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.

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.

Non-viral delivery of genome-editing nucleases for gene therapy.

PubMed

Wang, M; Glass, Z A; Xu, Q

2017-03-01

Manipulating the genetic makeup of mammalian cells using programmable nuclease-based genome-editing technology has recently evolved into a powerful avenue that holds great potential for treating genetic disorders. There are four types of genome-editing nucleases, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeat-associated nucleases such as Cas9. These nucleases have been harnessed to introduce precise and specific changes of the genome sequence at virtually any genome locus of interest. The therapeutic relevance of these genome-editing technologies, however, is challenged by the safe and efficient delivery of nuclease into targeted cells. Herein, we summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins for genome editing. In addition, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.

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

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.

Genome Editing in Rats Using TALE Nucleases.

PubMed

Tesson, Laurent; Remy, Séverine; Ménoret, Séverine; Usal, Claire; Thinard, Reynald; Savignard, Chloé; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

2016-01-01

The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

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.

Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula

PubMed Central

Lomate, Purushottam R.; Bonning, Bryony C.

2016-01-01

Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest. PMID:27282882

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

Gene targeting technologies in rats: zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats.

PubMed

Mashimo, Tomoji

2014-01-01

The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed. © 2013 The Author Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

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

Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN.

PubMed

Ul Ain, Qurrat; Chung, Jee Young; Kim, Yong-Hee

2015-05-10

Gene therapy by engineered nucleases is a genetic intervention being investigated for curing the hereditary disorders by targeting selected genes with specific nucleotides for establishment, suppression, abolishment of a function or correction of mutation. Here, we review the fast developing technology of targeted genome engineering using site specific programmable nucleases zinc finger nucleases (ZFNs), transcription activator like nucleases (TALENs) and cluster regulatory interspaced short palindromic repeat/CRISPR associated proteins (CRISPR/Cas) based RNA-guided DNA endonucleases (RGENs) and their different characteristics including pros and cons of genome modifications by these nucleases. We have further discussed different types of delivery methods to induce gene editing, novel development in genetic engineering other than nucleases and future prospects. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Mismatch cleavage by single-strand specific nucleases

PubMed Central

Till, Bradley J.; Burtner, Chris; Comai, Luca; Henikoff, Steven

2004-01-01

We have investigated the ability of single-strand specific (sss) nucleases from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single-nucleotide polymorphism analysis. The TILLING (Targeting Induced Local Lesions IN Genomes) mismatch cleavage protocol was used with the LI-COR gel detection system to assay cleavage of amplified heteroduplexes derived from a variety of induced mutations and naturally occurring polymorphisms. We found that purified nucleases derived from celery (CEL I), mung bean sprouts and Aspergillus (S1) were able to specifically cleave nearly all single base pair mismatches tested. Optimal nicking of heteroduplexes for mismatch detection was achieved using higher pH, temperature and divalent cation conditions than are routinely used for digestion of single-stranded DNA. Surprisingly, crude plant extracts performed as well as the highly purified preparations for this application. These observations suggest that diverse members of the S1 family of sss nucleases act similarly in cleaving non-specifically at bulges in heteroduplexes, and single-base mismatches are the least accessible because they present the smallest single-stranded region for enzyme binding. We conclude that a variety of sss nucleases and extracts can be effectively used for high-throughput mutation and polymorphism discovery. PMID:15141034

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

Design of a colicin E7 based chimeric zinc-finger nuclease

NASA Astrophysics Data System (ADS)

Németh, Eszter; Schilli, Gabriella K.; Nagy, Gábor; Hasenhindl, Christoph; Gyurcsik, Béla; Oostenbrink, Chris

2014-08-01

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

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.

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.

Generation of knockout rabbits using transcription activator-like effector nucleases.

PubMed

Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

2014-01-01

Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9

PubMed Central

Koo, Taeyoung; Lee, Jungjoon; Kim, Jin-Soo

2015-01-01

Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations. PMID:25985872

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.

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....

Enhanced gene disruption by programmable nucleases delivered by a minicircle vector.

PubMed

Dad, A-B K; Ramakrishna, S; Song, M; Kim, H

2014-11-01

Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors, which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group. Taken together, our results show that gene disruption using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases.

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.

Genetic correction using engineered nucleases for gene therapy applications.

PubMed

Li, Hongmei Lisa; Nakano, Takao; Hotta, Akitsu

2014-01-01

Genetic mutations in humans are associated with congenital disorders and phenotypic traits. Gene therapy holds the promise to cure such genetic disorders, although it has suffered from several technical limitations for decades. Recent progress in gene editing technology using tailor-made nucleases, such as meganucleases (MNs), zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and, more recently, CRISPR/Cas9, has significantly broadened our ability to precisely modify target sites in the human genome. In this review, we summarize recent progress in gene correction approaches of the human genome, with a particular emphasis on the clinical applications of gene therapy. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

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.

DNA aptamers against FokI nuclease domain for genome editing applications.

PubMed

Nishio, Maui; Matsumoto, Daisuke; Kato, Yoshio; Abe, Koichi; Lee, Jinhee; Tsukakoshi, Kaori; Yamagishi, Ayana; Nakamura, Chikashi; Ikebukuro, Kazunori

2017-07-15

Genome editing with site-specific nucleases (SSNs) can modify only the target gene and may be effective for gene therapy. The main limitation of genome editing for clinical use is off-target effects; excess SSNs in the cells and their longevity can contribute to off-target effects. Therefore, a controlled delivery system for SSNs is necessary. FokI nuclease domain (FokI) is a common DNA cleavage domain in zinc finger nuclease (ZFN) and transcription activator-like effector nuclease. Previously, we reported a zinc finger protein delivery system that combined aptamer-fused, double-strand oligonucleotides and nanoneedles. Here, we report the development of DNA aptamers that bind to the target molecules, with high affinity and specificity to the FokI. DNA aptamers were selected in six rounds of systematic evolution of ligands by exponential enrichment. Aptamers F6#8 and #71, which showed high binding affinity to FokI (K d =82nM, 74nM each), showed resistance to nuclease activity itself and did not inhibit nuclease activity. We immobilized the ZFN-fused GFP to nanoneedles through these aptamers and inserted the nanoneedles into HEK293 cells. We observed the release of ZFN-fused GFP from the nanoneedles in the presence of cells. Therefore, these aptamers are useful for genome editing applications such as controlled delivery of SSNs. Copyright © 2016 Elsevier B.V. All rights reserved.

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-...

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

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis.

PubMed

Almeida Garcia, Rayssa; Lima Pepino Macedo, Leonardo; Cabral do Nascimento, Danila; Gillet, François-Xavier; Moreira-Pinto, Clidia Eduarda; Faheem, Muhammad; Moreschi Basso, Angelina Maria; Mattar Silva, Maria Cristina; Grossi-de-Sa, Maria Fatima

2017-01-01

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.

The Genome of Gryllus bimaculatus Nudivirus Indicates an Ancient Diversification of Baculovirus-Related Nonoccluded Nudiviruses of Insects▿

PubMed Central

Wang, Yongjie; Kleespies, Regina G.; Huger, Alois M.; Jehle, Johannes A.

2007-01-01

The Gryllus bimaculatus nudivirus (GbNV) infects nymphs and adults of the cricket Gryllus bimaculatus (Orthoptera: Gryllidae). GbNV and other nudiviruses such as Heliothis zea nudivirus 1 (HzNV-1) and Oryctes rhinoceros nudivirus (OrNV) were previously called “nonoccluded baculoviruses” as they share some similar structural, genomic, and replication aspects with members of the family Baculoviridae. Their relationships to each other and to baculoviruses are elucidated by the sequence of the complete genome of GbNV, which is 96,944 bp, has an AT content of 72%, and potentially contains 98 predicted protein-coding open reading frames (ORFs). Forty-one ORFs of GbNV share sequence similarities with ORFs found in OrNV, HzNV-1, baculoviruses, and bacteria. Most notably, 15 GbNV ORFs are homologous to the baculovirus core genes, which are associated with transcription (lef-8, lef-9, lef-4, vlf-1, and lef-5), replication (dnapol), structural proteins (p74, pif-1, pif-2, pif-3, vp91, and odv-e56), and proteins of unknown function (38K, ac81, and 19kda). Homologues to these baculovirus core genes have been predicted in HzNV-1 as well. Six GbNV ORFs are homologous to nonconserved baculovirus genes dnaligase, helicase 2, rr1, rr2, iap-3, and desmoplakin. However, the remaining 57 ORFs revealed no homology or poor similarities to the current gene databases. No homologous repeat (hr) sequences but fourteen short direct repeat (dr) regions were detected in the GbNV genome. Gene content and sequence similarity suggest that the nudiviruses GbNV, HzNV-1, and OrNV form a monophyletic group of nonoccluded double-stranded DNA viruses, which separated from the baculovirus lineage before this radiated into dipteran-, hymenopteran-, and lepidopteran-specific clades of occluded nucleopolyhedroviruses and granuloviruses. The accumulated information on the GbNV genome suggests that nudiviruses form a highly diverse and phylogenetically ancient sister group of the baculoviruses, which have

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

Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity.

PubMed

Flenker, Katie S; Burghardt, Elliot L; Dutta, Nirmal; Burns, William J; Grover, Julia M; Kenkel, Elizabeth J; Weaver, Tyler M; Mills, James; Kim, Hyeon; Huang, Lingyan; Owczarzy, Richard; Musselman, Catherine A; Behlke, Mark A; Ford, Bradley; McNamara, James O

2017-06-07

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis

PubMed Central

Almeida Garcia, Rayssa; Lima Pepino Macedo, Leonardo; Cabral do Nascimento, Danila; Gillet, François-Xavier; Moreira-Pinto, Clidia Eduarda; Faheem, Muhammad; Moreschi Basso, Angelina Maria; Mattar Silva, Maria Cristina

2017-01-01

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests. PMID:29261729

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

Nuclease-mediated genome editing: At the front-line of functional genomics technology.

PubMed

Sakuma, Tetsushi; Woltjen, Knut

2014-01-01

Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

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

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

2008-01-20

DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His{sub 6}-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA andmore » that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.« less

Nucleases activities during French bean leaf aging and dark-induced senescence.

PubMed

Lambert, Rocío; Quiles, Francisco Antonio; Gálvez-Valdivieso, Gregorio; Piedras, Pedro

2017-11-01

During leaf senescence resources are managed, with nutrients mobilized from older leaves to new sink tissues. The latter implies a dilemma in terms of resource utilization, the leaf senescence should increase seed quality whereas delay in senescence should improve the seed yield. Increased knowledge about nutrient recycling during leaf senescence could lead to advances in agriculture and improved seed quality. Macromolecules mobilized during leaf senescence include proteins and nucleic acids. Although nucleic acids have been less well studied than protein degradation, they are possible reservoirs of nitrogen and phosphorous. The present study investigated nuclease activities and gene expression patterns of five members of the S1/P1 family in French bean (Phaseolus vulgaris L. cv.)Page: 2 during leaf senescence. An in-gel assay was used to detect nuclease activity during natural and dark-induced senescence, with single-stranded DNA (ssDNA) used as a substrate. The results revealed two nucleases (glycoproteins), with molecular masses of 34 and 39kDa in the senescent leaves. The nuclease activities were higher at a neutral than at an acidic pH. EDTA treatment inhibited the activities of the nucleases, and the addition of zinc resulted in the recovery of these activities. Both the 34 and 39kDa nucleases were able to use RNA and double-stranded DNA (dsDNA) as substrates, although their activities were low when dsDNA was used as a substrate. In addition, two ribonucleases with molecular masses of 14 and 16kDa, both of which could only utilize RNA as a substrate, were detected in the senescent leaves. Two members of the S1/P1 family, PVN2 and PVN5, were expressed under the experimental conditions, suggesting that these two genes were involved in senescence. The nuclease activity of the glycoproteins and gene expression were similar under both natural senescence and dark-induced senescence conditions. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights

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.

Hybrid nanosensor for colorimetric and ultrasensitive detection of nuclease contaminations

NASA Astrophysics Data System (ADS)

Cecere, Paola; Valentini, Paola; Pompa, Pier Paolo

2016-04-01

Nucleases are ubiquitous enzymes that degrade DNA or RNA, thus they can prejudice the good outcome of molecular biology experiments involving nucleic acids. We propose a colorimetric test for the naked-eye detection of nuclease contaminations. The system uses an hybrid nanosensor, based on gold nanoparticles functionalized with DNA probes. Our assay is rapid, instrument-free, simple and low-cost. Moreover, it reaches sensitivity equal or better than those of commercial kits, and presents a lot of advantageous aspects. Therefore, it is very competitive, with a real market potential. This test will be relevant in routine process monitoring in scientific laboratories, and in quality control in clinical laboratories and industrial processes, allowing the simultaneous detection of nucleases with different substrate specificities and large-scale screening.

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

Halophilic Nuclease from a Moderately Halophilic Micrococcus varians

PubMed Central

Kamekura, Masahiro; Onishi, Hiroshi

1974-01-01

The moderately halophilic bacterium Micrococcus varians, isolated from soy sauce mash, produced extracellular nuclease when cultivated aerobically in media containing 1 to 4 M NaCl or KCl. The enzyme, purified to an electrophoretically homogeneous state, had both ribonuclease and deoxyribonuclease activities. The nuclease had maximal activity in the presence of 2.9 M NaCl or 2.1 M KCl at 40 C. The enzymatic activity was lost by dialysis against low-salt buffer, whereas when the inactivated enzyme was dialyzed against 3.4 M NaCl buffer as much as 77% of the initial activity could be restored. Images PMID:4852218

Design principles for nuclease-deficient CRISPR-based transcriptional regulators

PubMed Central

Jensen, Michael K

2018-01-01

Abstract The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies. PMID:29726937

Design principles for nuclease-deficient CRISPR-based transcriptional regulators.

PubMed

Jensen, Michael K

2018-06-01

The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualisation and epigenetic studies of living organisms. In this review, the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and guide RNA (gRNA), but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discusses the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such, this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies.

Targeting of a Nuclease to Murine Leukemia Virus Capsids Inhibits Viral Multiplication

NASA Astrophysics Data System (ADS)

Natsoulis, Georges; Seshaiah, Partha; Federspiel, Mark J.; Rein, Alan; Hughes, Stephen H.; Boeke, Jef D.

1995-01-01

Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis.

PubMed

Pérez-Amador, M A; Abler, M L; De Rocher, E J; Thompson, D M; van Hoof, A; LeBrasseur, N D; Lers, A; Green, P J

2000-01-01

Nuclease I enzymes are responsible for the degradation of RNA and single-stranded DNA during several plant growth and developmental processes, including senescence. However, in the case of senescence the corresponding genes have not been reported. We describe the identification and characterization of BFN1 of Arabidopsis, and demonstrate that it is a senescence-associated nuclease I gene. BFN1 nuclease shows high similarity to the sequence of a barley nuclease induced during germination and a zinnia (Zinnia elegans) nuclease induced during xylogenesis. In transgenic plants overexpressing the BFN1 cDNA, a nuclease activity of about 38 kD was detected on both RNase and DNase activity gels. Levels of BFN1 mRNA were extremely low or undetectable in roots, leaves, and stems. In contrast, relatively high BFN1 mRNA levels were detected in flowers and during leaf and stem senescence. BFN1 nuclease activity was also induced during leaf and stem senescence. The strong response of the BFN1 gene to senescence indicated that it would be an excellent tool with which to study the mechanisms of senescence induction, as well as the role of the BFN1 enzyme in senescence using reverse genetic approaches in Arabidopsis.

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.

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

PubMed

Doke, M; Fukamachi, H; Morisaki, H; Arimoto, T; Kataoka, H; Kuwata, H

2017-08-01

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg 2+ and Ca 2+ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs. © 2016 The Authors Molecular Oral Microbiology Published by John Wiley & Sons Ltd.

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.

Purification and identification of a nuclease activity in embryo axes from French bean.

PubMed

Lambert, Rocío; Quiles, Francisco Antonio; Cabello-Díaz, Juan Miguel; Piedras, Pedro

2014-07-01

Plant nucleases are involved in nucleic acid degradation associated to programmed cell death processes as well as in DNA restriction, repair and recombination processes. However, the knowledge about the function of plant nucleases is limited. A major nuclease activity was detected by in-gel assay with whole embryonic axes of common bean by using ssDNA or RNA as substrate, whereas this activity was minimal in cotyledons. The enzyme has been purified to electrophoretic homogeneity from embryonic axes. The main biochemical properties of the purified enzyme indicate that it belongs to the S1/P1 family of nucleases. This was corroborated when this protein, after SDS-electrophoresis, was excised from the gel and further analysis by MALDI TOF/TOF allowed identification of the gene (PVN1) that codes this protein. The gene that codes the purified protein was identified. The expression of PVN1 gene was induced at the specific moment of radicle protrusion. The inclusion of inorganic phosphate to the imbibition media reduced the level of expression of this gene and the nuclease activity suggesting a relationship with the phosphorous status in French bean seedlings. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A comprehensive overview of computational resources to aid in precision genome editing with engineered nucleases.

PubMed

Periwal, Vinita

2017-07-01

Genome editing with engineered nucleases (zinc finger nucleases, TAL effector nucleases s and Clustered regularly inter-spaced short palindromic repeats/CRISPR-associated) has recently been shown to have great promise in a variety of therapeutic and biotechnological applications. However, their exploitation in genetic analysis and clinical settings largely depends on their specificity for the intended genomic target. Large and complex genomes often contain highly homologous/repetitive sequences, which limits the specificity of genome editing tools and could result in off-target activity. Over the past few years, various computational approaches have been developed to assist the design process and predict/reduce the off-target activity of these nucleases. These tools could be efficiently used to guide the design of constructs for engineered nucleases and evaluate results after genome editing. This review provides a comprehensive overview of various databases, tools, web servers and resources for genome editing and compares their features and functionalities. Additionally, it also describes tools that have been developed to analyse post-genome editing results. The article also discusses important design parameters that could be considered while designing these nucleases. This review is intended to be a quick reference guide for experimentalists as well as computational biologists working in the field of genome editing with engineered nucleases. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Repair of DNA-polypeptide crosslinks by human excision nuclease

NASA Astrophysics Data System (ADS)

Reardon, Joyce T.; Sancar, Aziz

2006-03-01

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases.

PubMed

Zhu, Lihua Julie; Lawrence, Michael; Gupta, Ankit; Pagès, Hervé; Kucukural, Alper; Garber, Manuel; Wolfe, Scot A

2017-05-15

Genome editing technologies developed around the CRISPR-Cas9 nuclease system have facilitated the investigation of a broad range of biological questions. These nucleases also hold tremendous promise for treating a variety of genetic disorders. In the context of their therapeutic application, it is important to identify the spectrum of genomic sequences that are cleaved by a candidate nuclease when programmed with a particular guide RNA, as well as the cleavage efficiency of these sites. Powerful new experimental approaches, such as GUIDE-seq, facilitate the sensitive, unbiased genome-wide detection of nuclease cleavage sites within the genome. Flexible bioinformatics analysis tools for processing GUIDE-seq data are needed. Here, we describe an open source, open development software suite, GUIDEseq, for GUIDE-seq data analysis and annotation as a Bioconductor package in R. The GUIDEseq package provides a flexible platform with more than 60 adjustable parameters for the analysis of datasets associated with custom nuclease applications. These parameters allow data analysis to be tailored to different nuclease platforms with different length and complexity in their guide and PAM recognition sequences or their DNA cleavage position. They also enable users to customize sequence aggregation criteria, and vary peak calling thresholds that can influence the number of potential off-target sites recovered. GUIDEseq also annotates potential off-target sites that overlap with genes based on genome annotation information, as these may be the most important off-target sites for further characterization. In addition, GUIDEseq enables the comparison and visualization of off-target site overlap between different datasets for a rapid comparison of different nuclease configurations or experimental conditions. For each identified off-target, the GUIDEseq package outputs mapped GUIDE-Seq read count as well as cleavage score from a user specified off-target cleavage score prediction

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

Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.

PubMed

Termglinchan, Vittavat; Seeger, Timon; Chen, Caressa; Wu, Joseph C; Karakikes, Ioannis

2017-01-01

Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

PubMed

Liu, Jia; Shui, Sai-Lan

2016-12-28

The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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

Engineering nucleases for gene targeting: safety and regulatory considerations.

PubMed

Pauwels, Katia; Podevin, Nancy; Breyer, Didier; Carroll, Dana; Herman, Philippe

2014-01-25

Nuclease-based gene targeting (NBGT) represents a significant breakthrough in targeted genome editing since it is applicable from single-celled protozoa to human, including several species of economic importance. Along with the fast progress in NBGT and the increasing availability of customized nucleases, more data are available about off-target effects associated with the use of this approach. We discuss how NBGT may offer a new perspective for genetic modification, we address some aspects crucial for a safety improvement of the corresponding techniques and we also briefly relate the use of NBGT applications and products to the regulatory oversight. Copyright © 2013 Elsevier B.V. All rights reserved.

[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.

Probing chromatin structure with nuclease sensitivity assays.

PubMed

Gregory, R I; Khosla, S; Feil, R

2001-01-01

To further our understanding of genomic imprinting it will be essential to identify key control elements, and to investigate their regulation by both epigenetic modifications (such as DNA methylation) and trans-acting factors. So far, sequence elements that regulate parental allele-specific gene expression have been identified in a number of imprinted loci, either because of their differential DNA methylation or through functional studies in transgenic mice (1,2). A systematic search for allele-specific chromatin features constitutes an alternative strategy to identify elements that regulate imprinting. The validity of such an in vivo chromatin approach derives from the fact that in several known imprinting control-elements, a specialized organization of chromatin characterized by nuclease hypersensitivity is present on only one of the two parental chromosome (3). For example, the differentially methylated 5 -portion of the human SNRPN gene-a sequence element that controls imprinting in the Prader-Willi and Angelman syndromes' domain on chromosome 15q11- q13-has strong DNase-I hypersensitive sites on the unmethylated paternal chromosome (4). A differentially methylated region that regulates the imprinting of H19 and that of the neighboring insulin-like growth factor-2 gene on mouse chromosome 7 was also found to have parental chromosome-specific hypersensitive sites (5,6). The precise nature of the allelic nuclease hypersensitivity in these and other imprinted loci remains to be determined in more detail, for example, by applying complementary chromatin methodologies (7,8). However, it is commonly observed that a nuclease hypersensitive site corresponds to a small region where nucleosomes are absent or partially disrupted.

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...

Improving Fab' fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA.

PubMed

Schofield, Desmond M; Sirka, Ernestas; Keshavarz-Moore, Eli; Ward, John M; Nesbeth, Darren N

2017-12-01

To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease. We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media.

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

PubMed

Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

2015-11-02

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

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

The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells. Baculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143 ori. This ori contains

Noninvasive imaging of Staphylococcus aureus infections with a nuclease-activated probe.

PubMed

Hernandez, Frank J; Huang, Lingyan; Olson, Michael E; Powers, Kristy M; Hernandez, Luiza I; Meyerholz, David K; Thedens, Daniel R; Behlke, Mark A; Horswill, Alexander R; McNamara, James O

2014-03-01

Technologies that enable the rapid detection and localization of bacterial infections in living animals could address an unmet need for infectious disease diagnostics. We describe a molecular imaging approach for the specific, noninvasive detection of S. aureus based on the activity of the S. aureus secreted nuclease, micrococcal nuclease (MN). Several short synthetic oligonucleotides, rendered resistant to mammalian serum nucleases by various chemical modifications and flanked with a fluorophore and quencher, were activated upon degradation by purified MN and in S. aureus culture supernatants. A probe consisting of a pair of deoxythymidines flanked by several 2'-O-methyl-modified nucleotides was activated in culture supernatants of S. aureus but not in culture supernatants of several other pathogenic bacteria. Systemic administration of this probe to mice bearing S. aureus muscle infections resulted in probe activation at the infection sites in an MN-dependent manner. This new bacterial imaging approach has potential clinical applicability for infections with S. aureus and several other medically important pathogens.

Evaluation of Biological and Physical Protection against Nuclease Degradation of Clay-Bound Plasmid DNA

PubMed Central

Demanèche, Sandrine; Jocteur-Monrozier, Lucile; Quiquampoix, Hervé; Simonet, Pascal

2001-01-01

In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules. PMID:11133458

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.

Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials.

PubMed

Bikard, David; Euler, Chad W; Jiang, Wenyan; Nussenzweig, Philip M; Goldberg, Gregory W; Duportet, Xavier; Fischetti, Vincent A; Marraffini, Luciano A

2014-11-01

Antibiotics target conserved bacterial cellular pathways or growth functions and therefore cannot selectively kill specific members of a complex microbial population. Here, we develop programmable, sequence-specific antimicrobials using the RNA-guided nuclease Cas9 (refs.1,2) delivered by a bacteriophage. We show that Cas9, reprogrammed to target virulence genes, kills virulent, but not avirulent, Staphylococcus aureus. Reprogramming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also show that CRISPR-Cas9 antimicrobials function in vivo to kill S. aureus in a mouse skin colonization model. This technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa

PubMed Central

Zhou, Chun; Pourmal, Sergei; Pavletich, Nikola P

2015-01-01

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5’ end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5’ but not 3’ end, explaining how Rpa regulates cleavage polarity. DOI: http://dx.doi.org/10.7554/eLife.09832.001 PMID:26491943

An ultrasensitive fluorescence method suitable for quantitative analysis of mung bean nuclease and inhibitor screening in vitro and vivo.

PubMed

Peng, Lan; Fan, Jialong; Tong, Chunyi; Xie, Zhenhua; Zhao, Chuan; Liu, Xuanming; Zhu, Yonghua; Liu, Bin

2016-09-15

Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5'-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10(-4) to 4×10(-2) with a detection limit of 1×10(-4) unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease in vitro and in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Selective Enhancement of Nucleases by Polyvalent DNA-Functionalized Gold Nanoparticles

PubMed Central

Prigodich, Andrew E.; Alhasan, Ali H.

2011-01-01

We demonstrate that polyvalent DNA-functionalized gold nanoparticles (DNA-Au NPs) selectively enhance Ribonuclease H (RNase H) activity, while inhibiting most biologically relevant nucleases. This combination of properties is particularly interesting in the context of gene regulation, since high RNase H activity results in rapid mRNA degradation and general nuclease inhibition results in high biological stability. We investigate the mechanism of selective RNase H activation and find that the high DNA density of DNA-Au NPs is responsible for this unusual behavior. This work adds to our understanding of polyvalent DNA-Au NPs as gene regulation agents, and suggests a new model for selectively controlling protein-nanoparticle interactions. PMID:21268581

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...

The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae.

PubMed

Blokesch, Melanie; Schoolnik, Gary K

2008-11-01

Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification.

The Extracellular Nuclease Dns and Its Role in Natural Transformation of Vibrio cholerae▿

PubMed Central

Blokesch, Melanie; Schoolnik, Gary K.

2008-01-01

Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification. PMID:18757542

[Isolation and purification of nonspecific nuclease of cyanobacterium Plectonema boryanum CALU 465].

PubMed

Tsymbal, N V; Samoĭlenko, V A; Syrchin, S A; Mendzhul, M I

2004-01-01

Nonspecific nuclease has been isolated from the cells of cyanobacterium Plectonema boryanum and purified to homogenic state. It has been established that the method of centrifugation of cell-free culture extract in the sucrose density gradient is efficient for the separation of pigment proteins and enzyme concentration. Under the successive use of two ion-exchangers the nuclease activity was determined in the concentration range of NaCl 0.065-0.085 M after separation of the cell-free cyanobacterium extract on the column with phosphocellulose in the range of 0.2-0.25 M, on the column with DEAE--Toyopearl respectively. The molecular mass of nuclease which is 40 kDa, has been determined by electrophoresis in polyacrylamide gel under denaturating conditions and gel-filtration on Sephadex G-100. It has been also established that the given enzyme is monosubunitary as to its structure.

Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

PubMed Central

Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

2016-01-01

The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

Au nanoparticles/hollow molybdenum disulfide microcubes based biosensor for microRNA-21 detection coupled with duplex-specific nuclease and enzyme signal amplification.

PubMed

Shuai, Hong-Lei; Huang, Ke-Jing; Chen, Ying-Xu; Fang, Lin-Xia; Jia, Meng-Pei

2017-03-15

An ultrasensitive electrochemical biosensor for detecting microRNAs is fabricated based on hollow molybdenum disulfide (MoS 2 ) microcubes. Duplex-specific nuclease, enzyme and electrochemical-chemical-chemical redox cycling are used for signal amplification. Hollow MoS 2 microcubes constructed by ultrathin nanosheets are synthesized by a facile template-assisted strategy and used as supporting substrate. For biosensor assembling, biotinylated ssDNA capture probes are first immobilized on Au nanoparticles (AuNPs)/MoS 2 modified electrode in order to combine with streptavidin-conjugated alkaline phosphatase (SA-ALP). When capture probes hybridize with miRNAs, duplex-specific nuclease cleaves the formative duplexes. At the moment, the biotin group strips from the electrode surface and SA-ALP is incapacitated to attach onto electrode. Then, ascorbic acids induce the electrochemical-chemical-chemical redox cycling to produce electrochemical response in the presence of ferrocene methanol and tris (2-carboxyethyl) phosphine. Under optimum conditions, the proposed biosensor shows a good linear relationship between the current variation and logarithm of the microRNAs concentration ranging from 0.1fM to 0.1pM with a detection limit of 0.086fM (S/N=3). Furthermore, the biosensor is successfully applied to detect target miRNA-21 in human serum samples. Copyright © 2016 Elsevier B.V. All rights reserved.

[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.

Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island

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

Pimkin, Maxim; Miller, C. Glenn; Blakesley, Lauryn

DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the proteinmore » identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.« less

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

Potentiometric sensing of nuclease activities and oxidative damage of single-stranded DNA using a polycation-sensitive membrane electrode.

PubMed

Ding, Jiawang; Qin, Wei

2013-09-15

A simple, general and label-free potentiometric method to measure nuclease activities and oxidative DNA damage in a homogeneous solution using a polycation-sensitive membrane electrode is reported. Protamine, a linear polyionic species, is used as an indicator to report the cleavage of DNA by nucleases such as restriction and nonspecific nucleases, and the damage of DNA induced by hydroxyl radicals. Measurements can be done with a titration mode or a direct detection mode. For the potentiometric titration mode, the enzymatic cleavage dramatically affects the electrostatical interaction between DNA and protamine and thus shifts the response curve for the potentiometric titration of the DNA with protamine. Under the optimized conditions, the enzyme activities can be sensed potentiometrically with detection limits of 2.7×10(-4)U/µL for S1 nuclease, and of 3.9×10(-4)U/µL for DNase I. For the direct detection mode, a biocomplex between protamine and DNA is used as a substrate. The nuclease of interest cleaves the DNA from the protamine/DNA complex into smaller fragments, so that free protamine is generated and can be detected potentiometrically via the polycation-sensitive membrane electrode. Using a direct measurement, the nuclease activities could be rapidly detected with detection limits of 3.2×10(-4)U/µL for S1 nuclease, and of 4.5×10(-4)U/µL for DNase I. Moreover, the proposed potentiometric assays demonstrate the potential applications in the detection of hydroxyl radicals. It is anticipated that the present potentiometric strategy will provide a promising platform for high-throughput screening of nucleases, reactive oxygen species and the drugs with potential inhibition abilities. Copyright © 2013 Elsevier B.V. All rights reserved.

Small Molecule Targeted Recruitment of a Nuclease to RNA.

PubMed

Costales, Matthew G; Matsumoto, Yasumasa; Velagapudi, Sai Pradeep; Disney, Matthew D

2018-06-06

The choreography between RNA synthesis and degradation is a key determinant in biology. Engineered systems such as CRISPR have been developed to rid a cell of RNAs. Here, we show that a small molecule can recruit a nuclease to a specific transcript, triggering its destruction. A small molecule that selectively binds the oncogenic microRNA(miR)-96 hairpin precursor was appended with a short 2'-5' poly(A) oligonucleotide. The conjugate locally activated endogenous, latent ribonuclease (RNase L), which selectively cleaved the miR-96 precursor in cancer cells in a catalytic and sub-stoichiometric fashion. Silencing miR-96 derepressed pro-apoptotic FOXO1 transcription factor, triggering apoptosis in breast cancer, but not healthy breast, cells. These results demonstrate that small molecules can be programmed to selectively cleave RNA via nuclease recruitment and has broad implications.

The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

PubMed Central

Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

2017-01-01

Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

A baculovirus (Bombyx mori nuclear polyhedrosis virus) repeat element functions as a powerful constitutive enhancer in transfected insect cells.

PubMed

Lu, M; Farrell, P J; Johnson, R; Iatrou, K

1997-12-05

It has been previously reported that baculovirus homologous regions, the regions of baculovirus genomes that contain the origins of DNA replication, can augment the expression of a small number of baculovirus genes in vitro. We are now reporting that a region of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) containing the homologous region 3 (HR3) acts as an enhancer for the promoter of a nonviral gene, the cytoplasmic actin gene of the silkmoth B. mori. Incorporation of the HR3 sequences of BmNPV into an actin promoter-based expression cassette results in an augmentation of transgene expression in transfected cells by two orders of magnitude relative to the control recombinant expression cassette. This increase is due to a corresponding increase in the rate of transcription from the actin promoter and not to replication of the expression cassette and occurs only when the HR3 element is linked to the expression cassette in cis. A comparable degree of enhancement in the activity of the silkworm actin promoter occurs also in heterologous lepidopteran cells. Concomitant supplementation of transfected cells with the BmIE1 trans-activator, which was previously shown to be capable of functioning in vitro as a transcriptional co-activator of the cytoplasmic actin gene promoter, results in more than a 1,000-fold increase in the level of expression of recombinant proteins placed under the control of the actin gene promoter. These findings provide the foundation for the development of a nonlytic insect cell expression system for continuous high-level expression of recombinant proteins. Such a system should provide levels of expression of recombinant proteins comparable to those obtained from baculovirus expression systems and should also have the additional advantage of continuous production in a cellular environment that, in contrast to that generated by a baculovirus infection, supports continuously proper posttranslational modifications of recombinant

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

Installing logic gates in permeability controllable polyelectrolyte-carbon nitride films for detecting proteases and nucleases.

