad folders, whose folding temperatures are much lower than those of random sequences. Imperfection in sequence repetition improves the folding properties of the bad folders while deteriorating those of the good folders. Our results may explain why nature has utilized repeated sequences for their versatility and especially to design functional proteins that are intrinsically unstructured at physiological temperatures.

Repeat Customer Success in Extension

ERIC Educational Resources Information Center

Bess, Melissa M.; Traub, Sarah M.

2013-01-01

Four multi-session research-based programs were offered by two Extension specialist in one rural Missouri county. Eleven participants who came to multiple Extension programs could be called "repeat customers." Based on the total number of participants for all four programs, 25% could be deemed as repeat customers. Repeat customers had…

Repeat migration and disappointment.

PubMed

Grant, E K; Vanderkamp, J

1986-01-01

This article investigates the determinants of repeat migration among the 44 regions of Canada, using information from a large micro-database which spans the period 1968 to 1971. The explanation of repeat migration probabilities is a difficult task, and this attempt is only partly successful. May of the explanatory variables are not significant, and the overall explanatory power of the equations is not high. In the area of personal characteristics, the variables related to age, sex, and marital status are generally significant and with expected signs. The distance variable has a strongly positive effect on onward move probabilities. Variables related to prior migration experience have an important impact that differs between return and onward probabilities. In particular, the occurrence of prior moves has a striking effect on the probability of onward migration. The variable representing disappointment, or relative success of the initial move, plays a significant role in explaining repeat migration probabilities. The disappointment variable represents the ratio of actural versus expected wage income in the year after the initial move, and its effect on both repeat migration probabilities is always negative and almost always highly significant. The repeat probabilities diminish after a year's stay in the destination region, but disappointment in the most recent year still has a bearing on the delayed repeat probabilities. While the quantitative impact of the disappointment variable is not large, it is difficult to draw comparisons since similar estimates are not available elsewhere.

Expanded complexity of unstable repeat diseases

PubMed Central

Polak, Urszula; McIvor, Elizabeth; Dent, Sharon Y.R.; Wells, Robert D.; Napierala, Marek

2015-01-01

Unstable Repeat Diseases (URDs) share a common mutational phenomenon of changes in the copy number of short, tandemly repeated DNA sequences. More than 20 human neurological diseases are caused by instability, predominantly expansion, of microsatellite sequences. Changes in the repeat size initiate a cascade of pathological processes, frequently characteristic of a unique disease or a small subgroup of the URDs. Understanding of both the mechanism of repeat instability and molecular consequences of the repeat expansions is critical to developing successful therapies for these diseases. Recent technological breakthroughs in whole genome, transcriptome and proteome analyses will almost certainly lead to new discoveries regarding the mechanisms of repeat instability, the pathogenesis of URDs, and will facilitate development of novel therapeutic approaches. The aim of this review is to give a general overview of unstable repeats diseases, highlight the complexities of these diseases, and feature the emerging discoveries in the field. PMID:23233240

Fostering repeat donations in Ghana.

PubMed

Owusu-Ofori, S; Asenso-Mensah, K; Boateng, P; Sarkodie, F; Allain, J-P

2010-01-01

Most African countries are challenged in recruiting and retaining voluntary blood donors by cost and other complexities and in establishing and implementing national blood policies. The availability of replacement donors who are a cheaper source of blood has not enhanced repeat voluntary donor initiatives. An overview of activities for recruiting and retaining voluntary blood donors was carried out. Donor records from mobile sessions were reviewed from 2002 to 2008. A total of 71,701 blood donations; 45,515 (63.5%) being voluntary donations with 11,680 (25%) repeat donations were collected during the study period. Donations from schools and colleges contributed a steady 60% of total voluntary whilst radio station blood drives increased contribution from 10 to 27%. Though Muslim population is less than 20%, blood collection was above the 30-donation cost-effectiveness threshold with a repeat donation trend reaching 60%. In contrast Christian worshippers provided <25 unit/session and 30% repeat donations. Repeat donation trends amongst school donors and radio blood drives were 20% and 70% respectively. Repeat donations rates have been variable amongst different blood donor groups in Kumasi, Ghana. The impact of community leaders in propagating altruism cannot be overemphasized. Programs aiming at motivating replacement donors to be repeat donors should be developed and assessed. Copyright 2009 The International Association for Biologicals. All rights reserved.

Genome Sequence of a Bombyx mori Nucleopolyhedrovirus Strain with Cubic Occlusion Bodies

PubMed Central

Cheng, Ruo-Lin; Xu, Yi-Peng

2012-01-01

Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical species of Baculoviridae. The complete genome sequence of a BmNPV strain with cubic occlusion bodies is reported here. The genome of this strain consists of 127,465 nucleotides with a G+C content of 40.36% and is 97.3% and 97.5% identical to those of BmNPV strain T3 and Bombyx mandarina NPV S1, respectively. Despite the abnormal polyhedra it forms, the polyhedrin gene of the BmNPV cubic strain is 100% identical to those of the other two strains. Baculovirus repeated ORFs and homologous repeat regions cause the major differences in genome size of these BmNPV isolates. PMID:22923803

Protection of chickens from Newcastle disease with a recombinant baculovirus subunit vaccine expressing the fusion and hemagglutinin-neuraminidase proteins

PubMed Central

Lee, Youn-Jeong; Sung, Haan-Woo; Choi, Jun-Gu; Lee, Eun-Kyoung; Yoon, Hachung; Kim, Jae-Hong

2008-01-01

Recombinant baculoviruses containing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein gene of the viscerotropic velogenic (vv) Newcastle disease virus (NDV) isolate, Kr-005/00, and a lentogenic La Sota strain of the NDV were constructed in an attempt to develop an effective subunit vaccine to the recent epizootic vvNDV. The level of protection was determined by evaluating the clinical signs, mortality, and virus shedding from the oropharynx and cloaca of chickens after a challenge with vvNDV Kr-005/00. The recombinant ND F (rND F) and recombinant HN (rND HN) glycoproteins derived from the velogenic strain provided good protection against the clinical signs and mortality, showing a 0.00 PI value and 100% protection after a booster immunization. On the other hand, the combined rND F + HN glycoprotein derived from the velogenic strain induced complete protection (0.00 PI value and 100% protection) and significantly reduced the amount of virus shedding even after a single immunization. The rND F and rND HN glycoproteins derived from the velogenic strain had a slightly, but not significantly, greater protective effect than the lentogenic strain. These results suggest that the combined rND F + HN glycoprotein derived from vvNDV can be an ideal subunit marker vaccine candidate in chickens in a future ND eradication program. PMID:18716451

The Complete Genome of a New Betabaculovirus from Clostera anastomosis

PubMed Central

Yin, Feifei; Zhu, Zheng; Liu, Xiaoping; Hou, Dianhai; Wang, Jun; Zhang, Lei; Wang, Manli; Kou, Zheng; Wang, Hualin; Deng, Fei; Hu, Zhihong

2015-01-01

Clostera anastomosis (Lepidoptera: Notodontidae) is a defoliating forest insect pest. Clostera anastomosis granulovirus-B (ClasGV-B) belonging to the genus Betabaculovirus of family Baculoviridae has been used for biological control of the pest. Here we reported the full genome sequence of ClasGV-B and compared it to other previously sequenced baculoviruses. The circular double-stranded DNA genome is 107,439 bp in length, with a G+C content of 37.8% and contains 123 open reading frames (ORFs) representing 93% of the genome. ClasGV-B contains 37 baculovirus core genes, 25 lepidopteran baculovirus specific genes, 19 betabaculovirus specific genes, 39 other genes with homologues to baculoviruses and 3 ORFs unique to ClasGV-B. Hrs appear to be absent from the ClasGV-B genome, however, two non-hr repeats were found. Phylogenetic tree based on 37 core genes from 73 baculovirus genomes placed ClasGV-B in the clade b of betabaculoviruses and was most closely related to Erinnyis ello GV (ErelGV). The gene arrangement of ClasGV-B also shared the strongest collinearity with ErelGV but differed from Clostera anachoreta GV (ClanGV), Clostera anastomosis GV-A (ClasGV-A, previously also called CaLGV) and Epinotia aporema GV (EpapGV) with a 20 kb inversion. ClasGV-B genome contains three copies of polyhedron envelope protein gene (pep) and phylogenetic tree divides the PEPs of betabaculoviruses into three major clades: PEP-1, PEP-2 and PEP/P10. ClasGV-B also contains three homologues of P10 which all harbor an N-terminal coiled-coil domain and a C-terminal basic sequence. ClasGV-B encodes three fibroblast growth factor (FGF) homologues which are conserved in all sequenced betabaculoviruses. Phylogenetic analysis placed these three FGFs into different groups and suggested that the FGFs were evolved at the early stage of the betabaculovirus expansion. ClasGV-B is different from previously reported ClasGV-A and ClanGV isolated from Notodontidae in sequence and gene arrangement

Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity.

PubMed

Swamy, N; Ghosh, S; Schneider, G B; Ray, R

2001-01-01

Vitamin D-binding protein (DBP) is a multi-functional serum protein that is converted to vitamin D-binding protein-macrophage activating factor (DBP-maf) by post-translational modification. DBP-maf is a new cytokine that mediates bone resorption by activating osteoclasts, which are responsible for resorption of bone. Defective osteoclast activation leads to disorders like osteopetrosis, characterized by excessive accumulation of bone mass. Previous studies demonstrated that two nonallelic mutations in the rat with osteopetrosis have independent defects in the cascade involved in the conversion of DBP to DBP-maf. The skeletal defects associated with osteopetrosis are corrected in these mutants with in vivo DBP-maf treatment. This study evaluates the effects of various forms of DBP-maf (native, recombinant, and 25-hydroxyvitamin D(3) bound) on osteoclast function in vitro in order to determine some of the structural requirements of this protein that relate to bone resorbing activities. Osteoclast activity was determined by evaluating pit formation using osteoclasts, isolated from the long bones of newborn rats, incubated on calcium phosphate coated, thin film, Ostologic MultiTest Slides. Incubation of osteoclasts with ex vivo generated native DBP-maf resulted in a dose dependent, statistically significant, activation of the osteoclasts. The activation was similar whether or not the vitamin D binding site of the DBP-maf was occupied. The level of activity in response to DBP-maf was greater than that elicited by optimal doses of other known stimulators (PTH and 1,25(OH(2)D(3)) of osteoclast function. Furthermore, another potent macrophage activating factor, interferon--gamma, had no effect on osteoclast activity. The activated form of a full length recombinant DBP, expressed in E. coli showed no activity in the in vitro assay. Contrary to this finding, baculovirus-expressed recombinant DBP-maf demonstrated significant osteoclast activating activity. The normal

DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

PubMed

Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

2003-03-01

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

Evolution of Protein Domain Repeats in Metazoa

PubMed Central

Schüler, Andreas; Bornberg-Bauer, Erich

2016-01-01

Repeats are ubiquitous elements of proteins and they play important roles for cellular function and during evolution. Repeats are, however, also notoriously difficult to capture computationally and large scale studies so far had difficulties in linking genetic causes, structural properties and evolutionary trajectories of protein repeats. Here we apply recently developed methods for repeat detection and analysis to a large dataset comprising over hundred metazoan genomes. We find that repeats in larger protein families experience generally very few insertions or deletions (indels) of repeat units but there is also a significant fraction of noteworthy volatile outliers with very high indel rates. Analysis of structural data indicates that repeats with an open structure and independently folding units are more volatile and more likely to be intrinsically disordered. Such disordered repeats are also significantly enriched in sites with a high functional potential such as linear motifs. Furthermore, the most volatile repeats have a high sequence similarity between their units. Since many volatile repeats also show signs of recombination, we conclude they are often shaped by concerted evolution. Intriguingly, many of these conserved yet volatile repeats are involved in host-pathogen interactions where they might foster fast but subtle adaptation in biological arms races. Key Words: protein evolution, domain rearrangements, protein repeats, concerted evolution. PMID:27671125

Topological characteristics of helical repeat proteins.

PubMed

Groves, M R; Barford, D

1999-06-01

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem repeats of an alpha-helical structural unit, creating extended superhelical structures that are ideally suited to create a protein recognition interface.

Quantifying ataxia: ideal trajectory analysis--a technical note

NASA Technical Reports Server (NTRS)

McPartland, M. D.; Krebs, D. E.; Wall, C. 3rd

2000-01-01

We describe a quantitative method to assess repeated stair stepping stability. In both the mediolateral (ML) and anterioposterior (AP) directions, the trajectory of the subject's center of mass (COM) was compared to an ideal sinusoid. The two identified sinusoids were unique in each direction but coupled. Two dimensionless numbers-the mediolateral instability index (IML) and AP instability index (IAP)-were calculated using the COM trajectory and ideal sinusoids for each subject with larger index values resulting from less stable performance. The COM trajectories of nine nonimpaired controls and six patients diagnosed with unilateral or bilateral vestibular labyrinth hypofunction were analyzed. The average IML and IAP values of labyrinth disorder patients were respectively 127% and 119% greater than those of controls (p<0.014 and 0.006, respectively), indicating that the ideal trajectory analysis distinguishes persons with labyrinth disorder from those without. The COM trajectories also identify movement inefficiencies attributable to vestibulopathy.

Repeated nightmares

MedlinePlus

Nightmares - repeated; Dream anxiety disorder ... Arnulf I, Nightmares and dream disturbances. In: Kryger M, Roth T, Dement WC, eds. Principles and Practice of Sleep Medicine . 6th ed. Philadelphia, PA: Elsevier; ...

Phenotypic characterization of individuals with 30-40 CAG repeats in the Huntington disease (HD) gene reveals HD cases with 36 repeats and apparently normal elderly individuals with 36-39 repeats

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

Rubinsztein, D.C.; Leggo, J.; Whittaker, J.L.

1996-07-01

Abnormal CAG expansions in the IT-15 gene are associated with Huntington disease (HD). In the diagnostic setting it is necessary to define the limits of the CAG size ranges on normal and HD-associated chromosomes. Most large analyses that defined the limits of the normal and pathological size ranges employed PCR assays, which included the CAG repeats and a CCG repeat tract that was thought to be invariant. Many of these experiments found an overlap between the normal and disease size ranges. Subsequent findings that the CCG repeats vary by 9 trinucleotide lengths suggested that the limits of the normal andmore » disease size ranges should be reevaluated with assays that exclude the CCG polymorphism. Since patients with between 30 and 40 repeats are rare, a consortium was assembled to collect such individuals. All 178 samples were reanalyzed in Cambridge by using assays specific for the CAG repeats. We have optimized methods for reliable sizing of CAG repeats and show cases that demonstrate the dangers of using PCR assays that include both the CAG and CCG polymorphisms. Seven HD patients had 36 repeats, which confirms that this allele is associated with disease. Individuals without apparent symptoms or signs of HD were found at 36 repeats (aged 74, 78, 79, and 87 years), 37 repeats (aged 69 years), 38 repeats (aged 69 and 90 years), and 39 repeats (aged 67, 90, and 95 years). The detailed case histories of an exceptional case from this series will be presented: a 95-year-old man with 39 repeats who did not have classical features of HD. The apparently healthy survival into old age of some individuals with 36-39 repeats suggests that the HD mutation may not always be fully penetrant. 26 refs., 3 figs., 1 tab.« less

47 CFR 97.205 - Repeater station.

Code of Federal Regulations, 2013 CFR

2013-10-01

... case, the licensee of the non-coordinated repeater has primary responsibility to resolve the interference. (d) A repeater may be automatically controlled. (e) Ancillary functions of a repeater that are available to users on the input channel are not considered remotely controlled functions of the station...

47 CFR 97.205 - Repeater station.

Code of Federal Regulations, 2014 CFR

2014-10-01

... case, the licensee of the non-coordinated repeater has primary responsibility to resolve the interference. (d) A repeater may be automatically controlled. (e) Ancillary functions of a repeater that are available to users on the input channel are not considered remotely controlled functions of the station...

47 CFR 97.205 - Repeater station.

Code of Federal Regulations, 2012 CFR

2012-10-01

... case, the licensee of the non-coordinated repeater has primary responsibility to resolve the interference. (d) A repeater may be automatically controlled. (e) Ancillary functions of a repeater that are available to users on the input channel are not considered remotely controlled functions of the station...

47 CFR 97.205 - Repeater station.

Code of Federal Regulations, 2011 CFR

2011-10-01

... case, the licensee of the non-coordinated repeater has primary responsibility to resolve the interference. (d) A repeater may be automatically controlled. (e) Ancillary functions of a repeater that are available to users on the input channel are not considered remotely controlled functions of the station...

47 CFR 97.205 - Repeater station.

Code of Federal Regulations, 2010 CFR

2010-10-01

... case, the licensee of the non-coordinated repeater has primary responsibility to resolve the interference. (d) A repeater may be automatically controlled. (e) Ancillary functions of a repeater that are available to users on the input channel are not considered remotely controlled functions of the station...

Synergistic anticancer effect of cisplatin and Chal-24 combination through IAP and c-FLIPL degradation, Ripoptosome formation and autophagy-mediated apoptosis

PubMed Central

Shi, Shaoqing; Wang, Qiong; Xu, Jennings; Jang, Jun-Ho; Padilla, Mabel T.; Nyunoya, Toru; Xing, Chengguo; Zhang, Lin; Lin, Yong

2015-01-01

Drug resistance is a major hurdle in anticancer chemotherapy. Combined therapy using drugs with distinct mechanisms of function may increase anticancer efficacy. We have recently identified the novel chalcone derivative, chalcone-24 (Chal-24), as a potential therapeutic that kills cancer cells through activation of an autophagy-mediated necroptosis pathway. In this report, we investigated if Chal-24 can be combined with the frontline genotoxic anticancer drug, cisplatin for cancer therapy. The combination of Chal-24 and cisplatin synergistically induced apoptotic cytotoxicity in lung cancer cell lines, which was dependent on Chal-24-induced autophagy. While cisplatin slightly potentiated the JNK/Bcl2/Beclin1 pathway for autophagy activation, its combination with Chal-24 strongly triggered proteasomal degradation of the cellular inhibitor of apoptosis proteins (c-IAPs) and formation of the Ripoptosome complex that contains RIP1, FADD and caspase 8. Furthermore, the cisplatin and Chal-24 combination induced dramatic degradation of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (cFLIPL) which suppresses Ripoptosome-mediated apoptosis activation. These results establish a novel mechanism for potentiation of anticancer activity with the combination of Chal-24 and cisplatin: to enhance apoptosis signaling through Ripoptosome formation and to release the apoptosis brake through c-FLIPL degradation. Altogether, our work suggests that the combination of Chal-24 and cisplatin could be employed to improve chemotherapy efficacy. PMID:25682199

Full protection against African horsesickness (AHS) in horses induced by baculovirus-derived AHS virus serotype 4 VP2, VP5 and VP7.

PubMed

Martínez-Torrecuadrada, J L; Díaz-Laviada, M; Roy, P; Sánchez, C; Vela, C; Sánchez-Vizcaíno, J M; Casal, J I

1996-06-01

African horsesickness virus serotype 4 (AHSV-4) outer capsid protein VP2, or VP2 and VP5 plus inner capsid protein VP7, derived from single or dual recombinant baculovirus expression vectors were used in different combinations to immunize horses. When the proteins were purified by affinity chromatography, the combination of all three proteins induced low levels of neutralizing antibodies and conferred protection against virulent virus challenge. However, purified VP2 or VP2 and VP5 in the absence of VP7 failed to induce neutralizing antibodies and protection. Immunization with non-purified proteins enhanced the titres of neutralizing antibodies. Again, the combination of the three proteins was able to confer total protection to immunized horses, which showed absence of viraemia. The antigenicity of recombinant VP2 was analysed with a collection of 30 MAbs. Both purified and unpurified recombinant VP2 proteins showed different antigenic patterns in comparison to that of VP2 on virions. An immunization experiment with four more horses confirmed these results. The vaccine described here would not only prevent the disease, but would drastically reduce the propagation of the virus by vectors.

[Use of the recombinant baculovirus BacVP6C for the construction of an internal positive control of rotavirus C].

PubMed

Abid-Ayadi, I; Guix, S; Pintó, R M; Bosch, A

2011-06-01

Unlike group A, a few studies have interested other groups of the rotavirus, especially in Tunisia. The role of rotavirus C (RVC) infection is underestimated because of its sporadic nature. The aim of our study was to develop rapid diagnostic procedures of RVC by using an internal positive control of reverse transcription PCR (RT-PCR). The internal positive control (386pb) was designed from the recombinant baculovirus BacVP6C containing the full length cDNA of the Cowden strain gene 5 (1353pb). A fragment of 596pb was amplified by PCR using the BacVP6C DNA ds as template. Then, a central part of 210pb was deleted and the remaining fragment (386pb) was cloned into pGEM-3Zf(+) plasmid between SP6 and T7 RNA polymerase promoters. The obtained recombinant plasmid "pIAM1" was then used for the generation of the internal positive control by in vitro transcription. The sensibility of the RT-PCR was about 3.66×10(5) molecules of RNA/μl. The use of a shorter positive control, as compared to the wild type, allows increased specificity of the RT-PCR reaction, and could be used for efficient diagnostic and surveillance of RVC-caused diseases. Copyright © 2009 Elsevier Masson SAS. All rights reserved.

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

PubMed

López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

2007-11-01

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.

Intracisternal A-Particle Element Transposition into the Murine β-Glucuronidase Gene Correlates with Loss of Enzyme Activity: a New Model for β-Glucuronidase Deficiency in the C3H Mouse†

PubMed Central

Gwynn, Babette; Lueders, Kira; Sands, Mark S.; Birkenmeier, Edward H.

1998-01-01

The severity of human mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, depends on the relative activity of the enzyme β-glucuronidase. Loss of β-glucuronidase activity can cause hydrops fetalis, with in utero or postnatal death of the patient. In this report, we show that β-glucuronidase activity is not detectable by a standard fluorometric assay in C3H/HeOuJ (C3H) mice homozygous for a new mutation, gusmps2J. These gusmps2J/gusmps2J mice are born and survive much longer than the previously characterized β-glucuronidase-null B6.C-H-2bm1/ByBir-gusmps (gusmps/gusmps) mice. Northern blot analysis of liver from gusmps2J/gusmps2J mice demonstrates a 750-bp reduction in size of β-glucuronidase mRNA. A 5.4-kb insertion in the Gus-sh nucleotide sequence from these mice was localized by Southern blot analysis to intron 8. The ends of the inserted sequences were cloned by inverse PCR and revealed an intracisternal A-particle (IAP) element inserted near the 3′ end of the intron. The sequence of the long terminal repeat (LTR) regions of the IAP most closely matches that of a composite LTR found in transposed IAPs previously identified in the C3H strain. The inserted IAP may contribute to diminished β-glucuronidase activity either by interfering with transcription or by destabilizing the message. The resulting phenotype is much less severe than that previously described in the gusmps/gusmps mouse and provides an opportunity to study MPS VII on a genetic background that clearly modulates disease severity. PMID:9774663

Two modes of the silk road pattern and their interannual variability simulated by LASG/IAP AGCM SAMIL2.0

NASA Astrophysics Data System (ADS)

Song, Fengfei; Zhou, Tianjun; Wang, Lu

2013-05-01

In this study, two modes of the Silk Road pattern were investigated using NCEP2 reanalysis data and the simulation produced by Spectral Atmospheric Circulation Model of IAP LASG, Version 2 (SAMIL2.0) that was forced by SST observation data. The horizontal distribution of both modes were reasonably reproduced by the simulation, with a pattern correlation coefficient of 0.63 for the first mode and 0.62 for the second mode. The wave train was maintained by barotropic energy conversion (denoted as CK) and baroclinic energy conversion (denoted as CP) from the mean flow. The distribution of CK was dominated by its meridional component (CK y ) in both modes. When integrated spatially, CK y was more efficient than its zonal component (CK x ) in the first mode but less in the second mode. The distribution and efficiency of CK were not captured well by SAMIL2.0. However, the model performed reasonably well at reproducing the distribution and efficiency of CP in both modes. Because CP is more efficient than CK, the spatial patterns of the Silk Road pattern were well reproduced. Interestingly, the temporal phase of the second mode was well captured by a single-member simulation. However, further analysis of other ensemble runs demonstrated that the successful reproduction of the temporal phase was a result of internal variability rather than a signal of SST forcing. The analysis shows that the observed temporal variations of both CP and CK were poorly reproduced, leading to the low accuracy of the temporal phase of the Silk Road pattern in the simulation.

Relationship between intra-abdominal pressure and trunk EMG.

PubMed

McGill, S M; Sharratt, M T

1990-05-01

Intra-abdominal pressure (IAP) has been proposed as an important mechanism in manual lifting and breathing mechanics. Direct (invasive) measures of IAP have required the swallowing of a radio transducer or insertion of a pressure sensor into the rectum or down the oesophagus to the stomach. The purpose of this study was to investigate the relationship between a non-invasive method (EMG) and IAP. Several tasks involving abdominal muscle activation were performed to assess whether or not IAP played a common role in these tasks. IAP and EMG from rectus abdominis, the abdominal obliques, intercostals and erector spinae were measured. Peak IAP reached 340 mmHg (valsalva) for one subject but most values were less than 100 mmHg for tasks other than valsalva. The IAP and EMG data provide some insight into the role of IAP during the performance of specific tasks. Peak IAP within 60 ms of the onset of vigorous abdominal activation indicated the importance of a very rapid pressure response to abdominal muscle activation. The correlations between various muscle EMG time histories and IAP exceeded 0·80 for only two activities (i.e. r(2) = 0·82 between the intercostals and IAP during valsalva manoeuvres). These data suggest that no unifying hypothesis exists to explain the role of IAP for a wide variety of movement tasks; rather, the role of IAP is task specific. Copyright © 1990. Published by Elsevier Ltd.

Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose.

PubMed

Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo; Wang, Junwei

2011-05-27

Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs. Copyright © 2011 Elsevier Inc. All rights reserved.

Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate.

PubMed

Moss, Angela K; Hamarneh, Sulaiman R; Mohamed, Mussa M Rafat; Ramasamy, Sundaram; Yammine, Halim; Patel, Palak; Kaliannan, Kanakaraju; Alam, Sayeda N; Muhammad, Nur; Moaven, Omeed; Teshager, Abeba; Malo, Nondita S; Narisawa, Sonoko; Millán, José Luis; Warren, H Shaw; Hohmann, Elizabeth; Malo, Madhu S; Hodin, Richard A

2013-03-15

Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP.

Intestinal alkaline phosphatase inhibits the proinflammatory nucleotide uridine diphosphate

PubMed Central

Hamarneh, Sulaiman R.; Mohamed, Mussa M. Rafat; Ramasamy, Sundaram; Yammine, Halim; Patel, Palak; Kaliannan, Kanakaraju; Alam, Sayeda N.; Muhammad, Nur; Moaven, Omeed; Teshager, Abeba; Malo, Nondita S.; Narisawa, Sonoko; Millán, José Luis; Warren, H. Shaw; Hohmann, Elizabeth; Malo, Madhu S.; Hodin, Richard A.

2013-01-01

Uridine diphosphate (UDP) is a proinflammatory nucleotide implicated in inflammatory bowel disease. Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor capable of inhibiting intestinal inflammation. We used the malachite green assay to show that IAP dephosphorylates UDP. To study the anti-inflammatory effect of IAP, UDP or other proinflammatory ligands (LPS, flagellin, Pam3Cys, or TNF-α) in the presence or absence of IAP were applied to cell cultures, and IL-8 was measured. UDP caused dose-dependent increase in IL-8 release by immune cells and two gut epithelial cell lines, and IAP treatment abrogated IL-8 release. Costimulation with UDP and other inflammatory ligands resulted in a synergistic increase in IL-8 release, which was prevented by IAP treatment. In vivo, UDP in the presence or absence of IAP was instilled into a small intestinal loop model in wild-type and IAP-knockout mice. Luminal contents were applied to cell culture, and cytokine levels were measured in culture supernatant and intestinal tissue. UDP-treated luminal contents induced more inflammation on target cells, with a greater inflammatory response to contents from IAP-KO mice treated with UDP than from WT mice. Additionally, UDP treatment increased TNF-α levels in intestinal tissue of IAP-KO mice, and cotreatment with IAP reduced inflammation to control levels. Taken together, these studies show that IAP prevents inflammation caused by UDP alone and in combination with other ligands, and the anti-inflammatory effect of IAP against UDP persists in mouse small intestine. The benefits of IAP in intestinal disease may be partly due to inhibition of the proinflammatory activity of UDP. PMID:23306083

Mechanical unfolding of an ankyrin repeat protein.

PubMed

Serquera, David; Lee, Whasil; Settanni, Giovanni; Marszalek, Piotr E; Paci, Emanuele; Itzhaki, Laura S

2010-04-07

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly alpha-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one repeat at a time. The peeling away of half an ankyrin repeat, or one or more ankyrin repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual ankyrin repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of ankyrin repeat proteins, as well as the differences between the mechanics of natural occurring ankyrin repeat proteins and those of designed consensus ankyin repeat and globular proteins. Copyright (c) 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Repeated vitrification/warming of human sperm gives better results than repeated slow programmable freezing

PubMed Central

Vutyavanich, Teraporn; Lattiwongsakorn, Worashorn; Piromlertamorn, Waraporn; Samchimchom, Sudarat

2012-01-01

In this study, we compared the effects of repeated freezing/thawing of human sperm by our in-house method of rapid freezing with slow programmable freezing. Sperm samples from 11 normozoospermic subjects were processed through density gradients and divided into three aliquots: non-frozen, rapid freezing and slow programmable freezing. Sperm in the rapid freezing group had better motility and viability than those in the slow freezing group (P<0.01) after the first, second and third cycles of freezing/thawing, but there was no difference in morphology. In the second experiment, rapid freezing was repeated three times in 20 subjects. The samples from each thawing cycle were evaluated for DNA fragmentation using the alkaline comet assay. DNA fragmentation began to increase considerably after the second cycle of freezing/thawing, but to a level that was not clinically important. In the third experiment, rapid freezing was done repeatedly in 10 subjects, until no motile sperm were observed after thawing. The median number of repeated freezing/thawing that yielded no motile sperm was seven (range: 5–8, mean: 6.8). In conclusion, we demonstrated that repeated freezing/thawing of processed semen using our rapid freezing method gave better results than standard slow programmable freezing. This method can help maximize the usage of precious cryopreserved sperm samples in assisted reproduction technology. PMID:23064685

A Discrete Ubiquitin-Mediated Network Regulates the Strength of NOD2 Signaling

PubMed Central

Tigno-Aranjuez, Justine T.; Bai, Xiaodong

2013-01-01

Dysregulation of NOD2 signaling is implicated in the pathology of various inflammatory diseases, including Crohn's disease, asthma, and sarcoidosis, making signaling proteins downstream of NOD2 potential therapeutic targets. Inhibitor-of-apoptosis (IAP) proteins, particularly cIAP1, are essential mediators of NOD2 signaling, and in this work, we describe a molecular mechanism for cIAP1's regulation in the NOD2 signaling pathway. While cIAP1 promotes RIP2's tyrosine phosphorylation and subsequent NOD2 signaling, this positive regulation is countered by another E3 ubiquitin ligase, ITCH, through direct ubiquitination of cIAP1. This ITCH-mediated ubiquitination leads to cIAP1's lysosomal degradation. Pharmacologic inhibition of cIAP1 expression in ITCH−/− macrophages attenuates heightened ITCH−/− macrophage muramyl dipeptide-induced responses. Transcriptome analysis, combined with pharmacologic inhibition of cIAP1, further defines specific pathways within the NOD2 signaling pathway that are targeted by cIAP1. This information provides genetic signatures that may be useful in repurposing cIAP1-targeted therapies to correct NOD2-hyperactive states and identifies a ubiquitin-regulated signaling network centered on ITCH and cIAP1 that controls the strength of NOD2 signaling. PMID:23109427

A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.

PubMed

Weyer, U; Possee, R D

1991-12-01

A baculovirus transfer vector, pAcUW3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins, into the Autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. The NA gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the HA gene was placed under the control of a copy of the p10 promoter at a site upstream of and in opposite orientation to the polyhedrin promoter. After infection of Spodoptera frugiperda cells with the recombinant virus, AcUW3HANA, both HA and NA were expressed in the very late phase of infection and were shown to be functional in appropriate assays. Immunofluorescence assays demonstrated their localization at the surface of infected insect cells. The expression of both foreign genes in the recombinant virus was found to be stable for at least 12 passages in cell culture.

Oxymatrine induces human pancreatic cancer PANC-1 cells apoptosis via regulating expression of Bcl-2 and IAP families, and releasing of cytochrome c

PubMed Central

2011-01-01

Background Oxymatrine, an isolated extract from traditional Chinese herb Sophora Flavescens Ait, has been traditionally used for therapy of anti-hepatitis B virus, anti-inflammation and anti-anaphylaxis. The present study was to investigate the anti-cancer effect of oxymatrine on human pancreatic cancer PANC-1 cells, and its possible molecular mechanism. Methods The effect of oxymatrine on the viability and apoptosis was examined by methyl thiazolyl tetrazolium and flow cytometry analysis. The expression of Bax, Bcl-2, Bcl-x (L/S), Bid, Bad, HIAP-1, HIAP-2, XIAP, NAIP, Livin and Survivin genes was accessed by RT-PCR. The levels of cytochrome c and caspase 3 protein were assessed by Western blotting. Results Oxymatrine inhibited cell viability and induced apoptosis of PANC-1 cells in a time- and dose-dependent manner. This was accompanied by down-regulated expression of Livin and Survivin genes while the Bax/Bcl-2 ratio was upregulated. Furthermore, oxymatrine treatment led to the release of cytochrome c and activation of caspase-3 proteins. Conclusion Oxymatrine can induce apoptotic cell death of human pancreatic cancer, which might be attributed to the regulation of Bcl-2 and IAP families, release of mitochondrial cytochrome c and activation of caspase-3. PMID:21714853

Intestinal alkaline phosphatase: novel functions and protective effects.

PubMed

Lallès, Jean-Paul

2014-02-01

Important protective roles of intestinal alkaline phosphatase (IAP)--including regulation of intestinal surface pH, absorption of lipids, detoxification of free nucleotides and bacterial lipopolysaccharide, attenuation of intestinal inflammation, and possible modulation of the gut microbiota--have been reviewed recently. IAP is modulated by numerous nutritional factors. The present review highlights new findings on the properties of IAP and extends the list of its protective functions. Critical assessment of data suggests that some IAP properties are a direct result of dephosphorylation of proinflammatory moieties, while others (e.g., gut barrier protection and microbiota shaping) may be secondary to IAP-mediated downregulation of inflammation. IAP and tissue-nonspecific alkaline phosphatase isoforms characterize the small intestine and the colon, respectively. Gastrointestinal administration of exogenous IAP ameliorates gut inflammation and favors gut tissue regeneration, whereas enteral and systemic IAP administration attenuates systemic inflammation only. Finally, the IAP gene family has a strong evolutionary link to food-driven changes in gastrointestinal tract anatomy and microbiota composition. Therefore, stimulation of IAP activity by dietary intervention is a goal for preserving gut homeostasis and health by minimizing low-grade inflammation. © 2013 International Life Sciences Institute.

Blockade of inhibitors of apoptosis (IAPs) in combination with tumor-targeted delivery of tumor necrosis factor-α leads to synergistic antitumor activity

PubMed Central

Yuan, Z; Syrkin, G; Adem, A; Geha, R; Pastoriza, J; Vrikshajanani, C; Smith, T; Quinn, T J; Alemu, G; Cho, H; Barrett, C J; Arap, W; Pasqualini, R; Libutti, S K

2013-01-01

In the current study, we examined whether the combination of tumor vasculature-targeted gene therapy with adeno-associated virus bacteriophage-tumor necrosis factor-α (AAVP-TNF-α) and/or the orally administered LCL161, an antagonist of inhibitors of apoptosis proteins (IAPs), enhanced antitumor efficacy without systemic toxicity. M21 human melanoma xenografts were grown subcutaneously in nude mice. Mice were treated according to one of four treatment regimens: AAVP-TNF-α alone (AAVP-TNF-α plus sodium acetate-acetic acid (NaAc) buffer) via tail vein injection; LCL161 alone (phosphate-buffered saline (PBS) plus LCL161) via oral gavage; AAVP-TNF-α plus LCL161; and PBS plus NaAc Buffer as a control group. Tumor volume, survival and toxicity were analyzed. AAVP trafficking and TNF-α production in vivo were detected on days 7 and 21 by real-time PCR, enzyme-linked immunosorbent assay and immunofluorescence. The levels of apoptosis and activation of caspases were assessed on days 7 and 21 by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) and immunofluorescence assays. Our results showed that the combination of AAVP-TNF-α and LCL161 significantly inhibited tumor growth and prolonged survival in mice with melanoma xenografts. The combination of AAVP-TNF-α and LCL161 was also significantly more effective than either agent alone, showing a synergistic effect without systemic toxicity. PMID:23154431

SCA8 Repeat Expansion: Large CTA/CTG Repeat Alleles Are More Common in Ataxic Patients, Including Those with SCA6

PubMed Central

Izumi, Yuishin; Maruyama, Hirofumi; Oda, Masaya; Morino, Hiroyuki; Okada, Takayuki; Ito, Hidefumi; Sasaki, Iwao; Tanaka, Hiroyasu; Komure, Osamu; Udaka, Fukashi; Nakamura, Shigenobu; Kawakami, Hideshi

2003-01-01

We analyzed the SCA8 CTA/CTG repeat in a large group of Japanese subjects. The frequency of large alleles (85–399 CTA/CTG repeats) was 1.9% in spinocerebellar ataxia (SCA), 0.4% in Parkinson disease, 0.3% in Alzheimer disease, and 0% in a healthy control group; the frequency was significantly higher in the group with SCA than in the control group. Homozygotes for large alleles were observed only in the group with SCA. In five patients with SCA from two families, a large SCA8 CTA/CTG repeat and a large SCA6 CAG repeat coexisted. Age at onset was correlated with SCA8 repeats rather than SCA6 repeats in these five patients. In one of these families, at least one patient showed only a large SCA8 CTA/CTG repeat allele, with no large SCA6 CAG repeat allele. We speculate that the presence of a large SCA8 CTA/CTG repeat allele influences the function of channels such as α1A-voltage–dependent calcium channel through changing or aberrant splicing, resulting in the development of cerebellar ataxia, especially in homozygous patients. PMID:12545428

SCA8 repeat expansion: large CTA/CTG repeat alleles are more common in ataxic patients, including those with SCA6.

PubMed

Izumi, Yuishin; Maruyama, Hirofumi; Oda, Masaya; Morino, Hiroyuki; Okada, Takayuki; Ito, Hidefumi; Sasaki, Iwao; Tanaka, Hiroyasu; Komure, Osamu; Udaka, Fukashi; Nakamura, Shigenobu; Kawakami, Hideshi

2003-03-01

We analyzed the SCA8 CTA/CTG repeat in a large group of Japanese subjects. The frequency of large alleles (85-399 CTA/CTG repeats) was 1.9% in spinocerebellar ataxia (SCA), 0.4% in Parkinson disease, 0.3% in Alzheimer disease, and 0% in a healthy control group; the frequency was significantly higher in the group with SCA than in the control group. Homozygotes for large alleles were observed only in the group with SCA. In five patients with SCA from two families, a large SCA8 CTA/CTG repeat and a large SCA6 CAG repeat coexisted. Age at onset was correlated with SCA8 repeats rather than SCA6 repeats in these five patients. In one of these families, at least one patient showed only a large SCA8 CTA/CTG repeat allele, with no large SCA6 CAG repeat allele. We speculate that the presence of a large SCA8 CTA/CTG repeat allele influences the function of channels such as alpha(1A)-voltage-dependent calcium channel through changing or aberrant splicing, resulting in the development of cerebellar ataxia, especially in homozygous patients.

Comparison of simple sequence repeats in 19 Archaea.

PubMed

Trivedi, S

2006-12-05

All organisms that have been studied until now have been found to have differential distribution of simple sequence repeats (SSRs), with more SSRs in intergenic than in coding sequences. SSR distribution was investigated in Archaea genomes where complete chromosome sequences of 19 Archaea were analyzed with the program SPUTNIK to find di- to penta-nucleotide repeats. The number of repeats was determined for the complete chromosome sequences and for the coding and non-coding sequences. Different from what has been found for other groups of organisms, there is an abundance of SSRs in coding regions of the genome of some Archaea. Dinucleotide repeats were rare and CG repeats were found in only two Archaea. In general, trinucleotide repeats are the most abundant SSR motifs; however, pentanucleotide repeats are abundant in some Archaea. Some of the tetranucleotide and pentanucleotide repeat motifs are organism specific. In general, repeats are short and CG-rich repeats are present in Archaea having a CG-rich genome. Among the 19 Archaea, SSR density was not correlated with genome size or with optimum growth temperature. Pentanucleotide density had an inverse correlation with the CG content of the genome.

Short Tandem Repeat DNA Internet Database

National Institute of Standards and Technology Data Gateway

SRD 130 Short Tandem Repeat DNA Internet Database (Web, free access)   Short Tandem Repeat DNA Internet Database is intended to benefit research and application of short tandem repeat DNA markers for human identity testing. Facts and sequence information on each STR system, population data, commonly used multiplex STR systems, PCR primers and conditions, and a review of various technologies for analysis of STR alleles have been included.

A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity.

PubMed

Malo, Madhu S

2015-12-01

Mice deficient in intestinal alkaline phosphatase (IAP) develop type 2 diabetes mellitus (T2DM). We hypothesized that a high level of IAP might be protective against T2DM in humans. We determined IAP levels in the stools of 202 diabetic patients and 445 healthy non-diabetic control people. We found that compared to controls, T2DM patients have approx. 50% less IAP (mean +/- SEM: 67.4 +/- 3.2 vs 35.3 +/- 2.5 U/g stool, respectively; p < 0.000001) indicating a protective role of IAP against T2DM. Multiple logistic regression analyses showed an independent association between the IAP level and diabetes status. With each 25 U/g decrease in stool IAP, there is a 35% increased risk of diabetes. The study revealed that obese people with high IAP (approx. 65 U/g stool) do not develop T2DM. Approx. 65% of the healthy population have < 65.0 U/g stool IAP, and predictably, these people might have 'the incipient metabolic syndrome', including 'incipient diabetes', and might develop T2DM and other metabolic disorders in the near future. In conclusion, high IAP levels appear to be protective against diabetes irrespective of obesity, and a 'temporal IAP profile' might be a valuable tool for predicting 'the incipient metabolic syndrome', including 'incipient diabetes'.

Nifty Nines and Repeating Decimals

ERIC Educational Resources Information Center

Brown, Scott A.

2016-01-01

The traditional technique for converting repeating decimals to common fractions can be found in nearly every algebra textbook that has been published, as well as in many precalculus texts. However, students generally encounter repeating decimal numerals earlier than high school when they study rational numbers in prealgebra classes. Therefore, how…

Repeated Prescribed Burning in Aspen

Treesearch

Donald A. Perala

1974-01-01

Infrequent burning weather, low flammability of the aspen-hardwood association, and prolific sprouting and seeding of shrubs and hardwoods made repeated dormant season burning a poor tool to convert good site aspen to conifers. Repeat fall burns for wildlife habitat maintenance is workable if species composition changes are not important.

Disease-associated repeat instability and mismatch repair.

PubMed

Schmidt, Monika H M; Pearson, Christopher E

2016-02-01

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential. Copyright © 2015 Elsevier B.V. All rights reserved.

Antagonism between apoptotic (Bax/Bcl-2) and anti-apoptotic (IAP) signals in human osteoblastic cells under vector-averaged gravity condition.

PubMed

Nakamura, Hiroshi; Kumei, Yasuhiro; Morita, Sadao; Shimokawa, Hitoyata; Ohya, Keiichi; Shinomiya, Kenichi

2003-12-01

A functional disorder associated with weightlessness is well documented in osteoblasts. The apototic features of this disorder are poorly understood. Harmful stress induces apoptosis in cells via mitochondria and/or Fas. The Bax triggers cytochrome c release from mitochondria, which can be blocked by the Bcl-2. Released cytochrome c then activates the initiator caspase, caspase-9, which can be blocked by the anti-apototic (IAP) family of molecules. The effector caspase, caspase-3, finally exerts DNA fragmentation. We conducted this study to examine the apoptotic effects of vector-averaged gravity on normal human osteoblastic cells. Cell culture flasks were incubated on the clinostat, which generated vector-averaged gravity condition (simulated microgravity) for 12, 24, 48, and 96 hours. Upon termination of clinostat cultures, the cell number and cell viability were assessed. DNA fragmentation was analyzed on the agarose-gel electrophoresis. The mRNA levels for Bax, Bcl-2, XIAP, and caspase-3 genes were analyzed by semi-quantitative RT-PCR. Twenty-four hours after starting clinostat rotation, the ratios of Bax/Bcl-2 mRNA levels (indicator of apoptosis) were significantly increased to 136% of the 1G static controls. However, the XIAP mRNA levels (anti-apoptotic molecule) were increased concomitantly to 138% of the 1G static controls. Thus, cell proliferation or cell viability was not affected by vector-averaged gravity. DNA fragmentation was not observed in clinostat group as well as in control group. Finally, the caspase-3 mRNA levels were not affected by vector-averaged gravity. Simulated microgravity might modulate some apoptotic signals upstream the mitochondrial pathway.

Autographa caljfornica nuclear polyhedrosis virus replication in non-permissive Lymantria dispar cell lines

Treesearch

Edward M. Dougherty; David Guzo; Kathleen S. Shields; Dwight E. Lynn; Susan K. Braun

1991-01-01

Autographa californica nuclear polyhedrosis virus (AcNPV) the prototypic group A baculovirus, has the widest reported host range of the baculoviruses and is considered to be one of the most virulent baculoviruses studied. The gypsy moth Lymantria dispar is not considered a natural host of AcNPV, however. To determine the factors...

The Effects of Repeated Writing and Repeated Revision Strategies on Composing Fluency of Learning Disabled Adolescents. Final Report.

ERIC Educational Resources Information Center

Harriman, Nancy E.; Gajar, Anna H.

The study investigated the use of repeated writings as a means of increasing written language fluency with 48 learning disabled students in senior high school resource room English classes. Subjects received one of four treatments for 4 days: repeated writing with structural cues, repeated revision with structural cues, writing on a new topic with…

Analysis of sequence repeats of proteins in the PDB.

PubMed

Mary Rajathei, David; Selvaraj, Samuel

2013-12-01

Internal repeats in protein sequences play a significant role in the evolution of protein structure and function. Applications of different bioinformatics tools help in the identification and characterization of these repeats. In the present study, we analyzed sequence repeats in a non-redundant set of proteins available in the Protein Data Bank (PDB). We used RADAR for detecting internal repeats in a protein, PDBeFOLD for assessing structural similarity, PDBsum for finding functional involvement and Pfam for domain assignment of the repeats in a protein. Through the analysis of sequence repeats, we found that identity of the sequence repeats falls in the range of 20-40% and, the superimposed structures of the most of the sequence repeats maintain similar overall folding. Analysis sequence repeats at the functional level reveals that most of the sequence repeats are involved in the function of the protein through functionally involved residues in the repeat regions. We also found that sequence repeats in single and two domain proteins often contained conserved sequence motifs for the function of the domain. Copyright © 2013 Elsevier Ltd. All rights reserved.

The baculovirus core gene ac83 is required for nucleocapsid assembly and per os infectivity of Autographa californica nucleopolyhedrovirus.

PubMed

Zhu, Shimao; Wang, Wei; Wang, Yan; Yuan, Meijin; Yang, Kai

2013-10-01

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac83 is a baculovirus core gene whose function in the AcMNPV life cycle is unknown. In the present study, an ac83-knockout AcMNPV (vAc83KO) was constructed to investigate the function of ac83 through homologous recombination in Escherichia coli. No budded virions were produced in vAc83KO-transfected Sf9 cells, although viral DNA replication was unaffected. Electron microscopy revealed that nucleocapsid assembly was aborted due to the ac83 deletion. Domain-mapping studies revealed that the expression of Ac83 amino acid residues 451 to 600 partially rescued the ability of AcMNPV to produce infectious budded virions. Bioassays indicated that deletion of the chitin-binding domain of Ac83 resulted in the failure of oral infection of Trichoplusia ni larvae by AcMNPV, but AcMNPV remained infectious following intrahemocoelic injection, suggesting that the domain is involved in the binding of occlusion-derived virions to the peritrophic membrane and/or to other chitin-containing insect tissues. It has been demonstrated that Ac83 is the only component with a chitin-binding domain in the per os infectivity factor complex on the occlusion-derived virion envelope. Interestingly, a functional inner nuclear membrane sorting motif, which may facilitate the localization of Ac83 to the envelopes of occlusion-derived virions, was identified by immunofluorescence analysis. Taken together, these results demonstrate that Ac83 plays an important role in nucleocapsid assembly and the establishment of oral infection.

Structural studies of CNG repeats

PubMed Central

Kiliszek, Agnieszka; Rypniewski, Wojciech

2014-01-01

CNG repeats (where N denotes one of the four natural nucleotides) are abundant in the human genome. Their tendency to undergo expansion can lead to hereditary diseases known as TREDs (trinucleotide repeat expansion disorders). The toxic factor can be protein, if the abnormal gene is expressed, or the gene transcript, or both. The gene transcripts have attracted much attention in the biomedical community, but their molecular structures have only recently been investigated. Model RNA molecules comprising CNG repeats fold into long hairpins whose stems generally conform to an A-type helix, in which the non-canonical N-N pairs are flanked by C-G and G-C pairs. Each homobasic pair is accommodated in the helical context in a unique manner, with consequences for the local helical parameters, solvent structure, electrostatic potential and potential to interact with ligands. The detailed three-dimensional profiles of RNA CNG repeats can be used in screening of compound libraries for potential therapeutics and in structure-based drug design. Here is a brief survey of the CNG structures published to date. PMID:24939898

A Complete and Accurate Ab Initio Repeat Finding Algorithm.

PubMed

Lian, Shuaibin; Chen, Xinwu; Wang, Peng; Zhang, Xiaoli; Dai, Xianhua

2016-03-01

It has become clear that repetitive sequences have played multiple roles in eukaryotic genome evolution including increasing genetic diversity through mutation, changes in gene expression and facilitating generation of novel genes. However, identification of repetitive elements can be difficult in the ab initio manner. Currently, some classical ab initio tools of finding repeats have already presented and compared. The completeness and accuracy of detecting repeats of them are little pool. To this end, we proposed a new ab initio repeat finding tool, named HashRepeatFinder, which is based on hash index and word counting. Furthermore, we assessed the performances of HashRepeatFinder with other two famous tools, such as RepeatScout and Repeatfinder, in human genome data hg19. The results indicated the following three conclusions: (1) The completeness of HashRepeatFinder is the best one among these three compared tools in almost all chromosomes, especially in chr9 (8 times of RepeatScout, 10 times of Repeatfinder); (2) in terms of detecting large repeats, HashRepeatFinder also performed best in all chromosomes, especially in chr3 (24 times of RepeatScout and 250 times of Repeatfinder) and chr19 (12 times of RepeatScout and 60 times of Repeatfinder); (3) in terms of accuracy, HashRepeatFinder can merge the abundant repeats with high accuracy.

Repeated Reading. What Works Clearinghouse Intervention Report

ERIC Educational Resources Information Center

What Works Clearinghouse, 2014

2014-01-01

"Repeated reading" is an academic practice that aims to increase oral reading fluency. "Repeated reading" can be used with students who have developed initial word reading skills but demonstrate inadequate reading fluency for their grade level. During "repeated reading," a student sits in a quiet location with a…

Interplay between intestinal alkaline phosphatase, diet, gut microbes and immunity.

PubMed

Estaki, Mehrbod; DeCoffe, Daniella; Gibson, Deanna L

2014-11-14

Intestinal alkaline phosphatase (IAP) plays an essential role in intestinal homeostasis and health through interactions with the resident microbiota, diet and the gut. IAP's role in the intestine is to dephosphorylate toxic microbial ligands such as lipopolysaccharides, unmethylated cytosine-guanosine dinucleotides and flagellin as well as extracellular nucleotides such as uridine diphosphate. IAP's ability to detoxify these ligands is essential in protecting the host from sepsis during acute inflammation and chronic inflammatory conditions such as inflammatory bowel disease. Also important in these complications is IAP's ability to regulate the microbial ecosystem by forming a complex relationship between microbiota, diet and the intestinal mucosal surface. Evidence reveals that diet alters IAP expression and activity and this in turn can influence the gut microbiota and homeostasis. IAP's ability to maintain a healthy gastrointestinal tract has accelerated research on its potential use as a therapeutic agent against a multitude of diseases. Exogenous IAP has been shown to have beneficial effects when administered during ulcerative colitis, coronary bypass surgery and sepsis. There are currently a handful of human clinical trials underway investigating the effects of exogenous IAP during sepsis, rheumatoid arthritis and heart surgery. In light of these findings IAP has been marked as a novel agent to help treat a variety of other inflammatory and infectious diseases. The purpose of this review is to highlight the essential characteristics of IAP in protection and maintenance of intestinal homeostasis while addressing the intricate interplay between IAP, diet, microbiota and the intestinal epithelium.

A High Level of Intestinal Alkaline Phosphatase Is Protective Against Type 2 Diabetes Mellitus Irrespective of Obesity☆

PubMed Central

Malo, Madhu S.

2015-01-01

Mice deficient in intestinal alkaline phosphatase (IAP) develop type 2 diabetes mellitus (T2DM). We hypothesized that a high level of IAP might be protective against T2DM in humans. We determined IAP levels in the stools of 202 diabetic patients and 445 healthy non-diabetic control people. We found that compared to controls, T2DM patients have approx. 50% less IAP (mean +/− SEM: 67.4 +/− 3.2 vs 35.3 +/− 2.5 U/g stool, respectively; p < 0.000001) indicating a protective role of IAP against T2DM. Multiple logistic regression analyses showed an independent association between the IAP level and diabetes status. With each 25 U/g decrease in stool IAP, there is a 35% increased risk of diabetes. The study revealed that obese people with high IAP (approx. 65 U/g stool) do not develop T2DM. Approx. 65% of the healthy population have < 65.0 U/g stool IAP, and predictably, these people might have ‘the incipient metabolic syndrome’, including ‘incipient diabetes’, and might develop T2DM and other metabolic disorders in the near future. In conclusion, high IAP levels appear to be protective against diabetes irrespective of obesity, and a ‘temporal IAP profile’ might be a valuable tool for predicting ‘the incipient metabolic syndrome’, including ‘incipient diabetes’. PMID:26844282

A novel approach to maintain gut mucosal integrity using an oral enzyme supplement.

PubMed

Hamarneh, Sulaiman R; Mohamed, Mussa M Rafat; Economopoulos, Konstantinos P; Morrison, Sara A; Phupitakphol, Tanit; Tantillo, Tyler J; Gul, Sarah S; Gharedaghi, Mohammad Hadi; Tao, Qingsong; Kaliannan, Kanakaraju; Narisawa, Sonoko; Millán, José L; van der Wilden, Gwendolyn M; Fagenholz, Peter J; Malo, Madhu S; Hodin, Richard A

2014-10-01

To determine the role of intestinal alkaline phosphatase (IAP) in enteral starvation-induced gut barrier dysfunction and to study its therapeutic effect as a supplement to prevent gut-derived sepsis. Critically ill patients are at increased risk for systemic sepsis and, in some cases, multiorgan failure leading to death. Years ago, the gut was identified as a major source for this systemic sepsis syndrome. Previously, we have shown that IAP detoxifies bacterial toxins, prevents endotoxemia, and preserves intestinal microbiotal homeostasis. WT and IAP-KO mice were used to examine gut barrier function and tight junction protein levels during 48-hour starvation and fed states. Human ileal fluid samples were collected from 20 patients postileostomy and IAP levels were compared between fasted and fed states. To study the effect of IAP supplementation on starvation-induced gut barrier dysfunction, WT mice were fasted for 48 hours +/- IAP supplementation in the drinking water. The loss of IAP expression is associated with decreased expression of intestinal junctional proteins and impaired barrier function. For the first time, we demonstrate that IAP expression is also decreased in humans who are deprived of enteral feeding. Finally, our data demonstrate that IAP supplementation reverses the gut barrier dysfunction and tight junction protein losses due to a lack of enteral feeding. IAP is a major regulator of gut mucosal permeability and is able to ameliorate starvation-induced gut barrier dysfunction. Enteral IAP supplementation may represent a novel approach to maintain bowel integrity in critically ill patients.

Intestinal alkaline phosphatase deficiency leads to dysbiosis and bacterial translocation in the newborn intestine.

PubMed

Fawley, Jason; Koehler, Shannon; Cabrera, Susan; Lam, Vy; Fredrich, Katherine; Hessner, Martin; Salzman, Nita; Gourlay, David

2017-10-01

Intestinal alkaline phosphatase (IAP) has been shown to help maintain intestinal homeostasis. Decreased expression of IAP has been linked with pediatric intestinal diseases associated with bacterial overgrowth and subsequent inflammation. We hypothesize that the absence of IAP leads to dysbiosis, with increased inflammation and permeability of the newborn intestine. Sprague-Dawley heterozygote IAP cross-matches were bred. Pups were dam fed ad lib and euthanized at weaning. The microbiotas of terminal ileum (TI) and colon was determined by quantitative real-time polymerase chain reaction (qRT-PCR) of subphylum-specific bacterial 16S ribosomal RNA. RT-PCR was performed on TI for inflammatory cytokines. Intestinal permeability was quantified by fluorescein isothiocyanate-dextran permeability and bacterial translocation by qRT-PCR for bacterial 16S ribosomal RNA in mesenteric lymph nodes. Statistical analysis was done by chi-square analysis. All three genotypes had similar concentrations of bacteria in the TI and colon. However, IAP knockout (IAP-KO) had significantly decreased diversity of bacterial species in their colonic stool compared with heterozygous and wild-type (WT). IAP-KO pups had a nonstatistically significant 3.9-fold increased inducible nitric oxide synthase messenger RNA expression compared with WT (IAP-KO, 3.92 ± 1.36; WT, 1.0 ± 0.27; P = 0.03). IAP-KO also had significantly increased bacterial translocation to mesenteric lymph nodes occurred in IAP-KO (IAP-KO, 7625 RFU/g ± 3469; WT, 4957 RFU/g ± 1552; P = 0.04). Furthermore, IAP-KO had increased permeability (IAP-KO, 0.297 mg/mL ± 0.2; WT, 0.189 mg/mL ± 0.15 P = 0.07), but was not statistically significant. Deficiency of IAP in the newborn intestine is associated with dysbiosis and increased inflammation, permeability, and bacterial translocation. Copyright © 2017 Elsevier Inc. All rights reserved.

Self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats.