PubMed

Chen, Lichan; Zeng, Xiaoting; Dandapat, Anirban; Chi, Yuwu; Kim, Donghwan

2015-09-01

Proteases and nucleases are enzymes heavily involved in many important biological processes, such as cancer initiation, progression, and metastasis; hence, they are indicative of potential diagnostic biomarkers. Here, we demonstrate a new label free and sensitive electrochemiluminescent (ECL) sensing strategy for protease and nuclease assays that utilize target-triggered desorption of programmable polyelectrolyte films assembled on graphite-like carbon nitride (g-C3N4) film to regulate the diffusion flux of a coreactant. Furthermore, we have built Boolean logic gates OR and AND into the polyelectrolyte films, capable of simultaneously sensing proteases and nucleases in a complicated system by breaking it into simple functions. The developed intelligent permeability controlled enzyme sensor may prove valuable in future medical diagnostics.

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.

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.

Early zygote-specific nuclease in mitochondria of the true slime mold Physarum polycephalum.

PubMed

Moriyama, Yohsuke; Yamazaki, Tomokazu; Nomura, Hideo; Sasaki, Narie; Kawano, Shigeyuki

2005-11-01

The active, selective digestion of mtDNA from one parent is a possible molecular mechanism for the uniparental inheritance of mtDNA. In Physarum polycephalum, mtDNA is packed by DNA-binding protein Glom, which packs mtDNA into rod-shaped mt-nucleoids. After the mating, mtDNA from one parent is selectively digested, and the Glom began to disperse. Dispersed Glom was retained for at least 6 h after mtDNA digestion, but disappeared completely by about 12 h after mixing two strains. We identified two novel nucleases using DNA zymography with native-PAGE and SDS-PAGE. One is a Ca2+-dependent, high-molecular-weight nuclease complex (about 670 kDa), and the other is a Mn2+-dependent, high-molecular-weight nuclease complex (440-670 kDa); the activity of the latter was detected as a Mn2+-dependent, 13-kDa DNase band on SDS-PAGE. All mitochondria isolated from myxamoebae had mt-nucleoids, whereas half of the mitochondria isolated from the zygotes at 12 h after mixing had lost the mt-nucleoids. The activity of the Mn2+-dependent nuclease in the isolated mitochondria was detected at least 8 h after mixing of two strains. The timing and localization of the Mn2+-dependent DNase activity matched the selective digestion of mtDNA.

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...

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair

PubMed Central

Meineke, Birthe; Shuman, Stewart

2012-01-01

Breakage of tRNALys(UUU) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNALys(UUU) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 5′-kinase and ligase functions. PMID:22101242

Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair.

PubMed

Meineke, Birthe; Shuman, Stewart

2012-01-01

Breakage of tRNA(Lys(UUU)) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNA(Lys(UUU)) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 5'-kinase and ligase functions.

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.

Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae.

PubMed

Moon, Andrea F; Gaudu, Philippe; Pedersen, Lars C

2014-11-01

The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.

Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases

PubMed Central

2014-01-01

Background The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. Results In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases. PMID:24997498

Functional Specificity of Extracellular Nucleases of Shewanella oneidensis MR-1

PubMed Central

Heun, Magnus; Binnenkade, Lucas; Kreienbaum, Maximilian

2012-01-01

Bacterial species such as Shewanella oneidensis MR-1 require extracellular nucleolytic activity for the utilization of extracellular DNA (eDNA) as a source of nutrients and for the turnover of eDNA as a structural matrix component during biofilm formation. We have previously characterized two extracellular nucleases of S. oneidensis MR-1, ExeM and ExeS. Although both are involved in biofilm formation, they are not specifically required for the utilization of eDNA as a nutrient. Here we identified and characterized EndA, a third extracellular nuclease of Shewanella. The heterologously overproduced and purified protein was highly active and rapidly degraded linear and supercoiled DNAs of various origins. Divalent metal ions (Mg2+ or Mn2+) were required for function. endA is cotranscribed with phoA, an extracellular phosphatase, and is not upregulated upon phosphostarvation. Deletion of endA abolished both extracellular degradation of DNA by S. oneidensis MR-1 and the ability to use eDNA as a sole source of phosphorus. PhoA is not strictly required for the exploitation of eDNA as a nutrient. The activity of EndA prevents the formation of large cell aggregates during planktonic growth. However, in contrast to the findings for ExeM, endA deletion had only minor effects on biofilm formation. The findings strongly suggest that the extracellular nucleases of S. oneidensis exert specific functions required under different conditions. PMID:22492434

Development of sequence-specific antimicrobials based on programmable CRISPR-Cas nucleases

PubMed Central

Bikard, David; Euler, Chad; Jiang, Wenyan; Nussenzweig, Philip M.; Goldberg, Gregory W.; Duportet, Xavier; Fischetti, Vincent A.; Marraffini, Luciano A.

2014-01-01

Antibiotics target conserved bacterial cellular pathways or growth functions and therefore cannot selectively kill specific members of a complex microbial population. Here, we develop programmable, sequence-specific antimicrobials using the RNA-guided nuclease Cas91, 2 delivered by a bacteriophage. We show that Cas9 re-programmed to target virulence genes kills virulent, but not avirulent, Staphylococcus aureus. Re-programming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes3, 4 and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also demonstrate the approach in vivo, showing its efficacy against S. aureus in a mouse skin colonization model. This new technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner. PMID:25282355

CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells.

PubMed

Heo, Young Tae; Quan, Xiaoyuan; Xu, Yong Nan; Baek, Soonbong; Choi, Hwan; Kim, Nam-Hyung; Kim, Jongpil

2015-02-01

Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption.

PubMed

Gopalappa, Ramu; Suresh, Bharathi; Ramakrishna, Suresh; Kim, Hyongbum Henry

2018-03-23

The use of paired Cas9 nickases instead of Cas9 nuclease drastically reduces off-target effects. Because both nickases must function for a nickase pair to make a double-strand break, the efficiency of paired nickases can intuitively be expected to be lower than that of either corresponding nuclease alone. Here, we carefully compared the gene-disrupting efficiency of Cas9 paired nickases with that of nucleases. Interestingly, the T7E1 assay and deep sequencing showed that on-target efficiency of paired D10A Cas9 nickases was frequently comparable, but sometimes higher than that of either corresponding nucleases in mammalian cells. As the underlying mechanism, we found that the HNH domain, which is preserved in the D10A Cas9 nickase, has higher activity than the RuvC domain in mammalian cells. In this study, we showed: (i) the in vivo cleavage efficiency of the HNH domain of Cas9 in mammalian cells is higher than that of the RuvC domain, (ii) paired Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. We envision that our findings which were overlooked in previous reports will serve as a new potential guideline for tool selection for CRISPR-Cas9-mediated gene disruption, facilitating efficient and precise genome editing.

A Novel Nuclease Activity that is Activated by Ca2+ Chelated to EGTA

PubMed Central

Dominguez, Kenneth; Ward, W. Steven

2010-01-01

Most nucleases require a divalent cation as a cofactor, usually Mg2+ or Ca2+, and are inhibited by the chelators EDTA and EGTA. We report the existence of a novel nuclease activity, initially identified in the luminal fluids of the mouse male reproductive tract but subsequently found in other tissues, that requires EGTA chelated to calcium to digest DNA. We refer to this unique enzyme as CEAN (Chelated EGTA Activated Nuclease). Using a fraction of vas deferens luminal fluid, plasmid DNA was degraded in the presence of excess Ca2+ (Ca2+:EGTA = 16) or excess EGTA (Ca2+:EGTA = 0.25), but required the presence of both. Higher levels of EGTA (Ca2+:EGTA = 0.10) prevented activity, suggesting that unchelated EGTA may be a competitive inhibitor. The EGTA-Ca2+ activation of CEAN is reversible as removing EGTA-Ca2+ stops ongoing DNA degradation, but adding EGTA-Ca2+ again reactivates the enzyme. This suggests the possibility that CEAN binds directly to EGTA-Ca2+. CEAN has a greater specificity for the chelator than for the divalent cation. Two other chelators, BAPTA and sodium citrate, do not activate CEAN in the presence of cation, but chelated EDTA does. EGTA chelated to other divalent cations such as Mn2+, Zn2+, and Cu2+ activate CEAN, but not Mg2+. The activity is lost upon boiling suggesting that it is a protein. These data suggest that EGTA and EDTA may not always prevent DNA from nuclease damage. PMID:19938954

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

PubMed Central

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J.; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-01-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%–5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. PMID:24989021

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

Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae

DOE PAGES

Moon, Andrea F.; Gaudu, Philippe; Pedersen, Lars C.

2014-11-01

The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae , facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structuremore » of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. Lastly, these structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.« less

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...

Electrostatic effects in unfolded staphylococcal nuclease

PubMed Central

Fitzkee, Nicholas C.; García-Moreno E, Bertrand

2008-01-01

Structure-based calculations of pK a values and electrostatic free energies of proteins assume that electrostatic effects in the unfolded state are negligible. In light of experimental evidence showing that this assumption is invalid for many proteins, and with increasing awareness that the unfolded state is more structured and compact than previously thought, a detailed examination of electrostatic effects in unfolded proteins is warranted. Here we address this issue with structure-based calculations of electrostatic interactions in unfolded staphylococcal nuclease. The approach involves the generation of ensembles of structures representing the unfolded state, and calculation of Coulomb energies to Boltzmann weight the unfolded state ensembles. Four different structural models of the unfolded state were tested. Experimental proton binding data measured with a variant of nuclease that is unfolded under native conditions were used to establish the validity of the calculations. These calculations suggest that weak Coulomb interactions are an unavoidable property of unfolded proteins. At neutral pH, the interactions are too weak to organize the unfolded state; however, at extreme pH values, where the protein has a significant net charge, the combined action of a large number of weak repulsive interactions can lead to the expansion of the unfolded state. The calculated pK a values of ionizable groups in the unfolded state are similar but not identical to the values in small peptides in water. These studies suggest that the accuracy of structure-based calculations of electrostatic contributions to stability cannot be improved unless electrostatic effects in the unfolded state are calculated explicitly. PMID:18227429

Genome Editing in Mice Using TALE Nucleases.

PubMed

Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

2016-01-01

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

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.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

PubMed

Remy, Séverine; Tesson, Laurent; Menoret, Séverine; Usal, Claire; De Cian, Anne; Thepenier, Virginie; Thinard, Reynald; Baron, Daniel; Charpentier, Marine; Renaud, Jean-Baptiste; Buelow, Roland; Cost, Gregory J; Giovannangeli, Carine; Fraichard, Alexandre; Concordet, Jean-Paul; Anegon, Ignacio

2014-08-01

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. © 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press.

Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases.

PubMed

Chen, Yen-Ting; Hsu, Chiao-Ling; Hou, Shao-Yi

2008-04-15

The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly.

Cold denaturation and 2H2O stabilization of a staphylococcal nuclease mutant.

PubMed Central

Antonino, L C; Kautz, R A; Nakano, T; Fox, R O; Fink, A L

1991-01-01

Cold denaturation is now recognized as a general property of proteins but has been observed only under destabilizing conditions, such as moderate denaturant concentration or low pH. By destabilizing the protein using site-directed mutagenesis, we have observed cold denaturation at pH 7.0 in the absence of denaturants in a mutant of staphylococcal nuclease, which we call NCA S28G for a hybrid protein between staphylococcal nuclease and concanavalin A in which there is the point mutation Ser-28----Gly. The temperature of maximum stability (tmax) as determined by circular dichroism (CD) was 18.1 degrees C, and the midpoints of the thermal unfolding transitions (tm) were 0.6 degrees C and 30.0 degrees C. These values may be compared with the tm of 52.5 degrees C for wild-type staphylococcal nuclease, for which no cold denaturation was observed under these conditions. When the stability of the mutant was examined in 2H2O by NMR, CD, or fluorescence, a substantial increase in the amount of folded protein at the tmax was noted as well as a decrease in tmax, reflecting increased stability. PMID:1652762

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

PubMed

Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

2017-08-25

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Mouse Spermatozoa Contain a Nuclease that Is Activated by Pretreatment with EGTA and Subsequent Calcium Incubation

PubMed Central

Boaz, Segal M.; Dominguez, Kenneth; Shaman, Jeffrey A.; Ward, W. Steven

2009-01-01

We demonstrated that mouse spermatozoa cleave their DNA into ~50 kb loop-sized fragments with topoisomerase IIB when treated with MnCl2 and CaCl2 in a process we term sperm chromatin fragmentation (SCF). SCF can be reversed by EDTA. A nuclease then further degrades the DNA in a process we term sperm DNA degradation (SDD). MnCl2 alone could elicit this activity, but CaCl2 had no effect. Here, we demonstrate the existence of a nuclease in the vas deferens that can be activated by EGTA to digest the sperm DNA by SDD. Spermatozoa were extracted with salt and dithiothreitol to remove protamines and then incubated with EGTA. Next, the EGTA was removed and divalent cations were added. We found that Mn+2, Ca+2, or Zn+2 could each activate SDD in spermatozoa but Mg+2 could not. When the reaction was slowed by incubation on ice, EGTA pretreatment followed by incubation in Ca+2 elicited the reversible fragmentation of sperm DNA evident in SCF. When the reactions were then incubated at 37°C they progressed to the more complete degradation of DNA by SDD. EDTA could also be used to activate the nuclease, but required a higher concentration than EGTA. This EGTA-activatable nuclease activity was found in each fraction of the vas deferens plasma: in the spermatozoa, in the surrounding fluid, and in the insoluble components in the fluid. These results suggest that this sperm nuclease is regulated by a mechanism that is sensitive to EGTA, possibly by removing inhibition of a calcium binding protein. PMID:17879959

GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases

PubMed Central

Nguyen, Nhu T.; Liebers, Matthew; Topkar, Ved V.; Thapar, Vishal; Wyvekens, Nicolas; Khayter, Cyd; Iafrate, A. John; Le, Long P.; Aryee, Martin J.; Joung, J. Keith

2014-01-01

CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called Genome-wide Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-Seq), relies on capture of double-stranded oligodeoxynucleotides into breaks Application of GUIDE-Seq to thirteen RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or ChIP-Seq. GUIDE-Seq also identified RGN-independent genomic breakpoint ‘hotspots’. Finally, GUIDE-Seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases prior to clinical use. PMID:25513782

Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp).

PubMed Central

Przykorska, A; el Adlouni, C; Keith, G; Szarkowski, J W; Dirheimer, G

1992-01-01

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions. Images PMID:1542562

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.

A novel mitochondrial nuclease-associated protein: a major executor of the programmed nuclear death in Tetrahymena thermophila.

PubMed

Osada, Eriko; Akematsu, Takahiko; Asano, Tomoya; Endoh, Hiroshi

2014-03-01

Programmed nuclear death (PND) in the ciliate Tetrahymena is an apoptosis-like phenomenon that occurs in a restricted space of cytoplasm during conjugation. In the process, only the parental macronucleus is selectively eliminated from the progeny cytoplasm, in conjunction with differentiation of new macronuclei for the next generation. For the last decade, mitochondria have been elucidated to be a crucial executioner like apoptosis: apoptosis-inducing factor and yet-unidentified nucleases localised in mitochondria are major factors for PND. To identify such nucleases, we performed a DNase assay in a PAGE (SDS-DNA-PAGE) using total mitochondrial proteins. Some proteins showed DNase activity, but particularly a 17 kDa protein exhibited the highest and predominant activity. Mass spectrometric analysis revealed a novel mitochondrial nuclease, named TMN1, whose homologue has been discovered only in the ciliate Paramecium tetraurelia, but not in other eukaryotes. Gene disruption of TMN1 led to a drastic reduction of mitochondrial nuclease activity and blocked nuclear degradation during conjugation, but did not affect accumulation of autophagic and lysosomal machinery around the parental macronucleus. These observations strongly suggest that the mitochondrial nuclease-associated protein plays a key role in PND as a major executor. Taking the novel protein specific to ciliates in consideration, Tetrahymena would have diverted a different protein from common apoptotic factors shared in eukaryotes to PND in the course of ciliate evolution. © 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

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...

megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

PubMed

Boissel, Sandrine; Jarjour, Jordan; Astrakhan, Alexander; Adey, Andrew; Gouble, Agnès; Duchateau, Philippe; Shendure, Jay; Stoddard, Barry L; Certo, Michael T; Baker, David; Scharenberg, Andrew M

2014-02-01

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.

Identification of Plasmodium falciparum DNA Repair Protein Mre11 with an Evolutionarily Conserved Nuclease Function

PubMed Central

Badugu, Sugith Babu; Nabi, Shaik Abdul; Vaidyam, Pratap; Laskar, Shyamasree; Bhattacharyya, Sunanda; Bhattacharyya, Mrinal Kanti

2015-01-01

The eukaryotic Meiotic Recombination protein 11 (Mre11) plays pivotal roles in the DNA damage response (DDR). Specifically, Mre11 senses and signals DNA double strand breaks (DSB) and facilitates their repair through effector proteins belonging to either homologous recombination (HR) or non-homologous end joining (NHEJ) repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11) that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11). Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N). PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium. PMID:25938776

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.

The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis

PubMed Central

Ipsaro, Jonathan J.; Haase, Astrid D.; Knott, Simon R.; Joshua-Tor, Leemor; Hannon, Gregory J.

2012-01-01

PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism that provides an essential protection for germ cell genomes against the activity of mobile genetic elements1. piRNA populations comprise a molecular definition of transposons that permits them to be distinguished from host genes and selectively silenced. piRNAs can be generated in two distinct ways. Primary piRNAs emanate from discrete genomic loci, termed piRNA clusters, and appear to be derived from long, single-stranded precursors2. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are likely formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner1,3. Secondary piRNAs arise during the adaptive ping-pong cycle, with their 5' termini being formed by the activity of PIWIs themselves2,4. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Zucchini, is a member of the phospholipase D family of phosphodiesterases, which includes both phospholipases and nucleases5–7. We have produced a dimeric, soluble fragment of the mouse Zucchini homolog (mZuc/PLD6) and have shown that it possesses single strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to PLD-family nucleases than to phospholipases. Considered together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs. PMID:23064227

Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

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

Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke

Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to constructmore » an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.« less

Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

PubMed

Patel, Sunita; Sasidhar, Yellamraju U

2007-10-01

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease. Copyright (c) 2007 European Peptide

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.

Biological availability and nuclease resistance extend the in vitro activity of a phosphorothioate-3'hydroxypropylamine oligonucleotide.

PubMed Central

Tam, R C; Li, Y; Noonberg, S; Hwang, D G; Lui, G; Hunt, C A; Garovoy, M R

1994-01-01

Augmented biological activity in vitro has been demonstrated in oligonucleotides (oligos) modified to provide nuclease resistance, to enhance cellular uptake or to increase target affinity. How chemical modification affects the duration of effect of an oligo with potent activity has not been investigated directly. We postulated that modification with internucleotide phosphorothioates and 3' alkylamine provided additional nuclease protection which could significantly extend the biological activity of a 26 mer, (T2). We showed this analog, sT2a, could maximally inhibit interferon gamma-induced HLA-DR mRNA synthesis and surface expression in both HeLa and retinal pigmented epithelial cells and could continue to be effective, in the absence of oligo, 15 days following initial oligo treatment; an effect not observed with its 3'amine counterpart, T2a. In vitro stability studies confirmed that sT2a conferred the greatest stability to nucleases and that cellular accumulation of 32P-sT2a in both cell types was also greater than other T2 oligos. Using confocal microscopy, we revealed that the intracellular distribution of sT2a favored greater nuclear accumulation and release of oligo from cytoplasmic vesicles; a pattern not observed with T2a. These results suggest that phosphorothioate-3'amine modification could increase the duration of effect of T2 oligo by altering nuclease resistance as well as intracellular accumulation and distribution; factors known to affect biological availability. Images PMID:8152930

Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases

PubMed Central

Butler, Nathaniel M.; Baltes, Nicholas J.; Voytas, Daniel F.; Douches, David S.

2016-01-01

Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species. PMID:27493650

Induction of an IAP antagonist in Culex quinquefasciatus larvae in response to infection by the baculovirus CuniNPV

USDA-ARS?s Scientific Manuscript database

CuniNPV is a member of the Dipteran–specific baculoviruses in the genus Deltabaculovirus that specifically infects mosquito larvae within the genus Culex while species of Aedes and Anopheles are refractory. Infections are restricted to the nuclei of larval midgut epithelial cells with transmission...

Modular Nuclease-Responsive DNA Three-Way Junction-Based Dynamic Assembly of a DNA Device and Its Sensing Application.

PubMed

Zhu, Jing; Wang, Lei; Xu, Xiaowen; Wei, Haiping; Jiang, Wei

2016-04-05

Here, we explored a modular strategy for rational design of nuclease-responsive three-way junctions (TWJs) and fabricated a dynamic DNA device in a "plug-and-play" fashion. First, inactivated TWJs were designed, which contained three functional domains: the inaccessible toehold and branch migration domains, the specific sites of nucleases, and the auxiliary complementary sequence. The actions of different nucleases on their specific sites in TWJs caused the close proximity of the same toehold and branch migration domains, resulting in the activation of the TWJs and the formation of a universal trigger for the subsequent dynamic assembly. Second, two hairpins (H1 and H2) were introduced, which could coexist in a metastable state, initially to act as the components for the dynamic assembly. Once the trigger initiated the opening of H1 via TWJs-driven strand displacement, the cascade hybridization of hairpins immediately switched on, resulting in the formation of the concatemers of H1/H2 complex appending numerous integrated G-quadruplexes, which were used to obtain label-free signal readout. The inherent modularity of this design allowed us to fabricate a flexible DNA dynamic device and detect multiple nucleases through altering the recognition pattern slightly. Taking uracil-DNA glycosylase and CpG methyltransferase M.SssI as models, we successfully realized the butt joint between the uracil-DNA glycosylase and M.SssI recognition events and the dynamic assembly process. Furthermore, we achieved ultrasensitive assay of nuclease activity and the inhibitor screening. The DNA device proposed here will offer an adaptive and flexible tool for clinical diagnosis and anticancer drug discovery.

Cancer translocations in human cells induced by zinc finger and TALE nucleases

PubMed Central

Piganeau, Marion; Ghezraoui, Hind; De Cian, Anne; Guittat, Lionel; Tomishima, Mark; Perrouault, Loic; René, Oliver; Katibah, George E.; Zhang, Lei; Holmes, Michael C.; Doyon, Yannick; Concordet, Jean-Paul; Giovannangeli, Carine; Jasin, Maria; Brunet, Erika

2013-01-01

Chromosomal translocations are signatures of numerous cancers and lead to expression of fusion genes that act as oncogenes. The wealth of genomic aberrations found in cancer, however, makes it challenging to assign a specific phenotypic change to a specific aberration. In this study, we set out to use genome editing with zinc finger (ZFN) and transcription activator-like effector (TALEN) nucleases to engineer, de novo, translocation-associated oncogenes at cognate endogenous loci in human cells. Using ZFNs and TALENs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant t(11;22)(q24;q12) and t(2;5)(p23;q35) translocations found in Ewing sarcoma and anaplastic large cell lymphoma (ALCL), respectively. We recovered both translocations with high efficiency, resulting in the expression of the EWSR1–FLI1 and NPM1–ALK fusions. Breakpoint junctions recovered after ZFN cleavage in human embryonic stem (ES) cell–derived mesenchymal precursor cells fully recapitulated the genomic characteristics found in tumor cells from Ewing sarcoma patients. This approach with tailored nucleases demonstrates that expression of fusion genes found in cancer cells can be induced from the native promoter, allowing interrogation of both the underlying mechanisms and oncogenic consequences of tumor-related translocations in human cells. With an analogous strategy, the ALCL translocation was reverted in a patient cell line to restore the integrity of the two participating chromosomes, further expanding the repertoire of genomic rearrangements that can be engineered by tailored nucleases. PMID:23568838

Design of nuclease-based target recycling signal amplification in aptasensors.

PubMed

Yan, Mengmeng; Bai, Wenhui; Zhu, Chao; Huang, Yafei; Yan, Jiao; Chen, Ailiang

2016-03-15

Compared with conventional antibody-based immunoassay methods, aptasensors based on nucleic acid aptamer have made at least two significant breakthroughs. One is that aptamers are more easily used for developing various simple and rapid homogeneous detection methods by "sample in signal out" without multi-step washing. The other is that aptamers are more easily employed for developing highly sensitive detection methods by using various nucleic acid-based signal amplification approaches. As many substances playing regulatory roles in physiology or pathology exist at an extremely low concentration and many chemical contaminants occur in trace amounts in food or environment, aptasensors for signal amplification contribute greatly to detection of such targets. Among the signal amplification approaches in highly sensitive aptasensors, the nuclease-based target recycling signal amplification has recently become a research focus because it shows easy design, simple operation, and rapid reaction and can be easily developed for homogenous assay. In this review, we summarized recent advances in the development of various nuclease-based target recycling signal amplification with the aim to provide a general guide for the design of aptamer-based ultrasensitive biosensing assays. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteases and nucleases involved in the biphasic digestion process of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae).

PubMed

Lomate, Purushottam R; Bonning, Bryony C

2018-07-01

Management of the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), an invasive, agricultural pest in the United States, has presented significant challenges. This polyphagous insect uses both extra-oral and gut-based digestion thwarting protein- or nucleotide-based control strategies. The objective of this study was to biochemically characterize the digestive enzymes (proteases and nucleases) from the saliva, salivary gland and the gut of H. halys. Enzyme profiles for the two tissues and saliva radically differ: The pH optimum for proteases in the gut was six, with cysteine proteases predominant. In contrast, the alkaline pH optima for protease activity in the salivary gland (8-10) and saliva (7) reflected abundant serine protease and cathepsin activities. RNase enzymes were most abundant in saliva, while dsRNase and DNase activities were higher in the salivary gland and saliva compared to those in the gut. These very different enzyme profiles highlight the biphasic digestive system used by this invasive species for efficient processing of plant nutrients. Knowledge of H. halys digestive physiology will allow for counteractive measures targeting digestive enzymes or for appropriate protection of protein- or nucleotide-based management options targeting this pest. © 2018 Wiley Periodicals, Inc.

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

Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases

PubMed Central

Fanslow, Danielle A.; Wirt, Stacey E.; Barker, Jenny C.; Connelly, Jon P.; Porteus, Matthew H.; Dann, Christina Tenenhaus

2014-01-01

Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse “GS” (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro. PMID:25409432

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

Cell wall-anchored nuclease of Streptococcus sanguinis contributes to escape from neutrophil extracellular trap-mediated bacteriocidal activity.

PubMed

Morita, Chisato; Sumioka, Ryuichi; Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg(2+) and Ca(2+) for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.

Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

PubMed Central

Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

2014-01-01

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg2+ and Ca2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression. PMID:25084357

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.

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

PubMed Central

Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D; Younan, Patrick M; Sommer, Karen; Kowalski, John P; Hale, Malika; Stoddard, Barry; Jarjour, Jordan; Astrakhan, Alexander; Kiem, Hans-Peter; Rawlings, David J

2016-01-01

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection. PMID:27741222

Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting

DOEpatents

Chen, Fanqing Frank; Liu, Gang L; Lee, Luke P

2013-10-29

This invention provides a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of nucleic acid (e.g., DNA) length changes and perform nucleic acid footprinting. In various embodiments the ruler comprises a nucleic acid attached to a nanoparticle, such that changes in the nucleic acid length are detectable using surface plasmon resonance. The nanoplasmonic ruler provides a fast and convenient platform for mapping nucleic acid-protein interactions, for nuclease activity monitoring, and for other footprinting related methods.

A Biochemical Approach to Understanding the Fanconi Anemia Pathway-Regulated Nucleases in Genome Maintenance for Preventing Bone Marrow Failure and Cancer

DTIC Science & Technology

2014-04-01

the Fanconi Anemia Pathway- Regulated Nucleases in Genome Maintenance for Preventing Bone Marrow Failure and Cancer PRINCIPAL INVESTIGATOR...GRANT NUMBER 4. TITLE AND SUBTITLE A Biochemical Approach to Understanding the Fanconi Anemia Pathway-Regulated Nucleases in Genome Maintenance for...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Fanconi anemia is the most prevalent inherited BMF syndromes, caused by mutations in

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.

Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

PubMed Central

Nerys-Junior, Arildo; Costa, Lendel C.; Braga-Dias, Luciene P.; Oliveira, Márcia; Rossi, Átila D.; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S.; Tanuri, Amilcar

2014-01-01

Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity. PMID:24688299

Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases.

PubMed

Nerys-Junior, Arildo; Costa, Lendel C; Braga-Dias, Luciene P; Oliveira, Márcia; Rossi, Atila D; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S; Tanuri, Amilcar

2014-03-01

Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease.

PubMed

Wolfs, Jason M; Hamilton, Thomas A; Lant, Jeremy T; Laforet, Marcon; Zhang, Jenny; Salemi, Louisa M; Gloor, Gregory B; Schild-Poulter, Caroline; Edgell, David R

2016-12-27

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

PubMed Central

Hodel, A.; Rice, L. M.; Simonson, T.; Fox, R. O.; Brünger, A. T.

1995-01-01

Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation. PMID:7613463

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

Label-Free Fluorescence Assay of S1 Nuclease and Hydroxyl Radicals Based on Water-Soluble Conjugated Polymers and WS₂ Nanosheets.

PubMed

Li, Junting; Zhao, Qi; Tang, Yanli

2016-06-13

We developed a new method for detecting S1 nuclease and hydroxyl radicals based on the use of water-soluble conjugated poly[9,9-bis(6,6-(N,N,N-trimethylammonium)-fluorene)-2,7-ylenevinylene-co-alt-2,5-dicyano-1,4-phenylene)] (PFVCN) and tungsten disulfide (WS₂) nanosheets. Cationic PFVCN is used as a signal reporter, and single-layer WS₂ is used as a quencher with a negatively charged surface. The ssDNA forms complexes with PFVCN due to much stronger electrostatic interactions between cationic PFVCN and anionic ssDNA, whereas PFVCN emits yellow fluorescence. When ssDNA is hydrolyzed by S1 nuclease or hydroxyl radicals into small fragments, the interactions between the fragmented DNA and PFVCN become weaker, resulting in PFVCN being adsorbed on the surface of WS₂ and the fluorescence being quenched through fluorescence resonance energy transfer. The new method based on PFVCN and WS₂ can sense S1 nuclease with a low detection limit of 5 × 10(-6) U/mL. Additionally, this method is cost-effective by using affordable WS₂ as an energy acceptor without the need for dye-labeled ssDNA. Furthermore, the method provides a new platform for the nuclease assay and reactive oxygen species, and provides promising applications for drug screening.

Label-free optical detection of single-base mismatches by the combination of nuclease and gold nanoparticles.

PubMed

Liu, Meiying; Yuan, Min; Lou, Xinhui; Mao, Hongju; Zheng, Dongmei; Zou, Ruxing; Zou, Nengli; Tang, Xiangrong; Zhao, Jianlong

2011-07-15

We report here an optical approach that enables highly selective and colorimetric single-base mismatch detection without the need of target modification, precise temperature control or stringent washes. The method is based on the finding that nucleoside monophosphates (dNMPs), which are digested elements of DNA, can better stabilize unmodified gold nanoparticles (AuNPs) than single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with the same base-composition and concentration. The method combines the exceptional mismatch discrimination capability of the structure-selective nucleases with the attractive optical property of AuNPs. Taking S1 nuclease as one example, the perfectly matched 16-base synthetic DNA target was distinctively differentiated from those with single-base mutation located at any position of the 16-base synthetic target. Single-base mutations present in targets with varied length up to 80-base, located either in the middle or near to the end of the targets, were all effectively detected. In order to prove that the method can be potentially used for real clinic samples, the single-base mismatch detections with two HBV genomic DNA samples were conducted. To further prove the generality of this method and potentially overcome the limitation on the detectable lengths of the targets of the S1 nuclease-based method, we also demonstrated the use of a duplex-specific nuclease (DSN) for color reversed single-base mismatch detection. The main limitation of the demonstrated methods is that it is limited to detect mutations in purified ssDNA targets. However, the method coupled with various convenient ssDNA generation and purification techniques, has the potential to be used for the future development of detector-free testing kits in single nucleotide polymorphism screenings for disease diagnostics and treatments. Copyright © 2011 Elsevier B.V. All rights reserved.

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

PubMed Central

Flowers, G. Parker; Timberlake, Andrew T.; Mclean, Kaitlin C.; Monaghan, James R.; Crews, Craig M.

2014-01-01

Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability. PMID:24764077

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

Comparison of culture and a novel 5' Taq nuclease assay for direct detection of Campylobacter fetus subsp. venerealis in clinical specimens from cattle.

PubMed

McMillen, Lyle; Fordyce, Geoffry; Doogan, Vivienne J; Lew, Ala E

2006-03-01

A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (chi2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport.

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.

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

Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

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

Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon

2012-03-16

The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosenmore » DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.« less

Prophylactic treatment with alkaline phosphatase in cardiac surgery induces endogenous alkaline phosphatase release.