PubMed

Li, Wenchao; Jin, Hongyan; Sui, Xiukun; Zhao, Zhanzhong; Yang, Chenghuai; Wang, Wenquan; Li, Junping; Li, Gang

2014-01-01

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA) vaccine candidates for the surveillance and eradication of PPR.

Repeat Radiosurgery for Trigeminal Neuralgia.

PubMed

Helis, Corbin A; Lucas, John T; Bourland, J Daniel; Chan, Michael D; Tatter, Stephen B; Laxton, Adrian W

2015-11-01

Repeat Gamma Knife radiosurgery (GKRS) is an established option for patients whose pain has recurred after the initial procedure, with reported success rates varying from 68% to 95%. Predictive factors for response to the repeat GKRS are ill-defined. This cohort study aimed to report the outcomes and factors predictive of success for patients who have undergone repeated GKRS for trigeminal neuralgia at Wake Forest University Baptist Medical Center. Between 1999 and 2013, 152 patients underwent repeat GKRS at Wake Forest, 125 of whom were available for long-term follow-up. A retrospective chart review and telephone interviews were conducted to determine background medical history, dosimetric data, outcomes, and adverse effects of the procedure. Eighty-four percent of patients achieved at least Barrow Neurological Institute (BNI) IIIb pain relief, with 46% achieving BNI I. The 1-, 3-, and 5-year rates of BNI I pain relief were 63%, 50%, and 37%, respectively. The 1-, 3-, and 5-year rates of BNI IIIb or better pain relief were 74%, 59%, and 46%, respectively. One patient experienced bothersome numbness and 2 patients developed anesthesia dolorosa. The dominant predictive factors for pain relief were facial numbness after the first GKRS and a positive pain response to the first GKRS. Repeat GKRS is an effective method of treating recurrent trigeminal neuralgia. Patients who have facial numbness after the first treatment and a positive pain response to the first GKRS are significantly more likely to respond well to the second treatment.

Repeat surfactant therapy for postsurfactant slump.

PubMed

Katz, L A; Klein, J M

2006-07-01

To evaluate repeat surfactant therapy for the treatment of respiratory failure associated with postsurfactant slump in extremely low birth weight infants (ELBW) by characterizing the population of premature infants who develop postsurfactant slump and measuring their response to a secondary course of surfactant therapy. A retrospective analysis of a cohort of all patients admitted over a 3-year period with birth weights <1000 g (ELBW infants). Information was collected by chart review and the patients were categorized into three distinct groups for analysis. Initial surfactant only, patients who received surfactant replacement therapy only for respiratory distress syndrome (RDS); repeat surfactant, patients who received both initial surfactant replacement for RDS and repeat surfactant therapy for postsurfactant slump (defined as respiratory failure after 6 days of age), and no surfactant, patients in whom no surfactant was ever administered. A respiratory severity score (RSS) was used to measure the severity of lung disease and response to surfactant therapy. Over 3 years, there were 165 ELBW infants who could develop postsurfactant slump and be eligible for repeat surfactant therapy. There were 39 infants who never received any surfactant therapy estimated gestational age (EGA) 27.7 +/- 1.7, birth weight 856 +/- 109 g) either at birth or after 6 days of life. There were 126 patients treated for RDS with initial surfactant replacement therapy (EGA 25.6 +/- 1.9 weeks, birth weight 713 +/- 179 g). Out of these RDS patients, 101 improved with an initial course of surfactant therapy (EGA 26 +/- 1.8, birth weight 751 +/- 143 g), but 25 (20% of the patients with RDS) developed postsurfactant slump and received a repeat course of surfactant therapy (EGA 24.7 +/- 1.2, birth weight 647 +/- 120 g). The repeat surfactant group (postsurfactant slump) was significantly more premature and had significantly lower birth weights compared to both the initial surfactant only group

Structural studies of CNG repeats.

PubMed

Kiliszek, Agnieszka; Rypniewski, Wojciech

2014-07-01

CNG repeats (where N denotes one of the four natural nucleotides) are abundant in the human genome. Their tendency to undergo expansion can lead to hereditary diseases known as TREDs (trinucleotide repeat expansion disorders). The toxic factor can be protein, if the abnormal gene is expressed, or the gene transcript, or both. The gene transcripts have attracted much attention in the biomedical community, but their molecular structures have only recently been investigated. Model RNA molecules comprising CNG repeats fold into long hairpins whose stems generally conform to an A-type helix, in which the non-canonical N-N pairs are flanked by C-G and G-C pairs. Each homobasic pair is accommodated in the helical context in a unique manner, with consequences for the local helical parameters, solvent structure, electrostatic potential and potential to interact with ligands. The detailed three-dimensional profiles of RNA CNG repeats can be used in screening of compound libraries for potential therapeutics and in structure-based drug design. Here is a brief survey of the CNG structures published to date. © Published by Oxford University Press on behalf of Nucleic Acids Research.

Some characteristics of repeated sickness absence

PubMed Central

Ferguson, David

1972-01-01

Ferguson, D. (1972).Brit. J. industr. Med.,29, 420-431. Some characteristics of repeated sickness absence. Several studies have shown that frequency of absence attributed to sickness is not distributed randomly but tends to follow the negative binomial distribution, and this has been taken to support the concept of `proneness' to such absence. Thus, the distribution of sickness absence resembles that of minor injury at work demonstrated over 50 years ago. Because the investigation of proneness to absence does not appear to have been reported by others in Australia, the opportunity was taken, during a wider study of health among telegraphists in a large communications undertaking, to analyse some characteristics of repeated sickness absence. The records of medically certified and uncertified sickness absence of all 769 telegraphists continuously employed in all State capitals over a two-and-a-half-year period were compared with those of 411 clerks and 415 mechanics and, in Sydney, 380 mail sorters and 80 of their supervisors. All telegraphists in Sydney, Melbourne, and Brisbane, and all mail sorters in Sydney, who were available and willing were later medically examined. From their absence pattern repeaters (employees who had had eight or more certified absences in two and a half years) were separated into three types based on a presumptive origin in chance, recurrent disease and symptomatic non-specific disorder. The observed distribution of individual frequency of certified absence over the full two-and-a-half-year period of study followed that expected from the univariate negative binomial, using maximum likelihood estimators, rather than the poisson distribution, in three of the four occupational groups in Sydney. Limited correlational and bivariate analysis supported the interpretation of proneness ascribed to the univariate fit. In the two groups studied, frequency of uncertified absence could not be fitted by the negative binomial, although the numbers of

Deletion of internal structured repeats increases the stability of a leucine-rich repeat protein, YopM

PubMed Central

Barrick, Doug

2011-01-01

Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins. PMID:21764506

Intestinal Alkaline Phosphatase Regulates Tight Junction Protein Levels

PubMed Central

Liu, Wei; Hu, Dong; Huo, Haizhong; Zhang, Weifeng; Adiliaghdam, Fatemeh; Morrison, Sarah; Ramirez, Juan M; Gul, Sarah S; Hamarneh, Sulaiman R; Hodin, Richard A

2017-01-01

BACKGROUND Intestinal alkaline phosphatase (IAP) plays a pivotal role in maintaining gut health and well-being. Oral supplementation with IAP in mice improves gut barrier function and prevents luminal proinflammatory factors from gaining access to the circulation. In this study, we sought to explore the relationship between IAP and tight junction protein (TJP) expression and function. STUDY DESIGN The effect of IAP deletion on TJP levels was studied in mouse embryonic fibroblasts (MEFs) generated from IAP-knockout and wild type mice. Regulation of TJPs by IAP was assayed in the human colon cancer Caco-2 and T84 cells by overexpressing the human IAP gene. Tight junction protein levels and localization were measured by using RT q-PCR and antibodies targeting the specific TJPs. Finally, the effect of IAP on inflammation-induced intestinal permeability was measured by in vitro trans-well epithelial electrical resistance (TEER). RESULTS Intestinal alkaline phosphatase gene deletion in MEFs resulted in significantly lower levels of ZO-1, ZO-2, and Occludin compared with levels in wild-type control cells; IAP over-expression in Caco-2 and T84 cells resulted in approximate 2-fold increases in the mRNA levels of ZO-1 and ZO-2. The IAP treatment ameliorated lipopolysaccharide-induced increased permeability in the Caco-2 trans-well system. Furthermore, IAP treatment preserved the localization of the ZO-1 and Occludin proteins during inflammation and was also associated with improved epithelial barrier function. CONCLUSIONS Intestinal alkaline phosphatase is a major regulator of gut mucosal permeability and appears to work at least partly through improving TJP levels and localization. These data provide a strong foundation to develop IAP as a novel therapy to maintain gut barrier function. PMID:27106638

Intestinal Alkaline Phosphatase Regulates Tight Junction Protein Levels.

PubMed

Liu, Wei; Hu, Dong; Huo, Haizhong; Zhang, Weifeng; Adiliaghdam, Fatemeh; Morrison, Sarah; Ramirez, Juan M; Gul, Sarah S; Hamarneh, Sulaiman R; Hodin, Richard A

2016-06-01

Intestinal alkaline phosphatase (IAP) plays a pivotal role in maintaining gut health and well-being. Oral supplementation with IAP in mice improves gut barrier function and prevents luminal proinflammatory factors from gaining access to the circulation. In this study, we sought to explore the relationship between IAP and tight junction protein (TJP) expression and function. The effect of IAP deletion on TJP levels was studied in mouse embryonic fibroblasts (MEFs) generated from IAP-knockout and wild type mice. Regulation of TJPs by IAP was assayed in the human colon cancer Caco-2 and T84 cells by overexpressing the human IAP gene. Tight junction protein levels and localization were measured by using RT q-PCR and antibodies targeting the specific TJPs. Finally, the effect of IAP on inflammation-induced intestinal permeability was measured by in vitro trans-well epithelial electrical resistance (TEER). Intestinal alkaline phosphatase gene deletion in MEFs resulted in significantly lower levels of ZO-1, ZO-2, and Occludin compared with levels in wild-type control cells; IAP overexpression in Caco-2 and T84 cells resulted in approximate 2-fold increases in the mRNA levels of ZO-1 and ZO-2. The IAP treatment ameliorated lipopolysaccharide-induced increased permeability in the Caco-2 trans-well system. Furthermore, IAP treatment preserved the localization of the ZO-1 and Occludin proteins during inflammation and was also associated with improved epithelial barrier function. Intestinal alkaline phosphatase is a major regulator of gut mucosal permeability and appears to work at least partly through improving TJP levels and localization. These data provide a strong foundation to develop IAP as a novel therapy to maintain gut barrier function. Copyright © 2016. Published by Elsevier Inc.

Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

PubMed

Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

2016-06-01

Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

PubMed

Levine, Min Z; Lewis, Melissa M; Rodriquez, Silvia; Jimenez, Juan A; Khan, Azra; Lin, Sehching; Garcia, Hector H; Gonzales, Armando E; Gilman, Robert H; Tsang, Victor C W

2007-04-01

Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

Digital repeat analysis; setup and operation.

PubMed

Nol, J; Isouard, G; Mirecki, J

2006-06-01

Since the emergence of digital imaging, there have been questions about the necessity of continuing reject analysis programs in imaging departments to evaluate performance and quality. As a marketing strategy, most suppliers of digital technology focus on the supremacy of the technology and its ability to reduce the number of repeats, resulting in less radiation doses given to patients and increased productivity in the department. On the other hand, quality assurance radiographers and radiologists believe that repeats are mainly related to positioning skills, and repeat analysis is the main tool to plan training needs to up-skill radiographers. A comparative study between conventional and digital imaging was undertaken to compare outcomes and evaluate the need for reject analysis. However, digital technology still being at its early development stages, setting a credible reject analysis program became the major task of the study. It took the department, with the help of the suppliers of the computed radiography reader and the picture archiving and communication system, over 2 years of software enhancement to build a reliable digital repeat analysis system. The results were supportive of both philosophies; the number of repeats as a result of exposure factors was reduced dramatically; however, the percentage of repeats as a result of positioning skills was slightly on the increase for the simple reason that some rejects in the conventional system qualifying for both exposure and positioning errors were classified as exposure error. The ability of digitally adjusting dark or light images reclassified some of those images as positioning errors.

High-speed and low-power repeater for VLSI interconnects

NASA Astrophysics Data System (ADS)

Karthikeyan, A.; Mallick, P. S.

2017-10-01

This paper proposes a repeater for boosting the speed of interconnects with low power dissipation. We have designed and implemented at 45 and 32 nm technology nodes. Delay and power dissipation performances are analyzed for various voltage levels at these technology nodes using Spice simulations. A significant reduction in delay and power dissipation are observed compared to a conventional repeater. The results show that the proposed high-speed low-power repeater has a reduced delay for higher load capacitance. The proposed repeater is also compared with LPTG CMOS repeater, and the results shows that the proposed repeater has reduced delay. The proposed repeater can be suitable for high-speed global interconnects and has the capacity to drive large loads.

The Role of Intestinal Alkaline Phosphatase in Inflammatory Disorders of Gastrointestinal Tract.

PubMed

Bilski, Jan; Mazur-Bialy, Agnieszka; Wojcik, Dagmara; Zahradnik-Bilska, Janina; Brzozowski, Bartosz; Magierowski, Marcin; Mach, Tomasz; Magierowska, Katarzyna; Brzozowski, Tomasz

2017-01-01

Over the past few years, the role of intestinal alkaline phosphatase (IAP) as a crucial mucosal defence factor essential for maintaining gut homeostasis has been established. IAP is an important apical brush border enzyme expressed throughout the gastrointestinal tract and secreted both into the intestinal lumen and into the bloodstream. IAP exerts its effects through dephosphorylation of proinflammatory molecules including lipopolysaccharide (LPS), flagellin, and adenosine triphosphate (ATP) released from cells during stressful events. Diminished activity of IAP could increase the risk of disease through changes in the microbiome, intestinal inflammation, and intestinal permeability. Exogenous IAP exerts a protective effect against intestinal and systemic inflammation in a variety of diseases and represents a potential therapeutic agent in diseases driven by gut barrier dysfunction such as IBD. The intestinal protective mechanisms are impaired in IBD patients due to lower synthesis and activity of endogenous IAP, but the pathomechanism of this enzyme deficiency remains unclear. IAP has been safely administered to humans and the human recombinant form of IAP has been developed. This review was designed to provide an update in recent research on the involvement of IAP in intestinal inflammatory processes with focus on IBD in experimental animal models and human patients.

The Role of Intestinal Alkaline Phosphatase in Inflammatory Disorders of Gastrointestinal Tract

PubMed Central

Wojcik, Dagmara; Zahradnik-Bilska, Janina; Mach, Tomasz

2017-01-01

Over the past few years, the role of intestinal alkaline phosphatase (IAP) as a crucial mucosal defence factor essential for maintaining gut homeostasis has been established. IAP is an important apical brush border enzyme expressed throughout the gastrointestinal tract and secreted both into the intestinal lumen and into the bloodstream. IAP exerts its effects through dephosphorylation of proinflammatory molecules including lipopolysaccharide (LPS), flagellin, and adenosine triphosphate (ATP) released from cells during stressful events. Diminished activity of IAP could increase the risk of disease through changes in the microbiome, intestinal inflammation, and intestinal permeability. Exogenous IAP exerts a protective effect against intestinal and systemic inflammation in a variety of diseases and represents a potential therapeutic agent in diseases driven by gut barrier dysfunction such as IBD. The intestinal protective mechanisms are impaired in IBD patients due to lower synthesis and activity of endogenous IAP, but the pathomechanism of this enzyme deficiency remains unclear. IAP has been safely administered to humans and the human recombinant form of IAP has been developed. This review was designed to provide an update in recent research on the involvement of IAP in intestinal inflammatory processes with focus on IBD in experimental animal models and human patients. PMID:28316376

Intestinal alkaline phosphatase in the colonic mucosa of children with inflammatory bowel disease

PubMed Central

Molnár, Kriszta; Vannay, Ádám; Szebeni, Beáta; Bánki, Nóra Fanni; Sziksz, Erna; Cseh, Áron; Győrffy, Hajnalka; Lakatos, Péter László; Papp, Mária; Arató, András; Veres, Gábor

2012-01-01

AIM: To investigate intestinal alkaline phosphatase (iAP) in the intestinal mucosa of children with inflammatory bowel disease (IBD). METHODS: Colonic biopsy samples were taken from 15 newly diagnosed IBD patients and from 10 healthy controls. In IBD patients, specimens were obtained both from inflamed and non-inflamed areas. The iAP mRNA and protein expression was determined by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Tissue localization of iAP and Toll-like receptor (TLR) 4 was investigated by immunofluorescent staining. RESULTS: The iAP protein level in the inflamed mucosa of children with Crohn’s disease (CD) and ulcerative colitis (UC) was significantly decreased when compared with controls (both P < 0.05). Similarly, we found a significantly decreased level of iAP protein in the inflamed mucosa in CD compared with non-inflamed mucosa in CD (P < 0.05). In addition, the iAP protein level in inflamed colonic mucosa in patients with UC was decreased compared with non-inflamed mucosa in patients with CD (P < 0.05). iAP protein levels in the non-inflamed mucosa of patients with CD were similar to controls. iAP mRNA expression in inflamed colonic mucosa of children with CD and UC was not significantly different from that in non-inflamed colonic mucosa with CD. Expression of iAP mRNA in patients with non-inflamed mucosa and in controls were similar. Co-localization of iAP with TLR4 showed intense staining with a dotted-like pattern. iAP was present in the inflamed and non-inflamed mucosa of patients with CD, UC, and in control biopsy specimens, irrespective of whether it was present in the terminal ileum or in the colon. However, the fluorescent signal of TLR4 was more pronounced in the colon compared with the terminal ileum in all groups studied. CONCLUSION: Lower than normal iAP protein levels in inflamed mucosa of IBD patients may indicate a role for iAP in inflammatory lesions in IBD. Based on our results

Intraabdominal pressure in women during CrossFit exercises and the effect of age and parity.

PubMed

Gephart, Laura Faye; Doersch, Karen M; Reyes, Michelle; Kuehl, Thomas J; Danford, Jill M

2018-07-01

To determine intraabdominal pressure (IAP) in women during CrossFit and to determine whether parity, age, or CrossFit experience affects IAP during CrossFit exercises, we evaluated 10 women: 5 experienced and active CrossFitters and 5 who were not regularly engaged in CrossFit. A Laborie urodynamics abdominal pressure probe with the Goby wireless system measured IAP during 10 repetitions of 13 different CrossFit exercises. Women had a mean age of 36 years. A significant difference was found between mean peak IAP of the 5 parous vs the 5 nulliparous women ( P = 0.009). Experience with CrossFit did not affect mean peak IAP achieved with exercise. In some exercises, there was a significant change in IAP as participants progressed through repetitions ( P = 0.003 for back squats and 0.04 for sit-ups). Participants achieved IAP values that were markedly higher than those previously published.

Angiopoietin-1-expressing adipose stem cells genetically modified with baculovirus nanocomplex: investigation in rat heart with acute infarction.

PubMed

Paul, Arghya; Nayan, Madhur; Khan, Afshan Afsar; Shum-Tim, Dominique; Prakash, Satya

2012-01-01

The objective of this study was to develop angiopoietin-1 (Ang1)-expressing genetically modified human adipose tissue derived stem cells (hASCs) for myocardial therapy. For this, an efficient gene delivery system using recombinant baculovirus complexed with cell penetrating transactivating transcriptional activator TAT peptide/deoxyribonucleic acid nanoparticles (Bac-NP), through ionic interactions, was used. It was hypothesized that the hybrid Bac- NP(Ang1) system can efficiently transduce hASCs and induces favorable therapeutic effects when transplanted in vivo. To evaluate this hypothesis, a rat model with acute myocardial infarction and intramyocardially transplanted Ang1-expressing hASCs (hASC-Ang1), genetically modified by Bac-NP(Ang1), was used. Ang1 is a crucial pro-angiogenic factor for vascular maturation and neovasculogenesis. The released hAng1 from hASC-Ang1 demonstrated profound mitotic and anti-apoptotic activities on endothelial cells and cardiomyocytes. The transplanted hASC-Ang1 group showed higher cell retention compared to hASC and control groups. A significant increase in capillary density and reduction in infarct sizes were noted in the infarcted hearts with hASC-Ang1 treatment compared to infarcted hearts treated with hASC or the untreated group. Furthermore, the hASC-Ang1 group showed significantly higher cardiac performance in echocardiography (ejection fraction 46.28% ± 6.3%, P < 0.001 versus control, n = 8) than the hASC group (36.35% ± 5.7%, P < 0.01, n = 8), 28 days post-infarction. The study identified Bac-NP complex as an advanced gene delivery vehicle for stem cells and demonstrated its potential to treat ischemic heart disease with high therapeutic index for combined stem cell-gene therapy strategy.

The respiratory pressure-abdominal volume curve in a porcine model.

PubMed

Regli, Adrian; De Keulenaer, Bart Leon; Singh, Bhajan; Hockings, Lisen Emma; Noffsinger, Bill; van Heerden, Peter Vernon

2017-12-01

Increasing intra-abdominal volume (IAV) can lead to intra-abdominal hypertension (IAH) or abdominal compartment syndrome. Both are associated with raised morbidity and mortality. IAH can increase airway pressures and impair ventilation. The relationship between increasing IAV and airway pressures is not known. We therefore assessed the effect of increasing IAV on airway and intra-abdominal pressures (IAP). Seven pigs (41.4 +/-8.5 kg) received standardized anesthesia and mechanical ventilation. A latex balloon inserted in the peritoneal cavity was inflated in 1-L increments until IAP exceeded 40 cmH 2 O. Peak airway pressure (pP AW ), respiratory compliance, and IAP (bladder pressure) were measured. Abdominal compliance was calculated. Different equations were tested that best described the measured pressure-volume curves. An exponential equation best described the measured pressure-volume curves. Raising IAV increased pP AW and IAP in an exponential manner. Increases in IAP were associated with parallel increases in pP AW with an approximate 40% transmission of IAP to pP AW . The higher the IAP, the greater IAV effected pP AW and IAP. The exponential nature of the effect of IAV on pP AW and IAP implies that, in the presence of high grades of IAH, small reductions in IAV can lead to significant reductions in airway and abdominal pressures. Conversely, in the presence of normal IAP levels, large increases in IAV may not affect airway and abdominal pressures.

Intestinal Alkaline Phosphatase: Potential Roles in Promoting Gut Health in Weanling Piglets and Its Modulation by Feed Additives - A Review.

PubMed

Melo, A D B; Silveira, H; Luciano, F B; Andrade, C; Costa, L B; Rostagno, M H

2016-01-01

The intestinal environment plays a critical role in maintaining swine health. Many factors such as diet, microbiota, and host intestinal immune response influence the intestinal environment. Intestinal alkaline phosphatase (IAP) is an important apical brush border enzyme that is influenced by these factors. IAP dephosphorylates bacterial lipopolysaccharides (LPS), unmethylated cytosine-guanosine dinucleotides, and flagellin, reducing bacterial toxicity and consequently regulating toll-like receptors (TLRs) activation and inflammation. It also desphosphorylates extracellular nucleotides such as uridine diphosphate and adenosine triphosphate, consequently reducing inflammation, modulating, and preserving the homeostasis of the intestinal microbiota. The apical localization of IAP on the epithelial surface reveals its role on LPS (from luminal bacteria) detoxification. As the expression of IAP is reported to be downregulated in piglets at weaning, LPS from commensal and pathogenic gram-negative bacteria could increase inflammatory processes by TLR-4 activation, increasing diarrhea events during this phase. Although some studies had reported potential IAP roles to promote gut health, investigations about exogenous IAP effects or feed additives modulating IAP expression and activity yet are necessary. However, we discussed in this paper that the critical assessment reported can suggest that exogenous IAP or feed additives that could increase its expression could show beneficial effects to reduce diarrhea events during the post weaning phase. Therefore, the main goals of this review are to discuss IAP's role in intestinal inflammatory processes and present feed additives used as growth promoters that may modulate IAP expression and activity to promote gut health in piglets.

Coexistence of 3G repeaters with LTE base stations.

PubMed

Yeo, Woon-Young; Lee, Sang-Min; Hwang, Gyung-Ho; Kim, Jae-Hoon

2013-01-01

Repeaters have been an attractive solution for mobile operators to upgrade their wireless networks at low cost and to extend network coverage effectively. Since the first LTE commercial deployment in 2009, many mobile operators have launched LTE networks by upgrading their 3G and legacy networks. Because all 3G frequency bands are shared with the frequency bands for LTE deployment and 3G mobile operators have an enormous number of repeaters, reusing 3G repeaters in LTE networks is definitely a practical and cost-efficient solution. However, 3G repeaters usually do not support spatial multiplexing with multiple antennas, and thus it is difficult to reuse them directly in LTE networks. In order to support spatial multiplexing of LTE, the role of 3G repeaters should be replaced with small LTE base stations or MIMO-capable repeaters. In this paper, a repeater network is proposed to reuse 3G repeaters in LTE deployment while still supporting multilayer transmission of LTE. Interestingly, the proposed network has a higher cluster throughput than an LTE network with MIMO-capable repeaters.

Coexistence of 3G Repeaters with LTE Base Stations

PubMed Central

Yeo, Woon-Young

2013-01-01

Repeaters have been an attractive solution for mobile operators to upgrade their wireless networks at low cost and to extend network coverage effectively. Since the first LTE commercial deployment in 2009, many mobile operators have launched LTE networks by upgrading their 3G and legacy networks. Because all 3G frequency bands are shared with the frequency bands for LTE deployment and 3G mobile operators have an enormous number of repeaters, reusing 3G repeaters in LTE networks is definitely a practical and cost-efficient solution. However, 3G repeaters usually do not support spatial multiplexing with multiple antennas, and thus it is difficult to reuse them directly in LTE networks. In order to support spatial multiplexing of LTE, the role of 3G repeaters should be replaced with small LTE base stations or MIMO-capable repeaters. In this paper, a repeater network is proposed to reuse 3G repeaters in LTE deployment while still supporting multilayer transmission of LTE. Interestingly, the proposed network has a higher cluster throughput than an LTE network with MIMO-capable repeaters. PMID:24459420

FRB 121102: A Starquake-induced Repeater?

NASA Astrophysics Data System (ADS)

Wang, Weiyang; Luo, Rui; Yue, Han; Chen, Xuelei; Lee, Kejia; Xu, Renxin

2018-01-01

Since its initial discovery, the fast radio burst (FRB) FRB 121102 has been found to be repeating with millisecond-duration pulses. Very recently, 14 new bursts were detected by the Green Bank Telescope during its continuous monitoring observations. In this paper, we show that the burst energy distribution has a power-law form which is very similar to the Gutenberg–Richter law of earthquakes. In addition, the distribution of burst waiting time can be described as a Poissonian or Gaussian distribution, which is consistent with earthquakes, while the aftershock sequence exhibits some local correlations. These findings suggest that the repeating FRB pulses may originate from the starquakes of a pulsar. Noting that the soft gamma-ray repeaters (SGRs) also exhibit such distributions, the FRB could be powered by some starquake mechanisms associated with the SGRs, including the crustal activity of a magnetar or solidification-induced stress of a newborn strangeon star. These conjectures could be tested with more repeating samples.

Effect of abdominal negative-pressure wound therapy on the measurement of intra-abdominal pressure.

PubMed

García, Alberto Federico; Sánchez, Álvaro Ignacio; Gutiérrez, Álvaro José; Bayona, Juan Gabriel; Naranjo, María Paula; Lago, Sebastián; Puyana, Juan Carlos

2018-07-01

In critically ill surgical patients undergoing abdominal negative-pressure wound therapy (NPWT), it remains uncertain whether or not intra-abdominal pressure (IAP) measurements should be obtained when NPWT is activated. We aimed to determine agreement between IAP measured with and without NPWT. In this analytic cross-sectional study, critically ill surgical adults (≥18 y) requiring abdominal NPWT for temporary abdominal closure after a damage control laparotomy were selected. Patients with urinary tract injuries or with pelvic packing were excluded. Paired IAP measures were performed in the same patient, with and without NPWT; two different operators performed the measures unaware of the other's result. Bland-Altman methods assessed the agreement between the two measures. Subgroup analyses (trauma and nontrauma) were performed. There were 198 IAP measures (99 pairs) in 38 patients. Mean IAP with and without NPWT were 8.33 (standard deviation 4.01) and 8.65 (standard deviation 4.04), respectively. Mean IAP difference was -0.323 (95% confidence interval -0.748 to 0.101), and reference range for difference was -4.579 to 3.932 (P = 0.864). From 112 IAP measures (56 pairs) in 21 trauma patients, mean IAP difference was -0.268 (95% confidence interval -0.867 to 0.331), and reference range for the difference was -4.740 to 4.204 (P = 0.427). There was no statistically significant disagreement in IAP measures. IAP could be measured with or without NPWT. In critically ill surgical patients with abdominal NPWT for temporary abdominal closure, monitoring and management of IAP either with or without NPWT is recommended. Copyright © 2018 Elsevier Inc. All rights reserved.

Chlamydia trachomatis can protect host cells against apoptosis in the absence of cellular Inhibitor of Apoptosis Proteins and Mcl-1.

PubMed

Ying, Songmin; Christian, Jan G; Paschen, Stefan A; Häcker, Georg

2008-01-01

Infection with Chlamydia protects mammalian host cells against apoptosis. Hypotheses have been proposed to explain this molecularly, including the up-regulation of host anti-apoptotic proteins such as cellular Inhibitor of Apoptosis Protein (IAP) 2 and the Bcl-2 protein Mcl-1. To test for the importance of these proteins, we used mouse embryonic fibroblasts from gene-targeted mice that were deficient in cIAP1, cIAP2, cIAP1/cIAP2, XIAP, or Mcl-1. Infection with Chlamydia trachomatis protected all cells equally well against apoptosis, which was induced either with tumour necrosis factor/cycloheximide (IAP-knock-out cells) or staurosporine (Mcl-1-knock-out). Therefore, these cellular anti-apoptotic proteins are not essential for apoptosis-protection by C. trachomatis.