PubMed

Kats, Suzanne; Brands, Ruud; Hamad, Mohamed A Soliman; Seinen, Willem; Scharnhorst, Volkher; Wulkan, Raymond W; Schönberger, Jacques P; Oeveren, Wim van

2012-02-01

Laboratory and clinical data have implicated endotoxin as an important factor in the inflammatory response to cardiopulmonary bypass. We assessed the effects of the administration of bovine intestinal alkaline phosphatase (bIAP), an endotoxin detoxifier, on alkaline phosphatase levels in patients undergoing coronary artery bypass grafting. A total of 63 patients undergoing coronary artery bypass grafting were enrolled and prospectively randomized. Bovine intestinal alkaline phosphatase (n=32) or placebo (n=31) was administered as an intravenous bolus followed by continuous infusion for 36 hours. The primary endpoint was to evaluate alkaline phosphatase levels in both groups and to find out if administration of bIAP to patients undergoing CABG would lead to endogenous alkaline phosphatase release. No significant adverse effects were identified in either group. In all the 32 patients of the bIAP-treated group, we found an initial rise of plasma alkaline phosphatase levels due to bolus administration (464.27±176.17 IU/L). A significant increase of plasma alkaline phosphatase at 4-6 hours postoperatively was observed (354.97±95.00 IU/L) as well. Using LHA inhibition, it was shown that this second peak was caused by the generation of tissue non specific alkaline phosphatase (TNSALP-type alkaline phosphatase). Intravenous bolus administration plus 8 hours continuous infusion of alkaline phosphatase in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass results in endogenous alkaline phosphatase release. This endogenous alkaline phosphatase may play a role in the immune defense system.

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

(NZ)CH...O contacts assist crystallization of a ParB-like nuclease.

PubMed

Shaw, Neil; Cheng, Chongyun; Tempel, Wolfram; Chang, Jessie; Ng, Joseph; Wang, Xin-Yu; Perrett, Sarah; Rose, John; Rao, Zihe; Wang, Bi-Cheng; Liu, Zhi-Jie

2007-07-07

The major bottleneck for determination of 3 D structures of proteins using X-rays is the production of diffraction quality crystals. Often proteins are subjected to chemical modification to improve the chances of crystallization Here, we report the successful crystallization of a nuclease employing a reductive methylation protocol. The key to crystallization was the successful introduction of 44 new cohesive (NZ) CH...O contacts (3.2-3.7 A) by the addition of 2 methyl groups to the side chain amine nitrogen (NZ) of 9 lysine residues of the nuclease. The new contacts dramatically altered the crystallization properties of the protein, resulting in crystals that diffracted to 1.2 A resolution. Analytical ultracentrifugation analysis and thermodynamics results revealed a more compact protein structure with better solvent exclusion of buried Trp residues in the folded state of the methylated protein, assisting crystallization. In this study, introduction of novel cohesive (NZ)CH...O contacts by reductive methylation resulted in the crystallization of a protein that had previously resisted crystallization in spite of extensive purification and crystallization space screening. Introduction of (NZ)CH...O contacts could provide a solution to crystallization problems for a broad range of protein targets.

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.

Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor.

PubMed

Chang, Ann; Khemlani, Adrina; Kang, HaeJoo; Proft, Thomas

2011-03-01

Streptococcus pyogenes nuclease A (SpnA) is a recently discovered DNase that plays a role in virulence as shown in a mouse infection model. SpnA is the only cell wall-anchored DNase found in S. pyogenes thus far and shows a unique protein architecture. The C-terminal nuclease domain contains highly conserved catalytic site and Mg(2+) binding site residues. However, expression of the SpnA nuclease domain alone resulted in a soluble, but enzymatically inactive protein. We found that at least two out of three oligonucleotide/oligosaccharide-binding fold motifs found in the N-terminal domain are required for SpnA activity, probably contributing to substrate binding. Using a combination of a spnA deletion mutant and a Lactococcus lactis'gain-of-function' mutant, we have shown that SpnA promotes survival in whole human blood and in neutrophil killing assays and this is, at least in part, achieved by the destruction of neutrophil extracellular traps (NETs). We observed higher frequencies for anti-SpnA antibodies in streptococcal disease patient sera (79%, n = 19) compared with sera from healthy donors (33%, n = 9) suggesting that SpnA is expressed during infection. Detection of anti-SpnA antibodies in patient serum might be useful for the diagnostic of post-streptococcal diseases, such as acute rheumatic fever or glomerulonephritis. © 2011 Blackwell Publishing Ltd.

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.

Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

NASA Astrophysics Data System (ADS)

Ponnuswamy, Nandhini; Bastings, Maartje M. C.; Nathwani, Bhavik; Ryu, Ju Hee; Chou, Leo Y. T.; Vinther, Mathias; Li, Weiwei Aileen; Anastassacos, Frances M.; Mooney, David J.; Shih, William M.

2017-05-01

DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.

Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

PubMed Central

Ponnuswamy, Nandhini; Bastings, Maartje M. C.; Nathwani, Bhavik; Ryu, Ju Hee; Chou, Leo Y. T.; Vinther, Mathias; Li, Weiwei Aileen; Anastassacos, Frances M.; Mooney, David J.; Shih, William M.

2017-01-01

DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications. PMID:28561045

Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation

PubMed Central

Seper, Andrea; Fengler, Vera H I; Roier, Sandro; Wolinski, Heimo; Kohlwein, Sepp D; Bishop, Anne L; Camilli, Andrew; Reidl, Joachim; Schild, Stefan

2011-01-01

Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae. PMID:22032623

Distinct Mechanisms of Nuclease-Directed DNA-Structure-Induced Genetic Instability in Cancer Genomes.

PubMed

Zhao, Junhua; Wang, Guliang; Del Mundo, Imee M; McKinney, Jennifer A; Lu, Xiuli; Bacolla, Albino; Boulware, Stephen B; Zhang, Changsheng; Zhang, Haihua; Ren, Pengyu; Freudenreich, Catherine H; Vasquez, Karen M

2018-01-30

Sequences with the capacity to adopt alternative DNA structures have been implicated in cancer etiology; however, the mechanisms are unclear. For example, H-DNA-forming sequences within oncogenes have been shown to stimulate genetic instability in mammals. Here, we report that H-DNA-forming sequences are enriched at translocation breakpoints in human cancer genomes, further implicating them in cancer etiology. H-DNA-induced mutations were suppressed in human cells deficient in the nucleotide excision repair nucleases, ERCC1-XPF and XPG, but were stimulated in cells deficient in FEN1, a replication-related endonuclease. Further, we found that these nucleases cleaved H-DNA conformations, and the interactions of modeled H-DNA with ERCC1-XPF, XPG, and FEN1 proteins were explored at the sub-molecular level. The results suggest mechanisms of genetic instability triggered by H-DNA through distinct structure-specific, cleavage-based replication-independent and replication-dependent pathways, providing critical evidence for a role of the DNA structure itself in the etiology of cancer and other human diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. 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.

Alteration of gene conversion tract length and associated crossing over during plasmid gap repair in nuclease-deficient strains of Saccharomyces cerevisiae.

PubMed

Symington, L S; Kang, L E; Moreau, S

2000-12-01

A plasmid gap repair assay was used to assess the role of three known nucleases, Exo1, Mre11 and Rad1, in the processing of DNA ends and resolution of recombination intermediates during double-strand gap repair. In this assay, alterations in end processing or branch migration are reflected by the frequency of co-conversion of a chromosomal marker 200 bp from the gap. Gap repair associated with crossing over results in integration at the homologous chromosomal locus, whereas the plasmid remains episomal for non-crossover repair events. In mre11 strains, the frequency of gap repair was reduced 3- to 10-fold and conversion tracts were shorter than in the wild-type strain, consistent with a role for this nuclease in processing double-strand breaks. However, conversion tracts were longer in a strain containing the nuclease deficient allele, mre11-H125N, suggesting increased end processing by redundant nucleases. The frequency of gap repair was reduced 2-fold in rad1 mutants and crossing over was reduced, consistent with a role for Rad1 in cleaving recombination intermediates. The frequency of gap repair was increased in exo1 mutants with a significant increase in crossing over. In exo1 mre11 double mutants gap repair was reduced to below the mre11 single mutant level.

Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata, but not in the desert locust, Schistocerca gregaria.

PubMed

Spit, Jornt; Philips, Annelies; Wynant, Niels; Santos, Dulce; Plaetinck, Geert; Vanden Broeck, Jozef

2017-02-01

The responsiveness towards orally delivered dsRNA and the potency of a subsequent environmental RNA interference (RNAi) response strongly differs between different insect species. While some species are very sensitive to dsRNA delivery through the diet, others are not. The underlying reasons for this may vary, but degradation of dsRNA by nucleases in the gut lumen is believed to play a crucial role. The Colorado potato beetle, Leptinotarsa decemlineata, is a voracious defoliator of potato crops worldwide, and is currently under investigation for novel control methods based on dsRNA treatments. Here we describe the identification and characterization of two nuclease genes exclusively expressed in the gut of this pest species. Removal of nuclease activity in adults increased the sensitivity towards dsRNA and resulted in improved protection of potato plants. A similar strategy in the desert locust, Schistocerca gregaria, for which we show a far more potent nuclease activity in the gut juice, did however not lead to an improvement of the RNAi response. Possible reasons for this are discussed. Taken together, the present data confirm a negative effect of nucleases in the gut on the environmental RNAi response, and further suggest that interfering with this activity is a strategy worth pursuing for improving RNAi efficacy in insect pest control applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mung bean nuclease: mode of action and specificity vs synthetic esters of 3′-nucleotides

PubMed Central

Kole, R.; Sierakowska, Halina; Szemplińska, Halina; Shugar, D.

1974-01-01

Mung bean nuclease hydrolyzes synthetic esters of 3′-nucleotides to nucleosides and phosphate esters; esters of 2′-nucleotides, and 2′→ 5′ internucleotide linkages, are resistant. Esters of ribonucleotides are cleaved at 100-fold the rate for deoxyribonucleotides, the increased rate being due to presence of the 2′-hydroxyl and not to differences in conformation. Introduction of a 5′-substituent leads to a 3-fold increase in rate. The rates of hydrolysis vary up to 10-fold with the nature of the base, in the order adenine > hypoxanthine > uracil; and up to 6-fold with the nature of the ester radical. This form of cleavage of esters of 3′-nucleotides is also characteristic for nuclease-3′-nucleotidase activities from potato tubers and wheat, suggesting that one type of enzyme is responsible for all these activities. PMID:10793750

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline.

PubMed

Sovová, Tereza; Kerins, Gerard; Demnerová, Kateřina; Ovesná, Jaroslava

2017-01-01

After induced mutagenesis and transgenesis, genome editing is the next step in the development of breeding techniques. Genome editing using site-directed nucleases - including meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system - is based on the mechanism of double strand breaks. The nuclease is directed to cleave the DNA at a specific place of the genome which is then repaired by natural repair mechanisms. Changes are introduced during the repair that are either accidental or can be targeted if a DNA template with the desirable sequence is provided. These techniques allow making virtually any change to the genome including specific DNA sequence changes, gene insertion, replacements or deletions with unprecedented precision and specificity while being less laborious and more straightforward compared to traditional breeding techniques or transgenesis. Therefore, the research in this field is developing quickly and, apart from model species, multiple studies have focused on economically important species and agronomically important traits that were the key subjects of this review. In plants, studies have been undertaken on disease resistance, herbicide tolerance, nutrient metabolism and nutritional value. In animals, the studies have mainly focused on disease resistance, meat production and allergenicity of milk. However, none of the promising studies has led to commercialization despite several patent applications. The uncertain legal status of genome-editing methods is one of the reasons for poor commercial development, as it is not clear whether the products would fall under the GMO regulation. We believe this issue should be clarified soon in order to allow promising methods to reach their full potential.

Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing.

PubMed

Miyaoka, Yuichiro; Berman, Jennifer R; Cooper, Samantha B; Mayerl, Steven J; Chan, Amanda H; Zhang, Bin; Karlin-Neumann, George A; Conklin, Bruce R

2016-03-31

Precise genome-editing relies on the repair of sequence-specific nuclease-induced DNA nicking or double-strand breaks (DSBs) by homology-directed repair (HDR). However, nonhomologous end-joining (NHEJ), an error-prone repair, acts concurrently, reducing the rate of high-fidelity edits. The identification of genome-editing conditions that favor HDR over NHEJ has been hindered by the lack of a simple method to measure HDR and NHEJ directly and simultaneously at endogenous loci. To overcome this challenge, we developed a novel, rapid, digital PCR-based assay that can simultaneously detect one HDR or NHEJ event out of 1,000 copies of the genome. Using this assay, we systematically monitored genome-editing outcomes of CRISPR-associated protein 9 (Cas9), Cas9 nickases, catalytically dead Cas9 fused to FokI, and transcription activator-like effector nuclease at three disease-associated endogenous gene loci in HEK293T cells, HeLa cells, and human induced pluripotent stem cells. Although it is widely thought that NHEJ generally occurs more often than HDR, we found that more HDR than NHEJ was induced under multiple conditions. Surprisingly, the HDR/NHEJ ratios were highly dependent on gene locus, nuclease platform, and cell type. The new assay system, and our findings based on it, will enable mechanistic studies of genome-editing and help improve genome-editing technology.

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster

PubMed Central

Simoni, Alekos; Siniscalchi, Carla; Chan, Yuk-Sang; Huen, David S.; Russell, Steven; Windbichler, Nikolai; Crisanti, Andrea

2014-01-01

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as ‘homing’ similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species. PMID:24803674

Surveyor nuclease detection of mutations and polymorphisms of mtDNA in children.

PubMed

Pilch, Jacek; Asman, Marek; Jamroz, Ewa; Kajor, Maciej; Kotrys-Puchalska, Elżbieta; Goss, Małgorzata; Krzak, Maria; Witecka, Joanna; Gmiński, Jan; Sieroń, Aleksander L

2010-11-01

Mitochondrial encephalomyopathies are complex disorders with wide range of clinical manifestations. Particularly time-consuming is the identification of mutations in mitochondrial DNA. A group of 20 children with clinical manifestations of mitochondrial encephalomyopathies was selected for molecular studies. The aims were (a) to identify mutations in mtDNA isolated from muscle and (b) to verify detected mutations in DNA isolated from blood, in order to assess the utility of a Surveyor nuclease assay kit for patient screening. The most common changes found were polymorphisms, including a few missense mutations altering the amino acid sequence of mitochondrial proteins. In two boys with MELAS (i.e., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), a mutation A→G3243 was detected in the tRNALeu gene of mtDNA isolated from muscle and blood. In one boy, the carrier status of his mother was confirmed, based on molecular analysis of DNA isolated from blood. A method using Surveyor nuclease allows systematic screening for small mutations in mtDNA, using as its source blood of the patients and asymptomatic carriers. The method still requires confirmation studying a larger group. In some patients, the use of this method should precede and might limit indications for traumatic muscle and skin biopsy. Copyright © 2010 Elsevier Inc. All rights reserved.

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.

Gene Editing With CRISPR/Cas9 RNA-Directed Nuclease.

PubMed

Doetschman, Thomas; Georgieva, Teodora

2017-03-03

Genetic engineering of model organisms and cultured cells has for decades provided important insights into the mechanisms underlying cardiovascular development and disease. In the past few years the development of several nuclease systems has broadened the range of model/cell systems that can be engineered. Of these, the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has become the favorite for its ease of application. Here we will review this RNA-guided nuclease system for gene editing with respect to its usefulness for cardiovascular studies and with an eye toward potential therapy. Studies on its off-target activity, along with approaches to minimize this activity will be given. The advantages of gene editing versus gene targeting in embryonic stem cells, including the breadth of species and cell types to which it is applicable, will be discussed. We will also cover its use in iPSC for research and possible therapeutic purposes; and we will review its use in muscular dystrophy studies where considerable progress has been made toward dystrophin correction in mice. The CRISPR/Ca9s system is also being used for high-throughput screening of genes, gene regulatory regions, and long noncoding RNAs. In addition, the CRISPR system is being used for nongene-editing purposes such as activation and inhibition of gene expression, as well as for fluorescence tagging of chromosomal regions and individual mRNAs to track their cellular location. Finally, an approach to circumvent the inability of post-mitotic cells to support homologous recombination-based gene editing will be presented. In conclusion, applications of the CRISPR/Cas system are expanding at a breath-taking pace and are revolutionizing approaches to gain a better understanding of human diseases. © 2017 American Heart Association, Inc.

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.

Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

PubMed Central

Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

2013-01-01

The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

Construction of a quadruple auxotrophic mutant of an industrial polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease.

PubMed

Zhang, Guo-Chang; Kong, In Iok; Kim, Heejin; Liu, Jing-Jing; Cate, Jamie H D; Jin, Yong-Su

2014-12-01

Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Inactivation of Hepatitis B Virus Replication in Cultured Cells and In Vivo with Engineered Transcription Activator-Like Effector Nucleases

PubMed Central

Bloom, Kristie; Ely, Abdullah; Mussolino, Claudio; Cathomen, Toni; Arbuthnot, Patrick

2013-01-01

Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open-reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in ~35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection. PMID:23883864

Development of synthetic selfish elements based on modular nucleases in Drosophila melanogaster.

PubMed

Simoni, Alekos; Siniscalchi, Carla; Chan, Yuk-Sang; Huen, David S; Russell, Steven; Windbichler, Nikolai; Crisanti, Andrea

2014-06-01

Selfish genes are DNA elements that increase their rate of genetic transmission at the expense of other genes in the genome and can therefore quickly spread within a population. It has been suggested that selfish elements could be exploited to modify the genome of entire populations for medical and ecological applications. Here we report that transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) can be engineered into site-specific synthetic selfish elements (SSEs) and demonstrate their transmission of up to 70% in the Drosophila germline. We show here that SSEs can spread via DNA break-induced homologous recombination, a process known as 'homing' similar to that observed for homing endonuclease genes (HEGs), despite their fundamentally different modes of DNA binding and cleavage. We observed that TALEN and ZFN have a reduced capability of secondary homing compared to HEG as their repetitive structure had a negative effect on their genetic stability. The modular architecture of ZFNs and TALENs allows for the rapid design of novel SSEs against specific genomic sequences making them potentially suitable for the genetic engineering of wild-type populations of animals and plants, in applications such as gene replacement or population suppression of pest species. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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...

Unifying the DNA End-processing Roles of the Artemis Nuclease

PubMed Central

Chang, Howard H. Y.; Watanabe, Go; Lieber, Michael R.

2015-01-01

Artemis is a member of the metallo-β-lactamase protein family of nucleases. It is essential in vertebrates because, during V(D)J recombination, the RAG complex generates hairpins when it creates the double strand breaks at V, D, and J segments, and Artemis is required to open the hairpins so that they can be joined. Artemis is a diverse endo- and exonuclease, and creating a unified model for its wide range of nuclease properties has been challenging. Here we show that Artemis resects iteratively into blunt DNA ends with an efficiency that reflects the AT-richness of the DNA end. GC-rich ends are not cut by Artemis alone because of a requirement for DNA end breathing (and confirmed using fixed pseudo-Y structures). All DNA ends are cut when both the DNA-dependent protein kinase catalytic subunit and Ku accompany Artemis but not when Ku is omitted. These are the first biochemical data demonstrating a Ku dependence of Artemis action on DNA ends of any configuration. The action of Artemis at blunt DNA ends is slower than at overhangs, consistent with a requirement for a slow DNA end breathing step preceding the cut. The AT sequence dependence, the order of strand cutting, the length of the cuts, and the Ku-dependence of Artemis action at blunt ends can be reconciled with the other nucleolytic properties of both Artemis and Artemis·DNA-PKcs in a model incorporating DNA end breathing of blunt ends to form transient single to double strand boundaries that have structural similarities to hairpins and fixed 5′ and 3′ overhangs. PMID:26276388

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

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.

PubMed

Canver, Matthew C; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily N; Needleman, Austen J; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D; Das, Partha Pratim; A Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel E; Orkin, Stuart H

2017-04-01

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

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.

Mineralogical, petrological and geochemical aspects of alkaline and alkaline-carbonatite associations from Brazil

NASA Astrophysics Data System (ADS)

Morbidelli, L.; Gomes, C. B.; Beccaluva, L.; Brotzu, P.; Conte, A. M.; Ruberti, E.; Traversa, G.

1995-12-01

A general description of Mesozoic and Tertiary (Fortaleza) Brazilian alkaline and alkaline-carbonatite districts is presented with reference to mineralogy, petrology, geochemistry and geochronology. It mainly refers to scientific results obtained during the last decade by an Italo-Brazilian research team. Alkaline occurrences are distributed across Brazilian territory from the southern (Piratini, Rio Grande do Sul State) to the northeastern (Fortaleza, Ceará State) regions and are mainly concentrated along the borders of the Paraná Basin generally coinciding with important tectonic lineaments. The most noteworthy characteristics of these alkaline and alkaline-carbonatite suites are: (i) prevalence of intrusive forms; (ii) abundance of cumulate assemblages (minor dunites, frequent clinopyroxenites and members of the ijolite series) and (iii) abundance of evolved rock-types. Many data demonstrate that crystal fractionation was the main process responsible for magma evolution of all Brazilian alkaline rocks. A hypothesis is proposed for the genesis of carbonatite liquids by immiscibility processes. The incidence of REE and trace elements for different major groups of lithotypes, belonging both to carbonatite-bearing and carbonatite-free districts, are documented. Sr and preliminary Nd isotopic data are indicative of a mantle origin for the least evolved magmas of all the studied occurrences. Mantle source material and melting models for the generation of the Brazilian alkaline magma types are also discussed.

Alkaline "Permanent" Paper.

ERIC Educational Resources Information Center

Pacey, Antony

1991-01-01

Discussion of paper manufacturing processes and their effects on library materials focuses on the promotion of alkaline "permanent" paper, with less acid, by Canadian library preservation specialists. Standards for paper acidity are explained; advantages of alkaline paper are described, including decreased manufacturing costs; and…

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5' flap DNA: basis of interstrand cross-link repair by FAN1

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

Gwon, Gwang Hyeon; Kim, Youngran; Liu, Yaqi

2014-10-15

Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5' flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domainmore » playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5' flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.« less

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

The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

PubMed Central

Zhang, Lingli; Nguyen, Hoang Chuong; Chipot, Ludovic; Piotrowski, Emilie; Bertrand, Claire

2014-01-01

Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance. PMID:24837284

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

SPECIES-SPECIFIC DETECTION OF THREE HUMAN-PATHOGENIC MICROSPORIDIAL SPECIES FROM THE GENUS ENCEPHALITOZOON VIA FLUOROGENIC 5' NUCLEASE PCR ASSAYS

EPA Science Inventory

This describes fluorogenic 5' nuclease PCR assays suitable for rapid, sensitive, quantitative, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cunicli and E. intestinalis. The assays utilize species-specific primer sets and a g...

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

PubMed

Kumar, S; Peng, X; Daley, J; Yang, L; Shen, J; Nguyen, N; Bae, G; Niu, H; Peng, Y; Hsieh, H-J; Wang, L; Rao, C; Stephan, C C; Sung, P; Ira, G; Peng, G

2017-04-17

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting

Alkaline pH sensor molecules.

PubMed

Murayama, Takashi; Maruyama, Ichiro N

2015-11-01

Animals can survive only within a narrow pH range. This requires continual monitoring of environmental and body-fluid pH. Although a variety of acidic pH sensor molecules have been reported, alkaline pH sensor function is not well understood. This Review describes neuronal alkaline pH sensors, grouped according to whether they monitor extracellular or intracellular alkaline pH. Extracellular sensors include the receptor-type guanylyl cyclase, the insulin receptor-related receptor, ligand-gated Cl- channels, connexin hemichannels, two-pore-domain K+ channels, and transient receptor potential (TRP) channels. Intracellular sensors include TRP channels and gap junction channels. Identification of molecular mechanisms underlying alkaline pH sensing is crucial for understanding how animals respond to environmental alkaline pH and how body-fluid pH is maintained within a narrow range. © 2015 Wiley Periodicals, Inc.

Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

PubMed Central

Zhao, Haiyan; Lin, Zihan; Lynn, Anna Y.; Varnado, Brittany; Beutler, John A.; Murelli, Ryan P.; Le Grice, Stuart F. J.; Tang, Liang

2015-01-01

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. PMID:26450964

Alkaline battery operational methodology

DOEpatents

Sholklapper, Tal; Gallaway, Joshua; Steingart, Daniel; Ingale, Nilesh; Nyce, Michael

2016-08-16

Methods of using specific operational charge and discharge parameters to extend the life of alkaline batteries are disclosed. The methods can be used with any commercial primary or secondary alkaline battery, as well as with newer alkaline battery designs, including batteries with flowing electrolyte. The methods include cycling batteries within a narrow operating voltage window, with minimum and maximum cut-off voltages that are set based on battery characteristics and environmental conditions. The narrow voltage window decreases available capacity but allows the batteries to be cycled for hundreds or thousands of times.

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.

Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection.

PubMed

Martel, Ralph R; Botros, Ihab W; Rounseville, Matthew P; Hinton, James P; Staples, Robin R; Morales, David A; Farmer, John B; Seligmann, Bruce E

2002-11-01

The principles and performance are described for the ArrayPlate mRNA assay, a multiplexed mRNA assay for high-throughput and high-content screening and drug development. THP-1 monocytes grown and subjected to compound treatments in 96-well plates were subjected to a multiplexed nuclease protection assay in situ. The nuclease protection assay destroyed all cell-derived mRNA, but left intact stoichiometric amounts of 16 target-specific oligonucleotide probes. Upon transfer of processed cell lysates to a microplate that contained a 16-element oligonucleotide array at the bottom of each well, the various probe species were separated by immobilization at predefined elements of the array. Quantitative detection of array-bound probes was by enzyme-mediated chemiluminescence. A high-resolution charge-coupled device imager was used for the simultaneous readout of all 1536 array elements in a 96-well plate. For the measurement of 16 genes in samples of 25000 cells, the average standard deviation from well to well within a plate was 8.6% of signal intensity and was 10.8% from plate to plate. Assay response was linear and reproducibility was constant for all detected genes in samples ranging from 1000 to 50000 cells. When THP-1 monocytes were differentiated with phorbol ester and subsequently activated with bacterial lipopolysaccharide that contained different concentrations of dexamethasone, dose-dependent effects of dexamethasone on the mRNA levels of several genes were observed.

Anodes for alkaline electrolysis

DOEpatents

Soloveichik, Grigorii Lev [Latham, NY

2011-02-01

A method of making an anode for alkaline electrolysis cells includes adsorption of precursor material on a carbonaceous material, conversion of the precursor material to hydroxide form and conversion of precursor material from hydroxide form to oxy-hydroxide form within the alkaline electrolysis cell.

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.

Apollo, an Artemis-related nuclease, interacts with TRF2 and protects human telomeres in S phase.

PubMed

van Overbeek, Megan; de Lange, Titia

2006-07-11

Human chromosome ends are protected by shelterin, an abundant six-subunit protein complex that binds specifically to the telomeric-repeat sequences, regulates telomere length, and ensures that chromosome ends do not elicit a DNA-damage response (reviewed in). Using mass spectrometry of proteins associated with the shelterin component Rap1, we identified an SMN1/PSO2 nuclease family member that is closely related to Artemis. We refer to this protein as Apollo and report that Apollo has the ability to localize to telomeres through an interaction with the shelterin component TRF2. Although its low abundance at telomeres indicates that Apollo is not a core component of shelterin, Apollo knockdown with RNAi resulted in senescence and the activation of a DNA-damage signal at telomeres as evidenced by telomere-dysfunction-induced foci (TIFs). The TIFs occurred primarily in S phase, suggesting that Apollo contributes to a processing step associated with the replication of chromosome ends. Furthermore, some of the metaphase chromosomes showed two telomeric signals at single-chromatid ends, suggesting an aberrant telomere structure. We propose that the Artemis-like nuclease Apollo is a shelterin accessory factor required for the protection of telomeres during or after their replication.

Surfactant-enhanced alkaline flooding: Buffering at intermediate alkaline pH

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

Rudin, J.; Wasan, D.T.

1993-11-01

The alkaline flooding process involves injecting alkaline agents into the reservoir to produce more oil than is produced through conventional waterflooding. The interaction of the alkali in the flood water with the naturally occurring acids in the reservoir oil results in in-situ formation of soaps, which are partially responsible for lowering IFT and improving oil recovery. The extent to which IFT is lowered depends on the specific oil and injection water properties. Numerous investigators have attempted to clarify the relationship between system chemical composition and IFT. An experimental investigation of buffered alkaline flooding system chemistry was undertaken to determine themore » influence of various species present on interfacial tension (IFT) as a function of pH and ionic strength. IFT was found to go through an ultralow minimum in certain pH ranges. This synergism results from simultaneous adsorption of un-ionized and ionized acid species on the interface.« less

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

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.

Effect of alkaline addition on anaerobic sludge digestion with combined pretreatment of alkaline and high pressure homogenization.

PubMed

Fang, Wei; Zhang, Panyue; Zhang, Guangming; Jin, Shuguang; Li, Dongyi; Zhang, Meixia; Xu, Xiangzhe

2014-09-01

To improve anaerobic digestion efficiency, combination pretreatment of alkaline and high pressure homogenization was applied to pretreat sewage sludge. Effect of alkaline dosage on anaerobic sludge digestion was investigated in detail. SCOD of sludge supernatant significantly increased with the alkaline dosage increase after the combined pretreatment because of sludge disintegration. Organics were significantly degraded after the anaerobic digestion, and the maximal SCOD, TCOD and VS removal was 73.5%, 61.3% and 43.5%, respectively. Cumulative biogas production, methane content in biogas and biogas production rate obviously increased with the alkaline dosage increase. Considering both the biogas production and alkaline dosage, the optimal alkaline dosage was selected as 0.04 mol/L. Relationships between biogas production and sludge disintegration showed that the accumulative biogas was mainly enhanced by the sludge disintegration. The methane yield linearly increased with the DDCOD increase as Methane yield (ml/gVS)=4.66 DDCOD-9.69. Copyright © 2014 Elsevier Ltd. All rights reserved.

Pediatric reference intervals for alkaline phosphatase.

PubMed

Zierk, Jakob; Arzideh, Farhad; Haeckel, Rainer; Cario, Holger; Frühwald, Michael C; Groß, Hans-Jürgen; Gscheidmeier, Thomas; Hoffmann, Reinhard; Krebs, Alexander; Lichtinghagen, Ralf; Neumann, Michael; Ruf, Hans-Georg; Steigerwald, Udo; Streichert, Thomas; Rascher, Wolfgang; Metzler, Markus; Rauh, Manfred

2017-01-01

Interpretation of alkaline phosphatase activity in children is challenging due to extensive changes with growth and puberty leading to distinct sex- and age-specific dynamics. Continuous percentile charts from birth to adulthood allow accurate consideration of these dynamics and seem reasonable for an analyte as closely linked to growth as alkaline phosphatase. However, the ethical and practical challenges unique to pediatric reference intervals have restricted the creation of such percentile charts, resulting in limitations when clinical decisions are based on alkaline phosphatase activity. We applied an indirect method to generate percentile charts for alkaline phosphatase activity using clinical laboratory data collected during the clinical care of patients. A total of 361,405 samples from 124,440 patients from six German tertiary care centers and one German laboratory service provider measured between January 2004 and June 2015 were analyzed. Measurement of alkaline phosphatase activity was performed on Roche Cobas analyzers using the IFCC's photometric method. We created percentile charts for alkaline phosphatase activity in girls and boys from birth to 18 years which can be used as reference intervals. Additionally, data tables of age- and sex-specific percentile values allow the incorporation of these results into laboratory information systems. The percentile charts provided enable the appropriate differential diagnosis of changes in alkaline phosphatase activity due to disease and changes due to physiological development. After local validation, integration of the provided percentile charts into result reporting facilitates precise assessment of alkaline phosphatase dynamics in pediatrics.

The Alkaline Diet: Is There Evidence That an Alkaline pH Diet Benefits Health?

PubMed Central

Schwalfenberg, Gerry K.

2012-01-01

This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy. Many books written in the lay literature on the alkaline diet were also reviewed and evaluated in light of the published medical literature. There may be some value in considering an alkaline diet in reducing morbidity and mortality from chronic diseases and further studies are warranted in this area of medicine. PMID:22013455

Characterization and quantification of biochar alkalinity.

PubMed

Fidel, Rivka B; Laird, David A; Thompson, Michael L; Lawrinenko, Michael

2017-01-01

Lack of knowledge regarding the nature of biochar alkalis has hindered understanding of pH-sensitive biochar-soil interactions. Here we investigate the nature of biochar alkalinity and present a cohesive suite of methods for its quantification. Biochars produced from cellulose, corn stover and wood feedstocks had significant low-pK a organic structural (0.03-0.34 meq g -1 ), other organic (0-0.92 meq g -1 ), carbonate (0.02-1.5 meq g -1 ), and other inorganic (0-0.26 meq g -1 ) alkalinities. All four categories of biochar alkalinity contributed to total biochar alkalinity and are therefore relevant to pH-sensitive soil processes. Total biochar alkalinity was strongly correlated with base cation concentration, but biochar alkalinity was not a simple function of elemental composition, soluble ash, fixed carbon, or volatile matter content. More research is needed to characterize soluble biochar alkalis other than carbonates and to establish predictive relationships among biochar production parameters and the composition of biochar alkalis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

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

Watanabe, Masahito; Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571; Umeyama, Kazuhiro

2010-11-05

Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor themore » exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.« less

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.

Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.

PubMed

Citorik, Robert J; Mimee, Mark; Lu, Timothy K

2014-11-01

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.

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

Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

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

Li, T; Huang, S; Zhao, XF

Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs tomore » target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.« less

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.

21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Leukocyte alkaline phosphatase test. 864.7660... Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used to identify the enzyme leukocyte alkaline phosphatase in neutrophilic granulocytes (granular...

21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Leukocyte alkaline phosphatase test. 864.7660... Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used to identify the enzyme leukocyte alkaline phosphatase in neutrophilic granulocytes (granular...