Do gamma-ray burst sources repeat?

NASA Technical Reports Server (NTRS)

Meegan, C. A.; Hartmann, D. H.; Brainerd, J. J.; Briggs, M.; Paciesas, W. S.; Pendleton, G.; Kouveliotou, C.; Fishman, G.; Blumenthal, G.; Brock, M.

1994-01-01

The demonstration of repeated gamma-ray bursts from an individual source would severely constrain burst source models. Recent reports of evidence for repetition in the first BATSE burst catalog have generated renewed interest in this issue. Here, we analyze the angular distribution of 585 bursts of the second BATSE catalog (Meegan et al. 1994). We search for evidence of burst recurrence using the nearest and farthest neighbor statistic ad the two-point angular correlation function. We find the data to be consistent with the hypothesis that burst sources do not repeat; however, a repeater fraction of up to about 20% of the bursts cannot be excluded.

Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

PubMed

López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

2009-09-01

A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.

Development of antiviral gene therapy for Monodon baculovirus using dsRNA loaded chitosan-dextran sulfate nanocapsule delivery system in Penaeus monodon post-larvae.

PubMed

Ramesh Kumar, D; Elumalai, Rajasegaran; Raichur, Ashok M; Sanjuktha, M; Rajan, J J; Alavandi, S V; Vijayan, K K; Poornima, M; Santiago, T C

2016-07-01

In the present study, a suitable carrier system was developed for the delivery of dsRNA into Penaeus monodon (P. monodon) post larvae to silence the Monodon baculovirus (MBV) structural gene of p74. The carrier system was developed by layer by layer adsorption of oppositely charged chitosan-dextran sulfate, on charged silica nanoparticles. The silica template was removedto produce multilayered hollow nanocapsules (CS-DS) that were utilized for dsRNA loading at an alkaline pH. The capsule's surface was modified by conjugating with shrimp feed for enhanced cellular uptake. In vivo cellular uptake of CS-DS/FITC loaded nanocapsules conjugated with feed was studied after oral administration into post-larvae. The results revealed that the encapsulated FITC was effectively delivered and exhibited a sustained release into the cytoplasm of shrimp post-larvae. The MBV challenge study for structural gene p74was conducted after 3-25 days of post infection (dpi) with respective CS-DS/dsRNA coated with feed. The results showed a significant survival rate of 86.63% and effective gene silencing in P. monodon. Our findings indicated that the delivery of dsRNA using shrimp feed coatedCS-DSnanocapsules could be a novel approach to prevent viral infections in shrimp. Copyright © 2016 Elsevier B.V. All rights reserved.

Crimean-Congo Hemorrhagic Fever Virus Gn Bioinformatic Analysis and Construction of a Recombinant Bacmid in Order to Express Gn by Baculovirus Expression System

PubMed Central

Rahpeyma, Mehdi; Fotouhi, Fatemeh; Makvandi, Manouchehr; Ghadiri, Ata; Samarbaf-Zadeh, Alireza

2015-01-01

Background Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the nairovirus, a genus in the Bunyaviridae family, which causes a life threatening disease in human. Currently, there is no vaccine against CCHFV and detailed structural analysis of CCHFV proteins remains undefined. The CCHFV M RNA segment encodes two viral surface glycoproteins known as Gn and Gc. Viral glycoproteins can be considered as key targets for vaccine development. Objectives The current study aimed to investigate structural bioinformatics of CCHFV Gn protein and design a construct to make a recombinant bacmid to express by baculovirus system. Materials and Methods To express the Gn protein in insect cells that can be used as antigen in animal model vaccine studies. Bioinformatic analysis of CCHFV Gn protein was performed and designed a construct and cloned into pFastBacHTb vector and a recombinant Gn-bacmid was generated by Bac to Bac system. Results Primary, secondary, and 3D structure of CCHFV Gn were obtained and PCR reaction with M13 forward and reverse primers confirmed the generation of recombinant bacmid DNA harboring Gn coding region under polyhedron promoter. Conclusions Characterization of the detailed structure of CCHFV Gn by bioinformatics software provides the basis for development of new experiments and construction of a recombinant bacmid harboring CCHFV Gn, which is valuable for designing a recombinant vaccine against deadly pathogens like CCHFV. PMID:26862379

Crimean-Congo Hemorrhagic Fever Virus Gn Bioinformatic Analysis and Construction of a Recombinant Bacmid in Order to Express Gn by Baculovirus Expression System.

PubMed

Rahpeyma, Mehdi; Fotouhi, Fatemeh; Makvandi, Manouchehr; Ghadiri, Ata; Samarbaf-Zadeh, Alireza

2015-11-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the nairovirus, a genus in the Bunyaviridae family, which causes a life threatening disease in human. Currently, there is no vaccine against CCHFV and detailed structural analysis of CCHFV proteins remains undefined. The CCHFV M RNA segment encodes two viral surface glycoproteins known as Gn and Gc. Viral glycoproteins can be considered as key targets for vaccine development. The current study aimed to investigate structural bioinformatics of CCHFV Gn protein and design a construct to make a recombinant bacmid to express by baculovirus system. To express the Gn protein in insect cells that can be used as antigen in animal model vaccine studies. Bioinformatic analysis of CCHFV Gn protein was performed and designed a construct and cloned into pFastBacHTb vector and a recombinant Gn-bacmid was generated by Bac to Bac system. Primary, secondary, and 3D structure of CCHFV Gn were obtained and PCR reaction with M13 forward and reverse primers confirmed the generation of recombinant bacmid DNA harboring Gn coding region under polyhedron promoter. Characterization of the detailed structure of CCHFV Gn by bioinformatics software provides the basis for development of new experiments and construction of a recombinant bacmid harboring CCHFV Gn, which is valuable for designing a recombinant vaccine against deadly pathogens like CCHFV.

Intestinal alkaline phosphatase: multiple biological roles in maintenance of intestinal homeostasis and modulation by diet.

PubMed

Lallès, Jean-Paul

2010-06-01

The diverse nature of intestinal alkaline phosphatase (IAP) functions has remained elusive, and it is only recently that four additional major functions of IAP have been revealed. The present review analyzes the earlier literature on the dietary factors modulating IAP activity in light of these new findings. IAP regulates lipid absorption across the apical membrane of enterocytes, participates in the regulation of bicarbonate secretion and of duodenal surface pH, limits bacterial transepithelial passage, and finally controls bacterial endotoxin-induced inflammation by dephosphorylation, thus detoxifying intestinal lipopolysaccharide. Many dietary components, including fat, protein, and carbohydrate, modulate IAP expression or activity and may be combined to sustain a high level of IAP activity. In conclusion, IAP has a pivotal role in intestinal homeostasis and its activity could be increased through the diet. This is especially true in pathological situations (e.g., inflammatory bowel diseases) in which the involvement of commensal bacteria is suspected and when intestinal AP is too low to detoxify a sufficient amount of bacterial lipopolysaccharide.

Intraabdominal pressure in women during CrossFit exercises and the effect of age and parity

PubMed Central

Gephart, Laura Faye; Doersch, Karen M.; Reyes, Michelle; Kuehl, Thomas J.; Danford, Jill M.

2018-01-01

ABSTRACT To determine intraabdominal pressure (IAP) in women during CrossFit and to determine whether parity, age, or CrossFit experience affects IAP during CrossFit exercises, we evaluated 10 women: 5 experienced and active CrossFitters and 5 who were not regularly engaged in CrossFit. A Laborie urodynamics abdominal pressure probe with the Goby wireless system measured IAP during 10 repetitions of 13 different CrossFit exercises. Women had a mean age of 36 years. A significant difference was found between mean peak IAP of the 5 parous vs the 5 nulliparous women (P = 0.009). Experience with CrossFit did not affect mean peak IAP achieved with exercise. In some exercises, there was a significant change in IAP as participants progressed through repetitions (P = 0.003 for back squats and 0.04 for sit-ups). Participants achieved IAP values that were markedly higher than those previously published. PMID:29904290

The network impact of hijacking a quantum repeater

NASA Astrophysics Data System (ADS)

Satoh, Takahiko; Nagayama, Shota; Oka, Takafumi; Van Meter, Rodney

2018-07-01

In quantum networking, repeater hijacking menaces the security and utility of quantum applications. To deal with this problem, it is important to take a measure of the impact of quantum repeater hijacking. First, we quantify the work of each quantum repeater with regards to each quantum communication. Based on this, we show the costs for repeater hijacking detection using distributed quantum state tomography and the amount of work loss and rerouting penalties caused by hijacking. This quantitative evaluation covers both purification-entanglement swapping and quantum error correction repeater networks. Naive implementation of the checks necessary for correct network operation can be subverted by a single hijacker to bring down an entire network. Fortunately, the simple fix of randomly assigned testing can prevent such an attack.

Biology and genomics of viruses within the genus Gammabaculovirus.

PubMed

Arif, Basil; Escasa, Shannon; Pavlik, Lillian

2011-11-01

Hymenoptera is a very large and ancient insect order encompassing bees, wasps, ants and sawflies. Fossil records indicate that they existed over 200 million years ago and about 100 million years before the appearance of Lepidoptera. Sawflies have been major pests in many parts of the world and some have caused serious forest defoliation in North America. All baculoviruses isolated from sawflies are of the single nucleocapsids phenotype and appear to replicate in midgut cells only. This group of viruses has been shown to be excellent pest control agents and three have been registered in Canada and Britain for this purpose. Sawfly baculoviruses contain the smallest genome of all baculoviruses sequenced so far. Gene orders among sequenced sawfly baculoviruses are co-linear but this is not shared with the genomes of lepidopteran baculoviruses. One distinguishing feature among all sequenced sawfly viruses is the lack of a gene encoding a membrane fusion protein, which brought into question the role of the budded virus phenotype in Gammabaculovirus biology.

LPA gene: interaction between the apolipoprotein(a) size ('kringle IV' repeat) polymorphism and a pentanucleotide repeat polymorphism influences Lp(a) lipoprotein level.

PubMed

Røsby, O; Berg, K

2000-01-01

In order to search for factors influencing the Lp(a) lipoprotein level, we have examined the apolipoprotein(a) (apo(a)) size polymorphism as well as a pentanucleotide (TTTTA) repeat polymorphism in the 5' control region of the LPA gene. Lp(a) lipoprotein levels were compared between individuals with different genotypes as defined by pulsed field gel electrophoresis of DNA plugs, and PCR of DNA samples followed by polyacrylamide gel electrophoresis. DNA plugs and DNA were prepared from blood samples collected from blood donors. Twenty-seven different K IV repeat alleles were observed in the 71 women and 92 men from which apo(a) size polymorphism results were obtained. Alleles encoding 26-32 Kringle IV repeats were the most frequent. Alleles encoding seven to 11 TTTTA repeats were detected in the 84 women and 122 men included in the pentanucleotide polymorphism study, and homozygosity for eight TTTTA repeats was the most common genotype. The eight TTTTA repeat allele occurred with almost any apo(a) allele. An inverse relationship between number of K IV repeats and Lp(a) concentration was confirmed. The contributions of the apo(a) size polymorphism and the pentanucleotide repeat polymorphism to the interindividual variance of Lp(a) lipoprotein concentrations were 9.7 and 3.5%, respectively (type IV sum of squares). Nineteen per cent of the variance in Lp(a) lipoprotein level appeared to be the result of the multiplication product (interaction) between the apo(a) size polymorphism and the pentanucleotide repeat polymorphism. The contribution of the apo(a) size polymorphism alone to the variation in Lp(a) lipoprotein level was lower than previously reported. However, the multiplicative interaction effect between the K IV repeat polymorphism and the pentanucleotide repeat polymorphism may be an important factor explaining the variation in Lp(a) lipoprotein levels among the populations.

Estimating the duration of intracellular action potentials in muscle fibres from single-fibre extracellular potentials.

PubMed

Rodríguez, Javier; Navallas, Javier; Gila, Luis; Dimitrova, Nonna Alexandrovna; Malanda, Armando

2011-04-30

In situ recording of the intracellular action potential (IAP) of human muscle fibres is not yet possible, and consequently, knowledge concerning certain IAP characteristics is still limited. According to the core-conductor theory, close to a fibre, a single fibre action potential (SFAP) can be assumed to be proportional to the IAP second derivative. Thus, we might expect to be able to derive some characteristics of the IAP, such as the duration of its spike, from the SFAP waveform. However, SFAP properties not only depend on the IAP shape but also on the fibre-to-electrode (radial) distance and other physiological properties of the fibre. In this paper we, first, propose an SFAP parameter (the negative phase duration, NPD) appropriate for estimating the IAP spike duration and, second, show that this parameter is largely independent of changes in radial distance and muscle fibre propagation velocity. Estimation of the IAP spike duration from a direct measurement taken from the SFAP waveform provides a possible way to enhance the accuracy of SFAP models. Because IAP spike duration is known to be sensitive to the effects of fatigue and calcium accumulation, the proposed SFAP parameter, the NPD, has potential value in electrodiagnosis and as an indicator of IAP profile changes due to peripheral fatigue. Copyright © 2011 Elsevier B.V. All rights reserved.

Role of memory errors in quantum repeaters

NASA Astrophysics Data System (ADS)

Hartmann, L.; Kraus, B.; Briegel, H.-J.; Dür, W.

2007-03-01

We investigate the influence of memory errors in the quantum repeater scheme for long-range quantum communication. We show that the communication distance is limited in standard operation mode due to memory errors resulting from unavoidable waiting times for classical signals. We show how to overcome these limitations by (i) improving local memory and (ii) introducing two operational modes of the quantum repeater. In both operational modes, the repeater is run blindly, i.e., without waiting for classical signals to arrive. In the first scheme, entanglement purification protocols based on one-way classical communication are used allowing to communicate over arbitrary distances. However, the error thresholds for noise in local control operations are very stringent. The second scheme makes use of entanglement purification protocols with two-way classical communication and inherits the favorable error thresholds of the repeater run in standard mode. One can increase the possible communication distance by an order of magnitude with reasonable overhead in physical resources. We outline the architecture of a quantum repeater that can possibly ensure intercontinental quantum communication.

Oral antibodies to human intestinal alkaline phosphatase reduce dietary phytate phosphate bioavailability in the presence of dietary 1α-hydroxycholecalciferol.

PubMed

Bobeck, Elizabeth A; Hellestad, Erica M; Helvig, Christian F; Petkovich, P Martin; Cook, Mark E

2016-03-01

While it is well established that active vitamin D treatment increases dietary phytate phosphate utilization, the mechanism by which intestinal alkaline phosphatase (IAP) participates in phytate phosphate use is less clear. The ability of human IAP (hIAP) oral antibodies to prevent dietary phytate phosphate utilization in the presence of 1α-hydroxycholecalciferol (1α-(OH) D3) in a chick model was investigated. hIAP specific chicken immunoglobulin Y (IgY) antibodies were generated by inoculating laying hens with 17 synthetic peptides derived from the human IAP amino acid sequence and harvesting egg yolk. Western blot analysis showed all antibodies recognized hIAP and 6 of the 8 antibodies selected showed modest inhibition of hIAP activity in vitro (6 to 33% inhibition). In chicks where dietary phosphate was primarily in the form of phytate, 4 selected hIAP antibodies inhibited 1α-(OH) D3-induced increases in blood phosphate, one of which, generated against selected peptide (MFPMGTPD), was as effective as sevelamer hydrochloride in preventing the 1α-(OH) D3-induced increase in blood phosphate, but ineffective in preventing an increase in body weight gain and bone ash induced by 1α-(OH) D3. These studies demonstrated that orally-delivered antibodies to IAP limit dietary phytate-phosphate utilization in chicks treated with 1α-(OH) D3, and implicate IAP as an important host enzyme in increasing phytate phosphate bioavailability in 1α-(OH) D3 fed chicks. © 2015 Poultry Science Association Inc.

Areas of high conservation value at risk by plant invaders in Georgia under climate change.

PubMed

Slodowicz, Daniel; Descombes, Patrice; Kikodze, David; Broennimann, Olivier; Müller-Schärer, Heinz

2018-05-01

Invasive alien plants (IAP) are a threat to biodiversity worldwide. Understanding and anticipating invasions allow for more efficient management. In this regard, predicting potential invasion risks by IAPs is essential to support conservation planning into areas of high conservation value (AHCV) such as sites exhibiting exceptional botanical richness, assemblage of rare, and threatened and/or endemic plant species. Here, we identified AHCV in Georgia, a country showing high plant richness, and assessed the susceptibility of these areas to colonization by IAPs under present and future climatic conditions. We used actual protected areas and areas of high plant endemism (identified using occurrences of 114 Georgian endemic plant species) as proxies for AHCV. Then, we assessed present and future potential distribution of 27 IAPs using species distribution models under four climate change scenarios and stacked single-species potential distribution into a consensus map representing IAPs richness. We evaluated present and future invasion risks in AHCV using IAPs richness as a metric of susceptibility. We show that the actual protected areas cover only 9.4% of the areas of high plant endemism in Georgia. IAPs are presently located at lower elevations around the large urban centers and in western Georgia. We predict a shift of IAPs toward eastern Georgia and higher altitudes and an increased susceptibility of AHCV to IAPs under future climate change. Our study provides a good baseline for decision makers and stakeholders on where and how resources should be invested in the most efficient way to protect Georgia's high plant richness from IAPs.

Deployment Repeatability

DTIC Science & Technology

2016-04-01

environment. Modeling is suitable for well- characterized parts, and stochastic modeling techniques can be used for sensitivity analysis and generating a...large cohort of trials to spot unusual cases. However, deployment repeatability is inherently a nonlinear phenomenon, which makes modeling difficult...recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the U.S. Air Force. 1. Test the flight model

Determination of repeatability of kinect sensor.

PubMed

Bonnechère, Bruno; Sholukha, Victor; Jansen, Bart; Omelina, Lubos; Rooze, Marcel; Van Sint Jan, Serge

2014-05-01

The Kinect™ (Microsoft™, Redmond, WA) sensor, originally developed for gaming purposes, may have interesting possibilities for other fields such as posture and motion assessment. The ability of the Kinect sensor to perform biomechanical measurements has previously been studied and shows promising results. However, interday repeatability of the device is still not known. This study assessed the intra- and interday repeatability of the Kinect sensor compared with a standard stereophotogrammetric device during posture assessment for measuring segment lengths. Forty subjects took part in the study. Five motionless captures were performed in one session to assess posture. Data were simultaneously recorded with both devices. Similar intraclass correlations coefficient (ICC) values were found for intraday (ICC=0.94 for the Kinect device and 0.98 for the stereophotogrammetric device) and interday (ICC=0.88 and 0.87, respectively) repeatability. Results of this study suggest that a cost-effective, easy-to-use, and portable single markerless camera offers the same repeatability during posture assessment as an expensive, time-consuming, and nontransportable marker-based device.

Do gamma-ray burst sources repeat?

NASA Technical Reports Server (NTRS)

Meegan, Charles A.; Hartmann, Dieter H.; Brainerd, J. J.; Briggs, Michael S.; Paciesas, William S.; Pendleton, Geoffrey; Kouveliotou, Chryssa; Fishman, Gerald; Blumenthal, George; Brock, Martin

1995-01-01

The demonstration of repeated gamma-ray bursts from an individual source would severely constrain burst source models. Recent reports (Quashnock and Lamb, 1993; Wang and Lingenfelter, 1993) of evidence for repetition in the first BATSE burst catalog have generated renewed interest in this issue. Here, we analyze the angular distribution of 585 bursts of the second BATSE catalog (Meegan et al., 1994). We search for evidence of burst recurrence using the nearest and farthest neighbor statistic and the two-point angular correlation function. We find the data to be consistent with the hypothesis that burst sources do not repeat; however, a repeater fraction of up to about 20% of the observed bursts cannot be excluded.

Dental Fear in Children with Repeated Tooth Injuries.

PubMed

Negovetić Vranić, Dubravka; Ivančić Jokić, Nataša; Bakarčić, Danko; Carek, Andreja; Rotim, Željko; Verzak, Željko

2016-06-01

Tooth injuries are serious clinical conditions. Some children experience dental trauma only once, while others are more prone to repeated tooth injuries. Repeated dental trauma occurs in 19.4% to 30% of patients. Pain and dental trauma are the most common reasons for fear and anxiety. The main objective of this study was to investigate how dental trauma, as well as repeated dental trauma affects the occurrence and development of dental fear in children. The study was conducted on a random sample of 147 subjects (88 boys and 59 girls) aged 5-8 and 9-12 years. Subjects in both age groups were divided into subroups without dental trauma, with one dental trauma and with repeated dental trauma. The validated Children’s Fear Survey Schedule – Dental Subscale was used on fear assessment. Results showed that only 12.2% of children without trauma, 33.3% with one trauma and 51.7% with repeated trauma were not afraid of injection. Older children had a significantly lower fear of injections, touch of an unknown person, choking, going to the hospital and people in white uniforms. Dentist was not the cause of fear in 65.5% of patients with repeated trauma. With each repeated injury of teeth, the degree of their fear of dental treatment was lower.

Erroneous Memories Arising from Repeated Attempts to Remember

ERIC Educational Resources Information Center

Henkel, Linda A.

2004-01-01

The impact of repeated and prolonged attempts at remembering on false memory rates was assessed in three experiments. Participants saw and imagined pictures and then made repeated recall attempts before taking a source memory test. Although the number of items recalled increased with repeated tests, the net gains were associated with more source…

Limitations on quantum key repeaters.

PubMed

Bäuml, Stefan; Christandl, Matthias; Horodecki, Karol; Winter, Andreas

2015-04-23

A major application of quantum communication is the distribution of entangled particles for use in quantum key distribution. Owing to noise in the communication line, quantum key distribution is, in practice, limited to a distance of a few hundred kilometres, and can only be extended to longer distances by use of a quantum repeater, a device that performs entanglement distillation and quantum teleportation. The existence of noisy entangled states that are undistillable but nevertheless useful for quantum key distribution raises the question of the feasibility of a quantum key repeater, which would work beyond the limits of entanglement distillation, hence possibly tolerating higher noise levels than existing protocols. Here we exhibit fundamental limits on such a device in the form of bounds on the rate at which it may extract secure key. As a consequence, we give examples of states suitable for quantum key distribution but unsuitable for the most general quantum key repeater protocol.

Identification, variation and transcription of pneumococcal repeat sequences

PubMed Central

2011-01-01

Background Small interspersed repeats are commonly found in many bacterial chromosomes. Two families of repeats (BOX and RUP) have previously been identified in the genome of Streptococcus pneumoniae, a nasopharyngeal commensal and respiratory pathogen of humans. However, little is known about the role they play in pneumococcal genetics. Results Analysis of the genome of S. pneumoniae ATCC 700669 revealed the presence of a third repeat family, which we have named SPRITE. All three repeats are present at a reduced density in the genome of the closely related species S. mitis. However, they are almost entirely absent from all other streptococci, although a set of elements related to the pneumococcal BOX repeat was identified in the zoonotic pathogen S. suis. In conjunction with information regarding their distribution within the pneumococcal chromosome, this suggests that it is unlikely that these repeats are specialised sequences performing a particular role for the host, but rather that they constitute parasitic elements. However, comparing insertion sites between pneumococcal sequences indicates that they appear to transpose at a much lower rate than IS elements. Some large BOX elements in S. pneumoniae were found to encode open reading frames on both strands of the genome, whilst another was found to form a composite RNA structure with two T box riboswitches. In multiple cases, such BOX elements were demonstrated as being expressed using directional RNA-seq and RT-PCR. Conclusions BOX, RUP and SPRITE repeats appear to have proliferated extensively throughout the pneumococcal chromosome during the species' past, but novel insertions are currently occurring at a relatively slow rate. Through their extensive secondary structures, they seem likely to affect the expression of genes with which they are co-transcribed. Software for annotation of these repeats is freely available from ftp://ftp.sanger.ac.uk/pub/pathogens/strep_repeats/. PMID:21333003

Apoptosis regulator through modulating IAP expression (ARIA) controls the PI3K/Akt pathway in endothelial and endothelial progenitor cells.

PubMed

Koide, Masahiro; Ikeda, Koji; Akakabe, Yoshiki; Kitamura, Youhei; Ueyama, Tomomi; Matoba, Satoaki; Yamada, Hiroyuki; Okigaki, Mitsuhiko; Matsubara, Hiroaki

2011-06-07

Endothelial and endothelial progenitor cells (ECs and EPCs) play a fundamental role in angiogenesis that is essential for numerous physiological and pathological processes. The phosphatase and tensin homolog (PTEN)/ phosphoinositide 3-kinase (PI3K) pathway has been implicated in angiogenesis, but the mechanism in the regulation of this pathway in ECs and EPCs is poorly understood. Here we show that ARIA (apoptosis regulator through modulating IAP expression), a transmembrane protein that we recently identified, regulates the PTEN/PI3K pathway in ECs and EPCs and controls developmental and postnatal angiogenesis in vivo. We found that ARIA is abundantly expressed in EPCs and regulates their angiogenic functions by modulating PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling. Genetic deletion of ARIA caused nonfatal bleeding during embryogenesis, in association with increased small vessel density and altered expression of various vascular growth factors including angiopoietins and VEGF receptors. Postnatal neovascularization induced by critical limb ischemia was substantially enhanced in ARIA-null mice, in conjunction with more bone marrow (BM)-derived ECs detected in ischemic muscles. Administration of PI3K or NO synthase inhibitor completely abolished the enhanced neovascularization in ARIA(-/-) mice. Mechanistically, we identified that ARIA interacts with PTEN at the intracellular domain independently of the PTEN phosphorylation in its C-terminal tail. Overexpressed ARIA increased PTEN in the membrane fraction, whereas ARIA-silencing reduced the membrane-associated PTEN, resulting in modified PI3K/Akt signaling. Taken together, our findings establish a previously undescribed mode of regulation of the PTEN/PI3K/Akt pathway by ARIA, and reveal a unique mechanism in the control of angiogenesis. These functions of ARIA might offer a unique therapeutic potential.

Reasons sought for repeat drinking and driving

DOT National Transportation Integrated Search

1996-06-01

This document summarizes the results of a study about why some individuals repeatedly drive while under the influence or intoxicated, even after being convicted of DWI and after experiencing sanctions. Qualified interviewers asked repeat offenders di...

A Gammaherpesviral Internal Repeat Contributes to Latency Amplification

PubMed Central

Thakur, Nagendra N.; El-Gogo, Susanne; Steer, Beatrix; Freimüller, Klaus; Waha, Andreas; Adler, Heiko

2007-01-01

Background Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. The genomes of gammaherpesviruses contain variable numbers of internal repeats whose precise role for in vivo pathogenesis is not well understood. Methodology/Principal Findings We used infection of laboratory mice with murine gammaherpesvirus 68 (MHV-68) to explore the biological role of the 40 bp internal repeat of MHV-68. We constructed several mutant viruses partially or completely lacking this repeat. Both in vitro and in vivo, the loss of the repeat did not substantially affect lytic replication of the mutant viruses. However, the extent of splenomegaly, which is associated with the establishment of latency, and the number of ex vivo reactivating and genome positive splenocytes were reduced. Since the 40 bp repeat is part of the hypothetical open reading frame (ORF) M6, it might function as part of M6 or as an independent structure. To differentiate between these two possibilities, we constructed an N-terminal M6STOP mutant, leaving the repeat structure intact but rendering ORF M6 unfunctional. Disruption of ORF M6 did neither affect lytic nor latent infection. In contrast to the situation in lytically infected NIH3T3 cells, the expression of the latency-associated genes K3 and ORF72 was reduced in the latently infected murine B cell line Ag8 in the absence of the 40 bp repeat. Conclusions/Significance These data suggest that the 40 bp repeat contributes to latency amplification and might be involved in the regulation of viral gene expression. PMID:17710133

SkyRepeater

DTIC Science & Technology

2005-01-01

MUSIC.consultant, Doug Lewis, host of WVGN’s Doug Lewis Show, cddick@viaccess.net Being For The Benefit of Mr. Kite, Beatles , Writer, lead vocal...and in the case of a repeater failure. Tested on both VHF and UHF bands, one person can setup in 15 minutes. Cost is approximately $2,000.00. Minimum...This was originally written as a term paper for the Center for Homeland Defense and Security at the Naval Postgraduate

R-loops: targets for nuclease cleavage and repeat instability.

PubMed

Freudenreich, Catherine H

2018-01-11

R-loops form when transcribed RNA remains bound to its DNA template to form a stable RNA:DNA hybrid. Stable R-loops form when the RNA is purine-rich, and are further stabilized by DNA secondary structures on the non-template strand. Interestingly, many expandable and disease-causing repeat sequences form stable R-loops, and R-loops can contribute to repeat instability. Repeat expansions are responsible for multiple neurodegenerative diseases, including Huntington's disease, myotonic dystrophy, and several types of ataxias. Recently, it was found that R-loops at an expanded CAG/CTG repeat tract cause DNA breaks as well as repeat instability (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Two factors were identified as causing R-loop-dependent breaks at CAG/CTG tracts: deamination of cytosines and the MutLγ (Mlh1-Mlh3) endonuclease, defining two new mechanisms for how R-loops can generate DNA breaks (Su and Freudenreich, Proc Natl Acad Sci USA 114, E8392-E8401, 2017). Following R-loop-dependent nicking, base excision repair resulted in repeat instability. These results have implications for human repeat expansion diseases and provide a paradigm for how RNA:DNA hybrids can cause genome instability at structure-forming DNA sequences. This perspective summarizes mechanisms of R-loop-induced fragility at G-rich repeats and new links between DNA breaks and repeat instability.

Evolution of genes and repeats in the Nimrod superfamily.

PubMed

Somogyi, Kálmán; Sipos, Botond; Pénzes, Zsolt; Kurucz, Eva; Zsámboki, János; Hultmark, Dan; Andó, István

2008-11-01

The recently identified Nimrod superfamily is characterized by the presence of a special type of EGF repeat, the NIM repeat, located right after a typical CCXGY/W amino acid motif. On the basis of structural features, nimrod genes can be divided into three types. The proteins encoded by Draper-type genes have an EMI domain at the N-terminal part and only one copy of the NIM motif, followed by a variable number of EGF-like repeats. The products of Nimrod B-type and Nimrod C-type genes (including the eater gene) have different kinds of N-terminal domains, and lack EGF-like repeats but contain a variable number of NIM repeats. Draper and Nimrod C-type (but not Nimrod B-type) proteins carry a transmembrane domain. Several members of the superfamily were claimed to function as receptors in phagocytosis and/or binding of bacteria, which indicates an important role in the cellular immunity and the elimination of apoptotic cells. In this paper, the evolution of the Nimrod superfamily is studied with various methods on the level of genes and repeats. A hypothesis is presented in which the NIM repeat, along with the EMI domain, emerged by structural reorganizations at the end of an EGF-like repeat chain, suggesting a mechanism for the formation of novel types of repeats. The analyses revealed diverse evolutionary patterns in the sequences containing multiple NIM repeats. Although in the Nimrod B and Nimrod C proteins show characteristics of independent evolution, many internal NIM repeats in Eater sequences seem to have undergone concerted evolution. An analysis of the nimrod genes has been performed using phylogenetic and other methods and an evolutionary scenario of the origin and diversification of the Nimrod superfamily is proposed. Our study presents an intriguing example how the evolution of multigene families may contribute to the complexity of the innate immune response.

Plant-mediated effects on an insect-pathogen interaction vary with intraspecific genetic variation in plant defences.

PubMed

Shikano, Ikkei; Shumaker, Ketia L; Peiffer, Michelle; Felton, Gary W; Hoover, Kelli

2017-04-01

Baculoviruses are food-borne microbial pathogens that are ingested by insects on contaminated foliage. Oxidation of plant-derived phenolics, activated by insect feeding, can directly interfere with infections in the gut. Since phenolic oxidation is an important component of plant resistance against insects, baculoviruses are suggested to be incompatible with plant defences. However, plants among and within species invest differently in a myriad of chemical and physical defences. Therefore, we hypothesized that among eight soybean genotypes, some genotypes would be able to maintain both high resistance against an insect pest and high efficacy of a baculovirus. Soybean constitutive (non-induced) and jasmonic acid (JA)-induced (anti-herbivore response) resistance was measured against the fall armyworm Spodoptera frugiperda (weight gain, leaf consumption and utilization). Indicators of phenolic oxidation were measured as foliar phenolic content and peroxidase activity. Levels of armyworm mortality inflicted by baculovirus (SfMNPV) did not vary among soybean genotypes when the virus was ingested with non-induced foliage. Ingestion of the virus on JA-induced foliage reduced armyworm mortality, relative to non-induced foliage, on some soybean genotypes. Baculovirus efficacy was lower when ingested with foliage that contained higher phenolic content and defensive properties that reduced armyworm weight gain and leaf utilization. However, soybean genotypes that defended the plant by reducing consumption rate and strongly deterred feeding upon JA-induction did not reduce baculovirus efficacy, indicating that these defences may be more compatible with baculoviruses to maximize plant protection. Differential compatibility of defence traits with the third trophic level highlights an important cost/trade-off associated with plant defence strategies.

Hypoxic Repeat Sprint Training Improves Rugby Player's Repeated Sprint but Not Endurance Performance

PubMed Central

Hamlin, Michael J.; Olsen, Peter D.; Marshall, Helen C.; Lizamore, Catherine A.; Elliot, Catherine A.

2017-01-01

This study aims to investigate the performance changes in 19 well-trained male rugby players after repeat-sprint training (six sessions of four sets of 5 × 5 s sprints with 25 s and 5 min of active recovery between reps and sets, respectively) in either normobaric hypoxia (HYP; n = 9; FIO2 = 14.5%) or normobaric normoxia (NORM; n = 10; FIO2 = 20.9%). Three weeks after the intervention, 2 additional repeat-sprint training sessions in hypoxia (FIO2 = 14.5%) was investigated in both groups to gauge the efficacy of using “top-up” sessions for previously hypoxic-trained subjects and whether a small hypoxic dose would be beneficial for the previously normoxic-trained group. Repeated sprint (8 × 20 m) and Yo-Yo Intermittent Recovery Level 1 (YYIR1) performances were tested twice at baseline (Pre 1 and Pre 2) and weekly after (Post 1–3) the initial intervention (intervention 1) and again weekly after the second “top-up” intervention (Post 4–5). After each training set, heart rate, oxygen saturation, and rate of perceived exertion were recorded. Compared to baseline (mean of Pre 1 and Pre 2), both the hypoxic and normoxic groups similarly lowered fatigue over the 8 sprints 1 week after the intervention (Post 1: −1.8 ± 1.6%, −1.5 ± 1.4%, mean change ± 90% CI in HYP and NORM groups, respectively). However, from Post 2 onwards, only the hypoxic group maintained the performance improvement compared to baseline (Post 2: −2.1 ± 1.8%, Post 3: −2.3 ± 1.7%, Post 4: −1.9 ± 1.8%, and Post 5: −1.2 ± 1.7%). Compared to the normoxic group, the hypoxic group was likely to have substantially less fatigue at Post 3–5 (−2.0 ± 2.4%, −2.2 ± 2.4%, −1.6 ± 2.4% Post 3, Post 4, Post 5, respectively). YYIR1 performances improved throughout the recovery period in both groups (13–37% compared to baseline) with unclear differences found between groups. The addition of two sessions of “top-up” training after intervention 1, had little effect on either

Informal Allopathic Provider Knowledge and Practice Regarding Hypertension in Urban and Rural Bangladesh

PubMed Central

Parr, John; Lindeboom, Wietze; Khanam, Masuma; Sanders, James; Koehlmoos, Tracey Pérez

2012-01-01

Objectives Describe informal allopathic practitioner (IAP) knowledge and practice about management of hypertension and identify gaps in IAP knowledge and practice amenable to interventions. Methods A cross sectional descriptive survey of 642 IAPs in Kamalapur (urban) and Mirsarai (rural) Bangladesh was conducted from March to April, 2011. Using a structured, pre-tested questionnaire sociodemographic, training, knowledge and practice data about management of hypertension was collected. Comparative statistics were preformed to show differences between urban and rural practitioners using SAS 8.0. Findings 99.4% of IAPs were male, mean age was 37.5 (12.5 SD) years. Greater than 65% correctly identified the upper limit of normal blood pressure. 50.2% underestimated lower limit of systolic hypertension. 79.8% allowed age to affect their treatment approach. As blood pressure increased, willingness to treat with medication decreased and tendency to refer increased. Sedative/sleeping pills, antidepressants, and beta blockers were the most commonly prescribed medications for prehypertension (58.7%, 50.3% and 53.7% respectively), stage I hypertension (55.0%, 38.6%, 49.8% respectively) and stage II hypertension (42.4%, 23.7%, and 28.8% respectively). Rural IAPs were more likely than urban IAPs to treat (84.7% vs 77.7%), order tests (27.1% vs 6.0%) and write prescriptions (60.4% vs 18.7%). Conclusion While IAPs are crucial to Bangladesh’s pluralistic healthcare system, gaps in knowledge and practice could cause unnecessary harm. To include IAPs in the public sector’s fight against the chronic disease epidemic, interventions aimed at standardizing IAPs knowledge and practice will be essential. Successfully utilizing IAPs will have beneficial implications not only for Bangladesh, but for all developing countries. PMID:23133546

Perceptions of impact: Invasive alien plants in the urban environment.

PubMed

Potgieter, Luke J; Gaertner, Mirijam; O'Farrell, Patrick J; Richardson, David M

2018-06-08

Many alien plant species are introduced to urban areas to create, augment or restore ecosystem services (ES). However, many of these species spread beyond original plantings, sometimes causing negative effects on existing ES or creating novel ecosystem disservices (EDS). An understanding of the perceptions of urban residents regarding invasive alien plants (IAPs) and the ES and EDS they provide is needed for the effective prioritisation of IAP management efforts in cities. Using the city of Cape Town, South Africa as a case study, we conducted questionnaire-based surveys (online and face-to-face) to determine the perceptions of urban residents regarding IAPs and their capacity to provide ES and EDS. Most urban residents perceive IAPs negatively (i.e. agreeing that they create EDS), but many recognise their importance in providing ES. Although most residents are not opposed to the management of IAPs, such actions are not perceived as a high priority relative to other environmental problems. Socio-demographic variables such as age, education, environmental awareness, and ethnicity shape urban residents' perceptions of IAPs. Older, more educated respondents were more likely to perceive IAPs negatively, while respondents with greater environmental awareness were aware of the benefits provided by IAPs. This study highlights the need to integrate public perceptions into the planning and management of IAPs and emphasises the importance of including ES assessments into the decision-making process, particularly in urban areas. Copyright © 2018 Elsevier Ltd. All rights reserved.

Intestinal alkaline phosphatase preserves the normal homeostasis of gut microbiota.

PubMed

Malo, M S; Alam, S Nasrin; Mostafa, G; Zeller, S J; Johnson, P V; Mohammad, N; Chen, K T; Moss, A K; Ramasamy, S; Faruqui, A; Hodin, S; Malo, P S; Ebrahimi, F; Biswas, B; Narisawa, S; Millán, J L; Warren, H S; Kaplan, J B; Kitts, C L; Hohmann, E L; Hodin, R A

2010-11-01

The intestinal microbiota plays a critical role in maintaining human health; however, the mechanisms governing the normal homeostatic number and composition of these microbes are largely unknown. Previously it was shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, functions as a gut mucosal defence factor limiting the translocation of gut bacteria to mesenteric lymph nodes. In this study the role of IAP in the preservation of the normal homeostasis of the gut microbiota was investigated. Bacterial culture was performed in aerobic and anaerobic conditions to quantify the number of bacteria in the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment length polymorphism, phylogenetic analyses and quantitative real-time PCR of subphylum-specific bacterial 16S rRNA genes were used to determine the compositional profiles of microbiotas. Oral supplementation of calf IAP (cIAP) was used to determine its effects on the recovery of commensal gut microbiota after antibiotic treatment and also on the colonisation of pathogenic bacteria. IAP-KO mice had dramatically fewer and also different types of aerobic and anaerobic microbes in their stools compared with WT mice. Oral supplementation of IAP favoured the growth of commensal bacteria, enhanced restoration of gut microbiota lost due to antibiotic treatment and inhibited the growth of a pathogenic bacterium (Salmonella typhimurium). IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.

Photometric Repeatability of Scanned Imagery: UVIS

NASA Astrophysics Data System (ADS)

Shanahan, Clare E.; McCullough, Peter; Baggett, Sylvia

2017-08-01

We provide the preliminary results of a study on the photometric repeatability of spatial scans of bright, isolated white dwarf stars with the UVIS channel of the Wide Field Camera 3 (WFC3) on the Hubble Space Telescope (HST). We analyze straight-line scans from the first pair of identical orbits of HST program 14878 to assess if sub 0.1% repeatability can be attained with WFC3/UVIS. This study is motivated by the desire to achieve better signal-to-noise in the UVIS contamination and stability monitor, in which observations of standard stars in staring mode have been taken from the installation of WFC3 in 2009 to the present to assess temporal photometric stability. Higher signal to noise in this program would greatly benefit the sensitivity to detect contamination, and to better characterize the observed small throughput drifts over time. We find excellent repeatability between identical visits of program 14878, with sub 0.1% repeatability achieved in most filters. These! results support the initiative to transition the staring mode UVIS contamination and photometric stability monitor from staring mode images to spatial scans.

Deployment Repeatability

DTIC Science & Technology

2016-08-31

photogrammetry to track a set of LEDS in two dimensions, a method that was able to resolve 2.2-mm lateral displacements of a 40-meter boom. This method... displacements . Sub-milliradian repeatability in MOIRE [This is a stand-in section for the MOIRE case study, which Dave Waller is getting through...The structure will most likely be under this stowed state for a long time under displacement paths and put this data into a computationally cheap 1

Androgen receptor CAG repeat polymorphisms in canine prostate cancer.

PubMed

Lai, C-L; L'Eplattenier, H; van den Ham, R; Verseijden, F; Jagtenberg, A; Mol, J A; Teske, E

2008-01-01

Relatively shorter lengths of the polymorphic polyglutamine repeat-1 of the androgen receptor (AR) have been associated with an increased risk of prostate cancer (PC) in humans. In the dog, there are 2 polymorphic CAG repeat (CAGr) regions. To investigate the relationship of CAGr length of the canine AR-gene and the development of PC. Thirty-two dogs with PC and 172 control dogs were used. DNA was extracted from blood. Both CAG repeats were amplified by polymerase chain reaction (PCR) and PCR products were sequenced. In dogs with PC, CAG-1 repeat length was shorter (P = .001) by an increased proportion of 10 repeats (P = .011) and no 12 repeats (P = .0017) than in the control dogs. No significant changes were found in CAG-3 length distribution. CAG-1 and CAG-3 polymorphisms proved not to be in linkage disequilibrium. Breed difference in allelic distribution was found in the control group. Of the prostate-disease sensitive breeds, a high percentage (64.5%) of the shortest haplotype 10/11 was found in the Doberman, whereas Beagles and German Pointers had higher haplotype 12/11 (47.1 and 50%). Bernese Mountain dogs and Bouvier dogs both shared a high percentage of 11 CAG-1 repeats and 13 CAG-3 repeats. Differences in (combined) allelic distributions among breeds were not significant. In this preliminary study, short CAG-1 repeats in the AR-gene were associated with an increased risk of developing canine PC. Although breed-specific differences in allelic distribution of CAG-1 and CAG-3 repeats were found, these could not be related to PC risk.

The Effects of Repeaters on Test Equating.

ERIC Educational Resources Information Center

Andrulis, Richard S.; And Others

1978-01-01

The effects of repeaters (testees included in both administrations of two forms of a test) on the test equating process are examined. It is shown that repeaters do effect test equating and tend to lower the cutoff point for passing the test. (JKS)

Development of analog watch with minute repeater

NASA Astrophysics Data System (ADS)

Okigami, Tomio; Aoyama, Shigeru; Osa, Takashi; Igarashi, Kiyotaka; Ikegami, Tomomi

A complementary metal oxide semiconductor with large scale integration was developed for an electronic minute repeater. It is equipped with the synthetic struck sound circuit to generate natural struck sound necessary for the minute repeater. This circuit consists of an envelope curve drawing circuit, frequency mixer, polyphonic mixer, and booster circuit made by using analog circuit technology. This large scale integration is a single chip microcomputer with motor drivers and input ports in addition to the synthetic struck sound circuit, and it is possible to make an electronic system of minute repeater at a very low cost in comparison with the conventional type.

Zero-determinant strategies in finitely repeated games.

PubMed

Ichinose, Genki; Masuda, Naoki

2018-02-07

Direct reciprocity is a mechanism for sustaining mutual cooperation in repeated social dilemma games, where a player would keep cooperation to avoid being retaliated by a co-player in the future. So-called zero-determinant (ZD) strategies enable a player to unilaterally set a linear relationship between the player's own payoff and the co-player's payoff regardless of the strategy of the co-player. In the present study, we analytically study zero-determinant strategies in finitely repeated (two-person) prisoner's dilemma games with a general payoff matrix. Our results are as follows. First, we present the forms of solutions that extend the known results for infinitely repeated games (with a discount factor w of unity) to the case of finitely repeated games (0 < w < 1). Second, for the three most prominent ZD strategies, the equalizers, extortioners, and generous strategies, we derive the threshold value of w above which the ZD strategies exist. Third, we show that the only strategies that enforce a linear relationship between the two players' payoffs are either the ZD strategies or unconditional strategies, where the latter independently cooperates with a fixed probability in each round of the game, proving a conjecture previously made for infinitely repeated games. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mission design for NISAR repeat-pass Interferometric SAR

NASA Astrophysics Data System (ADS)

Alvarez-Salazar, Oscar; Hatch, Sara; Rocca, Jennifer; Rosen, Paul; Shaffer, Scott; Shen, Yuhsyen; Sweetser, Theodore; Xaypraseuth, Peter

2014-10-01

The proposed spaceborne NASA-ISRO SAR (NISAR) mission would use the repeat-pass interferometric Synthetic Aperture Radar (InSAR) technique to measure the changing shape of Earth's surface at the centimeter scale in support of investigations in solid Earth and cryospheric sciences. Repeat-pass InSAR relies on multiple SAR observations acquired from nearly identical positions of the spacecraft as seen from the ground. Consequently, there are tight constraints on the repeatability of the orbit, and given the narrow field of view of the radar antenna beam, on the repeatability of the beam pointing. The quality and accuracy of the InSAR data depend on highly precise control of both orbital position and observatory pointing throughout the science observation life of the mission. This paper describes preliminary NISAR requirements and rationale for orbit repeatability and attitude control in order to meet science requirements. A preliminary error budget allocation and an implementation approach to meet these allocations are also discussed.

RTEL1 Inhibits Trinucleotide Repeat Expansions and Fragility

PubMed Central

Frizzell, Aisling; Nguyen, Jennifer H.G.; Petalcorin, Mark I.R.; Turner, Katherine D.; Boulton, Simon J.; Freudenreich, Catherine H.; Lahue, Robert S.

2018-01-01

SUMMARY Human RTEL1 is an essential, multifunctional helicase that maintains telomeres, regulates homologous recombination, and helps prevent bone marrow failure. Here, we show that RTEL1 also blocks trinucleotide repeat expansions, the causal mutation for 17 neurological diseases. Increased expansion frequencies of (CTG·CAG) repeats occurred in human cells following knockdown of RTEL1, but not the alternative helicase Fbh1, and purified RTEL1 efficiently unwound triplet repeat hairpins in vitro. The expansion-blocking activity of RTEL1 also required Rad18 and HLTF, homologs of yeast Rad18 and Rad5. These findings are reminiscent of budding yeast Srs2, which inhibits expansions, unwinds hairpins, and prevents triplet-repeat-induced chromosome fragility. Accordingly, we found expansions and fragility were suppressed in yeast srs2 mutants expressing RTEL1, but not Fbh1. We propose that RTEL1 serves as a human analog of Srs2 to inhibit (CTG·CAG) repeat expansions and fragility, likely by unwinding problematic hairpins. PMID:24561255

Isolation of human simple repeat loci by hybridization selection.

PubMed

Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Ligand binding by repeat proteins: natural and designed

PubMed Central

Grove, Tijana Z; Cortajarena, Aitziber L; Regan, Lynne

2012-01-01

Repeat proteins contain tandem arrays of small structural motifs. As a consequence of this architecture, they adopt non-globular, extended structures that present large, highly specific surfaces for ligand binding. Here we discuss recent advances toward understanding the functional role of this unique modular architecture. We showcase specific examples of natural repeat proteins interacting with diverse ligands and also present examples of designed repeat protein–ligand interactions. PMID:18602006

Tandem-repeat protein domains across the tree of life.

PubMed

Jernigan, Kristin K; Bordenstein, Seth R

2015-01-01

Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20-40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny. In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins. We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life. A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species.

The effectiveness of repeat lumbar transforaminal epidural steroid injections.

PubMed

Murthy, Naveen S; Geske, Jennifer R; Shelerud, Randy A; Wald, John T; Diehn, Felix E; Thielen, Kent R; Kaufmann, Timothy J; Morris, Jonathan M; Lehman, Vance T; Amrami, Kimberly K; Carter, Rickey E; Maus, Timothy P

2014-10-01

The aim of this study was to determine 1) if repeat lumbar transforaminal epidural steroid injections (TFESIs) resulted in recovery of pain relief, which has waned since an index injection, and 2) if cumulative benefit could be achieved by repeat injections within 3 months of the index injection. Retrospective observational study with statistical modeling of the response to repeat TFESI. Academic radiology practice. Two thousand eighty-seven single-level TFESIs were performed for radicular pain on 933 subjects. Subjects received repeat TFESIs >2 weeks and <1 year from the index injection. Hierarchical linear modeling was performed to evaluate changes in continuous and categorical pain relief outcomes after repeat TFESI. Subgroup analyses were performed on patients with <3 months duration of pain (acute pain), patients receiving repeat injections within 3 months (clustered injections), and in patients with both acute pain and clustered injections. Repeat TFESIs achieved pain relief in both continuous and categorical outcomes. Relative to the index injection, there was a minimal but statistically significant decrease in pain relief in modeled continuous outcome measures with subsequent injections. Acute pain patients recovered all prior benefit with a statistically significant cumulative benefit. Patients receiving clustered injections achieved statistically significant cumulative benefit, of greater magnitude in acute pain patients. Repeat TFESI may be performed for recurrence of radicular pain with the expectation of recovery of most or all previously achieved benefit; acute pain patients will likely recover all prior benefit. Repeat TFESIs within 3 months of the index injection can provide cumulative benefit. Wiley Periodicals, Inc.

Tandem-repeat protein domains across the tree of life

PubMed Central

Jernigan, Kristin K.

2015-01-01

Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20–40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny. In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins. We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life. A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species. PMID:25653910

Repeated, but Not Acute, Stress Suppresses Inflammatory Plasma Extravasation

NASA Astrophysics Data System (ADS)

Strausbaugh, Holly J.; Dallman, Mary F.; Levine, Jon D.

1999-12-01

Clinical findings suggest that inflammatory disease symptoms are aggravated by ongoing, repeated stress, but not by acute stress. We hypothesized that, compared with single acute stressors, chronic repeated stress may engage different physiological mechanisms that exert qualitatively different effects on the inflammatory response. Because inhibition of plasma extravasation, a critical component of the inflammatory response, has been associated with increased disease severity in experimental arthritis, we tested for a potential repeated stress-induced inhibition of plasma extravasation. Repeated, but not single, exposures to restraint stress produced a profound inhibition of bradykinin-induced synovial plasma extravasation in the rat. Experiments examining the mechanism of inhibition showed that the effect of repeated stress was blocked by adrenalectomy, but not by adrenal medullae denervation, suggesting that the adrenal cortex mediates this effect. Consistent with known effects of stress and with mediation by the adrenal cortex, restraint stress evoked repeated transient elevations of plasma corticosterone levels. This elevated corticosterone was necessary and sufficient to produce inhibition of plasma extravasation because the stress-induced inhibition was blocked by preventing corticosterone synthesis and, conversely, induction of repeated transient elevations in plasma corticosterone levels mimicked the effects of repeated stress. These data suggest that repetition of a mild stressor can induce changes in the physiological state of the animal that enable a previously innocuous stressor to inhibit the inflammatory response. These findings provide a potential explanation for the clinical association between repeated stress and aggravation of inflammatory disease symptoms and provide a model for study of the biological mechanisms underlying the stress-induced aggravation of chronic inflammatory diseases.

Study of repeater technology for advanced multifunctional communications satellites

NASA Technical Reports Server (NTRS)

1972-01-01

Investigations are presented concerning design concepts and implementation approaches for the satellite communication repeater subsystems of advanced multifunctional satellites. In such systems the important concepts are the use of multiple antenna beams, repeater switching (routing), and efficient spectrum utilization through frequency reuse. An information base on these techniques was developed and tradeoff analyses were made of repeater design concepts, with the work design taken in a broad sense to include modulation beam coverage patterns. There were five major areas of study: requirements analysis and processing; study of interbeam interference in multibeam systems; characterization of multiple-beam switching repeaters; estimation of repeater weight and power for a number of alternatives; and tradeoff analyses based on these weight and power data.

Effect of Repeat Copy Number on Variable-Number Tandem Repeat Mutations in Escherichia coli O157:H7

PubMed Central

Vogler, Amy J.; Keys, Christine; Nemoto, Yoshimi; Colman, Rebecca E.; Jay, Zack; Keim, Paul

2006-01-01

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 × 10−4 mutations/generation and a combined 28-locus rate of 6.4 × 10−4 mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2 = 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2 = 0.833, P < 0.0001) or excluded (r2 = 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data. PMID:16740932

Effect of repeat copy number on variable-number tandem repeat mutations in Escherichia coli O157:H7.

PubMed

Vogler, Amy J; Keys, Christine; Nemoto, Yoshimi; Colman, Rebecca E; Jay, Zack; Keim, Paul

2006-06-01

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x 10(-4) mutations/generation and a combined 28-locus rate of 6.4 x 10(-4) mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2= 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2= 0.833, P < 0.0001) or excluded (r2= 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.

Experimental intra-abdominal hypertension influences airway pressure limits for lung protective mechanical ventilation.

PubMed

Cortes-Puentes, Gustavo A; Cortes-Puentes, Luis A; Adams, Alexander B; Anderson, Christopher P; Marini, John J; Dries, David J

2013-06-01

Intra-abdominal hypertension (IAH) and abdominal compartment syndrome (ACS) may complicate monitoring of pulmonary mechanics owing to their impact on the respiratory system. However, recommendations for mechanical ventilation of patients with IAH/ACS and the interpretation of thoracoabdominal interactions remain unclear. Our study aimed to characterize the influence of elevated intra-abdominal pressure (IAP) and positive end-expiratory pressure (PEEP) on airway plateau pressure (PPLAT) and bladder pressure (PBLAD). Nine deeply anesthetized swine were mechanically ventilated via tracheostomy: volume-controlled mode at tidal volume (VT) of 10 mL/kg, frequency of 15, inspiratory-expiratory ratio of 1:2, and PEEP of 1 and 10 cm H2O (PEEP1 and PEEP10, respectively). A tracheostomy tube was placed in the peritoneal cavity, and IAP levels of 5, 10, 15, 20, and 25 mm Hg were applied, using a continuous positive airway pressure system. At each IAP level, PBLAD and airway pressure measurements were performed during both PEEP1 and PEEP10. PBLAD increased as experimental IAP rose (y = 0.83x + 0.5; R = 0.98; p < 0.001 at PEEP1). Minimal underestimation of IAP by PBLAD was observed (-2.5 ± 0.8 mm Hg at an IAP of 10-25 mm Hg). Applying PEEP10 did not significantly affect the correlation between experimental IAP and PBLAD. Approximately 50% of the PBLAD (in cm H2O) was reflected by changes in PPLAT, regardless of the PEEP level applied. Increasing IAP did not influence hemodynamics at any level of IAP generated. With minimal underestimation, PBLAD measurements closely correlated with experimentally regulated IAP, independent of the PEEP level applied. For each PEEP level applied, a constant proportion (approximately 50%) of measured PBLAD (in cm H2O) was reflected in PPLAT. A higher safety threshold for PPLAT should be considered in the setting of IAH/ACS as the clinician considers changes in VT. A strategy of reducing VT to cap PPLAT at widely recommended values may not be

40 CFR 141.858 - Repeat monitoring and E. coli requirements.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 24 2013-07-01 2013-07-01 false Repeat monitoring and E. coli....858 Repeat monitoring and E. coli requirements. (a) Repeat monitoring. (1) If a sample taken under... exceeded. (b) Escherichia coli (E. coli) testing. (1) If any routine or repeat sample is total coliform...

Intestinal alkaline phosphatase to treat necrotizing enterocolitis.

PubMed

Biesterveld, Ben E; Koehler, Shannon M; Heinzerling, Nathan P; Rentea, Rebecca M; Fredrich, Katherine; Welak, Scott R; Gourlay, David M

2015-06-15

Intestinal alkaline phosphatase (IAP) activity is decreased in necrotizing enterocolitis (NEC), and IAP supplementation prevents NEC development. It is not known if IAP given after NEC onset can reverse the course of the disease. We hypothesized that enteral IAP given after NEC induction would not reverse intestinal injury. NEC was induced in Sprague-Dawley pups by delivery preterm followed by formula feedings with lipopolysaccharide (LPS) and hypoxia exposure and continued up to 4 d. IAP was added to feeds on day 2 until being sacrificed on day 4. NEC severity was scored based on hematoxylin and eosin-stained terminal ileum sections, and AP activity was measured using a colorimetric assay. IAP and interleukin-6 expression were measured using real time polymerase chain reaction. NEC pups' alkaline phosphatase (AP) activity was decreased to 0.18 U/mg compared with controls of 0.57 U/mg (P < 0.01). Discontinuation of LPS and hypoxia after 2 d increased AP activity to 0.36 U/mg (P < 0.01). IAP supplementation in matched groups did not impact total AP activity or expression. Discontinuing LPS and hypoxia after NEC onset improved intestinal injury scores to 1.14 compared with continued stressors, score 2.25 (P < 0.01). IAP supplementation decreased interleukin-6 expression two-fold (P < 0.05), though did not reverse NEC intestinal damage (P = 0.5). This is the first work to demonstrate that removing the source of NEC improves intestinal damage and increases AP activity. When used as a rescue treatment, IAP decreased intestinal inflammation though did not impact injury making it likely that IAP is best used preventatively to those neonates at risk. Copyright © 2015 Elsevier Inc. All rights reserved.

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures.