21 CFR 864.7660 - Leukocyte alkaline phosphatase test.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Leukocyte alkaline phosphatase test. 864.7660... Leukocyte alkaline phosphatase test. (a) Identification. A leukocyte alkaline phosphatase test is a device used to identify the enzyme leukocyte alkaline phosphatase in neutrophilic granulocytes (granular...

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, David B.; Lao, Guifang

1998-01-01

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, D.B.; Lao, G.

1998-01-06

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

Application of halophilic nuclease H of Micrococcus varians subsp. halophilus to commercial production of flavoring agent 5'-GMP.

PubMed Central

Kamekura, M; Hamakawa, T; Onishi, H

1982-01-01

RNA was degraded at 60 degrees C for 24 h by halophilic nuclease H in supernatants from broth cultures of Micrococcus varians subsp. halophilus containing 12% NaCl. Since contaminating 5'-nucleotidase exhibited almost no activity under these conditions, the 5'-GMP formed could be recovered from the reaction mixture, and the yield was 805 mg from 5 g of RNA. PMID:6184020

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.

PubMed

Sather, Blythe D; Romano Ibarra, Guillermo S; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A; Scharenberg, Andrew M; Rawlings, David J

2015-09-30

Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties. Copyright © 2015, American Association for the Advancement of Science.

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.

Zinc finger nuclease technology: advances and obstacles in modelling and treating genetic disorders.

PubMed

Jabalameli, Hamid Reza; Zahednasab, Hamid; Karimi-Moghaddam, Amin; Jabalameli, Mohammad Reza

2015-03-01

Zinc finger nucleases (ZFNs) are engineered restriction enzymes designed to target specific DNA sequences within the genome. Assembly of zinc finger DNA-binding domain to a DNA-cleavage domain enables the enzyme machinery to target unique locus in the genome and invoke endogenous DNA repair mechanisms. This machinery offers a versatile approach in allele editing and gene therapy. Here we discuss the architecture of ZFNs and strategies for generating targeted modifications within the genome. We review advances in gene therapy and modelling of the disease using these enzymes and finally, discuss the practical obstacles in using this technology. Copyright © 2014 Elsevier B.V. All rights reserved.

Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

EPA Science Inventory

Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

Net alkalinity and net acidity 2: Practical considerations

USGS Publications Warehouse

Kirby, C.S.; Cravotta, C.A.

2005-01-01

The pH, alkalinity, and acidity of mine drainage and associated waters can be misinterpreted because of the chemical instability of samples and possible misunderstandings of standard analytical method results. Synthetic and field samples of mine drainage having various initial pH values and concentrations of dissolved metals and alkalinity were titrated by several methods, and the results were compared to alkalinity and acidity calculated based on dissolved solutes. The pH, alkalinity, and acidity were compared between fresh, unoxidized and aged, oxidized samples. Data for Pennsylvania coal mine drainage indicates that the pH of fresh samples was predominantly acidic (pH 2.5-4) or near neutral (pH 6-7); ??? 25% of the samples had pH values between 5 and 6. Following oxidation, no samples had pH values between 5 and 6. The Standard Method Alkalinity titration is constrained to yield values >0. Most calculated and measured alkalinities for samples with positive alkalinities were in close agreement. However, for low-pH samples, the calculated alkalinity can be negative due to negative contributions by dissolved metals that may oxidize and hydrolyze. The Standard Method hot peroxide treatment titration for acidity determination (Hot Acidity) accurately indicates the potential for pH to decrease to acidic values after complete degassing of CO2 and oxidation of Fe and Mn, and it indicates either the excess alkalinity or that required for neutralization of the sample. The Hot Acidity directly measures net acidity (= -net alkalinity). Samples that had near-neutral pH after oxidation had negative Hot Acidity; samples that had pH < 6.3 after oxidation had positive Hot Acidity. Samples with similar pH values before oxidation had dissimilar Hot Acidities due to variations in their alkalinities and dissolved Fe, Mn, and Al concentrations. Hot Acidity was approximately equal to net acidity calculated based on initial pH and dissolved concentrations of Fe, Mn, and Al minus the

Evaluation of Alkaline Cleaner Materials

NASA Technical Reports Server (NTRS)

Partz, Earl

1998-01-01

Alkaline cleaners used to process aluminum substrates have contained chromium as the corrosion inhibitor. Chromium is a hazardous substance whose use and control are described by environmental laws. Replacement materials that have the characteristics of chromated alkaline cleaners need to be found that address both the cleaning requirements and environmental impacts. This report will review environmentally friendly candidates evaluated as non-chromium alkaline cleaner replacements and methods used to compare those candidates one versus another. The report will also list characteristics used to select candidates based on their declared contents. It will also describe and evaluate methods used to discriminate among the large number of prospective candidates.

Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.

PubMed

Liu, Yibin; Song, Chen; Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Makrigiorgos, G Mike

2017-04-07

Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

DNA targeting specificity of RNA-guided Cas9 nucleases.

PubMed

Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

2013-09-01

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate.

PubMed Central

Trang, P; Hsu, A W; Liu, F

1999-01-01

RNase P ribozyme cleaves an RNA helix substrate which resembles the acceptor stem and T-stem structures of its natural tRNA substrate. By linking the ribozyme covalently to a sequence (guide sequence) complementary to a target RNA, the catalytic RNA can be converted into a sequence-specific ribozyme, M1GS RNA. We have previously shown that M1GS RNA can efficiently cleave the mRNA sequence encoding thymidine kinase (TK) of herpes simplex virus 1. In this study, a footprint procedure using different nucleases was carried out to map the regions of a M1GS ribozyme that potentially interact with the TK mRNA substrate. The ribozyme regions that are protected from nuclease degradation in the presence of the TK mRNA substrate include those that interact with the acceptor stem and T-stem, the 3' terminal CCA sequence and the cleavage site of a tRNA substrate. However, some of the protected regions (e.g. P13 and P14) are unique and not among those protected in the presence of a tRNA substrate. Identification of the regions that interact with a mRNA substrate will allow us to study how M1GS RNA recognizes a mRNA substrate and facilitate the development of mRNA-cleaving ribozymes for gene-targeting applications. PMID:10556315

Zinc-finger nucleases-based genome engineering to generate isogenic human cell lines.

PubMed

Dreyer, Anne-Kathrin; Cathomen, Toni

2012-01-01

Customized zinc-finger nucleases (ZFNs) have developed into a promising technology to precisely alter mammalian genomes for biomedical research, biotechnology, or human gene therapy. In the context of synthetic biology, the targeted integration of a transgene or reporter cassette into a "neutral site" of the human genome, such as the AAVS1 locus, permits the generation of isogenic human cell lines with two major advantages over standard genetic manipulation techniques: minimal integration site-dependent effects on the transgene and, vice versa, no functional perturbation of the host-cell transcriptome. Here we describe in detail how ZFNs can be employed to target integration of a transgene cassette into the AAVS1 locus and how to characterize the targeted cells by PCR-based genotyping.

Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding.

PubMed

Podevin, Nancy; Davies, Howard V; Hartung, Frank; Nogué, Fabien; Casacuberta, Josep M

2013-06-01

Conventional plant breeding exploits existing genetic variability and introduces new variability by mutagenesis. This has proven highly successful in securing food supplies for an ever-growing human population. The use of genetically modified plants is a complementary approach but all plant breeding techniques have limitations. Here, we discuss how the recent evolution of targeted mutagenesis and DNA insertion techniques based on tailor-made site-directed nucleases (SDNs) provides opportunities to overcome such limitations. Plant breeding companies are exploiting SDNs to develop a new generation of crops with new and improved traits. Nevertheless, some technical limitations as well as significant uncertainties on the regulatory status of SDNs may challenge their use for commercial plant breeding. Copyright © 2013 Elsevier Ltd. All rights reserved.

Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

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

Jemmerson, R.; Low, M.G.

1987-09-08

Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast,more » treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.« less

Increased river alkalinization in the Eastern U.S.

PubMed

Kaushal, Sujay S; Likens, Gene E; Utz, Ryan M; Pace, Michael L; Grese, Melissa; Yepsen, Metthea

2013-09-17

The interaction between human activities and watershed geology is accelerating long-term changes in the carbon cycle of rivers. We evaluated changes in bicarbonate alkalinity, a product of chemical weathering, and tested for long-term trends at 97 sites in the eastern United States draining over 260,000 km(2). We observed statistically significant increasing trends in alkalinity at 62 of the 97 sites, while remaining sites exhibited no significant decreasing trends. Over 50% of study sites also had statistically significant increasing trends in concentrations of calcium (another product of chemical weathering) where data were available. River alkalinization rates were significantly related to watershed carbonate lithology, acid deposition, and topography. These three variables explained ~40% of variation in river alkalinization rates. The strongest predictor of river alkalinization rates was carbonate lithology. The most rapid rates of river alkalinization occurred at sites with highest inputs of acid deposition and highest elevation. The rise of alkalinity in many rivers throughout the Eastern U.S. suggests human-accelerated chemical weathering, in addition to previously documented impacts of mining and land use. Increased river alkalinization has major environmental implications including impacts on water hardness and salinization of drinking water, alterations of air-water exchange of CO2, coastal ocean acidification, and the influence of bicarbonate availability on primary production.

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.

Coupling alkaline pre-extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic hydrolysis and fermentability.

PubMed

Liu, Tongjun; Williams, Daniel L; Pattathil, Sivakumar; Li, Muyang; Hahn, Michael G; Hodge, David B

2014-04-03

A two-stage chemical pretreatment of corn stover is investigated comprising an NaOH pre-extraction followed by an alkaline hydrogen peroxide (AHP) post-treatment. We propose that conventional one-stage AHP pretreatment can be improved using alkaline pre-extraction, which requires significantly less H2O2 and NaOH. To better understand the potential of this approach, this study investigates several components of this process including alkaline pre-extraction, alkaline and alkaline-oxidative post-treatment, fermentation, and the composition of alkali extracts. Mild NaOH pre-extraction of corn stover uses less than 0.1 g NaOH per g corn stover at 80°C. The resulting substrates were highly digestible by cellulolytic enzymes at relatively low enzyme loadings and had a strong susceptibility to drying-induced hydrolysis yield losses. Alkaline pre-extraction was highly selective for lignin removal over xylan removal; xylan removal was relatively minimal (~20%). During alkaline pre-extraction, up to 0.10 g of alkali was consumed per g of corn stover. AHP post-treatment at low oxidant loading (25 mg H2O2 per g pre-extracted biomass) increased glucose hydrolysis yields by 5%, which approached near-theoretical yields. ELISA screening of alkali pre-extraction liquors and the AHP post-treatment liquors demonstrated that xyloglucan and β-glucans likely remained tightly bound in the biomass whereas the majority of the soluble polymeric xylans were glucurono (arabino) xylans and potentially homoxylans. Pectic polysaccharides were depleted in the AHP post-treatment liquor relative to the alkaline pre-extraction liquor. Because the already-low inhibitor content was further decreased in the alkaline pre-extraction, the hydrolysates generated by this two-stage pretreatment were highly fermentable by Saccharomyces cerevisiae strains that were metabolically engineered and evolved for xylose fermentation. This work demonstrates that this two-stage pretreatment process is well suited for

Coupling alkaline pre-extraction with alkaline-oxidative post-treatment of corn stover to enhance enzymatic hydrolysis and fermentability

PubMed Central

2014-01-01

Background A two-stage chemical pretreatment of corn stover is investigated comprising an NaOH pre-extraction followed by an alkaline hydrogen peroxide (AHP) post-treatment. We propose that conventional one-stage AHP pretreatment can be improved using alkaline pre-extraction, which requires significantly less H2O2 and NaOH. To better understand the potential of this approach, this study investigates several components of this process including alkaline pre-extraction, alkaline and alkaline-oxidative post-treatment, fermentation, and the composition of alkali extracts. Results Mild NaOH pre-extraction of corn stover uses less than 0.1 g NaOH per g corn stover at 80°C. The resulting substrates were highly digestible by cellulolytic enzymes at relatively low enzyme loadings and had a strong susceptibility to drying-induced hydrolysis yield losses. Alkaline pre-extraction was highly selective for lignin removal over xylan removal; xylan removal was relatively minimal (~20%). During alkaline pre-extraction, up to 0.10 g of alkali was consumed per g of corn stover. AHP post-treatment at low oxidant loading (25 mg H2O2 per g pre-extracted biomass) increased glucose hydrolysis yields by 5%, which approached near-theoretical yields. ELISA screening of alkali pre-extraction liquors and the AHP post-treatment liquors demonstrated that xyloglucan and β-glucans likely remained tightly bound in the biomass whereas the majority of the soluble polymeric xylans were glucurono (arabino) xylans and potentially homoxylans. Pectic polysaccharides were depleted in the AHP post-treatment liquor relative to the alkaline pre-extraction liquor. Because the already-low inhibitor content was further decreased in the alkaline pre-extraction, the hydrolysates generated by this two-stage pretreatment were highly fermentable by Saccharomyces cerevisiae strains that were metabolically engineered and evolved for xylose fermentation. Conclusions This work demonstrates that this two

Identification and characterization of miRNAs and targets in flax (Linum usitatissimum) under saline, alkaline, and saline-alkaline stresses.

PubMed

Yu, Ying; Wu, Guangwen; Yuan, Hongmei; Cheng, Lili; Zhao, Dongsheng; Huang, Wengong; Zhang, Shuquan; Zhang, Liguo; Chen, Hongyu; Zhang, Jian; Guan, Fengzhi

2016-05-27

MicroRNAs (miRNAs) play a critical role in responses to biotic and abiotic stress and have been characterized in a large number of plant species. Although flax (Linum usitatissimum L.) is one of the most important fiber and oil crops worldwide, no reports have been published describing flax miRNAs (Lus-miRNAs) induced in response to saline, alkaline, and saline-alkaline stresses. In this work, combined small RNA and degradome deep sequencing was used to analyze flax libraries constructed after alkaline-salt stress (AS2), neutral salt stress (NSS), alkaline stress (AS), and the non-stressed control (CK). From the CK, AS, AS2, and NSS libraries, a total of 118, 119, 122, and 120 known Lus-miRNAs and 233, 213, 211, and 212 novel Lus-miRNAs were isolated, respectively. After assessment of differential expression profiles, 17 known Lus-miRNAs and 36 novel Lus-miRNAs were selected and used to predict putative target genes. Gene ontology term enrichment analysis revealed target genes that were involved in responses to stimuli, including signaling and catalytic activity. Eight Lus-miRNAs were selected for analysis using qRT-PCR to confirm the accuracy and reliability of the miRNA-seq results. The qRT-PCR results showed that changes in stress-induced expression profiles of these miRNAs mirrored expression trends observed using miRNA-seq. Degradome sequencing and transcriptome profiling showed that expression of 29 miRNA-target pairs displayed inverse expression patterns under saline, alkaline, and saline-alkaline stresses. From the target prediction analysis, the miR398a-targeted gene codes for a copper/zinc superoxide dismutase, and the miR530 has been shown to explicitly target WRKY family transcription factors, which suggesting that these two micRNAs and their targets may significant involve in the saline, alkaline, and saline-alkaline stress response in flax. Identification and characterization of flax miRNAs, their target genes, functional annotations, and gene

Posttranslational heterogeneity of bone alkaline phosphatase in metabolic bone disease.

PubMed

Langlois, M R; Delanghe, J R; Kaufman, J M; De Buyzere, M L; Van Hoecke, M J; Leroux-Roels, G G

1994-09-01

Bone alkaline phosphatase is a marker of osteoblast activity. In order to study the posttranscriptional modification (glycosylation) of bone alkaline phosphatase in bone disease, we investigated the relationship between mass and catalytic activity of bone alkaline phosphatase in patients with osteoporosis and hyperthyroidism. Serum bone alkaline phosphatase activity was measured after lectin precipitation using the Iso-ALP test kit. Mass concentration of bone alkaline phosphatase was determined with an immunoradiometric assay (Tandem-R Ostase). In general, serum bone alkaline phosphatase mass and activity concentration correlated well. The activity : mass ratio of bone alkaline phosphatase was low in hyperthyroidism. Activation energy of the reaction catalysed by bone alkaline phosphatase was high in osteoporosis and in hyperthyroidism. Experiments with neuraminidase digestion further demonstrated that the thermodynamic heterogeneity of bone alkaline phosphatase can be explained by a different glycosylation of the enzyme.

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

Myostatin gene mutated mice induced with tale nucleases.

PubMed

Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru

2015-01-01

Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection.

Alkaline polymer electrolyte membranes for fuel cell applications.

PubMed

Wang, Yan-Jie; Qiao, Jinli; Baker, Ryan; Zhang, Jiujun

2013-07-07

In this review, we examine the most recent progress and research trends in the area of alkaline polymer electrolyte membrane (PEM) development in terms of material selection, synthesis, characterization, and theoretical approach, as well as their fabrication into alkaline PEM-based membrane electrode assemblies (MEAs) and the corresponding performance/durability in alkaline polymer electrolyte membrane fuel cells (PEMFCs). Respective advantages and challenges are also reviewed. To overcome challenges hindering alkaline PEM technology advancement and commercialization, several research directions are then proposed.

Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.

PubMed

Li, Lixin; Piatek, Marek J; Atef, Ahmed; Piatek, Agnieszka; Wibowo, Anjar; Fang, Xiaoyun; Sabir, J S M; Zhu, Jian-Kang; Mahfouz, Magdy M

2012-03-01

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.

The antigenic surface of staphylococcal nuclease. II. Analysis of the N-1 epitope by site-directed mutagenesis.

PubMed

Smith, A M; Benjamin, D C

1991-02-15

Previous studies in our laboratory on the production and isolation of a panel of mAb to staphylococcal nuclease allowed us to define a series of eight overlapping epitopes. Using site-directed mutagenesis of the nuclease coding sequences we were able to map the nonoverlapping epitopes recognized by two members of this panel. In the study reported here, we report the generation and analysis of a number of single amino acid substitutions for seven surface residues predicted to lie within one of these two epitopes. Immunochemical analysis showed that one or more substitutions at each of these seven positions had a major effect on mAb binding, whereas other substitutions had none. Based on the nature of these substitutions and the chemical and physical properties of the variant molecules, we believe that any structural effects induced by these substitutions are local and do not result in long-range structural alterations that indirectly influence antibody reactivity. Therefore, we conclude that disruption of mAb binding can be directly attributed to changes in amino acid side chains and that not only are all seven of the residues studied part of the epitope but all seven make contact with the antibody combining site. These studies demonstrate the advantages of using site-directed mutagenesis to study antigen structure and emphasize the importance of constructing the examining multiple substitutions for any given amino acid.

Inhibition of aac(6′)-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria

PubMed Central

Soler Bistué, Alfonso J. C.; Martín, Fernando A.; Vozza, Nicolás; Ha, Hongphuc; Joaquín, Jonathan C.; Zorreguieta, Angeles; Tolmasky, Marcelo E.

2009-01-01

Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6′)-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6′)-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6′)-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect. PMID:19666539

Net alkalinity and net acidity 1: Theoretical considerations

USGS Publications Warehouse

Kirby, C.S.; Cravotta, C.A.

2005-01-01

Net acidity and net alkalinity are widely used, poorly defined, and commonly misunderstood parameters for the characterization of mine drainage. The authors explain theoretical expressions of 3 types of alkalinity (caustic, phenolphthalein, and total) and acidity (mineral, CO2, and total). Except for rarely-invoked negative alkalinity, theoretically defined total alkalinity is closely analogous to measured alkalinity and presents few practical interpretation problems. Theoretically defined "CO 2-acidity" is closely related to most standard titration methods with an endpoint pH of 8.3 used for determining acidity in mine drainage, but it is unfortunately named because CO2 is intentionally driven off during titration of mine-drainage samples. Using the proton condition/mass- action approach and employing graphs to illustrate speciation with changes in pH, the authors explore the concept of principal components and how to assign acidity contributions to aqueous species commonly present in mine drainage. Acidity is defined in mine drainage based on aqueous speciation at the sample pH and on the capacity of these species to undergo hydrolysis to pH 8.3. Application of this definition shows that the computed acidity in mg L -1 as CaCO3 (based on pH and analytical concentrations of dissolved FeII, FeIII, Mn, and Al in mg L -1):aciditycalculated=50{1000(10-pH)+[2(FeII)+3(FeIII)]/56+2(Mn)/ 55+3(Al)/27}underestimates contributions from HSO4- and H+, but overestimates the acidity due to Fe3+ and Al3+. However, these errors tend to approximately cancel each other. It is demonstrated that "net alkalinity" is a valid mathematical construction based on theoretical definitions of alkalinity and acidity. Further, it is shown that, for most mine-drainage solutions, a useful net alkalinity value can be derived from: (1) alkalinity and acidity values based on aqueous speciation, (2) measured alkalinity minus calculated acidity, or (3) taking the negative of the value obtained in a

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase*

PubMed Central

Arulanandam, Bernard P.; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J.; Chambers, James P.

2012-01-01

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related Gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella. PMID:22923614

Francisella DnaK inhibits tissue-nonspecific alkaline phosphatase.

PubMed

Arulanandam, Bernard P; Chetty, Senthilnath Lakshmana; Yu, Jieh-Juen; Leonard, Sean; Klose, Karl; Seshu, Janakiram; Cap, Andrew; Valdes, James J; Chambers, James P

2012-10-26

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Let's think in alkaline phosphatase at heart function.

PubMed

Martins, Maria João; Azevedo, Isabel

2010-10-08

In their recent paper, Cheung et al [B.M. Cheung, K.L. Ong, L.Y. Wong, Elevated serum alkaline phosphatase and peripheral arterial disease in the United States National Health and Nutrition Examination Survey 1999-2004. Int J Cardiol 2008 (Electronic publication ahead of print)] described a significant association between serum alkaline phosphatase levels and low ankle-brachial blood pressure index, a risk factor for cardiovascular pathology. We had verified that alkaline phosphatase is present at the rat heart, showing a distribution compatible with cardiomyocyte sarcoplasmic reticulum. Moreover, several drugs with cardiac effect were shown to interfere with heart alkaline phosphatase activity. We therefore propose that alkaline phosphatase may be a local regulator at heart function and a putative target for therapeutic interventions. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.

Alkaline sorbent injection for mercury control

DOEpatents

Madden, Deborah A.; Holmes, Michael J.

2003-01-01

A mercury removal system for removing mercury from combustion flue gases is provided in which alkaline sorbents at generally extremely low stoichiometric molar ratios of alkaline earth or an alkali metal to sulfur of less than 1.0 are injected into a power plant system at one or more locations to remove at least between about 40% and 60% of the mercury content from combustion flue gases. Small amounts of alkaline sorbents are injected into the flue gas stream at a relatively low rate. A particulate filter is used to remove mercury-containing particles downstream of each injection point used in the power plant system.

Alkaline sorbent injection for mercury control

DOEpatents

Madden, Deborah A.; Holmes, Michael J.

2002-01-01

A mercury removal system for removing mercury from combustion flue gases is provided in which alkaline sorbents at generally extremely low stoichiometric molar ratios of alkaline earth or an alkali metal to sulfur of less than 1.0 are injected into a power plant system at one or more locations to remove at least between about 40% and 60% of the mercury content from combustion flue gases. Small amounts of alkaline sorbents are injected into the flue gas stream at a relatively low rate. A particulate filter is used to remove mercury-containing particles downstream of each injection point used in the power plant system.

Inorganic-organic separators for alkaline batteries

NASA Technical Reports Server (NTRS)

Sheibley, D. W. (Inventor)

1978-01-01

A flexible separator is reported for use between the electrodes of Ni-Cd and Ni-Zn batteries using alkaline electrolytes. The separator was made by coating a porous substrate with a battery separator composition. The coating material included a rubber-based resin copolymer, a plasticizer and inorganic and organic fillers which comprised 55% by volume or less of the coating as finally dried. One or more of the filler materials, whether organic or inorganic, is preferably active with the alkaline electrolyte to produce pores in the separator coating. The plasticizer was an organic material which is hydrolyzed by the alkaline electrolyte to improve conductivity of the separator coating.

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.

Oil recovery by alkaline waterflooding

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

Cooke, C.E. Jr.; Williams, R.E.; Kolodzie, P.A.

1974-01-01

Flooding of oil containing organic acids with alkaline water under favorable conditions can result in recovery of around 50% of the residual oil left in a watered-out model. A high recovery efficiency results from the formation of a bank of viscous water-in-oil emulsion as surface active agents (soaps) are created by reactions of base in the water with the organic acids in the oil. The type and amount of organic acids in the oil, the pH and salt content of the water, and the amount of fines in the porous medium are the primary factors which determine the amount ofmore » additional oil recovered by this method. Interaction of alkaline water with reservoir rock largely determines the amount of chemical needed to flood a reservoir. Laboratory investigations using synthetic oils and crude oils show the importance of oil-water and liquid-solid interfacial properties to the results of an alkaline waterflood. A small field test demonstrated that emulsion banks can be formed in the reservoir and that chemical costs can be reasonable in selected reservoirs. Although studies have provided many qualitative guide lines for evaluating the feasibility of alkaline waterflooding, the economic attractiveness of the process must be considered on an individual reservoir.« less

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.

Alkaline Phosphatase: MedlinePlus Lab Test Information

MedlinePlus

... Test Information → Alkaline Phosphatase URL of this page: https://medlineplus.gov/labtests/alkalinephosphatase.html Alkaline Phosphatase To ... 2017 Mar 13]; [about 3 screens]. Available from: http://www.liverfoundation.org/abouttheliver/info/liverfunctiontests/ Centers for ...

21 CFR 862.1050 - Alkaline phosphatase or isoenzymes test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alkaline phosphatase or isoenzymes test system... Test Systems § 862.1050 Alkaline phosphatase or isoenzymes test system. (a) Identification. An alkaline phosphatase or isoenzymes test system is a device intended to measure alkaline phosphatase or its isoenzymes...

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

Acidity and alkalinity in mine drainage: Theoretical considerations

USGS Publications Warehouse

Kirby, Carl S.; Cravotta,, Charles A.

2004-01-01

Acidity, net acidity, and net alkalinity are widely used parameters for the characterization of mine drainage, but these terms are not well defined and are often misunderstood. Incorrect interpretation of acidity, alkalinity, and derivative terms can lead to inadequate treatment design or poor regulatory decisions. We briefly explain derivations of theoretical expressions of three types of alkalinities (caustic, phenolphthalein, and total) and acidities (mineral, CO2, and total). Theoretically defined total alkalinity is closely analogous to measured alkalinity and presents few practical interpretation problems. Theoretically defined “CO2- acidity” is closely related to most standard titration methods used for mine drainage with an endpoint pH of 8.3, but it presents numerous interpretation problems, and it is unfortunately named because CO2 is intentionally driven off during titration of mine-drainage samples. Using the proton condition/massaction approach and employing graphs for visualization, we explore the concept of principal components and how to assign acidity contributions to solution species, including aqueous complexes, commonly found in mine drainage. We define a comprehensive theoretical definition of acidity in mine drainage on the basis of aqueous speciation at the sample pH and the capacity of these species to undergo hydrolysis to pH 8.3. This definition indicates the computed acidity in milligrams per liter (mg L-1 ) as CaCO3 (based on pH and analytical concentrations of dissolved FeIII , FeII , Mn, and Al in mg L-1 ): Aciditycomputed = 50. (10(3-pH) + 3.CFeIII/55.8 + 2.CFeII/55.8 + 2.CMn/54.9 + 3.CAl/27.0) underestimates contributions from HSO4 - and H+ , but overestimates the acidity due to Fe3+. These errors tend to approximately cancel each other. We demonstrate that “net alkalinity” is a valid mathematical construction based on theoretical definitions of alkalinity and acidity. We demonstrate that, for most mine-drainage solutions, a

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein.

PubMed

Pawluk, April; Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F; Davidson, Alan R; Maxwell, Karen L

2017-12-12

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. IMPORTANCE The CRISPR-Cas immune system provides bacteria with resistance to invasion by potentially harmful viruses, plasmids, and other foreign mobile genetic elements. This study presents the first structural and mechanistic insight into a phage-encoded protein that inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa The interaction of this anti-CRISPR protein with the CRISPR-associated helicase/nuclease proteins Cas3 shuts down the CRISPR-Cas system and protects phages carrying this gene from destruction. This interaction also allows the repurposing of the endogenous type I-E CRISPR system into a programmable transcriptional repressor, providing a new biotechnological tool for genetic studies of bacteria encoding this type I-E CRISPR-Cas system. Copyright © 2017 Pawluk et al.

Field measurement of alkalinity and pH

USGS Publications Warehouse

Barnes, Ivan

1964-01-01

The behavior of electrometric pH equipment under field conditions departs from the behavior predicted from Nernst's law. The response is a linear function of pH, and hence measured pH values may be corrected to true pH if the instrument is calibrated with two reference solutions for each measurement. Alkalinity titrations may also be made in terms of true pH. Standard methods, such as colorimetric titrations, were rejected as unreliable or too cumbersome for rapid field use. The true pH of the end point of the alkalinity titration as a function of temperature, ionic strength, and total alkalinity has been calculated. Total alkalinity in potable waters is the most important factor influencing the end point pH, which varies from 5.38 (0 ? C, 5 ppm (parts per million) HC0a-) to 4.32 (300 ppm HC0a-,35 ? C), for the ranges of variables considered. With proper precautions, the pH may be determined to =i:0.02 pH and the alkalinity to =i:0.6 ppm HCO3- for many naturally occurring bodies of fresh water.

Diclofenac salts. III. Alkaline and earth alkaline salts.

PubMed

Fini, Adamo; Fazio, Giuseppe; Rosetti, Francesca; Angeles Holgado, M; Iruín, Ana; Alvarez-Fuentes, Josefa

2005-11-01

Diclofenac salts containing the alkaline and two earth alkaline cations have been prepared and characterized by scanning electron microscopy (SEM) and EDAX spectroscopy; and by thermal and thermogravimetric analysis (TGA): all of them crystallize as hydrate when precipitated from water. The salts dehydrate at room temperature and more easily on heating, but recovery the hydration, when placed in a humid environment. X-ray diffraction spectra suggest that on dehydration new peaks appear on diffractograms and the lattice of the salts partially looses crystallinity. This phenomenon is readily visible in the case of the calcium and magnesium salts, whose thermograms display a crystallization exotherm, before melting or decomposing at temperatures near or above 200 degrees C; these last salts appear to form solvates, when prepared from methanol. The thermogram of each salt shows a complex endotherm of dehydration about 100 degrees C; the calcium salt displays two endotherms, well separated at about 120 and 160 degrees C, which disappear after prolonged heating. Decomposition exotherms, before or soon after the melting, appear below 300 degrees C. The ammonium salt is thermally unstable and, when heated to start dehydration, dissociates and leaves acidic diclofenac.

Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance.

PubMed

Hayashi, Junsuke; Samezawa, Yusuke; Ochi, Yosuke; Wada, Shun-Ichi; Urata, Hidehito

2017-07-15

We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named "Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides" were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Tale nucleases--new tool for genome editing].

PubMed

Glazkova, D V; Shipulin, G A

2014-01-01

The ability to introduce targeted changes in the genome of living cells or entire organisms enables researchers to meet the challenges of basic life sciences, biotechnology and medicine. Knockdown of target genes in the zygotes gives the opportunity to investigate the functions of these genes in different organisms. Replacement of single nucleotide in the DNA sequence allows to correct mutations in genes and thus to cure hereditary diseases. Adding transgene to specific genomic.loci can be used in biotechnology for generation of organisms with certain properties or cell lines for biopharmaceutical production. Such manipulations of gene sequences in their natural chromosomal context became possible after the emergence of the technology called "genome editing". This technology is based on the induction of a double-strand break in a specific genomic target DNA using endonucleases that recognize the unique sequences in the genome and on subsequent recovery of DNA integrity through the use of cellular repair mechanisms. A necessary tool for the genome editing is a custom-designed endonuclease which is able to recognize selected sequences. The emergence of a new type of programmable endonucleases, which were constructed on the basis of bacterial proteins--TAL-effectors (Transcription activators like effector), has become an important stage in the development of technology and promoted wide spread of the genome editing. This article reviews the history of the discovery of TAL effectors and creation of TALE nucleases, and describes their advantages over zinc finger endonucleases that appeared earlier. A large section is devoted to description of genetic modifications that can be performed using the genome editing.

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.

Temperature Dependence of Mineral Solubility in Water. Part 3. Alkaline and Alkaline Earth Sulfates

NASA Astrophysics Data System (ADS)

Krumgalz, B. S.

2018-06-01

The databases of alkaline and alkaline earth sulfate solubilities in water at various temperatures were created using experimental data from the publications over about the last two centuries. Statistical critical evaluation of the created databases was produced since there were enough independent data sources to justify such evaluation. The reliable experimental data were adequately described by polynomial expressions over various temperature ranges. Using the Pitzer approach for ionic activity and osmotic coefficients, the thermodynamic solubility products for the discussed minerals have been calculated at various temperatures and represented by polynomial expressions.

Temperature Dependence of Mineral Solubility in Water. Part 2. Alkaline and Alkaline Earth Bromides

NASA Astrophysics Data System (ADS)

Krumgalz, B. S.

2018-03-01

Databases of alkaline and alkaline earth bromide solubilities in water at various temperatures were created using experimental data from publications over about the last two centuries. Statistical critical evaluation of the created databases was produced since there were enough independent data sources to justify such evaluation. The reliable experimental data were adequately described by polynomial expressions over various temperature ranges. Using the Pitzer approach for ionic activity and osmotic coefficients, the thermodynamic solubility products for the discussed bromide minerals have been calculated at various temperature intervals and also represented by polynomial expressions.

The modification of siRNA with 3' cholesterol to increase nuclease protection and suppression of native mRNA by select siRNA polyplexes.

PubMed

Ambardekar, Vishakha V; Han, Huai-Yun; Varney, Michelle L; Vinogradov, Serguei V; Singh, Rakesh K; Vetro, Joseph A

2011-02-01

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3' cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. Copyright © 2010 Elsevier Ltd. All rights reserved.

Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2

PubMed Central

Tsabar, Michael; Eapen, Vinay V.; Mason, Jennifer M.; Memisoglu, Gonen; Waterman, David P.; Long, Marcus J.; Bishop, Douglas K.; Haber, James E.

2015-01-01

In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5′ to 3′ end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. PMID:26019182

A Comparative Study: Taxonomic Grouping of Alkaline Protease Producing Bacilli.

PubMed

Tekin, Nilgun; Cihan, Arzu Coleri; Karaca, Basar; Cokmus, Cumhur

2017-03-30

Alkaline proteases have biotechnological importance due to their activity and stability at alkaline pH. 56 bacteria, capable of growing under alkaline conditions were isolated and their alkaline protease activities were carried out at different parameters to determine their optimum alkaline protease production conditions. Seven isolates were showed higher alkaline protease production capacity than the reference strains. The highest alkaline protease producing isolates (103125 U/g), E114 and C265, were identified as Bacillus licheniformis with 99.4% and Bacillus mojavensis 99.8% based on 16S rRNA gene sequence similarities, respectively. Interestingly, the isolates identified as Bacillus safensis were also found to be high alkaline protease producing strains. Genotypic characterizations of the isolates were also determined by using a wide range of molecular techniques (ARDRA, ITS-PCR, (GTG)5-PCR, BOX-PCR). These different techniques allowed us to differentiate the alkaliphilic isolates and the results were in concurrence with phylogenetic analyses of the 16S rRNA genes. While ITS-PCR provided the highest correlation with 16S rRNA groups, (GTG)5-PCR showed the highest differentiation at species and intra-species level. In this study, each of the biotechnologically valuable alkaline protease producing isolates was grouped into their taxonomic positions with multi-genotypic analyses.

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.

Zirconia coating stabilized super-iron alkaline cathodes

NASA Astrophysics Data System (ADS)

Yu, Xingwen; Licht, Stuart

A low-level zirconia coating significantly stabilizes high energy alkaline super-iron cathodes, and improves the energy storage capacity of super-iron batteries. Zirconia coating is derived from ZrCl 4 in an organic medium through the conversion of ZrCl 4 to ZrO 2. In alkaline battery system, ZrO 2 provides an intact shield for the cathode materials and the hydroxide shuttle through the coating sustains alkaline cathode redox chemistry. Most super-iron cathodes are solid-state stable, such as K 2FeO 4 and Cs 2FeO 4, but tend to be passivated in alkaline electrolyte due to the formation of Fe(III) over layer. Zirconia coating effectively enhances the stability of these super-iron cathodes. However, for solid-state unstable super-iron cathode (e.g. BaFeO 4), only a little stabilization effect of zirconia coating is observed.

Global Variability and Changes in Ocean Total Alkalinity from Aquarius Satellite

NASA Astrophysics Data System (ADS)

Fine, R. A.; Willey, D. A.; Millero, F. J., Jr.

2016-02-01

To document effects of ocean acidification it is important to have an understanding of the processes and parameters that influence alkalinity. Alkalinity is a gauge on the ability of seawater to neutralize acids. We use Aquarius satellite data, which allow unprecedented global mapping of surface total alkalinity as it correlates strongly with salinity and to a lesser extent with temperature. Spatial variability in total alkalinity and salinity exceed temporal variability, the latter includes seasonal and differences compared to climatological data. The northern hemisphere has more spatial and monthly variability in total alkalinity and salinity, while less variability in Southern Ocean alkalinity is due to less salinity variability and upwelling of waters enriched in alkalinity. Satellite alkalinity data are providing a global baseline that can be used for comparing with future carbon data, and for evaluating spatial and temporal variability and past trends. For the first time it is shown that recent satellite derived total alkalinity in the subtropics have increased as compared with climatological data; this is reflective of large scale changes in the global water cycle. Total alkalinity increases imply increased dissolution of calcareous minerals and difficulty for calcifying organisms to make their shells.

Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment.

PubMed

Kozlowska, Anna Karolina; Florczak, Anna; Smialek, Maciej; Dondajewska, Ewelina; Mackiewicz, Andrzej; Kortylewski, Marcin; Dams-Kozlowska, Hanna

2017-09-01

Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel

Process for extracting technetium from alkaline solutions

DOEpatents

Moyer, Bruce A.; Sachleben, Richard A.; Bonnesen, Peter V.

1995-01-01

A process for extracting technetium values from an aqueous alkaline solution containing at least one alkali metal hydroxide and at least one alkali metal nitrate, the at least one alkali metal nitrate having a concentration of from about 0.1 to 6 molar. The solution is contacted with a solvent consisting of a crown ether in a diluent for a period of time sufficient to selectively extract the technetium values from the aqueous alkaline solution. The solvent containing the technetium values is separated from the aqueous alkaline solution and the technetium values are stripped from the solvent.

Alkaline tolerant dextranase from streptomyces anulatus

DOEpatents

Decker, Stephen R.; Adney, William S.; Vinzant, Todd B.; Himmel, Michael E.

2003-01-01

A process for production of an alkaline tolerant dextranase enzyme comprises culturing a dextran-producing microorganism Streptomyces anulatus having accession no. ATCC PTA-3866 to produce an alkaline tolerant dextranase, Dex 1 wherein the protein in said enzyme is characterized by a MW of 63.3 kDa and Dex 2 wherein its protein is characterized by a MW of 81.8 kDa.

40 CFR 420.110 - Applicability; description of the alkaline cleaning subcategory.

Code of Federal Regulations, 2011 CFR

2011-07-01

... alkaline cleaning subcategory. 420.110 Section 420.110 Protection of Environment ENVIRONMENTAL PROTECTION... Alkaline Cleaning Subcategory § 420.110 Applicability; description of the alkaline cleaning subcategory... alkaline cleaning baths to remove mineral and animal fats or oils from the steel, and those rinsing...

40 CFR 420.110 - Applicability; description of the alkaline cleaning subcategory.

Code of Federal Regulations, 2010 CFR

2010-07-01

... alkaline cleaning subcategory. 420.110 Section 420.110 Protection of Environment ENVIRONMENTAL PROTECTION... Alkaline Cleaning Subcategory § 420.110 Applicability; description of the alkaline cleaning subcategory... alkaline cleaning baths to remove mineral and animal fats or oils from the steel, and those rinsing...

40 CFR 420.110 - Applicability; description of the alkaline cleaning subcategory.

Code of Federal Regulations, 2013 CFR

2013-07-01

... alkaline cleaning subcategory. 420.110 Section 420.110 Protection of Environment ENVIRONMENTAL PROTECTION... Alkaline Cleaning Subcategory § 420.110 Applicability; description of the alkaline cleaning subcategory... alkaline cleaning baths to remove mineral and animal fats or oils from the steel, and those rinsing...

40 CFR 420.110 - Applicability; description of the alkaline cleaning subcategory.

Code of Federal Regulations, 2012 CFR

2012-07-01

... alkaline cleaning subcategory. 420.110 Section 420.110 Protection of Environment ENVIRONMENTAL PROTECTION... Alkaline Cleaning Subcategory § 420.110 Applicability; description of the alkaline cleaning subcategory... alkaline cleaning baths to remove mineral and animal fats or oils from the steel, and those rinsing...

40 CFR 420.110 - Applicability; description of the alkaline cleaning subcategory.

Code of Federal Regulations, 2014 CFR

2014-07-01

... alkaline cleaning subcategory. 420.110 Section 420.110 Protection of Environment ENVIRONMENTAL PROTECTION... Alkaline Cleaning Subcategory § 420.110 Applicability; description of the alkaline cleaning subcategory... alkaline cleaning baths to remove mineral and animal fats or oils from the steel, and those rinsing...

Assessing ocean alkalinity for carbon sequestration

NASA Astrophysics Data System (ADS)

Renforth, Phil; Henderson, Gideon

2017-09-01

Over the coming century humanity may need to find reservoirs to store several trillions of tons of carbon dioxide (CO2) emitted from fossil fuel combustion, which would otherwise cause dangerous climate change if it were left in the atmosphere. Carbon storage in the ocean as bicarbonate ions (by increasing ocean alkalinity) has received very little attention. Yet recent work suggests sufficient capacity to sequester copious quantities of CO2. It may be possible to sequester hundreds of billions to trillions of tons of C without surpassing postindustrial average carbonate saturation states in the surface ocean. When globally distributed, the impact of elevated alkalinity is potentially small and may help ameliorate the effects of ocean acidification. However, the local impact around addition sites may be more acute but is specific to the mineral and technology. The alkalinity of the ocean increases naturally because of rock weathering in which >1.5 mol of carbon are removed from the atmosphere for every mole of magnesium or calcium dissolved from silicate minerals (e.g., wollastonite, olivine, and anorthite) and 0.5 mol for carbonate minerals (e.g., calcite and dolomite). These processes are responsible for naturally sequestering 0.5 billion tons of CO2 per year. Alkalinity is reduced in the ocean through carbonate mineral precipitation, which is almost exclusively formed from biological activity. Most of the previous work on the biological response to changes in carbonate chemistry have focused on acidifying conditions. More research is required to understand carbonate precipitation at elevated alkalinity to constrain the longevity of carbon storage. A range of technologies have been proposed to increase ocean alkalinity (accelerated weathering of limestone, enhanced weathering, electrochemical promoted weathering, and ocean liming), the cost of which may be comparable to alternative carbon sequestration proposals (e.g., $20-100 tCO2-1). There are still many

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.

The Modification of siRNA with 3′ Cholesterol to Increase Nuclease Protection and Suppression of Native mRNA by Select siRNA Polyplexes

PubMed Central

Ambardekar, Vishakha V.; Han, Huai-Yun; Varney, Michelle L.; Vinogradov, Serguei V.; Singh, Rakesh K.; Vetro, Joseph A.

2010-01-01

Polymer-siRNA complexes (siRNA polyplexes) are being actively developed to improve the therapeutic application of siRNA. A major limitation for many siRNA polyplexes, however, is insufficient mRNA suppression. Given that modifying the sense strand of siRNA with 3′ cholesterol (chol-siRNA) increases the activity of free nuclease-resistant siRNA in vitro and in vivo, we hypothesized that complexation of chol-siRNA can increase mRNA suppression by siRNA polyplexes. In this study, the characteristics and siRNA activity of self assembled polyplexes formed with chol-siRNA or unmodified siRNA were compared using three types of conventional, positively charged polymers: (i) biodegradable, cross-linked nanogels (BDNG) (ii) graft copolymers (PEI-PEG), and (iii) linear block copolymers (PLL10-PEG, and PLL50-PEG). Chol-siRNA did not alter complex formation or the resistance of polyplexes to siRNA displacement by heparin but increased nuclease protection by BDNG, PLL10-PEG, and PLL50-PEG polyplexes over polyplexes with unmodified siRNA. Chol-CYPB siRNA increased suppression of native CYPB mRNA in mammary microvascular endothelial cells (MVEC) by BDNG polyplexes (35%) and PLL10-PEG polyplexes (69%) over comparable CYPB siRNA polyplexes but had no effect on PEI-PEG or PLL50-PEG polyplexes. Overall, these results indicate that complexation of chol-siRNA increases nuclease protection and mRNA suppression by select siRNA polyplexes. These results also suggest that polycationic block length is an important factor in increasing mRNA suppression by PLL-PEG chol-siRNA polyplexes in mammary MVEC. PMID:21047680

From classical mutagenesis to nuclease-based breeding - directing natural DNA repair for a natural end-product.

PubMed

Pacher, Michael; Puchta, Holger

2017-05-01

Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases (SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based 'DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Isolating DNA from sexual assault cases: a comparison of standard methods with a nuclease-based approach

PubMed Central

2012-01-01

Background Profiling sperm DNA present on vaginal swabs taken from rape victims often contributes to identifying and incarcerating rapists. Large amounts of the victim’s epithelial cells contaminate the sperm present on swabs, however, and complicate this process. The standard method for obtaining relatively pure sperm DNA from a vaginal swab is to digest the epithelial cells with Proteinase K in order to solubilize the victim’s DNA, and to then physically separate the soluble DNA from the intact sperm by pelleting the sperm, removing the victim’s fraction, and repeatedly washing the sperm pellet. An alternative approach that does not require washing steps is to digest with Proteinase K, pellet the sperm, remove the victim’s fraction, and then digest the residual victim’s DNA with a nuclease. Methods The nuclease approach has been commercialized in a product, the Erase Sperm Isolation Kit (PTC Labs, Columbia, MO, USA), and five crime laboratories have tested it on semen-spiked female buccal swabs in a direct comparison with their standard methods. Comparisons have also been performed on timed post-coital vaginal swabs and evidence collected from sexual assault cases. Results For the semen-spiked buccal swabs, Erase outperformed the standard methods in all five laboratories and in most cases was able to provide a clean male profile from buccal swabs spiked with only 1,500 sperm. The vaginal swabs taken after consensual sex and the evidence collected from rape victims showed a similar pattern of Erase providing superior profiles. Conclusions In all samples tested, STR profiles of the male DNA fractions obtained with Erase were as good as or better than those obtained using the standard methods. PMID:23211019

A tailored biocatalyst achieved by the rational anchoring of imidazole groups on a natural polymer: furnishing a potential artificial nuclease by sustainable materials engineering.

PubMed

Ferreira, José G L; Grein-Iankovski, Aline; Oliveira, Marco A S; Simas-Tosin, Fernanda F; Riegel-Vidotti, Izabel C; Orth, Elisa S

2015-04-11

Foreseeing the development of artificial enzymes by sustainable materials engineering, we rationally anchored reactive imidazole groups on gum arabic, a natural biocompatible polymer. The tailored biocatalyst GAIMZ demonstrated catalytic activity (>10(5)-fold) in dephosphorylation reactions with recyclable features and was effective in cleaving plasmid DNA, comprising a potential artificial nuclease.

Alkaline quinone flow battery.

PubMed

Lin, Kaixiang; Chen, Qing; Gerhardt, Michael R; Tong, Liuchuan; Kim, Sang Bok; Eisenach, Louise; Valle, Alvaro W; Hardee, David; Gordon, Roy G; Aziz, Michael J; Marshak, Michael P

2015-09-25

Storage of photovoltaic and wind electricity in batteries could solve the mismatch problem between the intermittent supply of these renewable resources and variable demand. Flow batteries permit more economical long-duration discharge than solid-electrode batteries by using liquid electrolytes stored outside of the battery. We report an alkaline flow battery based on redox-active organic molecules that are composed entirely of Earth-abundant elements and are nontoxic, nonflammable, and safe for use in residential and commercial environments. The battery operates efficiently with high power density near room temperature. These results demonstrate the stability and performance of redox-active organic molecules in alkaline flow batteries, potentially enabling cost-effective stationary storage of renewable energy. Copyright © 2015, American Association for the Advancement of Science.

Technetium recovery from high alkaline solution

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

Nash, Charles A.

2016-07-12

Disclosed are methods for recovering technetium from a highly alkaline solution. The highly alkaline solution can be a liquid waste solution from a nuclear waste processing system. Methods can include combining the solution with a reductant capable of reducing technetium at the high pH of the solution and adding to or forming in the solution an adsorbent capable of adsorbing the precipitated technetium at the high pH of the solution.

Gene Editing in Human Lymphoid Cells: Role for Donor DNA, Type of Genomic Nuclease and Cell Selection Method.

PubMed

Zotova, Anastasia; Lopatukhina, Elena; Filatov, Alexander; Khaitov, Musa; Mazurov, Dmitriy

2017-11-02

Programmable endonucleases introduce DNA breaks at specific sites, which are repaired by non-homologous end joining (NHEJ) or homology recombination (HDR). Genome editing in human lymphoid cells is challenging as these difficult-to-transfect cells may also inefficiently repair DNA by HDR. Here, we estimated efficiencies and dynamics of knockout (KO) and knockin (KI) generation in human T and B cell lines depending on repair template, target loci and types of genomic endonucleases. Using zinc finger nuclease (ZFN), we have engineered Jurkat and CEM cells with the 8.2 kb human immunodeficiency virus type 1 (HIV-1) ∆Env genome integrated at the adeno-associated virus integration site 1 (AAVS1) locus that stably produce virus particles and mediate infection upon transfection with helper vectors. Knockouts generated by ZFN or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) double nicking techniques were comparably efficient in lymphoid cells. However, unlike polyclonal sorted cells, gene-edited cells selected by cloning exerted tremendous deviations in functionality as estimated by replication of HIV-1 and human T cell leukemia virus type 1 (HTLV-1) in these cells. Notably, the recently reported high-fidelity eCas9 1.1 when combined to the nickase mutation displayed gene-dependent decrease in on-target activity. Thus, the balance between off-target effects and on-target efficiency of nucleases, as well as choice of the optimal method of edited cell selection should be taken into account for proper gene function validation in lymphoid cells.

Functional identification of the non-specific nuclease from white spot syndrome virus

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

Li Li; Lin Shumei; Yanga Feng

2005-07-05

The product encoded by the wsv191 gene from shrimp white spot syndrome virus (WSSV) is homologous with non-specific nucleases (NSN) of other organisms. To functionally identify the protein, the wsv191 gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with 6His-tag at C-terminal. The fusion protein (termed as rWSSV-NSN) was purified using Ni-NTA affinity chromatography under denatured conditions, renatured and characterized by three methods. The results showed that rWSSV-NSN could hydrolyze both DNA and RNA. 5'-RACE result revealed that the transcription initiation site of the wsv191 gene was located at nucleotide residue G of the predictedmore » ATG triplet. Therefore, we concluded that the next ATG should be the genuine translation initiation codon of the wsv191 gene. Western blot analysis revealed that the molecular mass of natural WSSV-NSN was 37 kDa.« less

Ethanol production from bamboo using mild alkaline pre-extraction followed by alkaline hydrogen peroxide pretreatment.

PubMed

Yuan, Zhaoyang; Wen, Yangbing; Kapu, Nuwan Sella

2018-01-01

A sequential two-stage pretreatment process comprising alkaline pre-extraction and alkaline hydrogen peroxide pretreatment (AHP) was investigated to convert bamboo carbohydrates into bioethanol. The results showed that mild alkaline pre-extraction using 8% (w/w) sodium hydroxide (NaOH) at 100°C for 180min followed by AHP pretreatment with 4% (w/w) hydrogen peroxide (H 2 O 2 ) was sufficient to generate a substrate that could be efficiently digested with low enzyme loadings. Moreover, alkali pre-extraction enabled the use of lower H 2 O 2 charges in AHP treatment. Two-stage pretreatment followed by enzymatic hydrolysis with only 9FPU/g cellulose led to the recovery of 87% of the original sugars in the raw feedstock. The use of the pentose-hexose fermenting Saccharomyces cerevisiae SR8u strain enabled the utilization of 95.7% sugars in the hydrolysate to reach 4.6%w/v ethanol titer. The overall process also enabled the recovery of 62.9% lignin and 93.8% silica at high levels of purity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Calcic amphiboles in calc-alkaline and alkaline magmas: thermobarometric and chemometric empirical equations valid up to 1,130°C and 2.2 GPa

NASA Astrophysics Data System (ADS)

Ridolfi, Filippo; Renzulli, Alberto

2012-05-01

The following article presents constraints of the stability of Mg-rich (Mg/(Mg + Fe2+) > 0.5) calcic amphibole in both calc-alkaline and alkaline magmas, testing of previous thermobarometers, and formulation of new empirical equations that take into consideration a large amount of literature data (e.g. more than one thousand amphibole compositions among experimental and natural crystals). Particular care has been taken in choosing a large number of natural amphiboles and selecting quality experimental data from literature. The final database of experimental data, composed of 61 amphiboles synthesized in the ranges of 800-1,130°C and 130-2,200 MPa, indicates that amphibole crystallization occurs in a horn-like P- T stability field limited by two increasing curves (i.e. the thermal stability and an upper limit), which should start to bend back to higher pressures. Among calcic amphiboles, magnesiohornblendes and tschermakitic pargasites are only found in equilibrium with calc-alkaline melts and crystallize at relatively shallow conditions ( P up to ~1 GPa). Kaersutite and pargasite are species almost exclusively found in alkaline igneous products, while magnesiohastingsite is equally distributed in calc-alkaline and alkaline rocks. The reliability of previous amphibole applications was checked using the selected experimental database. The results of this testing indicate that none of the previous thermobarometers can be successfully used to estimate the P, T and fO2 in a wide range of amphibole crystallization conditions. Multivariate least-square analyses of experimental amphibole compositions and physico-chemical parameters allowed us to achieve a new thermobarometric model that gives reasonably low uncertainties ( T ± 23.5°C, P ± 11.5%, H2Omelt ± 0.78wt%) for calc-alkaline and alkaline magmas in a wide range of P- T conditions (up to 1,130°C and 2,200 MPa) and ΔNNO values (±0.37 log units) up to 500 MPa. The AK-[4]Al relation in amphibole can be readily

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.

Discomfort from an Alkaline Formulation Delivered Subcutaneously in Humans

PubMed Central

Ward, W. Kenneth; Castle, Jessica R.; Branigan, Deborah L.; Massoud, Ryan G.; Youssef, Joseph El

2013-01-01

Background and Objective There is a paucity of data regarding tolerability of alkaline drugs administered subcutaneously. The aim of this study was to assess the tolerability of alkaline preparations of human albumin delivered subcutaneously to healthy humans. Methods We compared the tolerability of neutral versus alkaline (pH 10) formulations of human albumin in ten volunteers. With an intent to minimize the time required to reach physiological pH after injection, the alkaline formulation was buffered with a low concentration of glycine (20 mmol/L). Each formulation was given at two rates: over 5 seconds and over 60 seconds. A six-point scale was used to assess discomfort. Results For slow injections, there was a significant difference between pH 7.4 and pH 10 injections (0.4 ± 0.2 vs 1.1 ± 0.2, mean ± SEM; p = 0.025), though the degree of discomfort at pH 10 injections was only ‘mild or slight’. For fast injections, the difference between neutral and alkaline formulations was of borderline significance. Inflammation and oedema, as judged by a physician, were very minimal for all injections, irrespective of pH. Conclusion For subcutaneous drug administration (especially when delivered slowly), there was more discomfort associated with alkaline versus neutral formulations of albumin, though the discomfort was mild. This study suggests that there is little discomfort and inflammation resulting from subcutaneous administration of protein drugs formulated with weak buffers at alkaline pH. PMID:22568666

Safety of an alkalinizing buffer designed for inhaled medications in humans.

PubMed

Davis, Michael D; Walsh, Brian K; Dwyer, Scott T; Combs, Casey; Vehse, Nico; Paget-Brown, Alix; Pajewski, Thomas; Hunt, John F

2013-07-01

Airway acidification plays a role in disorders of the pulmonary tract. We hypothesized that the inhalation of alkalinized glycine buffer would measurably alkalinize the airways without compromising lung function or causing adverse events. We evaluated the safety of an inhaled alkaline glycine buffer in both healthy subjects and in subjects with stable obstructive airway disease. This work includes 2 open-label safety studies. The healthy controls were part of a phase 1 safety study of multiple inhalations of low-dose alkaline glycine buffer; nebulized saline was used as a comparator in 8 of the healthy controls. Subsequently, a phase 2 study in subjects with stable obstructive airway disease was completed using a single nebulized higher-dose strategy of the alkaline inhalation. We studied 20 non-smoking adults (10 healthy controls and 10 subjects with obstructive airway disease), both at baseline and after inhalation of alkaline buffer. We used spirometry and vital signs as markers of clinical safety. We used changes in fraction of exhaled nitric oxide (NO) and exhaled breath condensate (EBC) pH as surrogate markers of airway pH modification. Alkaline glycine inhalation was tolerated by all subjects in both studies, with no adverse effects on spirometric parameters or vital signs. Airway alkalinization was confirmed by a median increase in EBC pH of 0.235 pH units (IQR 0.56-0.03, P = .03) in subjects after inhalation of the higher-dose alkaline buffer (2.5 mL of 100 mmol/L glycine). Alkalinization of airway lining fluid is accomplished with inhalation of alkaline glycine buffer and causes no adverse effects on pulmonary function or vital signs.

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.

Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly.

PubMed

Reyon, Deepak; Khayter, Cyd; Regan, Maureen R; Joung, J Keith; Sander, Jeffry D

2012-10-01

Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe detailed protocols for the serial, hierarchical assembly of TALENs that require neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. These restriction enzyme and ligation (REAL)-based protocols can be practiced using plasmid libraries and user-friendly, Web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. With the described platform of reagents, protocols, and software, researchers can easily engineer multiple TALENs within 2 weeks using standard cloning techniques. 2012 by John Wiley & Sons, Inc.

Nuclease digestion and mass spectrometric characterization of oligodeoxyribonucleotides containing 1,2-GpG, 1,2-ApG, and 1,3-GpXpG cisplatin intrastrand cross-links.

PubMed

Williams, Renee T; Nalbandian, Jenifer N; Tu, Audrey; Wang, Yinsheng

2013-05-01

The primary mode of action for cis-diamminedichloroplatinum (II), referred to as cisplatin, toward the treatment of solid malignancies is through formation of cross-links with DNA at purine sites, especially guanines. We prepared oligodeoxyribonucleotides (ODNs) containing a 1,2-GpG, 1,2-ApG, or 1,3-GpXpG cisplatin intrastrand cross-link and the corresponding ODNs modified with (15)N2-labeled cisplatin, and characterized these ODNs with electrospray ionization mass spectrometry (ESI-MS) and tandem MS (MS/MS). We also employed LC-MS/MS to characterize the digestion products of these ODNs after treatment with a cocktail of 4 enzymes (nuclease P1, phosphodiesterases I and II, and alkaline phosphatase). 1,2-GpG was released from the ODNs as a dinucleoside monophosphate or a dinucleotide. Analyses of the digestion products of ODNs containing a 1,2-GpG cross-link on the 5' or 3' terminus revealed that the dinucleotide carries a terminal 5' phosphate. On the other hand, digestion of the 1,3-GpXpG intrastrand cross-link yielded 3 dinucleoside products with 0, 1, or 2 phosphate groups. The availability of the ODNs carrying the stable isotope-labeled lesions, MS/MS analyses of the cisplatin-modified ODNs, and the characterization of the enzymatic digestion products of these ODNs set the stage for the future LC-MS/MS quantification of the 1,2-GpG, 1,2-ApG, and 1,3-GpXpG lesions in cellular DNA. Copyright © 2012 Elsevier B.V. All rights reserved.

Nuclease Digestion and Mass Spectrometric Characterization of Oligodeoxyribonucleotides Containing 1,2-GpG, 1,2-ApG, and 1,3-GpXpG Cisplatin Intrastrand Cross-links

PubMed Central

Williams, Renee T.; Nalbandian, Jenifer; Tu, Audrey; Wang, Yinsheng

2013-01-01

Background The primary mode of action for cis-diamminedichloroplatinum (II), referred to as cisplatin, towards the treatment of solid malignancies is through formation of cross-links with DNA at purine sites, especially guanines. Methods We prepared oligodeoxyribonucleotides (ODNs) containing a 1,2-GpG, 1,2-ApG, or 1,3-GpXpG cisplatin intrastrand cross-link and the corresponding ODNs modified with 15N2-labeled cisplatin, and characterized these ODNs with electrospray ionization mass spectrometry (ESI-MS) and tandem MS (MS/MS). We also employed LC-MS/MS to characterize the digestion products of these ODNs after treatment with a cocktail of 4 enzymes (nuclease P1, phosphodiesterases I and II, and alkaline phosphatase). Results 1,2-GpG was released from the ODNs as a dinucleoside monophosphate or a dinucleotide. Analyses of the digestion products of ODNs containing a 1,2-GpG cross-link on the 5′ or 3′ terminus revealed that the dinucleotide carries a terminal 5′ phosphate. On the other hand, digestion of the 1,3-GpXpG intrastrand cross-link yielded 3 dinucleoside products with 0, 1, or 2 phosphate groups. Results The availability of the ODNs carrying the stable isotope-labeled lesions, MS/MS analyses of the cisplatin-modified ODNs, and the characterization of the enzymatic digestion products of these ODNs set the stage for the future LC-MS/MS quantification of the 1,2-GpG, 1,2-ApG, and 1,3-GpXpG lesions in cellular DNA. PMID:23266768

TMC-1 mediates alkaline sensation in C. elegans through nociceptive neurons

PubMed Central

Wang, Xiang; Li, Guang; Liu, Jie; Liu, Jianfeng; Xu, X.Z. Shawn

2016-01-01

Noxious pH triggers pungent taste and nocifensive behavior. While the mechanisms underlying acidic pH sensation has been extensively characterized, little is known about how animals sense alkaline pH in the environment. TMC genes encode a family of evolutionarily conserved membrane proteins, whose functions are largely unknown. Here, we characterize C. elegans TMC-1 which was suggested to form a Na+-sensitive channel mediating salt chemosensation. Interestingly, we find that TMC-1 is required for worms to avoid noxious alkaline environment. Alkaline pH evokes an inward current in nociceptive neurons, which is primarily mediated by TMC-1 and to a lesser extent by the TRP channel OSM-9. However, unlike OSM-9 which is sensitive to both acidic and alkaline pH, TMC-1 is only required for alkali-activated current, revealing a specificity for alkaline sensation. Ectopic expression of TMC-1 confers alkaline sensitivity to alkali-insensitive cells. Our results identify an unexpected role for TMCs in alkaline sensation and nociception. PMID:27321925

40 CFR 721.9680 - Alkaline titania silica gel (generic name).

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Alkaline titania silica gel (generic... Specific Chemical Substances § 721.9680 Alkaline titania silica gel (generic name). (a) Chemical substance... alkaline titania silica gel (PMN P-95-529) is subject to reporting under this section for the significant...

40 CFR 721.9680 - Alkaline titania silica gel (generic name).

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Alkaline titania silica gel (generic... Specific Chemical Substances § 721.9680 Alkaline titania silica gel (generic name). (a) Chemical substance... alkaline titania silica gel (PMN P-95-529) is subject to reporting under this section for the significant...

40 CFR 721.9680 - Alkaline titania silica gel (generic name).

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Alkaline titania silica gel (generic... Specific Chemical Substances § 721.9680 Alkaline titania silica gel (generic name). (a) Chemical substance... alkaline titania silica gel (PMN P-95-529) is subject to reporting under this section for the significant...

40 CFR 721.9680 - Alkaline titania silica gel (generic name).

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Alkaline titania silica gel (generic... Specific Chemical Substances § 721.9680 Alkaline titania silica gel (generic name). (a) Chemical substance... alkaline titania silica gel (PMN P-95-529) is subject to reporting under this section for the significant...

40 CFR 721.9680 - Alkaline titania silica gel (generic name).

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Alkaline titania silica gel (generic... Specific Chemical Substances § 721.9680 Alkaline titania silica gel (generic name). (a) Chemical substance... alkaline titania silica gel (PMN P-95-529) is subject to reporting under this section for the significant...

Dynamic Properties of the Alkaline Vesicle Population at Hippocampal Synapses

PubMed Central

Röther, Mareike; Brauner, Jan M.; Ebert, Katrin; Welzel, Oliver; Jung, Jasmin; Bauereiss, Anna; Kornhuber, Johannes; Groemer, Teja W.

2014-01-01

In compensatory endocytosis, scission of vesicles from the plasma membrane to the cytoplasm is a prerequisite for intravesicular reacidification and accumulation of neurotransmitter molecules. Here, we provide time-resolved measurements of the dynamics of the alkaline vesicle population which appears upon endocytic retrieval. Using fast perfusion pH-cycling in live-cell microscopy, synapto-pHluorin expressing rat hippocampal neurons were electrically stimulated. We found that the relative size of the alkaline vesicle population depended significantly on the electrical stimulus size: With increasing number of action potentials the relative size of the alkaline vesicle population expanded. In contrast to that, increasing the stimulus frequency reduced the relative size of the population of alkaline vesicles. Measurement of the time constant for reacification and calculation of the time constant for endocytosis revealed that both time constants were variable with regard to the stimulus condition. Furthermore, we show that the dynamics of the alkaline vesicle population can be predicted by a simple mathematical model. In conclusion, here a novel methodical approach to analyze dynamic properties of alkaline vesicles is presented and validated as a convenient method for the detection of intracellular events. Using this method we show that the population of alkaline vesicles is highly dynamic and depends both on stimulus strength and frequency. Our results implicate that determination of the alkaline vesicle population size may provide new insights into the kinetics of endocytic retrieval. PMID:25079223

Alkaline β-fructofuranosidases of tuberous roots: Possible physiological function.

PubMed

Ricardo, C P

1974-12-01

Alkaline invertase of roots of carrot (Daucus carota L.) did not hydrolyze raffinose while the acid invertase from the same tissue showed with this sugar ca. 60% of the activity found with sucrose. The activity of the two invertases was inhibited by fructose to a different extent, the K i value being ca. 4×10(-2) M and 3×10(-1)M, respectively, for the alkaline and the acid invertases from the roots of both carrot and turnip (Brassica rapa L.). It is proposed that fructose inhibition of acid invertase is of no physiological significance but that, in contrast, hexoses might regulate the activity of alkaline invertase.Comparing several species and cultivars, it was found that the content of reducing sugars and the activity of alkaline invertase of mature tuberous roots showed a positive correlation. This indicates that alkaline invertase may participate in the regulation of the hexose level of the cell, as was previously suggested for sugar-cane. A scheme is presented which proposes a way of participation of alkaline invertase in such a regulation, assuming that this enzyme is located in the cytoplasm and acid invertase is membrane-bound and mainly located at the cell surface.

Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

NASA Technical Reports Server (NTRS)

Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

2000-01-01

The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

Contemporaneous eruption of calc-alkaline and alkaline lavas in a continental arc (Eastern Mexican Volcanic Belt): chemically heterogeneous but isotopically homogeneous source

NASA Astrophysics Data System (ADS)

Carrasco-Núñez, Gerardo; Righter, Kevin; Chesley, John; Siebert, Lee; Aranda-Gómez, José Jorge

2005-11-01

Nearly contemporaneous eruption of alkaline and calc-alkaline lavas occurred about 900 years BP from El Volcancillo paired vent, located behind the volcanic front in the Mexican Volcanic Belt (MVB). Emission of hawaiite (Toxtlacuaya) was immediately followed by calc-alkaline basalt (Río Naolinco). Hawaiites contain olivine microphenocrysts (Fo67-72), plagioclase (An56-60) phenocrysts, have 4-5 wt% MgO and 49.6-50.9 wt% SiO2. In contrast, calc-alkaline lavas contain plagioclase (An64-72) and olivine phenocrysts (Fo81-84) with spinel inclusions, and have 8-9 wt% MgO and 48.4-49.4 wt% SiO2. The most primitive lavas in the region (Río Naolinco and Cerro Colorado) are not as primitive as parental melts in other arcs, and could represent either (a) variable degrees of melting of a subduction modified, garnet-bearing depleted mantle source, followed by AFC process, or (b) melting of two distinct mantle sources followed by AFC processes. These two hypotheses are evaluated using REE, HFSE, and Sr, Os and Pb isotopic data. The Toxtlacuaya flow and the Y & I lavas can be generated by combined fractional crystallization and assimilation of gabbroic granulite, starting with a parental liquid similar to the Cerro Colorado basalt. Although calc-alkaline and alkaline magmas commonly occur together in other areas of the MVB, evidence for subduction component in El Volcancillo magmas is minimal and limited to <1%, which is a unique feature in this region further from the trench. El Volcancillo lavas were produced from two different magma batches: we surmise that the injection of calc-alkaline magma into an alkaline magma chamber triggered the eruption of hawaiites. Our results suggest that the subalkaline and hawaiitic lavas were formed by different degrees of partial melting of a similar, largely depleted mantle source, followed by later AFC processes. This model is unusual for arcs, where such diversity is usually explained by melting of heterogeneous (enriched and depleted) and

Site-Specific Editing of the Plasmodium falciparum Genome Using Engineered Zinc-Finger Nucleases

PubMed Central

Straimer, Judith; Lee, Marcus CS; Lee, Andrew H; Zeitler, Bryan; Williams, April E; Pearl, Jocelynn R; Zhang, Lei; Rebar, Edward J; Gregory, Philip D; Llinás, Manuel; Urnov, Fyodor D; Fidock, David A

2013-01-01

Malaria afflicts over 200 million people worldwide and its most lethal etiologic agent, Plasmodium falciparum, is evolving to resist even the latest-generation therapeutics. Efficient tools for genome-directed investigations of P. falciparum pathogenesis, including drug resistance mechanisms, are clearly required. Here we report rapid and targeted genetic engineering of this parasite, using zinc-finger nucleases (ZFNs) that produce a double-strand break in a user-defined locus and trigger homology-directed repair. Targeting an integrated egfp locus, we obtained gene deletion parasites with unprecedented speed (two weeks), both with and without direct selection. ZFNs engineered against the endogenous parasite gene pfcrt, responsible for chloroquine treatment escape, rapidly produced parasites that carried either an allelic replacement or a panel of specified point mutations. The efficiency, versatility and precision of this method will enable a diverse array of genome editing approaches to interrogate this human pathogen. PMID:22922501

Inhibition of rat corneal angiogenesis by a nuclease-resistant RNA aptamer specific for angiopoietin-2

PubMed Central

White, Rebekah R.; Shan, Siqing; Rusconi, Christopher P.; Shetty, Geetha; Dewhirst, Mark W.; Kontos, Christopher D.; Sullenger, Bruce A.

2003-01-01

Angiopoietin-2 (Ang2) appears to be a naturally occurring antagonist of the endothelial receptor tyrosine kinase Tie2, an important regulator of vascular stability. Destabilization of the endothelium by Ang2 is believed to potentiate the actions of proangiogenic growth factors. To investigate the specific role of Ang2 in the adult vasculature, we generated a nuclease-resistant RNA aptamer that binds and inhibits Ang2 but not the related Tie2 agonist, angiopoietin-1. Local delivery of this aptamer but not a partially scrambled mutant aptamer inhibited basic fibroblast growth factor-mediated neovascularization in the rat corneal micropocket angiogenesis assay. These in vivo data directly demonstrate that a specific inhibitor of Ang2 can act as an antiangiogenic agent. PMID:12692304

Hydroxide Self-Feeding High-Temperature Alkaline Direct Formate Fuel Cells.

PubMed

Li, Yinshi; Sun, Xianda; Feng, Ying

2017-05-22

Conventionally, both the thermal degradation of the anion-exchange membrane and the requirement of additional hydroxide for fuel oxidation reaction hinder the development of the high-temperature alkaline direct liquid fuel cells. The present work addresses these two issues by reporting a polybenzimidazole-membrane-based direct formate fuel cell (DFFC). Theoretically, the cell voltage of the high-temperature alkaline DFFC can be as high as 1.45 V at 90 °C. It has been demonstrated that a proof-of-concept alkaline DFFC without adding additional hydroxide yields a peak power density of 20.9 mW cm -2 , an order of magnitude higher than both alkaline direct ethanol fuel cells and alkaline direct methanol fuel cells, mainly because the hydrolysis of formate provides enough OH - ions for formate oxidation reaction. It was also found that this hydroxide self-feeding high-temperature alkaline DFFC shows a stable 100 min constant-current discharge at 90 °C, proving the conceptual feasibility. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

When can ocean acidification impacts be detected from decadal alkalinity measurements?

NASA Astrophysics Data System (ADS)

Carter, B. R.; Frölicher, T. L.; Dunne, J. P.; Rodgers, K. B.; Slater, R. D.; Sarmiento, J. L.

2016-04-01

We use a large initial condition suite of simulations (30 runs) with an Earth system model to assess the detectability of biogeochemical impacts of ocean acidification (OA) on the marine alkalinity distribution from decadally repeated hydrographic measurements such as those produced by the Global Ship-Based Hydrographic Investigations Program (GO-SHIP). Detection of these impacts is complicated by alkalinity changes from variability and long-term trends in freshwater and organic matter cycling and ocean circulation. In our ensemble simulation, variability in freshwater cycling generates large changes in alkalinity that obscure the changes of interest and prevent the attribution of observed alkalinity redistribution to OA. These complications from freshwater cycling can be mostly avoided through salinity normalization of alkalinity. With the salinity-normalized alkalinity, modeled OA impacts are broadly detectable in the surface of the subtropical gyres by 2030. Discrepancies between this finding and the finding of an earlier analysis suggest that these estimates are strongly sensitive to the patterns of calcium carbonate export simulated by the model. OA impacts are detectable later in the subpolar and equatorial regions due to slower responses of alkalinity to OA in these regions and greater seasonal equatorial alkalinity variability. OA impacts are detectable later at depth despite lower variability due to smaller rates of change and consistent measurement uncertainty.

Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

NASA Technical Reports Server (NTRS)

Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

1999-01-01

A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

Contemporaneous alkaline and calc-alkaline series in Central Anatolia (Turkey): Spatio-temporal evolution of a post-collisional Quaternary basaltic volcanism

NASA Astrophysics Data System (ADS)

Dogan-Kulahci, Gullu Deniz; Temel, Abidin; Gourgaud, Alain; Varol, Elif; Guillou, Hervé; Deniel, Catherine

2018-05-01

This study focuses on spatio-temporal evolution of basaltic volcanism in the Central Anatolian post-collisional Quaternary magmatic province which developed along a NE-SW orientation in Turkey. This magmatic province consists of the stratovolcanoes Erciyes (ES) and Hasandag (HS), and the basaltic volcanic fields of Obruk-Zengen (OZ) and Karapınar (KA). The investigated samples range between basic to intermediate in composition (48-56 wt% SiO2), and exhibit calc-alkaline affinity at ES whereas HS, OZ and KA are alkaline in composition. Based on new Ksbnd Ar ages and major element data, the oldest basaltic rock of ES is 1700 ± 40 ka old and exhibits alkaline character, whereas the youngest basaltic trachyandesite is 12 ± 5 ka old and calc-alkaline in composition. Most ES basaltic rocks are younger than 350 ka. All samples dated from HS are alkaline basalts, ranging from 543 ± 12 ka to 2 ± 7 ka old. With the exception of one basalt, all HS basalts are 100 ka or younger in age. Ksbnd Ar ages range from 797 ± 20 ka to 66 ± 7 ka from OZ. All the basalt samples are alkaline in character and are older than the HS alkaline basalts, with the exception of the youngest samples. The oldest and youngest basaltic samples from KA are 280 ± 7 ka and 163 ± 10 ka, respectively, and are calc-alkaline in character. Based on thermobarometric estimates samples from OZ exhibit the highest cpx-liqidus temperature and pressure. For all centers the calculated crystallization depths are between 11 and 28 km and increase from NE to SW. Multistage crystallization in magma chamber(s) located at different depths can explain this range in pressure. Harker variation diagrams coupled with least-squares mass balance calculations support fractional crystallization for ES and, to lesser extend for HS, OZ and KA. All basaltic volcanic rocks of this study are enriched in large-ion lithophile elements (LILE) and light rare earth elements (LREE). The lack of negative anomalies for high field

Osteoblast Differentiation on Collagen Scaffold with Immobilized Alkaline Phosphatase.

PubMed

Jafary, F; Hanachi, P; Gorjipour, K

2017-01-01

In tissue engineering, scaffold characteristics play an important role in the biological interactions between cells and the scaffold. Cell adhesion, proliferation, and activation depend on material properties used for the fabrication of scaffolds. In the present investigation, we used collagen with proper characteristics including mechanically stability, biodegradability and low antigenicity. Optimization of the scaffold was done by immobilization of alkaline phosphatase on the collagen surface via cross-linking method, because this enzyme is one of the most important markers of osteoblast, which increases inorganic phosphate concentration and promote mineralization of bone formation. Alkaline phosphatase was immobilized on a collagen surface by 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride, as a reagent. Then, rat mesenchymal stem cells were cultured in osteogenic medium in control and treated groups. The osteogenesis-related genes were compared between treatments (differentiated cells with immobilized alkaline phosphatase/collagen scaffold) and control groups (differentiated cells on collagen surface without alkaline phosphatase) on days 3 and 7 by quantitative real-time PCR (QIAGEN software). Several genes, including alkaline phosphatase, collagen type I and osteocalcine associated with calcium binding and mineralization, showed upregulation in expression during the first 3 days, whereas tumor necrosis factor-α, acting as an inhibitor of differentiation, was down-regulated during osteogenesis. Collagen scaffold with immobilized alkaline phosphatase can be utilized as a good candidate for enhancing the differentiation of osteoblasts from mesenchymal stem cells.

Structural Analysis of Alkaline β-Mannanase from Alkaliphilic Bacillus sp. N16-5: Implications for Adaptation to Alkaline Conditions

PubMed Central

Zhao, Yueju; Zhang, Yunhua; Cao, Yang; Qi, Jianxun; Mao, Liangwei; Xue, Yanfen; Gao, Feng; Peng, Hao; Wang, Xiaowei; Gao, George F.; Ma, Yanhe

2011-01-01

Significant progress has been made in isolating novel alkaline β-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these β-mannanases. We report the catalytic domain structure of an industrially important β-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (β/α)8-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 β-mannanases. It was revealed that BSP165 MAN and other subfamily 8 β-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to β-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline β-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (β/α)8-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pK a calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 β-mannanases and will help to further understanding of alkaline

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

VSL#3 probiotic upregulates intestinal mucosal alkaline sphingomyelinase and reduces inflammation.

PubMed

Soo, I; Madsen, K L; Tejpar, Q; Sydora, B C; Sherbaniuk, R; Cinque, B; Di Marzio, L; Cifone, M Grazia; Desimone, C; Fedorak, R N

2008-03-01

Alkaline sphingomyelinase, an enzyme found exclusively in bile and the intestinal brush border, hydrolyzes sphingomyelin into ceramide, sphingosine and sphingosine-1-phosphate, thereby inducing epithelial apoptosis. Reduced levels of alkaline sphingomyelinase have been found in premalignant and malignant intestinal epithelia and in ulcerative colitis tissue. Probiotic bacteria can be a source of sphingomyelinase. To determine the effect of VSL#3 probiotic therapy on mucosal levels of alkaline sphingomyelinase, both in a mouse model of colitis and in patients with ulcerative colitis. Interleukin-10 gene-deficient (IL10KO) and wild type control mice were treated with VSL#3 (10(9) colony-forming units per day) for three weeks, after which alkaline sphingomyelinase activity was measured in ileal and colonic tissue. As well, 15 patients with ulcerative colitis were treated with VSL#3 (900 billion bacteria two times per day for five weeks). Alkaline sphingomyelinase activity was measured through biopsies and comparison of ulcerative colitis disease activity index scores obtained before and after treatment. Lowered alkaline sphingomyelinase levels were seen in the colon (P=0.02) and ileum (P=0.04) of IL10KO mice, as compared with controls. Treatment of these mice with VSL#3 resulted in upregulation of mucosal alkaline sphingomyelinase activity in both the colon (P=0.04) and the ileum (P=0.01). VSL#3 treatment of human patients who had ulcerative colitis decreased mean (+/- SEM) ulcerative colitis disease activity index scores from 5.3+/-1.8946 to 0.70+/-0.34 (P=0.02) and increased mucosal alkaline sphingomyelinase activity. Mucosal alkaline sphingomyelinase activity is reduced in the intestine of IL10KO mice with colitis and in humans with ulcerative colitis. VSL#3 probiotic therapy upregulates mucosal alkaline sphingomyelinase activity.

Alkaline nanoparticle coatings improve resin bonding of 10-methacryloyloxydecyldihydrogenphosphate-conditioned zirconia.

PubMed

Qian, Mengke; Lu, Zhicen; Chen, Chen; Zhang, Huaiqin; Xie, Haifeng

Creating an alkaline environment prior to 10-methacryloyloxydecyldihydrogenphosphate (MDP) conditioning improves the resin bonding of zirconia. The present study evaluated the effects of four alkaline coatings with different water solubilities and pH values on resin bonding of MDP-conditioned zirconia. Two alkaline nanoparticle coatings were studied in particular. Thermodynamics calculations were performed to evaluate the strengths of MDP-tetragonal phase zirconia chemical bonds at different pH values. Zirconia surfaces with and without alkaline coatings were characterized by scanning electron microscope (SEM)/energy dispersive spectrometer and Fourier transform infrared spectroscopy; alkaline coatings included NaOH, Ca(OH) 2 , nano-MgO, and nano-Zr(OH) 4 . A shear bond strength (SBS) test was performed to evaluate the effects of the four alkaline coatings on bonding; the alkaline coatings were applied to the surfaces prior to conditioning the zirconia with MDP-containing primers. Gibbs free energies of the MDP-tetragonal zirconia crystal model coordination reaction in different pH environments were -583.892 (NaOH), -569.048 [Ca(OH) 2 ], -547.393 (MgO), and -530.279 kJ/mol [Zr(OH) 4 ]. Thermodynamic calculations indicated that the alkaline coatings improved bonding in the following order: NaOH > Ca(OH) 2 > MgO > Zr(OH) 4 . Statistical analysis of SBS tests showed a different result. SBSs were significantly different in groups that had different alkaline coatings, but it was not influenced by different primers. All four alkaline coatings increased SBS compared to control groups. Of the four coatings, nano-Zr(OH) 4 and -MgO showed higher SBS. Therefore, preparing nano-Zr(OH) 4 or -MgO coatings prior to conditioning with MDP-containing primers may potentially improve resin bonding of zirconia in the clinic.

Alkaline nanoparticle coatings improve resin bonding of 10-methacryloyloxydecyldihydrogenphosphate-conditioned zirconia

PubMed Central

Qian, Mengke; Lu, Zhicen; Chen, Chen; Zhang, Huaiqin; Xie, Haifeng

2016-01-01

Creating an alkaline environment prior to 10-methacryloyloxydecyldihydrogenphosphate (MDP) conditioning improves the resin bonding of zirconia. The present study evaluated the effects of four alkaline coatings with different water solubilities and pH values on resin bonding of MDP-conditioned zirconia. Two alkaline nanoparticle coatings were studied in particular. Thermodynamics calculations were performed to evaluate the strengths of MDP-tetragonal phase zirconia chemical bonds at different pH values. Zirconia surfaces with and without alkaline coatings were characterized by scanning electron microscope (SEM)/energy dispersive spectrometer and Fourier transform infrared spectroscopy; alkaline coatings included NaOH, Ca(OH)2, nano-MgO, and nano-Zr(OH)4. A shear bond strength (SBS) test was performed to evaluate the effects of the four alkaline coatings on bonding; the alkaline coatings were applied to the surfaces prior to conditioning the zirconia with MDP-containing primers. Gibbs free energies of the MDP-tetragonal zirconia crystal model coordination reaction in different pH environments were −583.892 (NaOH), −569.048 [Ca(OH)2], −547.393 (MgO), and −530.279 kJ/mol [Zr(OH)4]. Thermodynamic calculations indicated that the alkaline coatings improved bonding in the following order: NaOH > Ca(OH)2 > MgO > Zr(OH)4. Statistical analysis of SBS tests showed a different result. SBSs were significantly different in groups that had different alkaline coatings, but it was not influenced by different primers. All four alkaline coatings increased SBS compared to control groups. Of the four coatings, nano-Zr(OH)4 and -MgO showed higher SBS. Therefore, preparing nano-Zr(OH)4 or -MgO coatings prior to conditioning with MDP-containing primers may potentially improve resin bonding of zirconia in the clinic. PMID:27785013

Low-heat, mild alkaline pretreatment of switchgrass for anaerobic digestion.

PubMed

Jin, Guang; Bierma, Tom; Walker, Paul M

2014-01-01

This study examines the effectiveness of alkaline pretreatment under mild heat conditions (100°C or 212°F) on the anaerobic co-digestion of switchgrass. The effects of alkaline concentration, types of alkaline, heating time and rinsing were evaluated. In addition to batch studies, continuous-feed studies were performed in triplicate to identify potential digester operational problems caused by switchgrass co-digestion while accounting for uncertainty due to digester variability. Few studies have examined anaerobic digestion of switchgrass or the effects of mild heating to enhance alkaline pretreatment prior to biomass digestion. Results indicate that pretreatment can significantly enhance digestion of coarse-ground (≤ 0.78 cm particle size) switchgrass. Energy conversion efficiency as high as 63% was observed, and was comparable or superior to fine-grinding as a pretreatment method. The optimal NaOH concentration was found to be 5.5% (wt/wt alkaline/biomass) with a 91.7% moisture level. No evidence of operational problems such as solids build-up, poor mixing, or floating materials were observed. These results suggest the use of waste heat from a generator could reduce the concentration of alkaline required to adequately pretreat lignocellulosic feedstock prior to anaerobic digestion.

Cation exchange properties of zeolites in hyper alkaline aqueous media.

PubMed

Van Tendeloo, Leen; de Blochouse, Benny; Dom, Dirk; Vancluysen, Jacqueline; Snellings, Ruben; Martens, Johan A; Kirschhock, Christine E A; Maes, André; Breynaert, Eric

2015-02-03

Construction of multibarrier concrete based waste disposal sites and management of alkaline mine drainage water requires cation exchangers combining excellent sorption properties with a high stability and predictable performance in hyper alkaline media. Though highly selective organic cation exchange resins have been developed for most pollutants, they can serve as a growth medium for bacterial proliferation, impairing their long-term stability and introducing unpredictable parameters into the evolution of the system. Zeolites represent a family of inorganic cation exchangers, which naturally occur in hyper alkaline conditions and cannot serve as an electron donor or carbon source for microbial proliferation. Despite their successful application as industrial cation exchangers under near neutral conditions, their performance in hyper alkaline, saline water remains highly undocumented. Using Cs(+) as a benchmark element, this study aims to assess the long-term cation exchange performance of zeolites in concrete derived aqueous solutions. Comparison of their exchange properties in alkaline media with data obtained in near neutral solutions demonstrated that the cation exchange selectivity remains unaffected by the increased hydroxyl concentration; the cation exchange capacity did however show an unexpected increase in hyper alkaline media.

An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease.

PubMed

Wiemels, Richard E; Cech, Stephanie M; Meyer, Nikki M; Burke, Caleb A; Weiss, Andy; Parks, Anastacia R; Shaw, Lindsey N; Carroll, Ronan K

2017-01-01

Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus IMPORTANCE: Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently

A macro-enzyme cause of an isolated increase of alkaline phosphatase.

PubMed

Cervinski, Mark A; Lee, Hong Kee; Martin, Isabella W; Gavrilov, Dimitar K

2015-02-02

Macroenzyme complexes of serum enzymes and antibody can increase the circulating enzymatic activity and may lead to unnecessary additional testing and procedures. Laboratory physicians and scientists need to be aware of techniques to identify macroenzyme complexes when suspected. To investigate the possibility of a macro-alkaline phosphatase in the serum of a 74 year old male with persistently increased alkaline phosphatase we coupled a protein A/G agarose affinity chromatography technique with isoenzyme electrophoresis to look for the presence of macro-alkaline phosphatase. The majority of the alkaline phosphatase activity in the patient's serum sample was bound to the column and only a minor fraction (25%) of alkaline phosphatase activity was present in the column flow-through. The alkaline phosphatase activity was also found to co-elute with the immunoglobulins in the patient sample. The alkaline phosphatase activity in a control serum sample concurrently treated in the same manner did not bind to the column and was found in the column flow-through. The use of protein A/G agarose affinity chromatography is a rapid and simple method that can be applied to the investigation of other macro-enzyme complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

Alkalinity-salinity relationship in the Chesapeake Bay

NASA Astrophysics Data System (ADS)

Cintrón Del Valle, S. M.; Najjar, R.; Herrmann, M.; Goldberger, S.; Stets, E.

2016-12-01

Estuaries are a significant source of atmospheric CO2, a major greenhouse gas. However, it is not known whether the Chesapeake Bay, the largest estuary in the United States, is a source or sink of CO2. Extensive pH measurements in the Bay offer the possibility of estimating the air-water CO2 flux if robust relationships between alkalinity, the acid neutralizing capacity of a water body, and salinity can be established. Here we conduct a comprehensive analysis of the alkalinity-salinity relationship in the Chesapeake Bay based on more than 18,000 alkalinity measurements made between 1985 and 2015. It was found that seven segments of the Bay could be grouped into three different linear functions, suggesting that alkalinity is conserved in the Bay and has properties that change depending on the freshwater endmember (the riverine source). The highest freshwater endmember was 1.21 mol m-3 for the Potomac River, the lowest one was 0.41 mol m-3 for the York and Rappahannock Rivers, and an intermediate freshwater endmember was 0.79 mol m-3 for the remaining four segments. For some segments, most notably the Potomac River, the scatter of the data increases with decreasing salinity, which is due, in part, to seasonal and interannual variations in the freshwater endmember.

Alkaline Activator Impact on the Geopolymer Binders

NASA Astrophysics Data System (ADS)

Błaszczyński, Tomasz Z.; Król, Maciej R.

2017-10-01

Concrete structures are constantly moving in the direction of improving the durability. Durability depends on many factors, which are the composition of concrete mix, the usage of additives and admixtures and the place, where material will work and carry the load. The introduction of new geopolymer binders for geopolymer structures adds a new aspect that is type of used activator. This substance with strongly alkaline reaction is divided because of the physical state, the alkaline degree and above all the chemical composition. Taking into account, that at present the geopolymer binders are made essentially from waste materials or by-products from the combustion of coal or iron ore smelting, unambiguous determination of the effect of the activator on the properties of the geopolymer material requires a number of trials, researches and observation. This paper shows the influence of the most alkaline activators on the basic parameters of the durability of geopolymer binders. In this study there were used highly alkaline hydroxides, water glasses and granules, which are waste materials in a variety of processes taking place in chemical plants. As the substrate of geopolymer binders there were used fly ash which came from coal and high calcareous ash from the burning of lignite.

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

PubMed

Bi, Yanwei; Sun, Le; Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-05-01

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

High-Efficiency Targeted Editing of Large Viral Genomes by RNA-Guided Nucleases

PubMed Central

Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-01-01

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses. PMID:24788700

A method for making an alkaline battery electrode plate

NASA Technical Reports Server (NTRS)

Chida, K.; Ezaki, T.

1983-01-01

A method is described for making an alkaline battery electrode plate where the desired active substances are filled into a nickel foam substrate. In this substrate an electrolytic oxidation reduction occurs in an alkaline solution containing lithium hydroxide.

Elevated serum level of human alkaline phosphatase in obesity.

PubMed

Khan, Abdul Rehman; Awan, Fazli Rabbi; Najam, Syeda Sadia; Islam, Mehboob; Siddique, Tehmina; Zain, Maryam

2015-11-01

To investigate a correlation between serum alkaline phosphatase level and body mass index in human subjects. The comparative cross-sectional study was carried out at the National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan, from April 2012 to June 2013. Blood serum alkaline phosphatase levels were estimated and the subjects were divided into three sub-groups on the basis of their body mass. normal weight (<25kg/m2), overweight (25-27kg/m2) and obese (>27kg/m2) subjects. The serum samples were used for the estimation of clinically important biochemical parameters, using commercial kits on clinical chemistry analyser. Of the 197 subjects, 97(49%) were obese and 100(51%) were non-obese. The serum alkaline phosphatase level increased in obese (214±6.4 IU/L) compared to the non-obese subjects (184.5±5 IU/L). Furthermore, a significant linear relationship (r=0.3;p-0.0001) was found between serum alkaline phosphatase and body mass index. Other biochemical variables were not correlated to the body mass index. Over activity and higher amounts of alkaline phosphatase were linked to the development of obesity.

Polyvinyl alcohol battery separator containing inert filler. [alkaline batteries

NASA Technical Reports Server (NTRS)

Sheibley, D. W.; Hsu, L. C.; Manzo, M. A. (Inventor)

1981-01-01

A cross-linked polyvinyl alcohol battery separator is disclosed. A particulate filler, inert to alkaline electrolyte of an alkaline battery, is incorporated in the separator in an amount of 1-20% by weight, based on the weight of the polyvinyl alcohol, and is dispersed throughout the product. Incorporation of the filler enhances performance and increases cycle life of alkaline batteries when compared with batteries containing a similar separator not containing filler. Suitable fillers include titanates, silicates, zirconates, aluminates, wood floor, lignin, and titania. Particle size is not greater than about 50 microns.

DNA Oxidation Profiles of Copper Phenanthrene Chemical Nucleases

NASA Astrophysics Data System (ADS)

Molphy, Zara; Slator, Creina; Chatgilialoglu, Chryssostomos; Kellett, Andrew

2015-04-01

The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids - as demonstrated by metal-activated bleomycin - has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)]2+ (Cu-DPQ-Phen), [Cu(DPPZ)(phen)]2+ (Cu-DPPZ-Phen), and [{Cu(phen)2}2(μ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman’s reagent [Cu(phen)2]2+ throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilisers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals (•OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2'-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited - particularly along A-T rich chains - through oxidative damage of the template strands.

Production of bioethanol and value added compounds from wheat straw through combined alkaline/alkaline-peroxide pretreatment.

PubMed

Yuan, Zhaoyang; Wen, Yangbing; Li, Guodong

2018-07-01

An efficient scheme was developed for the conversion of wheat straw (WS) into bioethanol, silica and lignin. WS was pre-extracted with 0.2 mol/L sodium hydroxide at 30 °C for 5 h to remove about 91% of initial silica. Subsequently, the alkaline-pretreated solids were subjected to alkaline hydrogen peroxide (AHP) pretreatment with 40 mg hydrogen peroxide (H 2 O 2 )/g biomass at 50 °C for 7 h to prepare highly digestible substrate. The results of enzymatic hydrolysis demonstrated that the sequential alkaline-AHP pretreated WS was efficiently hydrolyzed at 10% (w/v) solids loading using an enzyme dosage of 10 mg protein/g glucan. The total sugar conversion of 92.4% was achieved. Simultaneous saccharification and co-fermentation (SSCF) was applied to produce ethanol from the two-stage pretreated substrate using Saccharomyces cerevisiae SR8u strain. Ethanol with concentration of 31.1 g/L was produced. Through the proposed process, about 86.4% and 54.1% of the initial silica and lignin were recovered, respectively. Copyright © 2018 Elsevier Ltd. All rights reserved.

Acid transformation of bauxite residue: Conversion of its alkaline characteristics.

PubMed

Kong, Xiangfeng; Li, Meng; Xue, Shengguo; Hartley, William; Chen, Chengrong; Wu, Chuan; Li, Xiaofei; Li, Yiwei

2017-02-15

Bauxite residue (BR) is a highly alkaline solid hazardous waste produced from bauxite processing for alumina production. Alkaline transformation appears to reduce the environmental risk of bauxite residue disposal areas (BRDAs) whilst potentially providing opportunities for the sustainable reuse and on-going management of BR. Mineral acids, a novel citric acid and a hybrid combination of acid-gypsum treatments were investigated for their potential to reduce residue pH and total alkalinity and transform the alkaline mineral phase. XRD results revealed that with the exception of andradite, the primary alkaline solid phases of cancrinite, grossular and calcite were transformed into discriminative products based on the transformation used. Supernatants separated from BR and transformed bauxite residue (TBR) displayed distinct changes in soluble Na, Ca and Al, and a reduction in pH and total alkalinity. SEM images suggest that mineral acid transformations promote macro-aggregate formation, and the positive promotion of citric acid, confirming the removal or reduction in soluble and exchangeable Na. NEXAFS analysis of Na K-edge revealed that the chemical speciation of Na in TBRs was consistent with BR. Three acid treatments and gypsum combination had no effect on Na speciation, which affects the distribution of Na revealed by sodium STXM imaging. Copyright © 2016 Elsevier B.V. All rights reserved.

Alkaline and non-aqueous proton-conducting pouch-cell batteries

DOEpatents

Young, Kwo-hsiung; Nei, Jean; Meng, Tiejun

2018-01-02

Provided are sealed pouch-cell batteries that are alkaline batteries or non-aqueous proton-conducing batteries. A pouch cell includes a flexible housing such as is used for pouch cell construction where the housing is in the form of a pouch, a cathode comprising a cathode active material suitable for use in an alkaline battery, an anode comprising an anode active material suitable for use in an alkaline battery, an electrolyte that is optionally an alkaline or proton-conducting electrolyte, and wherein the pouch does not include or require a safety vent or other gas absorbing or releasing system as the anode active material and the cathode active material do not increase the internal atmospheric pressure any more than 2 psig during cycling. The batteries provided function contrary to the art recognized belief that such battery systems were impossible due to unacceptable gas production during cycling.

Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.

PubMed

Reyon, Deepak; Maeder, Morgan L; Khayter, Cyd; Tsai, Shengdar Q; Foley, Jonathan E; Sander, Jeffry D; Joung, J Keith

2013-07-01

Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput. © 2013 by John Wiley & Sons, Inc.

Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) Assembly

PubMed Central

Reyon, Deepak; Maeder, Morgan L.; Khayter, Cyd; Tsai, Shengdar Q.; Foley, Jonathan E.; Sander, Jeffry D.; Joung, J. Keith

2013-01-01

Customized DNA-binding domains made using Transcription Activator-Like Effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in multiple different organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or down-regulate expression of endogenous genes in human cells and plants. Here we describe a detailed protocol for practicing the recently described Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multi-channel pipet. With the automated version of FLASH, a single researcher can construct up to 96 DNA fragments encoding various length TALE repeat arrays in one day and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in one week or less. Plas-mids required to practice FLASH are available by request from the Joung Lab (http://www.jounglab.org/). We also describe here improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) webserver (http://ZiFiTBeta.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high-throughput. PMID:23821439

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease.

PubMed

Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

2017-01-01

Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F' that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) ( P < 0.0001). According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample.

Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay.

PubMed

Li, Chia-Lung; Yang, Wei-Zen; Shi, Zhonghao; Yuan, Hanna S

2018-05-01

Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1-SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I•U and U•I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca 2+ -dependent ribonuclease, cleaving RNA chains at the 5'-side of the phosphodiester linkage to produce degraded fragments with 5'-hydroxyl and 3'-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I•U and U•I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca 2+ -binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay. © 2018 Li et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

PubMed Central

Ochiai, Hiroshi; Sakamoto, Naoaki; Fujita, Kazumasa; Nishikawa, Masatoshi; Suzuki, Ken-ichi; Matsuura, Shinya; Miyamoto, Tatsuo; Sakuma, Tetsushi; Shibata, Tatsuo; Yamamoto, Takashi

2012-01-01

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B–GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos. PMID:22711830

TaqMan 5′-Nuclease Human Immunodeficiency Virus Type 1 PCR Assay with Phage-Packaged Competitive Internal Control for High-Throughput Blood Donor Screening

PubMed Central

Drosten, C.; Seifried, E.; Roth, W. K.

2001-01-01

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5′-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5′-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the gag genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5′-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a ≥95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1. PMID:11724836

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.

40 CFR 434.40 - Applicability; description of the alkaline mine drainage subcategory.

Code of Federal Regulations, 2012 CFR

2012-07-01

... alkaline mine drainage subcategory. 434.40 Section 434.40 Protection of Environment ENVIRONMENTAL... BPT, BAT, BCT LIMITATIONS AND NEW SOURCE PERFORMANCE STANDARDS Alkaline Mine Drainage § 434.40 Applicability; description of the alkaline mine drainage subcategory. The provisions of this subpart are...

40 CFR 434.40 - Applicability; description of the alkaline mine drainage subcategory.

Code of Federal Regulations, 2013 CFR

2013-07-01

... alkaline mine drainage subcategory. 434.40 Section 434.40 Protection of Environment ENVIRONMENTAL... BPT, BAT, BCT LIMITATIONS AND NEW SOURCE PERFORMANCE STANDARDS Alkaline Mine Drainage § 434.40 Applicability; description of the alkaline mine drainage subcategory. The provisions of this subpart are...