PubMed Central

Bozzo, Gale G; Raghothama, Kashchandra G; Plaxton, William C

2004-01-01

An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286]. PMID:14521509

Cost-effectiveness of a potential group B streptococcal vaccine for pregnant women in the United States.

PubMed

Kim, Sun-Young; Nguyen, Chi; Russell, Louise B; Tomczyk, Sara; Abdul-Hakeem, Fatimah; Schrag, Stephanie J; Verani, Jennifer R; Sinha, Anushua

2017-10-27

In the U.S., intrapartum antibiotic prophylaxis (IAP) for pregnant women colonized with group B streptococcus (GBS) has reduced GBS disease in the first week of life (early-onset/EOGBS). Nonetheless, GBS remains a leading cause of neonatal sepsis, including 1000 late-onset (LOGBS) cases annually. A maternal vaccine under development could prevent EOGBS and LOGBS. Using a decision-analytic model, we compared the public health impact, costs, and cost-effectiveness of five strategies to prevent GBS disease in infants: (1) no prevention; (2) currently recommended screening/IAP; (3) maternal GBS immunization; (4) maternal immunization with IAP when indicated for unimmunized women; (5) maternal immunization plus screening/IAP for all women. We modeled a pentavalent vaccine covering serotypes 1a, 1b, II, III, and V, which cause almost all GBS disease. In the base case, screening/IAP alone prevents 46% of EOGBS compared to no prevention, at a cost of $70,275 per quality-adjusted life-year (QALY) from a healthcare and $51,249/QALY from a societal perspective (2013 US$). At coverage rates typical of maternal vaccines in the U.S., a pentavalent vaccine alone would not prevent as much disease as screening/IAP until its efficacy approached 90%, but would cost less per QALY. At vaccine efficacy of ≥70%, maternal immunization together with IAP for unimmunized women would prevent more disease than screening/IAP, at a similar cost/QALY. GBS maternal immunization, with IAP as indicated for unvaccinated women, could be an attractive alternative to screening/IAP if a pentavalent vaccine is sufficiently effective. Coverage, typically low for maternal vaccines, is key to the vaccine's public health impact. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hyaluronic acid-fabricated nanogold delivery of the inhibitor of apoptosis protein-2 siRNAs inhibits benzo[a]pyrene-induced oncogenic properties of lung cancer A549 cells

NASA Astrophysics Data System (ADS)

Lin, Chung-Ming; Kao, Wei-Chien; Yeh, Chun-An; Chen, Hui-Jye; Lin, Shinn-Zong; Hsieh, Hsien-Hsu; Sun, Wei-Shen; Chang, Chih-Hsuan; Hung, Huey-Shan

2015-03-01

Benzo[a]pyrene (BaP), a component of cooking oil fumes (COF), promotes lung cancer cell proliferation and survival via the induction of inhibitor of apoptosis protein-2 (IAP-2) proteins. Thus knockdown of IAP-2 would be a promising way to battle against lung cancer caused by COF. Functionalized gold nanoparticle (AuNP) is an effective delivery system for bio-active materials. Here, biocompatible hyaluronic acid (HA) was fabricated into nanoparticles to increase the target specificity by binding to CD44-over-expressed cancer cells. IAP-2-specific small-interfering RNA (siRNAs) or fluorescein isothiocyanate (FITC) were then incorporated into AuNP-HA. Conjugation of IAP-2 siRNA into AuNPs-HA was verified by the UV-vis spectrometer and Fourier transform infrared spectrometer. Further studies showed that AuNP-HA/FITC were effectively taken up by A549 cells through CD44-mediated endocytosis. Incubation of BaP-challenged cells with AuNP-HA-IAP-2 siRNAs silenced the expression of IAP-2, decreased cell proliferation and triggered pronounced cell apoptosis by the decrease in Bcl-2 protein and the increase in Bax protein as well as the active form of caspases-3. The BaP-elicited cell migration and enzymatic activity of the secreted matrix metalloproteinase-2 were also substantially suppressed by treatment with AuNP-HA-IAP-2 siRNAs. These results indicated that IAP-2 siRNAs can be efficiently delivered into A549 cells by functionalized AuNP-HA to repress the IAP-2 expression and BaP-induced oncogenic events, suggesting the potential therapeutic application of IAP-2 siRNA or other siRNA-conjugated AuNP-HA composites to COF-induced lung cancer and other gene-caused diseases in the future.

Parameter regimes for a single sequential quantum repeater

NASA Astrophysics Data System (ADS)

Rozpędek, F.; Goodenough, K.; Ribeiro, J.; Kalb, N.; Caprara Vivoli, V.; Reiserer, A.; Hanson, R.; Wehner, S.; Elkouss, D.

2018-07-01

Quantum key distribution allows for the generation of a secret key between distant parties connected by a quantum channel such as optical fibre or free space. Unfortunately, the rate of generation of a secret key by direct transmission is fundamentally limited by the distance. This limit can be overcome by the implementation of so-called quantum repeaters. Here, we assess the performance of a specific but very natural setup called a single sequential repeater for quantum key distribution. We offer a fine-grained assessment of the repeater by introducing a series of benchmarks. The benchmarks, which should be surpassed to claim a working repeater, are based on finite-energy considerations, thermal noise and the losses in the setup. In order to boost the performance of the studied repeaters we introduce two methods. The first one corresponds to the concept of a cut-off, which reduces the effect of decoherence during the storage of a quantum state by introducing a maximum storage time. Secondly, we supplement the standard classical post-processing with an advantage distillation procedure. Using these methods, we find realistic parameters for which it is possible to achieve rates greater than each of the benchmarks, guiding the way towards implementing quantum repeaters.

ATXN2 trinucleotide repeat length correlates with risk of ALS.

PubMed

Sproviero, William; Shatunov, Aleksey; Stahl, Daniel; Shoai, Maryam; van Rheenen, Wouter; Jones, Ashley R; Al-Sarraj, Safa; Andersen, Peter M; Bonini, Nancy M; Conforti, Francesca L; Van Damme, Philip; Daoud, Hussein; Del Mar Amador, Maria; Fogh, Isabella; Forzan, Monica; Gaastra, Ben; Gellera, Cinzia; Gitler, Aaron D; Hardy, John; Fratta, Pietro; La Bella, Vincenzo; Le Ber, Isabelle; Van Langenhove, Tim; Lattante, Serena; Lee, Yi-Chung; Malaspina, Andrea; Meininger, Vincent; Millecamps, Stéphanie; Orrell, Richard; Rademakers, Rosa; Robberecht, Wim; Rouleau, Guy; Ross, Owen A; Salachas, Francois; Sidle, Katie; Smith, Bradley N; Soong, Bing-Wen; Sorarù, Gianni; Stevanin, Giovanni; Kabashi, Edor; Troakes, Claire; van Broeckhoven, Christine; Veldink, Jan H; van den Berg, Leonard H; Shaw, Christopher E; Powell, John F; Al-Chalabi, Ammar

2017-03-01

We investigated a CAG trinucleotide repeat expansion in the ATXN2 gene in amyotrophic lateral sclerosis (ALS). Two new case-control studies, a British dataset of 1474 ALS cases and 567 controls, and a Dutch dataset of 1328 ALS cases and 691 controls were analyzed. In addition, to increase power, we systematically searched PubMed for case-control studies published after 1 August 2010 that investigated the association between ATXN2 intermediate repeats and ALS. We conducted a meta-analysis of the new and existing studies for the relative risks of ATXN2 intermediate repeat alleles of between 24 and 34 CAG trinucleotide repeats and ALS. There was an overall increased risk of ALS for those carrying intermediate sized trinucleotide repeat alleles (odds ratio 3.06 [95% confidence interval 2.37-3.94]; p = 6 × 10 -18 ), with an exponential relationship between repeat length and ALS risk for alleles of 29-32 repeats (R 2  = 0.91, p = 0.0002). No relationship was seen for repeat length and age of onset or survival. In contrast to trinucleotide repeat diseases, intermediate ATXN2 trinucleotide repeat expansion in ALS does not predict age of onset but does predict disease risk. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

The effect of repeated testing vs repeated practice on skills learning in undergraduate dental education.

PubMed

Sennhenn-Kirchner, S; Goerlich, Y; Kirchner, B; Notbohm, M; Schiekirka, S; Simmenroth, A; Raupach, T

2018-02-01

Recent studies in undergraduate medical education have demonstrated the advantage of repeated testing over repeated practice with regard to knowledge and skills retention. The aim of this study was to investigate whether this "testing effect" also applies to skills retention in undergraduate dental education. In this prospective, randomised controlled trial, fourth-year dental students at Göttingen University Medical Centre participated in a training session on surgical suturing in winter term 2014/2015. Following this, they were either assigned to two sessions of additional skills training (group A) or two sessions of skills assessment with feedback (group B). These sessions were spaced over a period of 4 weeks. Skills retention was assessed in a summative objective structured clinical examination (OSCE) at the end of term, that is 6 months after the initial teaching session. A total of 32 students completed the study. With regard to suturing, OSCE performance was significantly better in group B than group A (81.9±13.1% vs 63.0±15.4%; P=0.001; Cohen's d=1.33). There was no significant OSCE performance difference in the two groups with regard to other learning objectives that were addressed in the end-of-term examination. Thus, the group difference was specific to suturing skills. This is the first study to demonstrate that in dental education, repeated testing produces more favourable skills retention than repeated practice. Test-enhanced learning might be a viable concept for skills retention in undergraduate dentistry education. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Continuous Influx of Genetic Material from Host to Virus Populations

PubMed Central

Gilbert, Clément; Peccoud, Jean; Chateigner, Aurélien; Moumen, Bouziane

2016-01-01

Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT) of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86) can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69) belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors. PMID:26829124

Continuous Influx of Genetic Material from Host to Virus Populations.

PubMed

Gilbert, Clément; Peccoud, Jean; Chateigner, Aurélien; Moumen, Bouziane; Cordaux, Richard; Herniou, Elisabeth A

2016-02-01

Many genes of large double-stranded DNA viruses have a cellular origin, suggesting that host-to-virus horizontal transfer (HT) of DNA is recurrent. Yet, the frequency of these transfers has never been assessed in viral populations. Here we used ultra-deep DNA sequencing of 21 baculovirus populations extracted from two moth species to show that a large diversity of moth DNA sequences (n = 86) can integrate into viral genomes during the course of a viral infection. The majority of the 86 different moth DNA sequences are transposable elements (TEs, n = 69) belonging to 10 superfamilies of DNA transposons and three superfamilies of retrotransposons. The remaining 17 sequences are moth sequences of unknown nature. In addition to bona fide DNA transposition, we uncover microhomology-mediated recombination as a mechanism explaining integration of moth sequences into viral genomes. Many sequences integrated multiple times at multiple positions along the viral genome. We detected a total of 27,504 insertions of moth sequences in the 21 viral populations and we calculate that on average, 4.8% of viruses harbor at least one moth sequence in these populations. Despite this substantial proportion, no insertion of moth DNA was maintained in any viral population after 10 successive infection cycles. Hence, there is a constant turnover of host DNA inserted into viral genomes each time the virus infects a moth. Finally, we found that at least 21 of the moth TEs integrated into viral genomes underwent repeated horizontal transfers between various insect species, including some lepidopterans susceptible to baculoviruses. Our results identify host DNA influx as a potent source of genetic diversity in viral populations. They also support a role for baculoviruses as vectors of DNA HT between insects, and call for an evaluation of possible gene or TE spread when using viruses as biopesticides or gene delivery vectors.

40 CFR 141.858 - Repeat monitoring and E. coli requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 23 2014-07-01 2014-07-01 false Repeat monitoring and E. coli....858 Repeat monitoring and E. coli requirements. (a) Repeat monitoring. (1) If a sample taken under... volume repeat sample(s) in one or more sample containers of any size, as long as the total volume...

Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice

PubMed Central

Huang, Shanshan; Yang, Su; Guo, Jifeng; Yan, Sen; Gaertig, Marta A.; Li, Shihua; Li, Xiao-Jiang

2015-01-01

SUMMARY In polyglutamine (polyQ) diseases, large polyQ repeats cause juvenile cases with different symptoms than adult-onset patients, who carry smaller expanded polyQ repeats. The mechanisms behind the differential pathology mediated by different polyQ repeat lengths remain unknown. By studying knock-in mouse models of spinal cerebellar ataxia-17 (SCA17), we found that a large polyQ (105 glutamines) in the TATA box-binding protein (TBP) preferentially causes muscle degeneration and reduces the expression of muscle-specific genes. Direct expression of TBP with different polyQ repeats in mouse muscle revealed that muscle degeneration is mediated only by the large polyQ repeats. Different polyQ repeats differentially alter TBP’s interaction with neuronal and muscle-specific transcription factors. As a result, the large polyQ repeat decreases the association of MyoD with TBP and DNA promoters. Our findings suggest that specific alterations in protein interactions by large polyQ repeats may account for the unique pathology in juvenile polyQ diseases. PMID:26387956

47 CFR 90.247 - Mobile repeater stations.

Code of Federal Regulations, 2010 CFR

2010-10-01

... of the mobile unit and an automatic time-delay device that de-activates the transmitter after any... 47 Telecommunication 5 2010-10-01 2010-10-01 false Mobile repeater stations. 90.247 Section 90.247... MOBILE RADIO SERVICES Non-Voice and Other Specialized Operations § 90.247 Mobile repeater stations. A...

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

Yokoo, Masako; Fujita, Ryosuke; Innate Immunity Laboratory, Graduate School of Life Science and Creative Research Institution, Hokkaido University, Sapporo 001-0021

Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes locatedmore » between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.« less

The genome sequence of Agrotis segetum granulovirus, isolate AgseGV-DA, reveals a new Betabaculovirus species of a slow killing granulovirus.

PubMed

Gueli Alletti, Gianpiero; Eigenbrod, Marina; Carstens, Eric B; Kleespies, Regina G; Jehle, Johannes A

2017-06-01

The European isolate Agrotis segetum granulovirus DA (AgseGV-DA) is a slow killing, type I granulovirus due to low dose-mortality responses within seven days post infection and a tissue tropism of infection restricted solely to the fat body of infected Agrotis segetum host larvae. The genome of AgseGV-DA was completely sequenced and compared to the whole genome sequences of the Chinese isolates AgseGV-XJ and AgseGV-L1. All three isolates share highly conserved genomes. The AgseGV-DA genome is 131,557bp in length and encodes for 149 putative open reading frames, including 37 baculovirus core genes and the per os infectivity factor ac110. Comprehensive investigations of repeat regions identified one putative non-hr like origin of replication in AgseGV-DA. Phylogenetic analysis based on concatenated amino acid alignments of 37 baculovirus core genes as well as pairwise distances based on the nucleotide alignments of partial granulin, lef-8 and lef-9 sequences with deposited betabaculoviruses confirmed AgseGV-DA, AgseGV-XJ and AgseGV-L1 as representative isolates of the same Betabaculovirus species. AgseGV encodes for a distinct putative enhancin, distantly related to enhancins from other granuloviruses. Copyright © 2017. Published by Elsevier Inc.

Accumulate repeat accumulate codes

NASA Technical Reports Server (NTRS)

Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

2004-01-01

In this paper we propose an innovative channel coding scheme called 'Accumulate Repeat Accumulate codes' (ARA). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes, thus belief propagation can be used for iterative decoding of ARA codes on a graph. The structure of encoder for this class can be viewed as precoded Repeat Accumulate (RA) code or as precoded Irregular Repeat Accumulate (IRA) code, where simply an accumulator is chosen as a precoder. Thus ARA codes have simple, and very fast encoder structure when they representing LDPC codes. Based on density evolution for LDPC codes through some examples for ARA codes, we show that for maximum variable node degree 5 a minimum bit SNR as low as 0.08 dB from channel capacity for rate 1/2 can be achieved as the block size goes to infinity. Thus based on fixed low maximum variable node degree, its threshold outperforms not only the RA and IRA codes but also the best known LDPC codes with the dame maximum node degree. Furthermore by puncturing the accumulators any desired high rate codes close to code rate 1 can be obtained with thresholds that stay close to the channel capacity thresholds uniformly. Iterative decoding simulation results are provided. The ARA codes also have projected graph or protograph representation that allows for high speed decoder implementation.

Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome.

PubMed

Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

2002-01-01

In humans, there are four alkaline phosphatases, and each form exhibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnant with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60-80% of activity. Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome.

Kinetic comparison of tissue non-specific and placental human alkaline phosphatases expressed in baculovirus infected cells: application to screening for Down's syndrome

PubMed Central

Denier, Colette C; Brisson-Lougarre, Andrée A; Biasini, Ghislaine G; Grozdea, Jean J; Fournier, Didier D

2002-01-01

Background In humans, there are four alkaline phosphatases, and each form exibits a characteristic pattern of tissue distribution. The availability of an easy method to reveal their activity has resulted in large amount of data reporting correlations between variations in activity and illnesses. For example, alkaline phosphatase from neutrophils of mothers pregnent with a trisomy 21 fetus (Down's syndrome) displays significant differences both in its biochemical and immunological properties, and in its affinity for some specific inhibitors. Results To analyse these differences, the biochemical characteristics of two isozymes (non specific and placental alkaline phosphatases) were expressed in baculovirus infected cells. Comparative analysis of the two proteins allowed us to estimate the kinetic constants of denaturation and sensitivity to two inhibitors (L-p-bromotetramisole and thiophosphate), allowing better discrimination between the two enzymes. These parameters were then used to estimate the ratio of the two isoenzymes in neutrophils of pregnant mothers with or without a trisomy 21 fetus. It appeared that the placental isozyme represented 13% of the total activity of neutrophils of non pregnant women. This proportion did not significantly increase with normal pregnancy. By contrast, in pregnancies with trisomy 21 fetus, the proportion reached 60–80% of activity. Conclusion Over-expression of the placental isozyme compared with the tissue-nonspecific form in neutrophils of mother with a trisomy 21 fetus may explain why the characteristics of the alkaline phosphatase in these cells is different from normal. Application of this knowledge could improve the potential of using alkaline phosphatase measurements to screen for Down's syndrome. PMID:11818032

Indian Academy of Pediatrics (IAP) recommended immunization schedule for children aged 0 through 18 years--India, 2014 and updates on immunization.

PubMed

Vashishtha, Vipin M; Choudhury, Panna; Kalra, Ajay; Bose, Anuradha; Thacker, Naveen; Yewale, Vijay N; Bansal, C P; Mehta, Pravin J

2014-10-01

There is a need to review/revise recommendations about existing vaccines in light of recent developments in the field of vaccinology. Following an IAP ACVIP meeting on April 19 and 20, 2014, a draft of revised recommendations for the year 2014 and updates on certain vaccine formulations was prepared and circulated among the meeting participants to arrive at a consensus. To review and revise recommendations for 2014 Immunization timetable for pediatricians in office practice and issue statements on certain new and existing vaccine formulations. The major changes in the 2014 Immunization Timetable include two doses of MMR vaccine at 9 and 15 months of age, single dose recommendation for administration of live attenuated H2 strain hepatitis A vaccine, inclusion of two new situations in high-risk category of children in context with pre-exposure prophylaxis of rabies, creation of a new slot at 9-12 months of age for typhoid conjugate vaccine for primary immunization, and recommendation of two doses of human papilloma virus vaccines with a minimum interval of 6 months between doses for primary schedule of adolescent/preadolescent girls aged 9-14 years. There would not be any change to the committee's last year's (2013) recommendations on pertussis vaccination and administration schedule of monovalent human rotavirus vaccine. There is no need of providing additional doses of whole-cell pertussis vaccine to children who have earlier completed their primary schedule with acellular pertussis vaccine-containing products. A brief update on the new Indian Rotavirus vaccine, 116E is also provided. The committee has reviewed and offered its recommendations on the currently available pentavalent vaccine (DTwP+Hib+Hepatitis-B) combinations in Indian market. The comments and footnotes for several vaccines are also updated and revised.

Intestinal alkaline phosphatase and sodium butyrate may be beneficial in attenuating LPS-induced intestinal inflammation.

PubMed

Melo, A D B; Silveira, H; Bortoluzzi, C; Lara, L J; Garbossa, C A P; Preis, G; Costa, L B; Rostagno, M H

2016-10-17

In this study, we evaluated the effect of intestinal alkaline phosphatase (IAP) and sodium butyrate (NaBu) on lipopolysaccharide (LPS)-induced intestinal inflammation. Intestinal alkaline phosphatase and RelA/p65 (NF-κB) gene expressions in porcine jejunum explants were evaluated following exposure to sodium butyrate (NaBu) and essential oil from Brazilian red pepper (EO), alone or in combination with NaBu, as well as exogenous IAP with or without LPS challenge. Five piglets weighing approximately 20 kg each were sacrificed, and their jejunum were extracted. The tissues were segmented into 10 parts, which were exposed to 10 treatments. Gene expressions of IAP and RelA/p65 (NF-κB) in jejunal explants were evaluated via RT-PCR. We found that EO, NaBu, and exogenous IAP were able to up-regulate endogenous IAP and enhance RelA/p65 (NF-κB) gene expression. However, only NaBu and exogenous IAP down-regulated LPS-induced inflammatory response via RelA/p65 (NF-κB). In conclusion, we demonstrated that exogenous IAP and NaBu may be beneficial in attenuating LPS-induced intestinal inflammation.

RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL.

PubMed

Lawlor, Kate E; Khan, Nufail; Mildenhall, Alison; Gerlic, Motti; Croker, Ben A; D'Cruz, Akshay A; Hall, Cathrine; Kaur Spall, Sukhdeep; Anderton, Holly; Masters, Seth L; Rashidi, Maryam; Wicks, Ian P; Alexander, Warren S; Mitsuuchi, Yasuhiro; Benetatos, Christopher A; Condon, Stephen M; Wong, W Wei-Lynn; Silke, John; Vaux, David L; Vince, James E

2015-02-18

RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1β inflammatory responses independent of MLKL and necroptotic cell death.

A repeating fast radio burst

NASA Astrophysics Data System (ADS)

Spitler, L. G.; Scholz, P.; Hessels, J. W. T.; Bogdanov, S.; Brazier, A.; Camilo, F.; Chatterjee, S.; Cordes, J. M.; Crawford, F.; Deneva, J.; Ferdman, R. D.; Freire, P. C. C.; Kaspi, V. M.; Lazarus, P.; Lynch, R.; Madsen, E. C.; McLaughlin, M. A.; Patel, C.; Ransom, S. M.; Seymour, A.; Stairs, I. H.; Stappers, B. W.; van Leeuwen, J.; Zhu, W. W.

2016-03-01

Fast radio bursts are millisecond-duration astronomical radio pulses of unknown physical origin that appear to come from extragalactic distances. Previous follow-up observations have failed to find additional bursts at the same dispersion measure (that is, the integrated column density of free electrons between source and telescope) and sky position as the original detections. The apparent non-repeating nature of these bursts has led to the suggestion that they originate in cataclysmic events. Here we report observations of ten additional bursts from the direction of the fast radio burst FRB 121102. These bursts have dispersion measures and sky positions consistent with the original burst. This unambiguously identifies FRB 121102 as repeating and demonstrates that its source survives the energetic events that cause the bursts. Additionally, the bursts from FRB 121102 show a wide range of spectral shapes that appear to be predominantly intrinsic to the source and which vary on timescales of minutes or less. Although there may be multiple physical origins for the population of fast radio bursts, these repeat bursts with high dispersion measure and variable spectra specifically seen from the direction of FRB 121102 support an origin in a young, highly magnetized, extragalactic neutron star.

A repeating fast radio burst.

PubMed

Spitler, L G; Scholz, P; Hessels, J W T; Bogdanov, S; Brazier, A; Camilo, F; Chatterjee, S; Cordes, J M; Crawford, F; Deneva, J; Ferdman, R D; Freire, P C C; Kaspi, V M; Lazarus, P; Lynch, R; Madsen, E C; McLaughlin, M A; Patel, C; Ransom, S M; Seymour, A; Stairs, I H; Stappers, B W; van Leeuwen, J; Zhu, W W

2016-03-10

Fast radio bursts are millisecond-duration astronomical radio pulses of unknown physical origin that appear to come from extragalactic distances. Previous follow-up observations have failed to find additional bursts at the same dispersion measure (that is, the integrated column density of free electrons between source and telescope) and sky position as the original detections. The apparent non-repeating nature of these bursts has led to the suggestion that they originate in cataclysmic events. Here we report observations of ten additional bursts from the direction of the fast radio burst FRB 121102. These bursts have dispersion measures and sky positions consistent with the original burst. This unambiguously identifies FRB 121102 as repeating and demonstrates that its source survives the energetic events that cause the bursts. Additionally, the bursts from FRB 121102 show a wide range of spectral shapes that appear to be predominantly intrinsic to the source and which vary on timescales of minutes or less. Although there may be multiple physical origins for the population of fast radio bursts, these repeat bursts with high dispersion measure and variable spectra specifically seen from the direction of FRB 121102 support an origin in a young, highly magnetized, extragalactic neutron star.

RTEL1 inhibits trinucleotide repeat expansions and fragility.

PubMed

Frizzell, Aisling; Nguyen, Jennifer H G; Petalcorin, Mark I R; Turner, Katherine D; Boulton, Simon J; Freudenreich, Catherine H; Lahue, Robert S

2014-03-13

Human RTEL1 is an essential, multifunctional helicase that maintains telomeres, regulates homologous recombination, and helps prevent bone marrow failure. Here, we show that RTEL1 also blocks trinucleotide repeat expansions, the causal mutation for 17 neurological diseases. Increased expansion frequencies of (CTG⋅CAG) repeats occurred in human cells following knockdown of RTEL1, but not the alternative helicase Fbh1, and purified RTEL1 efficiently unwound triplet repeat hairpins in vitro. The expansion-blocking activity of RTEL1 also required Rad18 and HLTF, homologs of yeast Rad18 and Rad5. These findings are reminiscent of budding yeast Srs2, which inhibits expansions, unwinds hairpins, and prevents triplet-repeat-induced chromosome fragility. Accordingly, we found expansions and fragility were suppressed in yeast srs2 mutants expressing RTEL1, but not Fbh1. We propose that RTEL1 serves as a human analog of Srs2 to inhibit (CTG⋅CAG) repeat expansions and fragility, likely by unwinding problematic hairpins. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

[Convergent origin of repeats in genes coding for globular proteins. An analysis of the factors determining the presence of inverted and symmetrical repeats].

PubMed

Solov'ev, V V; Kel', A E; Kolchanov, N A

1989-01-01

The factors, determining the presence of inverted and symmetrical repeats in genes coding for globular proteins, have been analysed. An interesting property of genetical code has been revealed in the analysis of symmetrical repeats: the pairs of symmetrical codons corresponded to pairs of amino acids with mostly similar physical-chemical parameters. This property may explain the presence of symmetrical repeats and palindromes only in genes coding for beta-structural proteins-polypeptides, where amino acids with similar physical-chemical properties occupy symmetrical positions. A stochastic model of evolution of polynucleotide sequences has been used for analysis of inverted repeats. The modelling demonstrated that only limiting of sequences (uneven frequencies of used codons) is enough for arising of nonrandom inverted repeats in genes.

Immunization of mice with baculovirus-derived recombinant SV40 large tumour antigen induces protective tumour immunity to a lethal challenge with SV40-transformed cells.

PubMed Central

Shearer, M H; Bright, R K; Lanford, R E; Kennedy, R C

1993-01-01

In this study, we examined the humoral immune responses and in vivo tumour immunity induced by baculovirus recombinant simian virus 40 (SV40) large tumour antigen (rSV40 T-ag). BALB/c mice immunized with rSV40 T-ag produced antibody responses that recognized SV40 large tumour antigen (T-ag) by ELISA. Analysis of these anti-SV40 T-ag responses indicated that the antibodies recognized epitopes associated with both the carboxy and amino terminus of SV40 T-ag. This pattern of SV40 T-ag epitope recognition was similar to that observed in anti-SV40 T-ag responses induced by inoculation with irradiated SV40-transformed cells. Mice immunized with either rSV40 T-ag or with the inactivated transformed cells were protected from a subsequent in vivo lethal tumour challenge with live SV40-transformed cells. These studies suggest that humoral immune responses induced by rSV40 T-ag are similar in epitope specificity to that induced by inactivated SV40-transformed cells. In addition, recombinant tumour-specific antigens from papovaviruses, such as SV40, can be used to induce tumour immunity which protects from a subsequent lethal tumour challenge. This study may provide insight into the use of recombinant tumour antigens as putative tumour vaccines and in the development of active immunotherapeutic strategies for treating virus-induced cancers. PMID:7679059

47 CFR 90.247 - Mobile repeater stations.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 47 Telecommunication 5 2014-10-01 2014-10-01 false Mobile repeater stations. 90.247 Section 90.247... MOBILE RADIO SERVICES Non-Voice and Other Specialized Operations § 90.247 Mobile repeater stations. A mobile station authorized to operate on a mobile service frequency above 25 MHz may be used as a mobile...

47 CFR 90.247 - Mobile repeater stations.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 47 Telecommunication 5 2013-10-01 2013-10-01 false Mobile repeater stations. 90.247 Section 90.247... MOBILE RADIO SERVICES Non-Voice and Other Specialized Operations § 90.247 Mobile repeater stations. A mobile station authorized to operate on a mobile service frequency above 25 MHz may be used as a mobile...

47 CFR 90.247 - Mobile repeater stations.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 5 2011-10-01 2011-10-01 false Mobile repeater stations. 90.247 Section 90.247... MOBILE RADIO SERVICES Non-Voice and Other Specialized Operations § 90.247 Mobile repeater stations. A mobile station authorized to operate on a mobile service frequency above 25 MHz may be used as a mobile...

47 CFR 90.247 - Mobile repeater stations.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 47 Telecommunication 5 2012-10-01 2012-10-01 false Mobile repeater stations. 90.247 Section 90.247... MOBILE RADIO SERVICES Non-Voice and Other Specialized Operations § 90.247 Mobile repeater stations. A mobile station authorized to operate on a mobile service frequency above 25 MHz may be used as a mobile...

Oxidative stress adaptation with acute, chronic, and repeated stress.

PubMed

Pickering, Andrew M; Vojtovich, Lesya; Tower, John; A Davies, Kelvin J

2013-02-01

Oxidative stress adaptation, or hormesis, is an important mechanism by which cells and organisms respond to, and cope with, environmental and physiological shifts in the level of oxidative stress. Most studies of oxidative stress adaption have been limited to adaptation induced by acute stress. In contrast, many if not most environmental and physiological stresses are either repeated or chronic. In this study we find that both cultured mammalian cells and the fruit fly Drosophila melanogaster are capable of adapting to chronic or repeated stress by upregulating protective systems, such as their proteasomal proteolytic capacity to remove oxidized proteins. Repeated stress adaptation resulted in significant extension of adaptive responses. Repeated stresses must occur at sufficiently long intervals, however (12-h or more for MEF cells and 7 days or more for flies), for adaptation to be successful, and the levels of both repeated and chronic stress must be lower than is optimal for adaptation to acute stress. Regrettably, regimens of adaptation to both repeated and chronic stress that were successful for short-term survival in Drosophila nevertheless also caused significant reductions in life span for the flies. Thus, although both repeated and chronic stress can be tolerated, they may result in a shorter life. Copyright © 2012 Elsevier Inc. All rights reserved.

Childhood remembered: Reports of both unique and repeated events.

PubMed

Peterson, Carole; Baker-Ward, Lynne; Grovenstein, Tiffany N

2016-01-01

To explore the significance of repeated memories for individuals' personal histories, we compared the characteristics of young adults' unique and repeated memories of childhood experiences. Memory type (unique vs. repeated) was a within-participant variable. In Experiment 1, college-age participants generated as many early memories as possible in 4 minutes; in Experiment 2, another sample provided complete reports of five early memories in each condition. In both experiments, participants rated the vividness, biographical importance and personal meaning of each memory and labelled the accompanying emotion. Unique memories were more vivid than repeated memories as well as more likely to include negative emotion, regardless of the method of reporting. Most importantly, college students rated their memories for unique and repeated events as equivalently infused with personal meaning. Analysis of the content of the memories reported in Experiment 2 established that unique and repeated memories did not differ in word count or percentages of perceptual terms or words indicating positive affect, although unique memories contained a greater percentage of negative affect. Additional analyses of content provided evidence for differences in the functions served by unique and repeated memories. The results have implications for the study of autobiographical memory and for identifying over-general memories.

Environmental stress induces trinucleotide repeat mutagenesis in human cells.

PubMed

Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A; Yotnda, Patricia; Wilson, John H

2015-03-24

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-the cause of multiple human diseases-have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential.

Security of Quantum Repeater Network Operation

DTIC Science & Technology

2016-10-03

readily in quantum networks than in classical networks. Our presentation at the SENT workshop attracted the attention of computer and network researchers...AFRL-AFOSR-JP-TR-2016-0079 Security of Quantum Repeater Network Operation Rodney Van Meter KEIO UNIVERSITY Final Report 10/03/2016 DISTRIBUTION A...To)  29 May 2014 to 28 May 2016 4. TITLE AND SUBTITLE Security of Quantum Repeater Network Operation 5a.  CONTRACT NUMBER 5b.  GRANT NUMBER FA2386

Repeatability of DTI-based skeletal muscle fiber tracking

PubMed Central

Heemskerk, Anneriet M.; Sinha, Tuhin K.; Wilson, Kevin J.; Ding, Zhaohua; Damon, Bruce M.

2015-01-01

Diffusion tensor imaging (DTI)-based muscle fiber tracking enables the measurement of muscle architectural parameters, such as pennation angle (θ) and fiber tract length (Lft), throughout the entire muscle. Little is known, however, about the repeatability of either the muscle architectural measures or the underlying diffusion measures. Therefore, the goal of this study was to investigate the repeatability of DTI fiber tracking-based measurements and θ and Lft. Four DTI acquisitions were performed on two days that allowed for between acquisition, within day, and between day analyses. The eigenvalues and fractional anisotropy were calculated at the maximum cross-sectional area of, and fiber tracking was performed in, the tibialis anterior muscle of nine healthy subjects. The between acquisitions condition had the highest repeatability for the DTI indices and the architectural parameters. The overall inter class correlation coefficients (ICC’s) were greater than 0.6 for both θ and Lft and the repeatability coefficients were θ <10.2° and Lft < 50 mm. In conclusion, under the experimental and data analysis conditions used, the repeatability of the diffusion measures is very good and repeatability of the architectural measurements is acceptable. Therefore, this study demonstrates the feasibility for longitudinal studies of alterations in muscle architecture using DTI-based fiber tracking, under similar noise conditions and with similar diffusion characteristics. PMID:20099372

Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota.

PubMed

Bates, Jennifer M; Akerlund, Janie; Mittge, Erika; Guillemin, Karen

2007-12-13

Vertebrates harbor abundant lipopolysaccharide (LPS) in their gut microbiota. Alkaline phosphatases can dephosphorylate and detoxify the endotoxin component of LPS. Here, we show that expression of the zebrafish intestinal alkaline phosphatase (Iap), localized to the intestinal lumen brush border, is induced during establishment of the gut microbiota. Iap-deficient zebrafish are hypersensitive to LPS toxicity and exhibit the excessive intestinal neutrophil influx characteristic of wild-type zebrafish exposed to LPS. Both of these Iap mutant phenotypes are dependent on Myd88 and Tumor Necrosis Factor Receptor (Tnfr), proteins also involved in LPS sensitivity in mammals. When reared germ-free, the intestines of Iap-deficient zebrafish are devoid of neutrophils. Together, these findings demonstrate that the endogenous microbiota establish the normal homeostatic level of neutrophils in the zebrafish intestine through a process involving Iap, Myd88, and Tnfr. Thus, by preventing inflammatory responses, Iap plays a crucial role in promoting mucosal tolerance to resident gut bacteria.

Optimization of infrared two-color multicycle field synthesis for intense-isolated-attosecond-pulse generation

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

Lan Pengfei; Takahashi, Eiji J.; Midorikawa, Katsumi

2010-11-15

We present the optimization of the two-color synthesis method for generating an intense isolated attosecond pulse (IAP) in the multicycle regime. By mixing an infrared assistant pulse with a Ti:sapphire main pulse, we show that an IAP can be produced using a multicycle two-color pulse with a duration longer than 30 fs. We also discuss the influence of the carrier-envelope phase (CEP) and the relative intensity on the generation of IAPs. By optimizing the wavelength of the assistant field, IAP generation becomes insensitive to the CEP slip. Therefore, the optimized two-color method enables us to relax the requirements of pulsemore » duration and easily produce the IAP with a conventional multicycle laser pulse. In addition, it enables us to markedly suppress the ionization of the harmonic medium. This is a major advantage for efficiently generating intense IAPs from a neutral medium by applying the appropriate phase-matching and energy-scaling techniques.« less

The Ubiquitin-associated Domain of Cellular Inhibitor of Apoptosis Proteins Facilitates Ubiquitylation*

PubMed Central

Budhidarmo, Rhesa; Day, Catherine L.

2014-01-01

The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases. PMID:25065467

Algorithm to find distant repeats in a single protein sequence

PubMed Central

Banerjee, Nirjhar; Sarani, Rangarajan; Ranjani, Chellamuthu Vasuki; Sowmiya, Govindaraj; Michael, Daliah; Balakrishnan, Narayanasamy; Sekar, Kanagaraj

2008-01-01

Distant repeats in protein sequence play an important role in various aspects of protein analysis. A keen analysis of the distant repeats would enable to establish a firm relation of the repeats with respect to their function and three-dimensional structure during the evolutionary process. Further, it enlightens the diversity of duplication during the evolution. To this end, an algorithm has been developed to find all distant repeats in a protein sequence. The scores from Point Accepted Mutation (PAM) matrix has been deployed for the identification of amino acid substitutions while detecting the distant repeats. Due to the biological importance of distant repeats, the proposed algorithm will be of importance to structural biologists, molecular biologists, biochemists and researchers involved in phylogenetic and evolutionary studies. PMID:19052663

GFP-based fluorescence assay for CAG repeat instability in cultured human cells.

PubMed

Santillan, Beatriz A; Moye, Christopher; Mittelman, David; Wilson, John H

2014-01-01

Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

Repeated human leukocyte antigen mismatches in lung re-transplantation.

PubMed

Sommer, Wiebke; Hallensleben, Michael; Ius, Fabio; Kühn, Christian; Tudorache, Igor; Avsar, Murat; Salman, Jawad; Siemeni, Thierry; Greer, Mark; Gottlieb, Jens; Boethig, Dietmar; Blasczyk, Rainer; Haverich, Axel; Warnecke, Gregor

2017-02-01

The role of HLA-sensitization in the absence of detectable DSA in lung re-transplantation is unclear. Antigens of the second donor matching the HLA typing of the first donor are considered 'unacceptable', by some tissue typing laboratories, especially in kidney re-transplantation. Thus, we performed a retrospective analysis of all lung re-transplantations focussing on the impact of HLA-homologies between the first and the second donor ('unacceptable' antigens; repeated HLA mismatch) on patient and graft survival. A total of 132 lung re-transplantations were performed at our centre between 1985 and 2014, of which 120 with complete HLA data were analysed. 55.8% of the recipients received re-transplants with repeated HLA mismatched antigens whereas 43.2% of the re-transplants were transplanted without repeated HLA mismatched antigens. Postoperative survival showed no difference between re-transplant procedures with or without repeated HLA mismatches (p=0.99). While neither homologies on the HLA-A, -B, -C, or -DR locus, nor the addition of several locus homologies (p=0.72) had an impact on survival, unexpectedly, repeated HLA mismatching on the HLA-DQ locus was correlated with better survival. Re-transplantations with repeated HLA mismatches did not result in more development of CLAD as compared to recipients without repeated HLA mismatches (p=0.99). Neither the number of repeated HLA mismatched antigens (p=0.52) nor the HLA locus (HLA-A(p=0.34), HLA-B(p=0.97), HLA-C (p=0.80), HLA-DR(p=0.49) and HLA-DQ(p=0.07)) had an impact on the development of CLAD after re-transplantation. Transplantation with repeated HLA mismatches due to sensitization by a previous transplantation in the absence of detectable HLA-antibodies does not have a negative impact on patient or graft survival. Copyright © 2016 Elsevier B.V. All rights reserved.

Repeat Gamma Knife Radiosurgery for Trigeminal Neuralgia

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

Aubuchon, Adam C., E-mail: acaubuchon@gmail.com; Chan, Michael D.; Lovato, James F.

2011-11-15

Purpose: Repeat gamma knife stereotactic radiosurgery (GKRS) for recurrent or persistent trigeminal neuralgia induces an additional response but at the expense of an increased incidence of facial numbness. The present series summarized the results of a repeat treatment series at Wake Forest University Baptist Medical Center, including a multivariate analysis of the data to identify the prognostic factors for treatment success and toxicity. Methods and Materials: Between January 1999 and December 2007, 37 patients underwent a second GKRS application because of treatment failure after a first GKRS treatment. The mean initial dose in the series was 87.3 Gy (range, 80-90).more » The mean retreatment dose was 84.4 Gy (range, 60-90). The dosimetric variables recorded included the dorsal root entry zone dose, pons surface dose, and dose to the distal nerve. Results: Of the 37 patients, 81% achieved a >50% pain relief response to repeat GKRS, and 57% experienced some form of trigeminal dysfunction after repeat GKRS. Two patients (5%) experienced clinically significant toxicity: one with bothersome numbness and one with corneal dryness requiring tarsorraphy. A dorsal root entry zone dose at repeat treatment of >26.6 Gy predicted for treatment success (61% vs. 32%, p = .0716). A cumulative dorsal root entry zone dose of >84.3 Gy (72% vs. 44%, p = .091) and a cumulative pons surface dose of >108.5 Gy (78% vs. 44%, p = .018) predicted for post-GKRS numbness. The presence of any post-GKRS numbness predicted for a >50% decrease in pain intensity (100% vs. 60%, p = .0015). Conclusion: Repeat GKRS is a viable treatment option for recurrent trigeminal neuralgia, although the patient assumes a greater risk of nerve dysfunction to achieve maximal pain relief.« less

Proteolytic Processing and Assembly of gag and gag-pol Proteins of TED, a Baculovirus-Associated Retrotransposon of the Gypsy Family

PubMed Central

Hajek, Kathryn L.; Friesen, Paul D.

1998-01-01

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55gag) is cleaved to produce a single VLP structural protein, p37gag. Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55gag cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195gag-pol. The PR cleavage site within Pr55gag was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55gag truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55gag abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37gag provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging. PMID:9765414

Proteolytic processing and assembly of gag and gag-pol proteins of TED, a baculovirus-associated retrotransposon of the gypsy family.

PubMed

Hajek, K L; Friesen, P D

1998-11-01

TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55(gag)) is cleaved to produce a single VLP structural protein, p37(gag). Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55(gag) cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195(gag-pol). The PR cleavage site within Pr55(gag) was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55(gag) truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55(gag) abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37(gag) provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.

Expression of human electron transfer flavoprotein-ubiquinone oxidoreductase from a baculovirus vector: kinetic and spectral characterization of the human protein.

PubMed

Simkovic, Martin; Degala, Gregory D; Eaton, Sandra S; Frerman, Frank E

2002-06-15

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulphur flavoprotein and a component of an electron-transfer system that links 10 different mitochondrial flavoprotein dehydrogenases to the mitochondrial bc1 complex via electron transfer flavoprotein (ETF) and ubiquinone. ETF-QO is an integral membrane protein, and the primary sequences of human and porcine ETF-QO were deduced from the sequences of the cloned cDNAs. We have expressed human ETF-QO in Sf9 insect cells using a baculovirus vector. The cDNA encoding the entire protein, including the mitochondrial targeting sequence, was present in the vector. We isolated a membrane-bound form of the enzyme that has a molecular mass identical with that of the mature porcine protein as determined by SDS/PAGE and has an N-terminal sequence that is identical with that predicted for the mature holoenzyme. These data suggest that the heterologously expressed ETF-QO is targeted to mitochondria and processed to the mature, catalytically active form. The detergent-solubilized protein was purified by ion-exchange and hydroxyapatite chromatography. Absorption and EPR spectroscopy and redox titrations are consistent with the presence of flavin and iron-sulphur centres that are very similar to those in the equivalent porcine and bovine proteins. Additionally, the redox potentials of the two prosthetic groups appear similar to those of the other eukaryotic ETF-QO proteins. The steady-state kinetic constants of human ETF-QO were determined with ubiquinone homologues, a ubiquinone analogue, and with human wild-type ETF and a Paracoccus-human chimaeric ETF as varied substrates. The results demonstrate that this expression system provides sufficient amounts of human ETF-QO to enable crystallization and mechanistic investigations of the iron-sulphur flavoprotein.

Diversity and evolution of centromere repeats in the maize genome.

PubMed

Bilinski, Paul; Distor, Kevin; Gutierrez-Lopez, Jose; Mendoza, Gabriela Mendoza; Shi, Jinghua; Dawe, R Kelly; Ross-Ibarra, Jeffrey

2015-03-01

Centromere repeats are found in most eukaryotes and play a critical role in kinetochore formation. Though centromere repeats exhibit considerable diversity both within and among species, little is understood about the mechanisms that drive centromere repeat evolution. Here, we use maize as a model to investigate how a complex history involving polyploidy, fractionation, and recent domestication has impacted the diversity of the maize centromeric repeat CentC. We first validate the existence of long tandem arrays of repeats in maize and other taxa in the genus Zea. Although we find considerable sequence diversity among CentC copies genome-wide, genetic similarity among repeats is highest within these arrays, suggesting that tandem duplications are the primary mechanism for the generation of new copies. Nonetheless, clustering analyses identify similar sequences among distant repeats, and simulations suggest that this pattern may be due to homoplasious mutation. Although the two ancestral subgenomes of maize have contributed nearly equal numbers of centromeres, our analysis shows that the majority of all CentC repeats derive from one of the parental genomes, with an even stronger bias when examining the largest assembled contiguous clusters. Finally, by comparing maize with its wild progenitor teosinte, we find that the abundance of CentC likely decreased after domestication, while the pericentromeric repeat Cent4 has drastically increased.

The evolution of filamin-a protein domain repeat perspective.

PubMed

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

2012-09-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

Repeatability and Reproducibility of Decisions by Latent Fingerprint Examiners

PubMed Central

Ulery, Bradford T.; Hicklin, R. Austin; Buscaglia, JoAnn; Roberts, Maria Antonia

2012-01-01

The interpretation of forensic fingerprint evidence relies on the expertise of latent print examiners. We tested latent print examiners on the extent to which they reached consistent decisions. This study assessed intra-examiner repeatability by retesting 72 examiners on comparisons of latent and exemplar fingerprints, after an interval of approximately seven months; each examiner was reassigned 25 image pairs for comparison, out of total pool of 744 image pairs. We compare these repeatability results with reproducibility (inter-examiner) results derived from our previous study. Examiners repeated 89.1% of their individualization decisions, and 90.1% of their exclusion decisions; most of the changed decisions resulted in inconclusive decisions. Repeatability of comparison decisions (individualization, exclusion, inconclusive) was 90.0% for mated pairs, and 85.9% for nonmated pairs. Repeatability and reproducibility were notably lower for comparisons assessed by the examiners as “difficult” than for “easy” or “moderate” comparisons, indicating that examiners' assessments of difficulty may be useful for quality assurance. No false positive errors were repeated (n = 4); 30% of false negative errors were repeated. One percent of latent value decisions were completely reversed (no value even for exclusion vs. of value for individualization). Most of the inter- and intra-examiner variability concerned whether the examiners considered the information available to be sufficient to reach a conclusion; this variability was concentrated on specific image pairs such that repeatability and reproducibility were very high on some comparisons and very low on others. Much of the variability appears to be due to making categorical decisions in borderline cases. PMID:22427888

Security of Quantum Repeater Network Operation

DTIC Science & Technology

2016-10-03

AFRL-AFOSR-JP-TR-2016-0079 Security of Quantum Repeater Network Operation Rodney Van Meter KEIO UNIVERSITY Final Report 10/03/2016 DISTRIBUTION A...To)  29 May 2014 to 28 May 2016 4. TITLE AND SUBTITLE Security of Quantum Repeater Network Operation 5a.  CONTRACT NUMBER 5b.  GRANT NUMBER FA2386...ABSTRACT Much of the work on quantum networks , both entangled and unentangled, has been about the uses of quantum networks to enhance end- host security

Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering

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

Naing, Swe-Htet; Oliver, Ryan C.; Weiss, Kevin L.

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, andmore » octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D 2O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. In conclusion, our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.« less

Solution Structure of an Intramembrane Aspartyl Protease via Small Angle Neutron Scattering

DOE PAGES

Naing, Swe-Htet; Oliver, Ryan C.; Weiss, Kevin L.; ...

2018-02-06

Intramembrane aspartyl proteases (IAPs) comprise one of four families of integral membrane proteases that hydrolyze substrates within the hydrophobic lipid bilayer. IAPs include signal peptide peptidase, which processes remnant signal peptides from nascent polypeptides in the endoplasmic reticulum, and presenilin, the catalytic component of the γ-secretase complex that processes Notch and amyloid precursor protein. Despite their broad biomedical reach, basic structure-function relationships of IAPs remain active areas of research. Characterization of membrane-bound proteins is notoriously challenging due to their inherently hydrophobic character. For IAPs, oligomerization state in solution is one outstanding question, with previous proposals for monomer, dimer, tetramer, andmore » octamer. Here we used small angle neutron scattering (SANS) to characterize n-dodecyl-β-D-maltopyranoside (DDM) detergent solutions containing and absent a microbial IAP ortholog. A unique feature of SANS is the ability to modulate the solvent composition to mask all but the enzyme of interest. The signal from the IAP was enhanced by deuteration and, uniquely, scattering from DDM and buffers were matched by the use of both tail-deuterated DDM and D 2O. The radius of gyration calculated for IAP and the corresponding ab initio consensus model are consistent with a monomer. The model is slightly smaller than the crystallographic IAP monomer, suggesting a more compact protein in solution compared with the crystal lattice. In conclusion, our study provides direct insight into the oligomeric state of purified IAP in surfactant solution, and demonstrates the utility of fully contrast-matching the detergent in SANS to characterize other intramembrane proteases and their membrane-bound substrates.« less

More complicated than it looks: The vagaries of calculating intra-abdominal pressure

PubMed Central

Hamad, Nadia M.; Shaw, Janet M.; Nygaard, Ingrid E.; Coleman, Tanner J.; Hsu, Yvonne; Egger, Marlene; Hitchcock, Robert W.

2013-01-01

Activities thought to induce high intra-abdominal pressure (IAP), such as lifting weights, are restricted in women with pelvic floor disorders. Standardized procedures to assess IAP during activity are lacking and typically only focus on maximal IAP, variably defined. Our intent in this methods paper is to establish the best strategies for calculating maximal IAP and to add area under the curve and first moment of the area as potentially useful measures in understanding biologic effects of IAP. Thirteen women completed a range of activities while wearing an intra-vaginal pressure transducer. We first analyzed various strategies heuristically using data from 3 women. The measure that appeared to best represent maximal IAP was an average of the three, five or ten highest values, depending on activity, determined using a top down approach, with peaks at least 1 second apart using algorithms written for Matlab computer software, we then compared this strategy with others commonly reported in the literature quantitatively using data from 10 additional volunteers. Maximal IAP calculated using the top down approach differed for some, but not all, activities compared to the single highest peak or to averaging all peaks. We also calculated area under the curve, which allows for a time component, and first moment of the area, which maintains the time component while weighting pressure amplitude. We validated methods of assessing IAP using computer-generated sine waves. We offer standardized methods for assessing maximal, area under the curve and first moment of the area for IAP to improve future reporting and application of this clinically relevant measure in exercise science. PMID:23439349

GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells

PubMed Central

Santillan, Beatriz A.; Moye, Christopher; Mittelman, David; Wilson, John H.

2014-01-01

Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries. PMID:25423602

Exploring the repeat protein universe through computational protein design

DOE PAGES

Brunette, TJ; Parmeggiani, Fabio; Huang, Po-Ssu; ...

2015-12-16

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. In this paper, we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix–loop–helix–loop structural motif. Eighty-three designs with sequences unrelatedmore » to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Finally, our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.« less

Environmental stress induces trinucleotide repeat mutagenesis in human cells

PubMed Central

Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A.; Yotnda, Patricia; Wilson, John H.

2015-01-01

The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)—the cause of multiple human diseases—have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

Effectiveness of different avian influenza (H5) vaccination regimens in layer chickens on the humoral immune response and interferon-alpha signalling immune marker.

PubMed

Hamad, Mustafa; Amen, Omar; Mahmoud, Mohamed; Hassanin, Ola; Saif-Edin, Mostafa

2018-06-01

Avian influenza (AI) vaccines are widely used to control and eliminate the ongoing avian influenza virus epidemic in Egypt. A strict vaccination policy with inactivated AI vaccines has been widely applied, however the virus still circulating, evolving and causing great negative impact to the poultry sector in Egypt. Therefore, an updated poultry vaccination policy using different vaccine technologies might be valuable as an innovative additional control strategy of AIV in Egypt. In the present study, the effectiveness of different avian influenza (AI) vaccination schedules was evaluated in 300 commercial layer chicks (ISA White) using either the oil-emulsion baculovirus-H5-prototype vaccine (baculovirus-H5 prototype) or turkey herpesvirus (HVT) vector vaccine containing the hemagglutinin (HA) gene from H5N1 strain (rHVT-H5), applied alone or in combination and in different settings. Vaccination with either two injections of the baculovirus-H5 prototype, a single injection of rHVT-H5 or priming with rHVT-H5 at 1 day old followed by boosting with the baculovirus-H5 prototype induced AI-HI protective antibody responses starting as early as 3 to 4 weeks of age and lasting up to the end of the rearing period (16 weeks). A single vaccination with the baculovirus-H5 prototype did not generate a protective antibody titre for the entire rearing period. Furthermore, the present study elucidated that vaccination once or twice with the baculovirus-H5 vaccine prototype activated the chicken interferon-alpha (Ch-IFN-alpha) signalling pathway via transduction of antiviral components, e.g., Mx1 and IRF7. Birds immunized once with rHVT-H5 at 1 day old did not show activation of the Mx1 and IRF7 transcripts; however, following boosting with the baculovirus-H5 prototype vaccine, up-regulation of Mx1 and IRF7 was observed. Based on our findings, it can be concluded that either reinforcement with two injections of the baculovirus-H5 prototype or prime-boost vaccination (rHVT-H5 at

Repeated high-intensity exercise in professional rugby union.

PubMed

Austin, Damien; Gabbett, Tim; Jenkins, David

2011-07-01

The aim of the present study was to describe the frequency, duration, and nature of repeated high-intensity exercise in Super 14 rugby union. Time-motion analysis was used during seven competition matches over the 2008 and 2009 Super 14 seasons; five players from each of four positional groups (front row forwards, back row forwards, inside backs, and outside backs) were assessed (20 players in total). A repeated high-intensity exercise bout was considered to involve three or more sprints, and/or tackles and/or scrum/ruck/maul activities within 21 s during the same passage of play. The range of repeated high-intensity exercise bouts for each group in a match was as follows: 11-18 for front row forwards, 11-21 for back row forwards, 13-18 for inside backs, and 2-11 for outside backs. The durations of the most intense repeated high-intensity exercise bouts for each position ranged from 53 s to 165 s and the minimum recovery periods between repeated high-intensity exercise bouts ranged from 25 s for the back row forwards to 64 s for the front row forwards. The present results show that repeated high-intensity exercise bouts vary in duration and activities relative to position but all players in a game will average at least 10 changes in activity in the most demanding bouts and complete at least one tackle and two sprints. The most intense periods of activity are likely to last as long as 120 s and as little as 25 s recovery may separate consecutive repeated high-intensity exercise bouts. The present findings can be used by coaches to prepare their players for the most demanding passages of play likely to be experienced in elite rugby union.

Strategy When Faced with Failure: Persistence and Degree Attainment of Course Repeaters versus Non-Repeaters. AIR 2002 Forum Paper.

ERIC Educational Resources Information Center

Fenton, Kathleen S.

Graduation and persistence rates were compared for 184 students, 92 of whom had repeated multiple courses or at least 1 course 3 times. A control group of 92 nonrepeating students was drawn from the remaining 303 students of the entire 1996 cohort. There was no difference between the graduation rate of repeaters and nonrepeaters. The persistence…

Accumulate-Repeat-Accumulate-Accumulate Codes

NASA Technical Reports Server (NTRS)

Divsalar, Dariush; Dolinar, Samuel; Thorpe, Jeremy

2007-01-01

Accumulate-repeat-accumulate-accumulate (ARAA) codes have been proposed, inspired by the recently proposed accumulate-repeat-accumulate (ARA) codes. These are error-correcting codes suitable for use in a variety of wireless data-communication systems that include noisy channels. ARAA codes can be regarded as serial turbolike codes or as a subclass of low-density parity-check (LDPC) codes, and, like ARA codes they have projected graph or protograph representations; these characteristics make it possible to design high-speed iterative decoders that utilize belief-propagation algorithms. The objective in proposing ARAA codes as a subclass of ARA codes was to enhance the error-floor performance of ARA codes while maintaining simple encoding structures and low maximum variable node degree.

Alu repeats: A source for the genesis of primate microsatellites

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

Arcot, S.S.; Batzer, M.A.; Wang, Zhenyuan

1995-09-01

As a result of their abundance, relatively uniform distribution, and high degree of polymorphism, microsatellites and minisatellites have become valuable tools in genetic mapping, forensic identity testing, and population studies. In recent years, a number of microsatellite repeats have been found to be associated with Alu interspersed repeated DNA elements. The association of an Alu element with a microsatellite repeat could result from the integration of an Alu element within a preexisting microsatellite repeat. Alternatively, Alu elements could have a direct role in the origin of microsatellite repeats. Errors introduced during reverse transcription of the primary transcript derived from anmore » Alu {open_quotes}master{close_quote} gene or the accumulation of random mutations in the middle A-rich regions and oligo(dA)-rich tails of Alu elements after insertion and subsequent expansion and contraction of these sequences could result in the genesis of a microsatellite repeat. We have tested these hypotheses by a direct evolutionary comparison of the sequences of some recent Alu elements that are found only in humans and are absent from nonhuman primates, as well as some older Alu elements that are present at orthologous positions in a number of nonhuman primates. The origin of {open_quotes}young{close_quotes} Alu insertions, absence of sequences that resemble microsatellite repeats at the orthologous loci in chimpanzees, and the gradual expansion of microsatellite repeats in some old Alu repeats at orthologous positions within the genomes of a number of nonhuman primates suggest that Alu elements are a source for the genesis of primate microsatellite repeats. 48 refs., 5 figs., 3 tabs.« less

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles.

PubMed

Yamaji, Hideki

2014-03-01

Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus-insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus-insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus-insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.

Repeated annual influenza vaccination and vaccine effectiveness: review of evidence.