40 CFR 434.40 - Applicability; description of the alkaline mine drainage subcategory.

Code of Federal Regulations, 2014 CFR

2014-07-01

... alkaline mine drainage subcategory. 434.40 Section 434.40 Protection of Environment ENVIRONMENTAL... BPT, BAT, BCT LIMITATIONS AND NEW SOURCE PERFORMANCE STANDARDS Alkaline Mine Drainage § 434.40 Applicability; description of the alkaline mine drainage subcategory. The provisions of this subpart are...

40 CFR 434.40 - Applicability; description of the alkaline mine drainage subcategory.

Code of Federal Regulations, 2010 CFR

2010-07-01

... alkaline mine drainage subcategory. 434.40 Section 434.40 Protection of Environment ENVIRONMENTAL..., BCT LIMITATIONS AND NEW SOURCE PERFORMANCE STANDARDS Alkaline Mine Drainage § 434.40 Applicability; description of the alkaline mine drainage subcategory. The provisions of this subpart are applicable to...

40 CFR 434.40 - Applicability; description of the alkaline mine drainage subcategory.

Code of Federal Regulations, 2011 CFR

2011-07-01

... alkaline mine drainage subcategory. 434.40 Section 434.40 Protection of Environment ENVIRONMENTAL..., BCT LIMITATIONS AND NEW SOURCE PERFORMANCE STANDARDS Alkaline Mine Drainage § 434.40 Applicability; description of the alkaline mine drainage subcategory. The provisions of this subpart are applicable to...

Orogenic potassic mafic magmatism, a product of alkaline-peraluminous mixing ? Variscan 'calc-alkaline' rocks from the Central Iberian and Ossa Morena Zones, Central Spain.

NASA Astrophysics Data System (ADS)

Scarrow, Jane H.; Cambeses, Aitor; Bea, Fernando; Montero, Pilar; Molina, José F.; Moreno, Juan Antonio

2013-04-01

Orogenic magmatic rocks provide information about mantle and crust melt-generation and -interaction processes. In this context, minor potassic mafic stocks which are formed of enriched mantle and crustal components and are common as late-orogenic intrusions in granitic plutons give insight into the timing of new crust formation and crustal recycling. Potassic mafic stocks are prevalent, albeit low volume, constituents of granite batholiths all through the European Variscan (350-280 Ma). In the Central Iberia Zone, Spanish Central System, crustal-melt, S-type, granitoid plutons are intruded by minor concomitant ultramafic-intermediate appinitic-vaugneritic stocks. Notwithstanding their whole-rock calc-alkaline composition, the stocks apparently did not have a subduction-related origin. Recent studies have attributed their genesis to mixing of alkaline mantle and peraluminous crustal melts. Their primary alkaline character, as indicated by amphibole and biotite mineral chemistry data, points, rather, towards an extension-related genesis. In the Ossa Morena Zone, south of the Central Iberian Zone, the igneous rocks also have a whole-rock calc-alkaline composition which has been considered to be the result of northward subduction of the South Portuguese Zone. Nevertheless, identification of a 'sill' of significant volume of mafic magma in the middle crust, the ´IBERSEIS reflective body', in a seismic profile across the Ossa Morena and South Portuguese Zones has cast doubt upon the calc-alkaline magmatism-subduction model; leading, instead, to the magmatism being attributed to intra-orogenic extension related to a mantle plume active from 340 Ma to 330 Ma. The aim here, then, is to reinvestigate the petrogenesis and age of the calc-alkaline rocks of the Ossa Morena Zone to determine their tectonomagmatic context be it subduction-, plume- or extension-related, and establish what they may reveal about mantle-crust interactions. Focussing, initially, on the Valencia del

Alkaline flooding for enhanced oil recovery

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

Gittler, W.E.

1983-09-01

There are over 12 active projects of varying size using one of 3 major types of alkaline agents. These include sodium silicate, caustic soda, and soda ash. Among the largest pilots currently is the THUMS project in the Wilmington field, California. Plans called for the injection of a 4% weight concentration of sodium orthosilicate over a 60% PV. Through the first 3 yr, over 27 million bbl of chemicals have been injected. Gulf Oil is operating several alkaline floods, one of which is located off shore in the Quarantine Bay field, Louisiana. In this pilot, sodium hydroxide in a weightmore » concentration of 5 to 12% is being injected. Belco Petroleum Corp. has reported that their pilot operating in the Isenhour Unit in Wyoming is using a .5% weight concentration of soda ash in conjunction with a polymer. Other uses for alkaline agents in chemical flooding include the use of silicate as a preflush or sacrificial agent in micellar/polymer and surfactant recovery systems. In addition, caustic has been tested in the surface-mixed caustic emulsion process while orthosilicate has been tested in a recovery method known as mobility-controlled caustic floods.« less

Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

PubMed Central

Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

2016-01-01

Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass.

PubMed

Davidson, Jesse A; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan; Wischmeyer, Paul E; Klawitter, Jelena

2016-01-01

Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post-operative alkaline phosphatase activity leads to

Alkaline Capacitors Based on Nitride Nanoparticles

NASA Technical Reports Server (NTRS)

Aldissi, Matt

2003-01-01

High-energy-density alkaline electrochemical capacitors based on electrodes made of transition-metal nitride nanoparticles are undergoing development. Transition- metal nitrides (in particular, Fe3N and TiN) offer a desirable combination of high electrical conductivity and electrochemical stability in aqueous alkaline electrolytes like KOH. The high energy densities of these capacitors are attributable mainly to their high capacitance densities, which, in turn, are attributable mainly to the large specific surface areas of the electrode nanoparticles. Capacitors of this type could be useful as energy-storage components in such diverse equipment as digital communication systems, implanted medical devices, computers, portable consumer electronic devices, and electric vehicles.

Method of increasing the sulfation capacity of alkaline earth sorbents

DOEpatents

Shearer, J.A.; Turner, C.B.; Johnson, I.

1980-03-13

A system and method for increasing the sulfation capacity of alkaline earth carbonates to scrub sulfur dioxide produced during the fluidized bed combustion of coal in which partially sulfated alkaline earth carbonates are hydrated in a fluidized bed to crack the sulfate coating and convert the alkaline earth oxide to the hydroxide. Subsequent dehydration of the sulfate-hydroxide to a sulfate-oxide particle produces particles having larger pore size, increased porosity, decreased grain size and additional sulfation capacity. A continuous process is disclosed.

Method of increasing the sulfation capacity of alkaline earth sorbents

DOEpatents

Shearer, John A.; Turner, Clarence B.; Johnson, Irving

1982-01-01

A system and method for increasing the sulfation capacity of alkaline earth carbonates to scrub sulfur dioxide produced during the fluidized bed combustion of coal in which partially sulfated alkaline earth carbonates are hydrated in a fluidized bed to crack the sulfate coating and convert the alkaline earth oxide to the hydroxide. Subsequent dehydration of the sulfate-hydroxide to a sulfate-oxide particle produces particles having larger pore size, increased porosity, decreased grain size and additional sulfation capacity. A continuous process is disclosed.

Increasing Alkalinity Export from Large Russian Arctic Rivers

NASA Astrophysics Data System (ADS)

Drake, T.; Zhulidov, A. V.; Gurtovaya, T. Y.; Spencer, R. G.

2017-12-01

Riverine carbonate alkalinity (HCO3- and CO32-) sourced from chemical weathering of minerals on land represents a significant sink for atmospheric CO2 over geologic timescales. The flux of alkalinity from rivers in the Arctic depends on precipitation, permafrost extent and thaw, groundwater flow paths, and surface vegetation, all of which are changing under a warming climate. Here we show that over the past four decades, the export of alkalinity from the Ob' and Yenisei Rivers has more than doubled. The increase is likely due to a combination of increasing precipitation and permafrost thaw in the watersheds, which lengthens hydrologic flow paths and increases residence time in soils. These trends have broad implications for the rate of carbon sequestration on land and the delivery of buffering capacity to the Arctic Ocean.

Serum creatinine and alkaline phosphatase levels are associated with severe chronic periodontitis.

PubMed

Caúla, A L; Lira-Junior, R; Tinoco, E M B; Fischer, R G

2015-12-01

Periodontitis may alter systemic homeostasis and influence creatinine and alkaline phosphatase levels. Therefore, the aim of this study was to evaluate the relationship between severe chronic periodontitis and serum creatinine and alkaline phosphatase levels. One hundred patients were evaluated, 66 with severe chronic periodontitis (test group) and 34 periodontally healthy controls (control group). Medical, demographic and periodontal parameters were registered. Blood sample was collected after an overnight fast and serum creatinine and alkaline phosphatase levels were determined. There were significant differences between test and control groups in ethnicity, gender and educational level (p < 0.05). Patients with periodontitis showed a lower mean creatinine level (p < 0.05) and higher mean alkaline phosphatase level (p < 0.001) than the control group. There were significant correlations between periodontal parameters and serum creatinine and alkaline phosphatase levels. Severe chronic periodontitis was associated to lower creatinine and higher alkaline phosphatase levels. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quantitation of Alkaline Phosphatase Isoenzymes Using Agarose Containing Wheat Germ Lectin

DTIC Science & Technology

1989-07-01

Gunshot M 40 179 150 16 13 0 Altered Mental Status M 39 1683 1650 0 33 0 Anemia, Chronic Dis. F 82 220 156 27 37 0 Arthritis , Rheumatoid M 70 79 26 0 53 0...34 5 Alkaline Phosphatase Isoenzyme Study on 210 Patients with Elevated Total Alkaline Phosphatase...46 6 Alkaline Phosphatase Isoenzyme Clinical Correlation Summary on 6 Distinct Disease Categories Containing 131 Patients

Ocean alkalinity and the Cretaceous/Tertiary boundary

NASA Technical Reports Server (NTRS)

Caldeira, K. G.; Rampino, Michael R.

1988-01-01

A biogeochemical cycle model resolving ocean carbon and alkalinity content is applied to the Maestrichtian and Danian. The model computes oceanic concentrations and distributions of Ca(2+), Mg(2+), and Sigma-CO2. From these values an atmospheric pCO2 value is calculated, which is used to estimate rates of terrestrial weathering of calcite, dolomite, and calcium and magnesium silicates. Metamorphism of carbonate rocks and the subsequent outgassing of CO2 to the atmosphere are parameterized in terms of carbonate rock reservoir sizes, total land area, and a measure of overall tectonic activity, the sea-floor generation rate. The ocean carbon reservoir computed by the model is used with Deep Sea Drilling Project (DSDP) C-13 data to estimate organic detrital fluxes under a variety of ocean mixing rate assumptions. Using Redfield ratios, the biogenic detrital flux estimate is used to partition the ocean carbon and alkalinity reservoirs between the mixed layer and deep ocean. The calcite flux estimate and carbonate ion concentrations are used to determine the rate of biologically mediated CaCO3 titration. Oceanic productivity was severely limited for approximately 500 kyr following the K/T boundary resulting in significant increases in total ocean alkalinity. As productivity returned to the ocean, excess carbon and alkalinity was removed from the ocean as CaCO3. Model runs indicate that this resulted in a transient imbalance in the other direction. Ocean chemistry returned to near-equilibrium by about 64 mybp.

Block Copolymers for Alkaline Fuel Cell Membrane Materials

DTIC Science & Technology

2014-07-30

temperature fuel cells including proton exchange membrane fuel cell ( PEMFC ) and alkaline fuel cell (AFC) with operation temperature usually lower than 120...advantages over proton exchange membrane fuel cells ( PEMFCs ) resulting in the popularity of AFCs in the US space program.[8-11] The primary benefit AFC...offered over PEMFC is better electrochemical kinetics on the anode and cathode under the alkaline environment, which results in the ability to use

OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines.

PubMed

Schmid-Burgk, Jonathan L; Schmidt, Tobias; Gaidt, Moritz M; Pelka, Karin; Latz, Eicke; Ebert, Thomas S; Hornung, Veit

2014-10-01

The application of designer nucleases allows the induction of DNA double-strand breaks (DSBs) at user-defined genomic loci. Due to imperfect DNA repair mechanisms, DSBs can lead to alterations in the genomic architecture, such as the disruption of the reading frame of a critical exon. This can be exploited to generate somatic knockout cell lines. While high genome editing activities can be achieved in various cellular systems, obtaining cell clones that contain all-allelic frameshift mutations at the target locus of interest remains a laborious task. To this end, we have developed an easy-to-follow deep sequencing workflow and the evaluation tool OutKnocker (www.OutKnocker.org), which allows convenient, reliable, and cost-effective identification of knockout cell lines. © 2014 Schmid-Burgk et al.; Published by Cold Spring Harbor Laboratory Press.

The Chemistry of Paper Preservation Part 4. Alkaline Paper.

ERIC Educational Resources Information Center

Carter, Henry A.

1997-01-01

Discusses the problem of the inherent instability of paper due to the presence of acids that catalyze the hydrolytic degradation of cellulose. Focuses on the chemistry involved in the sizing of both acid and alkaline papers and the types of fillers used. Discusses advantages and problems of alkaline papermaking. Contains 48 references. (JRH)

Removal of plutonium and americium from alkaline waste solutions

DOEpatents

Schulz, Wallace W.

1979-01-01

High salt content, alkaline waste solutions containing plutonium and americium are contacted with a sodium titanate compound to effect removal of the plutonium and americium from the alkaline waste solution onto the sodium titanate and provide an effluent having a radiation level of less than 10 nCi per gram alpha emitters.

Alkaline earth filled nickel skutterudite antimonide thermoelectrics

DOEpatents

Singh, David Joseph

2013-07-16

A thermoelectric material including a body centered cubic filled skutterudite having the formula A.sub.xFe.sub.yNi.sub.zSb.sub.12, where A is an alkaline earth element, x is no more than approximately 1.0, and the sum of y and z is approximately equal to 4.0. The alkaline earth element includes guest atoms selected from the group consisting of Be, Mb, Ca, Sr, Ba, Ra and combinations thereof. The filled skutterudite is shown to have properties suitable for a wide variety of thermoelectric applications.

40 CFR 721.4668 - Hydrated alkaline earth metal salts of metalloid oxyanions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Hydrated alkaline earth metal salts of... Specific Chemical Substances § 721.4668 Hydrated alkaline earth metal salts of metalloid oxyanions. (a... hydrated alkaline earth metal salts of metalloid oxyanions (PMN P-94-1557) is subject to reporting under...

40 CFR 721.4668 - Hydrated alkaline earth metal salts of metalloid oxyanions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Hydrated alkaline earth metal salts of... Specific Chemical Substances § 721.4668 Hydrated alkaline earth metal salts of metalloid oxyanions. (a... hydrated alkaline earth metal salts of metalloid oxyanions (PMN P-94-1557) is subject to reporting under...

40 CFR 721.4668 - Hydrated alkaline earth metal salts of metalloid oxyanions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Hydrated alkaline earth metal salts of... Specific Chemical Substances § 721.4668 Hydrated alkaline earth metal salts of metalloid oxyanions. (a... hydrated alkaline earth metal salts of metalloid oxyanions (PMN P-94-1557) is subject to reporting under...

40 CFR 721.4668 - Hydrated alkaline earth metal salts of metalloid oxyanions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Hydrated alkaline earth metal salts of... Specific Chemical Substances § 721.4668 Hydrated alkaline earth metal salts of metalloid oxyanions. (a... hydrated alkaline earth metal salts of metalloid oxyanions (PMN P-94-1557) is subject to reporting under...

40 CFR 721.4668 - Hydrated alkaline earth metal salts of metalloid oxyanions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Hydrated alkaline earth metal salts of... Specific Chemical Substances § 721.4668 Hydrated alkaline earth metal salts of metalloid oxyanions. (a... hydrated alkaline earth metal salts of metalloid oxyanions (PMN P-94-1557) is subject to reporting under...

Reductive dechlorination of carbon tetrachloride using buffered alkaline ascorbic acid.

PubMed

Lin, Ya-Ting; Liang, Chenju

2015-10-01

Alkaline ascorbic acid (AA) was recently discovered as a novel in-situ chemical reduction (ISCR) reagent for remediating chlorinated solvents in the subsurface. For this ISCR process, the maintenance of an alkaline pH is essential. This study investigated the possibility of the reduction of carbon tetrachloride (CT) using alkaline AA solution buffered by phosphate and by NaOH. The results indicated that CT was reduced by AA, and chloroform (CF) was a major byproduct at a phosphate buffered pH of 12. However, CT was completely reduced by AA in 2M NaOH without CF formation. In the presence of iron/soil minerals, iron could be reduced by AA and Fe(2+) tends to precipitate on the mineral surface to accelerate CT degradation. A simultaneous transfer of hydrogenolysis and dichloroelimination would occur under phosphate buffered pH 12. This implies that a high alkaline environment is a crucial factor for maintaining the dominant pathway of two electron transfer from dianionic AA to dehydroascorbic acid, and to undergo dichloroelimination of CT. Moreover, threonic acid and oxalic acid were identified to be the major AA decomposition products in alkaline solutions. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transition from alkaline to calc-alkaline volcanism during evolution of the Paleoproterozoic Francevillian basin of eastern Gabon (Western Central Africa)

NASA Astrophysics Data System (ADS)

Thiéblemont, Denis; Bouton, Pascal; Préat, Alain; Goujou, Jean-Christian; Tegyey, Monique; Weber, Francis; Ebang Obiang, Michel; Joron, Jean Louis; Treuil, Michel

2014-11-01

We report new geochemical data for the volcanic and subvolcanic rocks associated with the evolution of the Francevillian basin of eastern Gabon during Paleoproterozoic times (c. 2.1-2 Ga). Filling of this basin has proceeded through four main sedimentary or volcano-sedimentary episodes, namely FA, FB, FC and FD. Volcanism started during the FB episode being present only in the northern part of the basin (Okondja sub-basin). This volcanism is ultramafic to trachytic in composition and displays a rather constant alkaline geochemical signature. This signature is typical of a within-plate environment, consistent with the rift-setting generally postulated for the Francevillian basin during the FB period. Following FB, the FC unit is 10-20 m-thick silicic horizon (jasper) attesting for a massive input of silica in the basin. Following FC, the FD unit is a c. 200-400 m-thick volcano-sedimentary sequence including felsic tuffs and epiclastic rocks. The geochemical signatures of these rocks are totally distinct from those of the FB alkaline lavas. High Th/Ta and La/Ta ratios attest for a calc-alkaline signature and slight fractionation between heavy rare-earth suggests melting at a rather low pressure. Such characteristics are comparable to those of felsic lavas associated with the Taupo zone of New Zealand, a modern ensialic back-arc basin. Following FD, the FE detrital unit is defined only in the Okondja region, probably associated with a late-stage collapse of the northern part of the basin. It is suggested that the alkaline to calc-alkaline volcanic transition reflects the evolution of the Francevillian basin from a diverging to a converging setting, in response to the onset of converging movements in the Eburnean Belt of Central Africa.

Dairy products and the French paradox: Could alkaline phosphatases play a role?

PubMed

Lallès, Jean-Paul

2016-07-01

The French paradox - high saturated fat consumption but low incidence of cardiovascular disease (CVD) and mortality - is still unresolved and continues to be a matter of debate and controversy. Recently, it was hypothesised that the high consumption of dairy products, and especially cheese by the French population might contribute to the explanation of the French paradox, in addition to the "(red) wine" hypothesis. Most notably this would involve milk bioactive peptides and biomolecules from cheese moulds. Here, we support the "dairy products" hypothesis further by proposing the "alkaline phosphatase" hypothesis. First, intestinal alkaline phosphatase (IAP), a potent endogenous anti-inflammatory enzyme, is directly stimulated by various components of milk (e.g. casein, calcium, lactose and even fat). This enzyme dephosphorylates and thus detoxifies pro-inflammatory microbial components like lipopolysaccharide, making them unable to trigger inflammatory responses and generate chronic low-grade inflammation leading to insulin resistance, glucose intolerance, type-2 diabetes, metabolic syndrome and obesity, known risk factors for CVD. Various vitamins present in high amounts in dairy products (e.g. vitamins A and D; methyl-donors: folate and vitamin B12), and also fermentation products such as butyrate and propionate found e.g. in cheese, all stimulate intestinal alkaline phosphatase. Second, moulded cheeses like Roquefort contain fungi producing an alkaline phosphatase. Third, milk itself contains a tissue nonspecific isoform of alkaline phosphatase that may function as IAP. Milk alkaline phosphatase is present in raw milk and dairy products increasingly consumed in France. It is deactivated by pasteurization but it can partially reactivate after thermal treatment. Experimental consolidation of the "alkaline phosphatase" hypothesis will require further work including: systematic alkaline phosphatase activity measurements in dairy products, live dairy ferments and

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.

Alkalinity production in intertidal sands intensified by lugworm bioirrigation.

PubMed

Rao, Alexandra M F; Malkin, Sairah Y; Montserrat, Francesc; Meysman, Filip J R

2014-07-05

Porewater profiles and sediment-water fluxes of oxygen, nutrients, pH, calcium, alkalinity, and sulfide were measured in intertidal sandflat sediments from the Oosterschelde mesotidal lagoon (The Netherlands). The influence of bioturbation and bioirrigation by the deep-burrowing polychaete Arenicola marina on the rates and sources of benthic alkalinity generation was examined by comparing measurements in intact and defaunated sediment cores before and after the addition of A. marina in summer and fall 2011. Higher organic matter remineralization rates, shallower O 2 penetration, and greater sediment-water solute fluxes were observed in summer, consistent with higher sediment community metabolic rates at a higher temperature. Lugworm activity stimulated porewater exchange (5.1 × in summer, 1.9 × in fall), organic matter remineralization (6.2 × in summer, 1.9 × in fall), aerobic respiration (2.4 × in summer, 2.1 × in fall), alkalinity release (4.7 × in summer, 4.0 × in fall), nutrient regeneration, and iron cycling. The effects of lugworm activity on net sediment-water fluxes were similar but more pronounced in summer than in fall. Alkalinity release in fall was entirely driven by metabolic carbonate dissolution, while this process explained between 22 and 69% of total alkalinity production in summer, indicating the importance of other processes in this season. By enhancing organic matter remineralization and the reoxidation of reduced metabolites by the sediment microbial community, lugworm activity stimulated the production of dissolved inorganic carbon and metabolic acidity, which in turn enhanced metabolic CaCO 3 dissolution efficiency. In summer, evidence of microbial long distance electron transport (LDET) was observed in defaunated sediment. Thus, alkalinity production by net carbonate dissolution was likely supplemented by anaerobic respiration and LDET in summer.

Chapter A6. Section 6.6. Alkalinity and Acid Neutralizing Capacity

USGS Publications Warehouse

Rounds, Stewart A.; Wilde, Franceska D.

2002-01-01

Alkalinity (determined on a filtered sample) and Acid Neutralizing Capacity (ANC) (determined on a whole-water sample) are measures of the ability of a water sample to neutralize strong acid. Alkalinity and ANC provide information on the suitability of water for uses such as irrigation, determining the efficiency of wastewater processes, determining the presence of contamination by anthropogenic wastes, and maintaining ecosystem health. In addition, alkalinity is used to gain insights on the chemical evolution of an aqueous system. This section of the National Field Manual (NFM) describes the USGS field protocols for alkalinity/ANC determination using either the inflection-point or Gran function plot methods, including calculation of carbonate species, and provides guidance on equipment selection.

De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline Stress.

PubMed

An, Yi-Min; Song, Li-Li; Liu, Ying-Rui; Shu, Yong-Jun; Guo, Chang-Hong

2016-01-01

Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a widely cultivated perennial forage legume with some tolerance to biotic and abiotic stresses, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0 day (control), 1 day (short-term treatment), and 7 days (long-term treatment) using ion torrent sequencing technology. A transcriptome database dataset of 53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially expressed in the short-term and long-term treatment, respectively. Gene ontology analysis revealed 14 highly enriched pathways and demonstrated the differential response of metabolic pathways between the short-term and long-term treatment. The expression levels of 109 and 96 transcription factors were significantly altered significantly after 1 day and 7 days of treatment, respectively. Specific responses of peroxidase, flavonoids, and the light pathway component indicated that the antioxidant capacity was one of the central mechanisms of saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially expressed genes examined by real time PCR, the expression levels of eight genes, including inositol transporter, DNA binding protein, raffinose synthase, ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans glucosylase, and a NAC transcription factor, exhibited different patterns in response to saline and alkaline stress. The expression levels of the NAC transcription factor and glutathione S-transferase were altered significantly under saline stress and saline-alkaline stress; they were upregulated under saline-alkaline stress and downregulated under salt stress. Physiology assays showed an increased concentration of reactive oxygen

De Novo Transcriptional Analysis of Alfalfa in Response to Saline-Alkaline Stress

PubMed Central

An, Yi-Min; Song, Li-Li; Liu, Ying-Rui; Shu, Yong-Jun; Guo, Chang-Hong

2016-01-01

Saline-alkaline stress, caused by high levels of harmful carbonate salts and high soil pH, is a major abiotic stress that affects crop productivity. Alfalfa is a widely cultivated perennial forage legume with some tolerance to biotic and abiotic stresses, especially to saline-alkaline stress. To elucidate the mechanism underlying plant saline-alkaline tolerance, we conducted transcriptome analysis of whole alfalfa seedlings treated with saline-alkaline solutions for 0 day (control), 1 day (short-term treatment), and 7 days (long-term treatment) using ion torrent sequencing technology. A transcriptome database dataset of 53,853 unigenes was generated, and 2,286 and 2,233 genes were differentially expressed in the short-term and long-term treatment, respectively. Gene ontology analysis revealed 14 highly enriched pathways and demonstrated the differential response of metabolic pathways between the short-term and long-term treatment. The expression levels of 109 and 96 transcription factors were significantly altered significantly after 1 day and 7 days of treatment, respectively. Specific responses of peroxidase, flavonoids, and the light pathway component indicated that the antioxidant capacity was one of the central mechanisms of saline-alkaline stress tolerance response in alfalfa. Among the 18 differentially expressed genes examined by real time PCR, the expression levels of eight genes, including inositol transporter, DNA binding protein, raffinose synthase, ferritin, aldo/keto reductase, glutathione S-transferase, xyloglucan endotrans glucosylase, and a NAC transcription factor, exhibited different patterns in response to saline and alkaline stress. The expression levels of the NAC transcription factor and glutathione S-transferase were altered significantly under saline stress and saline-alkaline stress; they were upregulated under saline-alkaline stress and downregulated under salt stress. Physiology assays showed an increased concentration of reactive oxygen

Extracellular Alkalinization as a Defense Response in Potato Cells.

PubMed

Moroz, Natalia; Fritch, Karen R; Marcec, Matthew J; Tripathi, Diwaker; Smertenko, Andrei; Tanaka, Kiwamu

2017-01-01

A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-derived elicitors in a dose- and time-dependent manner. We also assessed the defense response against a variety of potato pathogens, such as protists ( Phytophthora infestans and Spongospora subterranea ) and fungi ( Verticillium dahliae and Colletotrichum coccodes ). Our results show that extracellular pH increases within 30 min in proportion to the number of pathogen spores added. Consistently with the alkalinization effect, the higher transcription level of several defense-related genes and production of reactive oxygen species was observed. Our results demonstrate that the alkalinization response is an effective marker to study early stages of defense response in potatoes.

A Novel Prokaryotic Green Fluorescent Protein Expression System for Testing Gene Editing Tools Activity Like Zinc Finger Nuclease

PubMed Central

Sabzehei, Faezeh; Kouhpayeh, Shirin; Dastjerdeh, Mansoureh Shahbazi; Khanahmad, Hossein; Salehi, Rasoul; Naderi, Shamsi; Taghizadeh, Razieh; Rabiei, Parisa; Hejazi, Zahra; Shariati, Laleh

2017-01-01

Background: Gene editing technology has created a revolution in the field of genome editing. The three of the most famous tools in gene editing technology are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated systems. As their predictable nature, it is necessary to assess their efficiency. There are some methods for this purpose, but most of them are time labor and complicated. Here, we introduce a new prokaryotic reporter system, which makes it possible to evaluate the efficiency of gene editing tools faster, cheaper, and simpler than previous methods. Materials and Methods: At first, the target sites of a custom ZFN, which is designed against a segment of ampicillin resistance gene, were cloned on both sides of green fluorescent protein (GFP) gene to construct pPRO-GFP. Then pPRO-GFP was transformed into Escherichia coli TOP10F’ that contains pZFN (contains expression cassette of a ZFN against ampicillin resistant gene), or p15A-KanaR as a negative control. The transformed bacteria were cultured on three separate media that contained ampicillin, kanamycin, and ampicillin + kanamycin; then the resulted colonies were assessed by flow cytometry. Results: The results of flow cytometry showed a significant difference between the case (bacteria contain pZFN) and control (bacteria contain p15A, KanaR) in MFI (Mean Fluorescence Intensity) (P < 0.0001). Conclusion: According to ZFN efficiency, it can bind and cut the target sites, the bilateral cutting can affect the intensity of GFP fluorescence. Our flow cytometry results showed that this ZFN could reduce the intensity of GFP color and colony count of bacteria in media containing amp + kana versus control sample. PMID:29285485

Systematic review of the association between dietary acid load, alkaline water and cancer.

PubMed

Fenton, Tanis R; Huang, Tian

2016-06-13

To evaluate the evidence for a causal relationship between dietary acid/alkaline and alkaline water for the aetiology and treatment of cancer. A systematic review was conducted on published and grey literature separately for randomised intervention and observational studies with either varying acid-base dietary intakes and/or alkaline water with any cancer outcome or for cancer treatment. Incidence of cancer and outcomes of cancer treatment. 8278 citations were identified, and 252 abstracts were reviewed; 1 study met the inclusion criteria and was included in this systematic review. No randomised trials were located. No studies were located that examined dietary acid or alkaline or alkaline water for cancer treatment. The included study was a cohort study with a low risk of bias. This study revealed no association between the diet acid load with bladder cancer (OR=1.15: 95% CI 0.86 to 1.55, p=0.36). No association was found even among long-term smokers (OR=1.72: 95% CI 0.96 to 3.10, p=0.08). Despite the promotion of the alkaline diet and alkaline water by the media and salespeople, there is almost no actual research to either support or disprove these ideas. This systematic review of the literature revealed a lack of evidence for or against diet acid load and/or alkaline water for the initiation or treatment of cancer. Promotion of alkaline diet and alkaline water to the public for cancer prevention or treatment is not justified. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Two Year Field Study to Evaluate the Efficacy of Mamestra brassicae Nucleopolyhedrovirus Combined with Proteins Derived from Xestia c-nigrum Granulovirus

PubMed Central

Goto, Chie; Mukawa, Shigeyuki; Mitsunaga, Takayuki

2015-01-01

Japan has only three registered baculovirus biopesticides despite its long history of studies on insect viruses. High production cost is one of the main hindrances for practical use of baculoviruses. Enhancement of insecticidal effect is one possible way to overcome this problem, so there have been many attempts to develop additives for baculoviruses. We found that alkaline soluble proteins of capsules (GVPs) of Xestia c-nigrum granulovirus can increase infectivity of some viruses including Mamestra brassicae nucleopolyhedrovirus (MabrNPV), and previously reported that MabrNPV mixed with GVPs was highly infectious to three important noctuid pests of vegetables in the following order, Helicoverpa armigera, M. brassicae, and Autographa nigrisigna. In this study, small-plot experiments were performed to assess concentrations of MabrNPV and GVPs at three cabbage fields and a broccoli field for the control of M. brassicae. In the first experiment, addition of GVPs (10 µg/mL) to MabrNPV at 106 OBs/mL resulted in a significant increase in NPV infection (from 53% to 66%). In the second experiment, the enhancing effect of GVP on NPV infection was confirmed at 10-times lower concentrations of MabrNPV. In the third and fourth experiments, a 50% reduction in GVPs (from 10 µg/mL to 5 µg/mL) did not result in a lowering of infectivity of the formulations containing MabrNPV at 105 OBs/mL. These results indicate that GVPs are promising additives for virus insecticides. PMID:25760139

Hydrothermal Alkalinity in Central Nepal Rivers

NASA Astrophysics Data System (ADS)

Evans, M. J.; Derry, L. A.

2002-12-01

Numerous hot springs flow along the base of the Himalayan front, at or near the Main Central Thrust, in the Narayani drainage of central Nepal. The springs are found in a narrow zone characterized by rapid uplift and high incision rates. In this zone, hot rocks are brought to the near-surface where they interact with meteoric waters to produce the hydrothermal system. Water-rock interaction produces springs with high solute loads (TDS up to 8000 mg/L.) The springs drive significant chemical anomalies (e.g. Cl, Na, K and Ge) in the rivers that flow through the hydrothermal zone In order to quantify the impact the springs have on the river chemistry, the spring discharge must be estimated. Direct measurement of the spring discharge is difficult, as the springs often flow within the stream bed itself or are inaccessible. We take advantage of the wide disparity in stream vs. hydrothermal [Ge] to calculate spring discharge by chemical mass balance. The hot springs have [Ge] up to 684 nmol/kg and Ge/Si ratios from 200 to 1000 μmol/mol while river waters have [Ge] near 0.15 nmol/kg and Ge/Si ratios near 0.5 μmol/mol, typical of non-polluted rivers. The discharge calculated from the Ge mass balance for individual springs ranges from 0.03 x 106 to 5.6 x 106 m3/yr, and accounts for a small percentage of the total river discharge (0.03% to 1.9%). The hot spring discharge for all of central Nepal is around 1.5x108 m3/yr, 0.5% of the Narayani river discharge. Distinguishing between silicate and carbonate sources is important to assessing the role of weathering on atmospheric CO2 levels and the relative contributions of silicate and carbonate alkalinity in central Nepal rivers are still not well resolved. The hot springs derive up to 100% of their alkalinity from silicate sources. Using the discharge estimates for the springs, we find that the sum of the silicate alkalinity fluxes from all the spring systems is 2.8 x 108 mol/yr. This implies that the hot springs deliver around

Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

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

Bach, Christian; Sherman, William; Pallis, Jani

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable toolsmore » to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.« less

Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

DOE PAGES

Bach, Christian; Sherman, William; Pallis, Jani; ...