PubMed

Belongia, Edward A; Skowronski, Danuta M; McLean, Huong Q; Chambers, Catharine; Sundaram, Maria E; De Serres, Gaston

2017-07-01

Studies in the 1970s and 1980s signaled concern that repeated influenza vaccination could affect vaccine protection. The antigenic distance hypothesis provided a theoretical framework to explain variability in repeat vaccination effects based on antigenic similarity between successive vaccine components and the epidemic strain. Areas covered: A meta-analysis of vaccine effectiveness studies from 2010-11 through 2014-15 shows substantial heterogeneity in repeat vaccination effects within and between seasons and subtypes. When negative effects were observed, they were most pronounced for H3N2, especially in 2014-15 when vaccine components were unchanged and antigenically distinct from the epidemic strain. Studies of repeated vaccination across multiple seasons suggest that vaccine effectiveness may be influenced by more than one prior season. In immunogenicity studies, repeated vaccination blunts the hemagglutinin antibody response, particularly for H3N2. Expert commentary: Substantial heterogeneity in repeated vaccination effects is not surprising given the variation in study populations and seasons, and the variable effects of antigenic distance and immunological landscape in different age groups and populations. Caution is required in the interpretation of pooled results across multiple seasons, since this can mask important variation in repeat vaccination effects between seasons. Multi-season clinical studies are needed to understand repeat vaccination effects and guide recommendations.

Clinical oversight and the avoidance of repeat induced abortion.

PubMed

Jacovetty, Erica L; Clare, Camille A; Squire, Mary-Beatrice; Kubal, Keshar P; Liou, Sherry; Inchiosa, Mario A

2018-06-03

To evaluate the impact of patient counseling, demographics, and contraceptive methods on repeat induced abortion in women attending family planning clinics. A retrospective chart review of repeat induced abortions was performed. The analysis included patients with an initial induced abortion obtained between January 1, 2001, and March 31, 2014, at New York City Health + Hospitals/Metropolitan. The duration of involvement in the family planning program, the use of contraceptive interventions, and 18 patient factors were analyzed for their correlation with the incidence of repeat induced abortions per year of follow-up. A decreased rate of repeat induced abortions was associated with a longer duration of clinical oversight (r 2 =0.449, P<0.001), a higher contraceptive efficacy score (r=0.280, P=0.025), and a larger number of clinic visits for contraception (r=0.333, P=0.007). A continuum of contact with all of the services of a family planning clinic demonstrated a strong efficacy to limit repeat induced abortions. By determining the patient characteristics that most influence repeat induced abortion rates, providers can best choose the most efficacious method of contraception available. © 2018 International Federation of Gynecology and Obstetrics.

Microevolution of Pandemic Vibrio parahaemolyticus Assessed by the Number of Repeat Units in Short Sequence Tandem Repeat Regions

PubMed Central

García, Katherine; Gavilán, Ronnie G.; Höfle, Manfred G.; Martínez-Urtaza, Jaime; Espejo, Romilio T.

2012-01-01

The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10−4 mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered. PMID:22292049

Genetic variation and virulence of Autographa californica multiple nucleopolyhedrovirus and Trichoplusia ni single nucleopolyhedrovirus isolates

USDA-ARS?s Scientific Manuscript database

To determine the genetic diversity within the baculovirus species Autographa calfornica multiple nucleopolyhedrovirus (AcMNPV; Baculoviridae: Alphabaculovirus), a PCR-based method was used to identify and classify baculoviruses found in virus samples from the lepidopteran host species A. californi...

Intestinal alkaline phosphatase is protective to the preterm rat pup intestine.

PubMed

Heinzerling, Nathan P; Liedel, Jennifer L; Welak, Scott R; Fredrich, Katherine; Biesterveld, Ben E; Pritchard, Kirkwood A; Gourlay, David M

2014-06-01

Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates, with a mortality rate between 10 and 50%. The onset of necrotizing enterocolitis is highly variable and associated with numerous risk factors. Prior research has shown that enteral supplementation with intestinal alkaline phosphatase (IAP) decreases the severity of NEC. The aim of this study is to investigate whether IAP is protective to the preterm intestine in the presence of formula feeding and in the absence of NEC. Preterm rat pups were fed formula with or without supplementation with IAP, and intestine was obtained on day of life 3 for analysis of IAP activity, mRNA expression of TNFα, IL-6 and iNOS and permeability and cytokine expression after LPS exposure. There was no difference in the absolute and intestine specific alkaline phosphatase activity in both groups. Rat pups fed IAP had decreased mRNA expression of the inflammatory cytokines TNFα, IL-6 and iNOS. Pups supplemented with IAP had decreased permeability and inflammatory cytokine expression after exposure to LPS ex vivo when compared to formula fed controls. Our results support that IAP is beneficial to preterm intestine and decreases intestinal injury and inflammation caused by LPS. Copyright © 2014 Elsevier Inc. All rights reserved.

Intestinal Alkaline Phosphatase Is Protective to the Preterm Rat Pup Intestine

PubMed Central

Heinzerling, Nathan P.; Liedel, Jennifer L.; Welak, Scott R.; Fredrich, Katherine; Biesterveld, Ben E.; Pritchard, Kirkwood A.; Gourlay, David M.

2014-01-01

Background Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates, with a mortality rate between 10 and 50%. The onset of necrotizing enterocolitis is highly variable and associated with numerous risk factors. Prior research has shown enteral supplementation with intestinal alkaline phosphatase (IAP) decreases the severity of NEC. The aim of this study is to investigate whether IAP is protective to the preterm intestine in the presence of formula feeding and in the absence of NEC. Methods Preterm rat pups were fed formula with or without supplementation with IAP, and intestine was obtained on day of life 3 for analysis of IAP activity, mRNA expression of TNF-a, IL-6 and iNOS and permeability and cytokine expression after LPS. exposure. Results There was no difference in the absolute and intestine specific alkaline phosphatase activity in both groups. Rat pups fed IAP had decreased mRNA expression of the inflammatory cytokines TNFα, IL-6 and iNOS. Pups supplemented with IAP had decreased permeability and inflammatory cytokine expression after exposure to LPS ex vivo when compared to formula fed controls. Conclusions Our results support that IAP is beneficial to preterm intestine and decreases intestinal injury and inflammation caused by LPS. PMID:24888842

In Vitro Expansion of CAG, CAA, and Mixed CAG/CAA Repeats.

PubMed

Figura, Grzegorz; Koscianska, Edyta; Krzyzosiak, Wlodzimierz J

2015-08-11

Polyglutamine diseases, including Huntington's disease and a number of spinocerebellar ataxias, are caused by expanded CAG repeats that are located in translated sequences of individual, functionally-unrelated genes. Only mutant proteins containing polyglutamine expansions have long been thought to be pathogenic, but recent evidence has implicated mutant transcripts containing long CAG repeats in pathogenic processes. The presence of two pathogenic factors prompted us to attempt to distinguish the effects triggered by mutant protein from those caused by mutant RNA in cellular models of polyglutamine diseases. We used the SLIP (Synthesis of Long Iterative Polynucleotide) method to generate plasmids expressing long CAG repeats (forming a hairpin structure), CAA-interrupted CAG repeats (forming multiple unstable hairpins) or pure CAA repeats (not forming any secondary structure). We successfully modified the original SLIP protocol to generate repeats of desired length starting from constructs containing short repeat tracts. We demonstrated that the SLIP method is a time- and cost-effective approach to manipulate the lengths of expanded repeat sequences.

Whole-animal metabolic rate is a repeatable trait: a meta-analysis.

PubMed

Nespolo, Roberto F; Franco, Marcela

2007-06-01

Repeatability studies are gaining considerable interest among physiological ecologists, particularly in traits affected by high environmental/residual variance, such as whole-animal metabolic rate (MR). The original definition of repeatability, known as the intraclass correlation coefficient, is computed from the components of variance obtained in a one-way ANOVA on several individuals from which two or more measurements are performed. An alternative estimation of repeatability, popular among physiological ecologists, is the Pearson product-moment correlation between two consecutive measurements. However, despite the more than 30 studies reporting repeatability of MR, so far there is not a definite synthesis indicating: (1) whether repeatability changes in different types of animals; (2) whether some kinds of metabolism are more repeatable than others; and most important, (3) whether metabolic rate is significantly repeatable. We performed a meta-analysis to address these questions, as well as to explore the historical trend in repeatability studies. Our results show that metabolic rate is significantly repeatable and its effect size is not statistically affected by any of the mentioned factors (i.e. repeatability of MR does not change in different species, type of metabolism, time between measurements, and number of individuals). The cumulative meta-analysis revealed that repeatability studies in MR have already reached an asymptotical effect size with no further change either in its magnitude and/or variance (i.e. additional studies will not contribute significantly to the estimator). There was no evidence of strong publication bias.

Optimizing working space in porcine laparoscopy: CT measurement of the effects of intra-abdominal pressure.

PubMed

Vlot, John; Wijnen, Rene; Stolker, Robert Jan; Bax, Klaas

2013-05-01

Several factors may affect volume and dimensions of the working space in laparoscopic surgery. The precise impact of these factors has not been well studied. In a porcine model, we used computed tomographic (CT) scanning for measuring working space volume and distances. In a first series of experiments, we studied the relationship between intra-abdominal pressure (IAP) and working space. Eleven 20 kg pigs were studied under standardized anesthesia and volume-controlled ventilation. Cardiorespiratory parameters were monitored continuously, and blood gas samples were taken at different IAP levels. Respiratory rate was increased when ETCO₂ exceeded 7 kPa. Breath-hold CT scans were made at IAP levels of 0, 5, 10, and 15 mmHg. Insufflator volumes were compared to CT-measured volumes. Maximum dimensions of pneumoperitoneum were measured on reconstructed CT images. Respiratory rate had to be increased in three animals. Mild hypercapnia and acidosis occurred at 15 mmHg IAP. Peak inspiratory pressure rose significantly at 10 and 15 mmHg. CT-measured volume increased relatively by 93 % from 5 to 10 mmHg IAP and by 19 % from 10 to 15 mmHg IAP. Comparing CT volumes to insufflator volumes gave a bias of 76 mL. The limits of agreement were -0.31 to +0.47, a range of 790 mL. The internal anteroposterior diameter increased by 18 % by increasing IAP from 5 to 10 mmHg and by 5 % by increasing IAP from 10 to 15 mmHg. At 15 mmHg, the total relative increase of the pubis-diaphragm distance was only 6 %. Abdominal width did not increase. CT allows for precise calculation of the actual CO₂ pneumoperitoneum volume, whereas the volume of CO₂ released by the insufflator does not. Increasing IAP up to 10 mmHg achieved most gain in volume and in internal anteroposterior diameter. At an IAP of 10 mmHg, higher peak inspiratory pressure was significantly elevated.

Loss of inhibitor of apoptosis proteins as a determinant of polyamine analog-induced apoptosis in human melanoma cells.

PubMed

Chen, Ying; Kramer, Debora L; Li, Fengzhi; Porter, Carl W

2003-08-07

We have previously shown that the clinically relevant polyamine analog N1,N11-diethylnorspermine (DENSPM) causes rapid apoptosis in human melanoma SK-MEL-28 cells via a series of events that include mitochondrial release of cytochrome c and activation of the caspase cascade. Upstream to these events, DENSPM downregulates polyamine biosynthesis and potently upregulates polyamine catabolism at the level of spermidine/spermine N1-acetyltransferase (SSAT). In searching for downstream effectors that either contribute to or abrogate the apoptotic response, we observed that DENSPM treatment of SK-MEL-28 cells for 30 h led to cytosolic release of Smac/Diablo, a mitochondrial protein known to bind and inhibit the function of inhibitor of apoptosis proteins (IAPs). Subsequently, we found that DENSPM markedly lowered survivin and ML-IAP protein (but not XIAP) levels by 18 h via an apparently Smac/Diablo-independent pathway. Proteasome inhibitors fully prevented survivin and ML-IAP protein loss as well as apoptosis, suggesting that the proteasome-mediated degradation of survivin and ML-IAP is causally linked to the cellular outcome. We also observed that structural analogs of DENSPM which differentially induced SSAT and apoptosis lowered survivin and ML-IAP levels in a manner that correlated with enzyme activity. The linkage between IAPs and SSAT was more directly established by the finding that selective prevention of SSAT induction by small interfering RNA prevented survivin and ML-IAP loss as well as apoptosis during DENSPM treatment. Among the melanoma cell lines (SK-MEL-28, MALME-3M, A375 and LOX), survivin degradation correlated temporally with the onset of DENSPM induced apoptosis or growth inhibition. By contrast, ML-IAP degradation occurred only during rapid apoptosis seen in SK-MEL-28 cells. These data suggest a sequence of events whereby DENSPM induction of SSAT leads to loss of IAP proteins and a more fulminate apoptotic response. The findings implicate survivin

Repeatability study of replicate crash tests: A signal analysis approach.

PubMed

Seppi, Jeremy; Toczyski, Jacek; Crandall, Jeff R; Kerrigan, Jason

2017-10-03

To provide an objective basis on which to evaluate the repeatability of vehicle crash test methods, a recently developed signal analysis method was used to evaluate correlation of sensor time history data between replicate vehicle crash tests. The goal of this study was to evaluate the repeatability of rollover crash tests performed with the Dynamic Rollover Test System (DRoTS) relative to other vehicle crash test methods. Test data from DRoTS tests, deceleration rollover sled (DRS) tests, frontal crash tests, frontal offset crash tests, small overlap crash tests, small overlap impact (SOI) crash tests, and oblique crash tests were obtained from the literature and publicly available databases (the NHTSA vehicle database and the Insurance Institute for Highway Safety TechData) to examine crash test repeatability. Signal analysis of the DRoTS tests showed that force and deformation time histories had good to excellent repeatability, whereas vehicle kinematics showed only fair repeatability due to the vehicle mounting method for one pair of tests and slightly dissimilar mass properties (2.2%) in a second pair of tests. Relative to the DRS, the DRoTS tests showed very similar or higher levels of repeatability in nearly all vehicle kinematic data signals with the exception of global X' (road direction of travel) velocity and displacement due to the functionality of the DRoTS fixture. Based on the average overall scoring metric of the dominant acceleration, DRoTS was found to be as repeatable as all other crash tests analyzed. Vertical force measures showed good repeatability and were on par with frontal crash barrier forces. Dynamic deformation measures showed good to excellent repeatability as opposed to poor repeatability seen in SOI and oblique deformation measures. Using the signal analysis method as outlined in this article, the DRoTS was shown to have the same or better repeatability of crash test methods used in government regulatory and consumer evaluation test

Evolution of vulnerability of communities facing repeated hazards

PubMed Central

Guikema, Seth D.; Zhu, Laiyin; Igusa, Takeru

2017-01-01

The decisions that individuals make when recovering from and adapting to repeated hazards affect a region’s vulnerability in future hazards. As such, community vulnerability is not a static property but rather a dynamic property dependent on behavioral responses to repeated hazards and damage. This paper is the first of its kind to build a framework that addresses the complex interactions between repeated hazards, regional damage, mitigation decisions, and community vulnerability. The framework enables researchers and regional planners to visualize and quantify how a community could evolve over time in response to repeated hazards under various behavioral scenarios. An illustrative example using parcel-level data from Anne Arundel County, Maryland—a county that experiences fairly frequent hurricanes—is presented to illustrate the methodology and to demonstrate how the interplay between individual choices and regional vulnerability is affected by the region’s hurricane experience. PMID:28953893

Criminal Behavior and Repeat Violent Trauma: A Case-Control Study.

PubMed

Nanney, John T; Conrad, Erich J; McCloskey, Michael; Constans, Joseph I

2015-09-01

Repeat violent injury is common among young urban men and is increasingly a focus of trauma center-based injury prevention efforts. Though understanding risk factors for repeat violent injury may be critical in designing such interventions, this knowledge is limited. This study aims to determine which criminal behaviors, both before and after the initial trauma, predict repeat violent trauma. Gun, violent, and drug crimes are expected to increase risk of subsequent violent injury among victims of violence. A case-control design examined trauma registry and publicly available criminal data for all male patients aged <40 years presenting for violent trauma between April 2006 and December 2011 (N=1,142) to the sole Level 1 trauma center in a city with high rates of violence. Logistic regression was used to determine criminal behaviors predictive of repeat violent injury. Data were obtained and analyzed between January 2013 and June 2014. Regarding crimes committed before the first injury, only drug crime (OR=5.32) predicted repeat violent trauma. With respect to crimes committed after the initial injury, illegal gun possession (OR=2.70) predicted repeat victimization. Initiating gun (OR=3.53) or drug crime (OR=5.12) was associated with increased risk. Prior drug involvement may identify young male victims of violence as at high risk of repeat violent injury. Gun carrying and initiating drug involvement after the initial injury may increase risk of repeat injury and may be important targets for interventions aimed at preventing repeat violent trauma. Published by Elsevier Inc.

ACCA phosphopeptide recognition by the BRCT repeats of BRCA1.

PubMed

Ray, Hind; Moreau, Karen; Dizin, Eva; Callebaut, Isabelle; Venezia, Nicole Dalla

2006-06-16

The tumour suppressor gene BRCA1 encodes a 220 kDa protein that participates in multiple cellular processes. The BRCA1 protein contains a tandem of two BRCT repeats at its carboxy-terminal region. The majority of disease-associated BRCA1 mutations affect this region and provide to the BRCT repeats a central role in the BRCA1 tumour suppressor function. The BRCT repeats have been shown to mediate phospho-dependant protein-protein interactions. They recognize phosphorylated peptides using a recognition groove that spans both BRCT repeats. We previously identified an interaction between the tandem of BRCA1 BRCT repeats and ACCA, which was disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We recently showed that BRCA1 modulates ACCA activity through its phospho-dependent binding to ACCA. To delineate the region of ACCA that is crucial for the regulation of its activity by BRCA1, we searched for potential phosphorylation sites in the ACCA sequence that might be recognized by the BRCA1 BRCT repeats. Using sequence analysis and structure modelling, we proposed the Ser1263 residue as the most favourable candidate among six residues, for recognition by the BRCA1 BRCT repeats. Using experimental approaches, such as GST pull-down assay with Bosc cells, we clearly showed that phosphorylation of only Ser1263 was essential for the interaction of ACCA with the BRCT repeats. We finally demonstrated by immunoprecipitation of ACCA in cells, that the whole BRCA1 protein interacts with ACCA when phosphorylated on Ser1263.

Automated quality checks on repeat prescribing.

PubMed Central

Rogers, Jeremy E; Wroe, Christopher J; Roberts, Angus; Swallow, Angela; Stables, David; Cantrill, Judith A; Rector, Alan L

2003-01-01

BACKGROUND: Good clinical practice in primary care includes periodic review of repeat prescriptions. Markers of prescriptions that may need review have been described, but manually checking all repeat prescriptions against the markers would be impractical. AIM: To investigate the feasibility of computerising the application of repeat prescribing quality checks to electronic patient records in United Kingdom (UK) primary care. DESIGN OF STUDY: Software performance test against benchmark manual analysis of cross-sectional convenience sample of prescribing documentation. SETTING: Three general practices in Greater Manchester, in the north west of England, during a 4-month period in 2001. METHOD: A machine-readable drug information resource, based on the British National Formulary (BNF) as the 'gold standard' for valid drug indications, was installed in three practices. Software raised alerts for each repeat prescribed item where the electronic patient record contained no valid indication for the medication. Alerts raised by the software in two practices were analysed manually. Clinical reaction to the software was assessed by semi-structured interviews in three practices. RESULTS: There was no valid indication in the electronic medical records for 14.8% of repeat prescribed items. Sixty-two per cent of all alerts generated were incorrect. Forty-three per cent of all incorrect alerts were as a result of errors in the drug information resource, 44% to locally idiosyncratic clinical coding, 8% to the use of the BNF without adaptation as a gold standard, and 5% to the inability of the system to infer diagnoses that, although unrecorded, would be 'obvious' to a clinical reading the record. The interviewed clinicians supported the goals of the software. CONCLUSION: Using electronic records for secondary decision support purposes will benefit from (and may require) both more consistent electronic clinical data collection across multiple sites, and reconciling clinicians

Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (αRep) based on thermostable HEAT-like repeats.

PubMed

Urvoas, Agathe; Guellouz, Asma; Valerio-Lepiniec, Marie; Graille, Marc; Durand, Dominique; Desravines, Danielle C; van Tilbeurgh, Herman; Desmadril, Michel; Minard, Philippe

2010-11-26

Repeat proteins have a modular organization and a regular architecture that make them attractive models for design and directed evolution experiments. HEAT repeat proteins, although very common, have not been used as a scaffold for artificial proteins, probably because they are made of long and irregular repeats. Here, we present and validate a consensus sequence for artificial HEAT repeat proteins. The sequence was defined from the structure-based sequence analysis of a thermostable HEAT-like repeat protein. Appropriate sequences were identified for the N- and C-caps. A library of genes coding for artificial proteins based on this sequence design, named αRep, was assembled using new and versatile methodology based on circular amplification. Proteins picked randomly from this library are expressed as soluble proteins. The biophysical properties of proteins with different numbers of repeats and different combinations of side chains in hypervariable positions were characterized. Circular dichroism and differential scanning calorimetry experiments showed that all these proteins are folded cooperatively and are very stable (T(m) >70 °C). Stability of these proteins increases with the number of repeats. Detailed gel filtration and small-angle X-ray scattering studies showed that the purified proteins form either monomers or dimers. The X-ray structure of a stable dimeric variant structure was solved. The protein is folded with a highly regular topology and the repeat structure is organized, as expected, as pairs of alpha helices. In this protein variant, the dimerization interface results directly from the variable surface enriched in aromatic residues located in the randomized positions of the repeats. The dimer was crystallized both in an apo and in a PEG-bound form, revealing a very well defined binding crevice and some structure flexibility at the interface. This fortuitous binding site could later prove to be a useful binding site for other low molecular mass

Instability of expanded CAG/CAA repeats in spinocerebellar ataxia type 17.

PubMed

Gao, Rui; Matsuura, Tohru; Coolbaugh, Mary; Zühlke, Christine; Nakamura, Koichiro; Rasmussen, Astrid; Siciliano, Michael J; Ashizawa, Tetsuo; Lin, Xi

2008-02-01

Trinucleotide repeat expansions are dynamic mutations causing many neurological disorders, and their instability is influenced by multiple factors. Repeat configuration seems particularly important, and pure repeats are thought to be more unstable than interrupted repeats. But direct evidence is still lacking. Here, we presented strong support for this hypothesis from our studies on spinocerebellar ataxia type 17 (SCA17). SCA17 is a typical polyglutamine disease caused by CAG repeat expansion in TBP (TATA binding protein), and is unique in that the pure expanded polyglutamine tract is coded by either a simple configuration with long stretches of pure CAGs or a complex configuration containing CAA interruptions. By small pool PCR (SP-PCR) analysis of blood DNA from SCA17 patients of distinct racial backgrounds, we quantitatively assessed the instability of these two types of expanded alleles coding similar length of polyglutamine expansion. Mutation frequency in patients harboring pure CAG repeats is 2-3 folds of those with CAA interruptions. Interestingly, the pure CAG repeats showed both expansion and deletion while the interrupted repeats exhibited mostly deletion at a significantly lower frequency. These data strongly suggest that repeat configuration is a critical determinant for instability, and CAA interruptions might serve as a limiting element for further expansion of CAG repeats in SCA17 locus, suggesting a molecular basis for lack of anticipation in SCA17 families with interrupted CAG expansion.

Repeatless and repeat-based centromeres in potato: implications for centromere evolution.

PubMed

Gong, Zhiyun; Wu, Yufeng; Koblízková, Andrea; Torres, Giovana A; Wang, Kai; Iovene, Marina; Neumann, Pavel; Zhang, Wenli; Novák, Petr; Buell, C Robin; Macas, Jirí; Jiang, Jiming

2012-09-01

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.

Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation.

PubMed

Rouble, Andrew N; Hefler, Joshua; Mamady, Hapsatou; Storey, Kenneth B; Tessier, Shannon N

2013-01-01

In the context of normal cell turnover, apoptosis is a natural phenomenon involved in making essential life and death decisions. Apoptotic pathways balance signals which promote cell death (pro-apoptotic pathways) or counteract these signals (anti-apoptotic pathways). We proposed that changes in anti-apoptotic proteins would occur during mammalian hibernation to aid cell preservation during prolonged torpor under cellular conditions that are highly injurious to most mammals (e.g. low body temperatures, ischemia). Immunoblotting was used to analyze the expression of proteins associated with pro-survival in six tissues of thirteen-lined ground squirrels, Ictidomys tridecemlineatus. The brain showed a concerted response to torpor with significant increases in the levels of all anti-apoptotic targets analyzed (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) as well as enhanced phosphorylation of Bcl-2 at S70 and T56. Heart responded similarly with most anti-apoptotic proteins elevated significantly during torpor except for Bcl-xL and xIAP that decreased and Mcl-1 that was unaltered. In liver, BI-1 increased whereas cIAP1/2 decreased. In kidney, there was an increase in BI-1, cIAP and xIAP but decreases in Bcl-xL and p-Bcl-2(T56) content. In brown adipose tissue, protein levels of BI-1, cIAP1/2, and xIAP decreased significantly during torpor (compared with euthermia) whereas Bcl-2, Bcl-xL, Mcl-1 were unaltered; however, Bcl-2 showed enhanced phosphorylation at Thr56 but not at Ser70. In skeletal muscle, only xIAP levels changed significantly during torpor (an increase). The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart and brain where coordinated enhancement of anti-apoptotic proteins occurred in response to torpor.

Anti-apoptotic signaling as a cytoprotective mechanism in mammalian hibernation

PubMed Central

Mamady, Hapsatou; Tessier, Shannon N.

2013-01-01

In the context of normal cell turnover, apoptosis is a natural phenomenon involved in making essential life and death decisions. Apoptotic pathways balance signals which promote cell death (pro-apoptotic pathways) or counteract these signals (anti-apoptotic pathways). We proposed that changes in anti-apoptotic proteins would occur during mammalian hibernation to aid cell preservation during prolonged torpor under cellular conditions that are highly injurious to most mammals (e.g. low body temperatures, ischemia). Immunoblotting was used to analyze the expression of proteins associated with pro-survival in six tissues of thirteen-lined ground squirrels, Ictidomys tridecemlineatus. The brain showed a concerted response to torpor with significant increases in the levels of all anti-apoptotic targets analyzed (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) as well as enhanced phosphorylation of Bcl-2 at S70 and T56. Heart responded similarly with most anti-apoptotic proteins elevated significantly during torpor except for Bcl-xL and xIAP that decreased and Mcl-1 that was unaltered. In liver, BI-1 increased whereas cIAP1/2 decreased. In kidney, there was an increase in BI-1, cIAP and xIAP but decreases in Bcl-xL and p-Bcl-2(T56) content. In brown adipose tissue, protein levels of BI-1, cIAP1/2, and xIAP decreased significantly during torpor (compared with euthermia) whereas Bcl-2, Bcl-xL, Mcl-1 were unaltered; however, Bcl-2 showed enhanced phosphorylation at Thr56 but not at Ser70. In skeletal muscle, only xIAP levels changed significantly during torpor (an increase). The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart and brain where coordinated enhancement of anti-apoptotic proteins occurred in response to torpor. PMID:23638364

Short intronic repeat sequences facilitate circular RNA production.

PubMed

Liang, Dongming; Wilusz, Jeremy E

2014-10-15

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery "backsplices" and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼ 30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3' end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. © 2014 Liang and Wilusz; Published by Cold Spring Harbor Laboratory Press.

Short intronic repeat sequences facilitate circular RNA production

PubMed Central

Liang, Dongming

2014-01-01

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery “backsplices” and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3′ end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. PMID:25281217

Pathways and intermediates in forced unfolding of spectrin repeats.

PubMed

Altmann, Stephan M; Grünberg, Raik G; Lenne, Pierre-François; Ylänne, Jari; Raae, Arnt; Herbert, Kristina; Saraste, Matti; Nilges, Michael; Hörber, J K Heinrich

2002-08-01

Spectrin repeats are triple-helical coiled-coil domains found in many proteins that are regularly subjected to mechanical stress. We used atomic force microscopy technique and steered molecular dynamics simulations to study the behavior of a wild-type spectrin repeat and two mutants. The experiments indicate that spectrin repeats can form stable unfolding intermediates when subjected to external forces. In the simulations the unfolding proceeded via a variety of pathways. Stable intermediates were associated to kinking of the central helix close to a proline residue. A mutant stabilizing the central helix showed no intermediates in experiments, in agreement with simulation. Spectrin repeats may thus function as elastic elements, extendable to intermediate states at various lengths.

47 CFR 95.1311 - Repeater operations and signal boosters prohibited.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 47 Telecommunication 5 2012-10-01 2012-10-01 false Repeater operations and signal boosters... § 95.1311 Repeater operations and signal boosters prohibited. MURS stations are prohibited from operating as a repeater station or as a signal booster. This prohibition includes store-and-forward packet...

47 CFR 95.1311 - Repeater operations and signal boosters prohibited.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 47 Telecommunication 5 2011-10-01 2011-10-01 false Repeater operations and signal boosters... § 95.1311 Repeater operations and signal boosters prohibited. MURS stations are prohibited from operating as a repeater station or as a signal booster. This prohibition includes store-and-forward packet...

47 CFR 95.1311 - Repeater operations and signal boosters prohibited.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 47 Telecommunication 5 2013-10-01 2013-10-01 false Repeater operations and signal boosters... § 95.1311 Repeater operations and signal boosters prohibited. MURS stations are prohibited from operating as a repeater station or as a signal booster. This prohibition includes store-and-forward packet...