2014-01-01

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable toolsmore » to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.« less

Acidity and Alkalinity in mine drainage: Practical considerations

USGS Publications Warehouse

Cravotta, III, Charles A.; Kirby, Carl S.

2004-01-01

In this paper, we emphasize that the Standard Method hot peroxide treatment procedure for acidity determination (hot acidity) directly measures net acidity or net alkalinity, but that more than one water-quality measure can be useful as a measure of the severity of acid mine drainage. We demonstrate that the hot acidity is related to the pH, alkalinity, and dissolved concentrations of Fe, Mn, and Al in fresh mine drainage. We show that the hot acidity accurately indicates the potential for pH to decrease to acidic values after complete oxidation of Fe and Mn, and it indicates the excess alkalinity or that required for neutralization of the sample. We show that the hot acidity method gives consistent, interpretable results on fresh or aged samples. Regional data for mine-drainage quality in Pennsylvania indicated the pH of fresh samples was predominantly acidic (pH 2.5 to 4) or near neutral (pH 6 to 7); approximately 25 percent of the samples had intermediate pH values. This bimodal frequency distribution of pH was distinctive for fully oxidized samples; oxidized samples had acidic or near-neutral pH, only. Samples that had nearneutral pH after oxidation had negative hot acidity; samples that had acidic pH after oxidation had positive hot acidity. Samples with comparable pH values had variable hot acidities owing to variations in their alkalinities and dissolved Fe, Mn, and Al concentrations. The hot acidity was comparable to net acidity computed on the basis of initial pH and concentrations of Fe, Mn, and Al minus the initial alkalinity. Acidity computed from the pH and dissolved metals concentrations, assuming equivalents of 2 per mole of Fe and Mn and 3 per mole of Al, was comparable to that computed on the basis of aqueous species and FeII/FeIII. Despite changes in the pH, alkalinity, and metals concentrations, the hot acidities were comparable for fresh and aged samples. Thus, meaningful “net” acidity can be determined from a measured hot acidity or by

The origin of life in alkaline hydrothermal vents

NASA Astrophysics Data System (ADS)

Sojo, V.; Herschy, B.; Whicher, A.; Camprubí, E.; Lane, N.

2016-12-01

The origin of life remains one of Science's greatest unresolved questions. The answer will no doubt involve almost all the basic disciplines, including Physics, Chemistry, Astronomy, Geology, and Biology. Chiefly, it is the link between the latter two that must be elucidated: how geochemistry gave rise to biochemistry. Serpentinizing systems such as alkaline hydrothermal vents offer the most robust combination of conditions to have hosted the origin of life on the early Earth, while bearing many parallels to modern living cells. Stark gradients of concentration, pH, oxidation/reduction, and temperature provided the ability to synthesise and concentrate organic products, drive polymerisation reactions, and develop an autotrophic lifestyle independent of foreign sources of organics. In the oxygen-depleted waters of the Hadean, alkaline vents would have acted as electrochemical flow reactors, in which alkaline fluids saturated in H2 mixed with the relatively acidic CO2-rich waters of the ocean, through interconnected micropores made of thin inorganic walls containing catalytic Fe(Ni)S minerals. Perhaps not coincidentally, the unit cells of these Fe(Ni)S minerals closely resemble the active sites of crucial ancestral bioenergetic enzymes. Meanwhile, differences in pH across the thin barriers produced natural proton gradients similar to those used for carbon fixation in modern archaea and bacteria. At the earliest stages, the problem of the origin of life is the problem of the origin of carbon fixation. I will discuss work over the last decade that suggests several possible hypotheses for how simple one-carbon molecules could have given rise to more complex organics, particularly within a serpentinizing alkaline hydrothermal vent. I will discuss the perplexing differences in carbon and energy metabolism in methanogenic archaea and acetogenic bacteria, thought to be the earliest representatives of each domain, to propose a possible ancestral mechanism of CO2 reduction in

Role of the Group 2 Mrp sodium/proton antiporter in rapid response to high alkaline shock in the alkaline- and salt-tolerant Dietzia sp. DQ12-45-1b.

PubMed

Fang, Hui; Qin, Xiao-Yu; Zhang, Kai-Duan; Nie, Yong; Wu, Xiao-Lei

2018-04-01

The six- and seven-subunit Na + /H + antiporters (Mrp) are widely distributed in bacteria. They are reported to be integral for pH homeostasis in alkaliphilic bacteria when adapting to high pH environments. In this study, operons encoding for the six-subunit Na + /H + antiporters were found in the genomes of all studied Dietzia strains, which have different alkaline-resistant abilities. Disruption of the operon in the strain Dietzia sp. DQ12-45-1b which leads to declined growth in presence of hypersaline and alkaline conditions suggested that the six-subunit Na + /H + antiporter played an important role in hypersaline and alkaline resistance. Although the complexes DqMrp from DQ12-45-1b (strain with high alkaline resistance) and DaMrp from D. alimentaria 72 T (strain with low alkaline resistance) displayed Na + (Li + )/H + antiport activities, they functioned optimally at different pH levels (9.0 for DQ12-45-1b and 8.0 for 72 T ). While both antiporters functioned properly to protect Escherichia coli cells from salt shock, only the DqMrp-containing strain survived the high alkaline shock. Furthermore, real-time PCR results showed that the expression of mrpA and mrpD induced only immediately after DQ12-45-1b cells were subjected to the alkaline shock. These results suggested that the expression of DqMrp might be induced by a pH gradient across the cell membrane, and DqMrp mainly functioned at an early stage to respond to the alkaline shock.

Isonicotinohydrazones as inhibitors of alkaline phosphatase and ecto-5'-nucleotidase.

PubMed

Channar, Pervaiz Ali; Shah, Syed Jawad Ali; Hassan, Sidra; Nisa, Zaib Un; Lecka, Joanna; Sévigny, Jean; Bajorath, Jürgen; Saeed, Aamer; Iqbal, Jamshed

2017-03-01

A series of isonicotinohydrazide derivatives was synthesized and tested against recombinant human and rat ecto-5'-nucleotidases (h-e5'NT and r-e5'NT) and alkaline phosphatase isozymes including both bovine tissue-non-specific alkaline phosphatase (b-TNAP) and tissue-specific calf intestinal alkaline phosphatase (c-IAP). These enzymes are implicated in vascular calcifications, hypophosphatasia, solid tumors, and cancers, such as colon, lung, breast, pancreas, and ovary. All tested compounds were active against both enzymes. The most potent inhibitor of h-e5'NT was derivative (E)-N'-(1-(3-(4-fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)ethylidene)isonicotinohydrazide (3j), whereas derivative (E)-N'-(4-hydroxy-3-methoxybenzylidene)isonicotinohydrazide (3g) exhibited significant inhibitory activity against r-e5'NT. In addition, the derivative (E)-N'-(4'-chlorobenzylidene)isonicotinohydrazide (3a) was most potent inhibitor against calf intestinal alkaline phosphatase and the derivative (E)-N'-(4-hydroxy-3-methoxybenzylidene)isonicotinohydrazide (3g) was found to be most potent inhibitor of bovine tissue-non-specific alkaline phosphatase. Furthermore, putative binding modes of potent compounds against e5'NT (human and rat e5'NT) and AP (including b-TNAP and c-IAP) were determined computationally. © 2016 John Wiley & Sons A/S.

[Effects of alkaline fertilizer on cadmium content in rice and paddy soil.

PubMed

Zhang, Liang Liang; Fan, Xiao Lin; Zhang, Li Dan; Liu, Fang

2016-03-01

A field plot trial was conducted at Songbai Village, Chenzhou, Hunan Province to exa-mine the effects of alkaline fertilizer with and without combination application of bacterial regent on cadmium accumulation in rice and paddy soil. Compared with the conventional fertilizer, the alkaline fertilizer reduced the available cadmium content in soil at the tillering, filling and the harvest stages by 8.3%, 6.7% and 16.4% respectively. Compared with the Cd content in soil before transplanting, it was reduced by 7.2% at harvest time in alkaline fertilizer treatment, while increased by 11.0% in the conventional fertilizer treatment. The available cadmium content in soil was increased by 1.2% to 23.3% by bacterial regent. Compared with the conventional fertilizer, the cadmium content of root, stem sheath, leaf, grain and brown rice of the alkaline fertilizer treatment reduced by 54.9%, 56.6%, 41.8%, 62.7% and 67.6% respectively. The alkaline fertilizer treatment combined with bacterial regent increased the cadmium content of brown rice by 63.2%. It was concluded that the alkaline fertilizer could significantly reduce the available cadmium content in both soil and the different organs of the rice, however, when it was combined with bacterial regent together, the effect of alkaline fertilizer to reduce Cd content of brown rice would be declined.

Alkaline polymer electrolyte fuel cells stably working at 80 °C

NASA Astrophysics Data System (ADS)

Peng, Hanqing; Li, Qihao; Hu, Meixue; Xiao, Li; Lu, Juntao; Zhuang, Lin

2018-06-01

Alkaline polymer electrolyte fuel cells are a new class of polymer electrolyte fuel cells that fundamentally enables the use of nonprecious metal catalysts. The cell performance mostly relies on the quality of alkaline polymer electrolytes, including the ionic conductivity and the chemical/mechanical stability. For a long time, alkaline polymer electrolytes are thought to be too weak in stability to allow the fuel cell to be operated at elevated temperatures, e.g., above 60 °C. In the present work, we report a progress in the state-of-the-art alkaline polymer electrolyte fuel cell technology. By using a newly developed alkaline polymer electrolyte, quaternary ammonia poly (N-methyl-piperidine-co-p-terphenyl), which simultaneously possesses high ionic conductivity and excellent chemical/mechanical stability, the fuel cell can now be stably operated at 80 °C with high power density. The peak power density reaches ca. 1.5 W/cm2 at 80 °C with Pt/C catalysts used in both the anode and the cathode. The cell works stably in a period of study over 100 h.

Coupling the Alkaline-Surfactant-Polymer Technology and The Gelation Technology to Maximize Oil Production

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

Malcolm Pitts; Jie Qi; Dan Wilson

2005-10-01

Gelation technologies have been developed to provide more efficient vertical sweep efficiencies for flooding naturally fractured oil reservoirs or more efficient areal sweep efficiency for those with high permeability contrast ''thief zones''. The field proven alkaline-surfactant-polymer technology economically recovers 15% to 25% OOIP more oil than waterflooding from swept pore space of an oil reservoir. However, alkaline-surfactant-polymer technology is not amenable to naturally fractured reservoirs or those with thief zones because much of injected solution bypasses target pore space containing oil. This work investigates whether combining these two technologies could broaden applicability of alkaline-surfactant-polymer flooding into these reservoirs. A priormore » fluid-fluid report discussed interaction of different gel chemical compositions and alkaline-surfactant-polymer solutions. Gel solutions under dynamic conditions of linear corefloods showed similar stability to alkaline-surfactant-polymer solutions as in the fluid-fluid analyses. Aluminum-polyacrylamide, flowing gels are not stable to alkaline-surfactant-polymer solutions of either pH 10.5 or 12.9. Chromium acetate-polyacrylamide flowing and rigid flowing gels are stable to subsequent alkaline-surfactant-polymer solution injection. Rigid flowing chromium acetate-polyacrylamide gels maintained permeability reduction better than flowing chromium acetate-polyacrylamide gels. Silicate-polyacrylamide gels are not stable with subsequent injection of either a pH 10.5 or a 12.9 alkaline-surfactant-polymer solution. Chromium acetate-xanthan gum rigid gels are not stable to subsequent alkaline-surfactant-polymer solution injection. Resorcinol-formaldehyde gels were stable to subsequent alkaline-surfactant-polymer solution injection. When evaluated in a dual core configuration, injected fluid flows into the core with the greatest effective permeability to the injected fluid. The same gel stability trends to

Cementitious porous pavement in stormwater quality control: pH and alkalinity elevation.

PubMed

Kuang, Xuheng; Sansalone, John

2011-01-01

A certain level of alkalinity acts as a buffer and maintains the pH value in a stable range in water bodies. With rapid urban development, more and more acidic pollutants flow to watersheds with runoff and drop alkalinity to a very low level and ultimately degrade the water environment. Cementitious porous pavement is an effective tool for stormwater acidic neutralization. When stormwater infiltrates cement porous pavement (CPP) materials, alkalinity and pH will be elevated due to the basic characteristics of cement concrete. The elevated alkalinity will neutralize acids in water bodies and maintain the pH in a stable level as a buffer. It is expected that CPP materials still have a certain capability of alkalinity elevation after years of service, which is important for CPP as an effective tool for stormwater management. However, few previous studies have reported on how CPP structures would elevate runoff alkalinity and pH after being exposed to rainfall-runoff for years. In this study, three groups of CPP specimens, all exposed to rainfall-runoff for 3 years, were used to test the pH and alkalinity elevation properties. It was found that runoff pH values were elevated from 7.4 to the range of 7.8-8.6 after infiltrating through the uncoated specimens, and from 7.4 to 8.5-10.7 after infiltrating through aluminum-coated specimens. Runoff alkalinity elevation efficiencies are 11.5-14.5% for uncoated specimens and 42.2% for coated specimens. The study shows that CPP is an effective passive unit operation for stormwater acid neutralization in our built environment.

Anaerobic digestion of thermal-alkaline-pretreated cephalosporin bacterial residues for methane production.

PubMed

Li, Guixia; Zhong, Weizhang; Wang, Rui; Chen, Jiaqi; Li, Zaixing

2017-08-01

Optimum anaerobic conditions of cephalosporin bacterial residues after thermal-alkaline pretreatment were determined by orthogonal experiments. And through biochemical methane potential tests (BMPs) for cephalosporin bacterial residues, the ability for bacterial degradation of cephalosporin was also evaluated. The thermal-alkaline pretreatment with the optimum values of 6% NaOH at 105 °C for 15 min significantly improved digestion performance. With the thermal-alkaline pretreatment, the specific methane yield of the pretreated cephalosporin bacterial residue increased by 254.79% compared with that of the un-pretreated cephalosporin bacterial residue. The results showed that anaerobic digestion of thermal-alkaline-pretreated cephalosporin bacterial residues could be one of the options for efficient methane production and waste treatment. This work investigates the thermal-alkaline pretreatment of cephalosporin bacterial residues, which can increase their methane yield by 254.79% compared with no pretreatment. The digestion performance is significantly improved under the condition of 6% NaOH at 105 °C for 15 min. The results show that anaerobic digestion of thermal-alkaline-pretreated cephalosporin bacterial residues could be one of the options for efficient methane production and waste treatment.

Increased performance of hydrogen production in microbial electrolysis cells under alkaline conditions.

PubMed

Rago, Laura; Baeza, Juan A; Guisasola, Albert

2016-06-01

This work reports the first successful enrichment and operation of alkaline bioelectrochemical systems (microbial fuel cells, MFC, and microbial electrolysis cells, MEC). Alkaline (pH=9.3) bioelectrochemical hydrogen production presented better performance (+117%) compared to conventional neutral conditions (2.6 vs 1.2 litres of hydrogen gas per litre of reactor per day, LH2·L(-1)REACTOR·d(-1)). Pyrosequencing results of the anodic biofilm showed that while Geobacter was mainly detected under conventional neutral conditions, Geoalkalibacter sp. was highly detected in the alkaline MFC (21%) and MEC (48%). This is the first report of a high enrichment of Geoalkalibacter from an anaerobic mixed culture using alkaline conditions in an MEC. Moreover, Alkalibacter sp. was highly present in the anodic biofilm of the alkaline MFC (37%), which would indicate its potentiality as a new exoelectrogen. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a 5'-nuclease real-time PCR assay targeting fliP for the rapid identification of Burkholderia mallei in clinical samples.

PubMed

Tomaso, Herbert; Scholz, Holger C; Al Dahouk, Sascha; Eickhoff, Meike; Treu, Thomas M; Wernery, Renate; Wernery, Ulrich; Neubauer, Heinrich

2006-02-01

Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. We developed a B. mallei-specific 5'-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. Compared with conventional PCR, our rapid 5'-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.

Sensitive detection of microRNAs based on the conversion of colorimetric assay into electrochemical analysis with duplex-specific nuclease-assisted signal amplification

PubMed Central

Xia, Ning; Liu, Ke; Zhou, Yingying; Li, Yuanyuan; Yi, Xinyao

2017-01-01

miRNAs have emerged as new biomarkers for the detection of a wide variety of cancers. By employing duplex-specific nuclease for signal amplification and gold nanoparticles (AuNPs) as the carriers of detection probes, a novel electrochemical assay of miRNAs was performed. The method is based on conversion of the well-known colorimetric assay into electrochemical analysis with enhanced sensitivity. DNA capture probes immobilized on the electrode surface and ferrocene (Fc)-labeled DNA detection probes (denoted “Fc-DNA-Fc”) presented in the solution induced the assembly of positively charged AuNPs on the electrode surface through the electrostatic interaction. As a result, a large number of Fc-DNA-Fc molecules were attached on the electrode surface, thus amplifying the electrochemical signal. When duplex-specific nuclease was added to recycle the process of miRNA-initiated digestion of the immobilized DNA probes, Fc-DNA-Fc-induced assembly of AuNPs on the electrode surface could not occur. This resulted in a significant fall in the oxidation current of Fc. The current was found to be inversely proportional to the concentration of miRNAs in the range of 0–25 fM, and a detection limit of 0.1 fM was achieved. Moreover, this work presents a new method for converting colorimetric assays into sensitive electrochemical analyses, and thus would be valuable for design of novel chemical/biosensors. PMID:28761341

Zinc finger nuclease-mediated precision genome editing of an endogenous gene in hexaploid bread wheat (Triticum aestivum) using a DNA repair template.

PubMed

Ran, Yidong; Patron, Nicola; Kay, Pippa; Wong, Debbie; Buchanan, Margaret; Cao, Ying-Ying; Sawbridge, Tim; Davies, John P; Mason, John; Webb, Steven R; Spangenberg, German; Ainley, William M; Walsh, Terence A; Hayden, Matthew J

2018-05-07

Sequence-specific nucleases have been used to engineer targeted genome modifications in various plants. While targeted gene knockouts resulting in loss of function have been reported with relatively high rates of success, targeted gene editing using an exogenously supplied DNA repair template and site-specific transgene integration has been more challenging. Here, we report the first application of zinc finger nuclease (ZFN)-mediated, nonhomologous end-joining (NHEJ)-directed editing of a native gene in allohexaploid bread wheat to introduce, via a supplied DNA repair template, a specific single amino acid change into the coding sequence of acetohydroxyacid synthase (AHAS) to confer resistance to imidazolinone herbicides. We recovered edited wheat plants having the targeted amino acid modification in one or more AHAS homoalleles via direct selection for resistance to imazamox, an AHAS-inhibiting imidazolinone herbicide. Using a cotransformation strategy based on chemical selection for an exogenous marker, we achieved a 1.2% recovery rate of edited plants having the desired amino acid change and a 2.9% recovery of plants with targeted mutations at the AHAS locus resulting in a loss-of-function gene knockout. The latter results demonstrate a broadly applicable approach to introduce targeted modifications into native genes for nonselectable traits. All ZFN-mediated changes were faithfully transmitted to the next generation. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Energetics of Amino Acid Synthesis in Alkaline Hydrothermal Environments

NASA Astrophysics Data System (ADS)

Kitadai, Norio

2015-12-01

Alkaline hydrothermal systems have received considerable attention as candidates for the origin and evolution of life on the primitive Earth. Nevertheless, sufficient information has not yet been obtained for the thermodynamic properties of amino acids, which are necessary components for life, at high temperatures and alkaline pH. These properties were estimated using experimental high-temperature volume and heat capacity data reported in the literature for several amino acids, together with correlation algorithms and the revised Helgeson-Kirkham-Flowers (HKF) equations of state. This approach enabled determination of a complete set of the standard molal thermodynamic data and the revised HKF parameters for the 20 protein amino acids in their zwitterionic and ionization states. The obtained dataset was then used to evaluate the energetics of amino acid syntheses from simple inorganic precursors (CO2, H2, NH3 and H2S) in a simulated alkaline hydrothermal system on the Hadean Earth. Results show that mixing between CO2-rich seawater and the H2-rich hydrothermal fluid can produce energetically favorable conditions for amino acid syntheses, particularly in the lower-temperature region of such systems. Together with data related to the pH and temperature dependences of the energetics of amino acid polymerizations presented in earlier reports, these results suggest the following. Hadean alkaline hydrothermal settings, where steep pH and temperature gradients may have existed between cool, slightly acidic Hadean ocean water and hot, alkaline hydrothermal fluids at the vent-ocean interface, may be energetically the most suitable environment for the synthesis and polymerization of amino acids.

Energetics of Amino Acid Synthesis in Alkaline Hydrothermal Environments.

PubMed

Kitadai, Norio

2015-12-01

Alkaline hydrothermal systems have received considerable attention as candidates for the origin and evolution of life on the primitive Earth. Nevertheless, sufficient information has not yet been obtained for the thermodynamic properties of amino acids, which are necessary components for life, at high temperatures and alkaline pH. These properties were estimated using experimental high-temperature volume and heat capacity data reported in the literature for several amino acids, together with correlation algorithms and the revised Helgeson-Kirkham-Flowers (HKF) equations of state. This approach enabled determination of a complete set of the standard molal thermodynamic data and the revised HKF parameters for the 20 protein amino acids in their zwitterionic and ionization states. The obtained dataset was then used to evaluate the energetics of amino acid syntheses from simple inorganic precursors (CO2, H2, NH3 and H2S) in a simulated alkaline hydrothermal system on the Hadean Earth. Results show that mixing between CO2-rich seawater and the H2-rich hydrothermal fluid can produce energetically favorable conditions for amino acid syntheses, particularly in the lower-temperature region of such systems. Together with data related to the pH and temperature dependences of the energetics of amino acid polymerizations presented in earlier reports, these results suggest the following. Hadean alkaline hydrothermal settings, where steep pH and temperature gradients may have existed between cool, slightly acidic Hadean ocean water and hot, alkaline hydrothermal fluids at the vent-ocean interface, may be energetically the most suitable environment for the synthesis and polymerization of amino acids.

Development of an alkaline/surfactant/polymer compositional reservoir simulator

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

Bhuyan, D.

1989-01-01

The mathematical formulation of a generalized three-dimensional compositional reservoir simulator for high-pH chemical flooding processes is presented in this work. The model assumes local thermodynamic equilibrium with respect to both reaction chemistry and phase behavior and calculates equilibrium electrolyte and phase compositions as a function of time and position. The reaction chemistry considers aqueous electrolytic chemistry, precipitation/dissolution of minerals, ion exchange reactions on matrix surface, reaction of acidic components of crude oil with the bases in the aqueous solution and cation exchange reactions with the micelles. The simulator combines this detailed reaction chemistry associated with these processes with the extensivemore » physical and flow property modeling schemes of an existing chemical flood simulator (UTCHEM) to model the multiphase, multidimensional displacement processes. The formulation of the chemical equilibrium model is quite general and is adaptable to simulate a variety of chemical descriptions. In addition to its use in the simulation of high-pH chemical flooding processes, the model will find application in the simulation of other reactive flow problems like the ground water contamination, reinjection of produced water, chemical waste disposal, etc. in one, two or three dimensions and under multiphase flow conditions. In this work, the model is used to simulate several hypothetical cases of high-pH chemical floods, which include cases from a simple alkaline preflush of a micellar/polymer flood to surfactant enhanced alkaline-polymer flooding and the results are analyzed. Finally, a few published alkaline, alkaline-polymer and surfactant-alkaline-polymer corefloods are simulated and compared with the experimental results.« less

Analysis of serum corticosteroid-induced alkaline phosphatase isoenzyme in dogs with hepatobiliary diseases.

PubMed

Kojima, K; Ohno, K; Kanemoto, H; Goto-Koshino, Y; Fukushima, K; Tsujimoto, H

2017-05-01

To reveal the relationship between canine corticosteroid-induced alkaline phosphatase isoenzyme activity and hepatobiliary diseases. Retrospective analysis of the relationship between serum corticosteroid-induced alkaline phosphatase activity and diagnosis, serum cortisol concentration and alanine transferase activity in dogs with hepatobiliary diseases. Dogs with a history of glucocorticoid administration were excluded. Seventy-two dogs with hepatobiliary diseases were analysed. The serum corticosteroid-induced alkaline phosphatase concentration was increased in dogs with hepatobiliary diseases. There was no correlation between serum cortisol concentration and serum corticosteroid-induced alkaline phosphatase percentage or activity. Dogs with hepatobiliary disease can exhibit high serum alkaline phosphatase activity even if the dogs have not been administrated glucocorticoids and the serum cortisol concentration is normal. © 2017 British Small Animal Veterinary Association.

Analysis of differentially expressed genes and adaptive mechanisms of Prunus triloba Lindl. under alkaline stress.

PubMed

Liu, Jia; Wang, Yongqing; Li, Qingtian

2017-01-01

Prunus triloba Lindl. is a naturally salt-alkaline-tolerant plant with several unique characteristics, and it can be used as the rootstock of Chinese plum ( Prunus salicina Lindl.) in saline-alkaline soils. To comprehensively investigate the alkaline acclimation mechanisms in P. triloba , a series of analyses were conducted under alkaline stress, including analyses of the kinetics of molecular and physiological changes, and leaf microstructure. To understand the kinetics of molecular changes under short-term alkaline stress, we used Illumina HiSeq 2500 platform to identify alkaline stress-related differentially expressed genes (DEGs) in P. triloba . Approximately 53.0 million high-quality clean reads were generated from 59.6 million raw reads, and a total of 124,786 unigenes were obtained after de novo assembly of P. triloba transcriptome data. After alkaline stress treatment, a total of 8948 unigenes were identified as DEGs. Based on these DEGs, a Gene Ontology (GO) enrichment analysis was conducted, suggesting that 28 genes may play an important role in the early alkaline stress response. In addition, analysis of DEGs with the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that pathways were significant at different treatment time points. A significant positive correlation was found between the quantitative real-time PCR (qRT-PCR) results and the RNA-Seq data for seven alkaline-related genes, confirming the reliability of the RNA-Seq results. Based on physiological analysis of P. triloba in response to long-term alkaline stress, we found that the internal microstructures of the leaves of P. triloba changed to adapt to long-term alkaline stress. Various physiological indexes indicated that the degree of membrane injury increased with increasing duration of alkaline stress, affecting photosynthesis in P. triloba seedlings. This represents the first investigation into the physiology and transcriptome of P. triloba in response to alkaline stress. The results

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions.

PubMed

Gonzales-Siles, Lucia; Karlsson, Roger; Kenny, Diarmuid; Karlsson, Anders; Sjöling, Åsa

2017-01-07

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB 5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the β-Barrel Assembly Machinery (BAM) complex were up-regulated. ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.

Coupling the Alkaline-Surfactant-Polymer Technology and the Gelation Technology to Maximize Oil Production

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

Malcolm Pitts; Jie Qi; Dan Wilson

2005-12-01

Gelation technologies have been developed to provide more efficient vertical sweep efficiencies for flooding naturally fractured oil reservoirs or reservoirs with different sand lenses with high permeability contrast. The field proven alkaline-surfactant-polymer technology economically recovers 15% to 25% OOIP more crude oil than waterflooding froin swept pore space of an oil reservoir. However, alkaline-surfactant-polymer technology is not amenable to naturally fractured reservoirs or reservoirs with high permeability contrast zones because much of injected solution bypasses target pore space containing oil. This work investigates whether combining these two technologies could broaden applicability of alkaline-surfactant-polymer flooding into these reservoirs. Fluid-fluid interaction withmore » different gel chemical compositions and alkaline-surfactant-polymer solution with pH values ranging from 9.2 to 12.9 have been tested. Aluminum-polyacrylamide gels are not stable to alkaline-surfactant-polymer solutions at any pH. Chromium-polyacrylamide gels with polymer to chromium ion ratios of 25 or greater were stable to alkaline-surfactant-polymer solutions if solution pH was 10.6 or less. When the polymer to chromium ion was 15 or less, chromium-polyacrylamide gels were stable to alkaline-surfactant-polymer solutions with pH values up to 12.9. Chromium-xanthan gum gels were stable to alkaline-surfactant-polymer solutions with pH values of 12.9 at the polymer to chromium ion ratios tested. Silicate-polyacrylamide, resorcinol-formaldehyde, and sulfomethylated resorcinol-formaldehyde gels were also stable to alkaline-surfactant-polymer solutions with pH values ranging from 9.2 to 12.9. Iron-polyacrylamide gels were immediately destroyed when contacted with any of the alkaline-surfactant-polymer solutions with pH values ranging from 9.2 to 12.9. Gel solutions under dynamic conditions of linear corefloods showed similar stability to alkaline-surfactant-polymer solutions

Coupling the Alkaline-Surfactant-Polymer Technology and The Gelation Technology to Maximize Oil Production

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

Malcolm Pitts; Jie Qi; Dan Wilson

2005-12-01

Gelation technologies have been developed to provide more efficient vertical sweep efficiencies for flooding naturally fractured oil reservoirs or reservoirs with different sand lenses with high permeability contrast. The field proven alkaline-surfactant-polymer technology economically recovers 15% to 25% OOIP more crude oil than waterflooding from swept pore space of an oil reservoir. However, alkaline-surfactant-polymer technology is not amenable to naturally fractured reservoirs or reservoirs with high permeability contrast zones because much of injected solution bypasses target pore space containing oil. This work investigates whether combining these two technologies could broaden applicability of alkaline-surfactant-polymer flooding into these reservoirs. Fluid-fluid interaction withmore » different gel chemical compositions and alkaline-surfactant-polymer solution with pH values ranging from 9.2 to 12.9 have been tested. Aluminum-polyacrylamide gels are not stable to alkaline-surfactant-polymer solutions at any pH. Chromium-polyacrylamide gels with polymer to chromium ion ratios of 25 or greater were stable to alkaline-surfactant-polymer solutions if solution pH was 10.6 or less. When the polymer to chromium ion was 15 or less, chromium-polyacrylamide gels were stable to alkaline-surfactant-polymer solutions with pH values up to 12.9. Chromium-xanthan gum gels were stable to alkaline-surfactant-polymer solutions with pH values of 12.9 at the polymer to chromium ion ratios tested. Silicate-polyacrylamide, resorcinol-formaldehyde, and sulfomethylated resorcinol-formaldehyde gels were also stable to alkaline-surfactant-polymer solutions with pH values ranging from 9.2 to 12.9. Iron-polyacrylamide gels were immediately destroyed when contacted with any of the alkaline-surfactant-polymer solutions with pH values ranging from 9.2 to 12.9. Gel solutions under dynamic conditions of linear corefloods showed similar stability to alkaline-surfactant-polymer solutions

Effect of aluminum phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria.

PubMed

Ramalingam, N; Prasanna, B Gowtham

2006-09-01

The impact of insoluble phosphorus such as aluminum and rock phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria was assessed. Polyurethane foam immobilized Nodularia recorded the highest alkaline phosphatase activity of 9.04 (m. mol p-nitrophenol released h(-1) mg(-1) protein) in vitro. A higher concentration of aluminum phosphate was recorded a 25% reduction in alkaline phosphatase activity, ammonia content, and available phosphorus in culture filtrate of polyurethane foam immobilized cyanobacteria. In general, immobilized cyanobacteria exhibited a higher alkaline phosphatase activity in rock phosphate than aluminum phosphate.

Alternative Alkaline Conditioning of Amidoxime Based Adsorbent for Uranium Extraction from Seawater

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

Das, S.; Liao, W. -P.; Flicker Byers, M.

2016-04-20

Alkaline conditioning of the amidoxime based adsorbents is a significant step in the preparation of the adsorbent for uranium uptake from seawater. The effects of various alkaline conditioning parameters such as the type of alkaline reagent, reaction temperature, and reaction time were investigated with respect to uranium adsorption capacity from simulated seawater (spiked with 8 ppm uranium) and natural seawater (from Sequim Bay, WA). An adsorbent (AF1) was prepared at the Oak Ridge National Laboratory by radiation-induced graft polymerization (RIGP) with acrylonitrile and itaconic acid onto high-surface-area polyethylene fibers. For the AF1 adsorbent, sodium hydroxide emerged as a better reagentmore » for alkaline conditioning over potassium hydroxide, which has typically been used in previous studies, because of higher uranium uptake capacity and lower cost over the other candidate alkaline reagents investigated in this study. Use of sodium hydroxide in place of potassium hydroxide is shown to result in a 21-30% decrease in the cost of uranium recovery.« less

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

PubMed Central

Shah, Megha; Mejdani, Marios; Calmettes, Charles; Moraes, Trevor F.

2017-01-01

ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor. PMID:29233895

LEM-3 is a midbody-tethered DNA nuclease that resolves chromatin bridges during late mitosis.

PubMed

Hong, Ye; Sonneville, Remi; Wang, Bin; Scheidt, Viktor; Meier, Bettina; Woglar, Alexander; Demetriou, Sarah; Labib, Karim; Jantsch, Verena; Gartner, Anton

2018-02-20

Faithful chromosome segregation and genome maintenance requires the removal of all DNA bridges that physically link chromosomes before cells divide. Using C. elegans embryos we show that the LEM-3/Ankle1 nuclease defines a previously undescribed genome integrity mechanism by processing DNA bridges right before cells divide. LEM-3 acts at the midbody, the structure where abscission occurs at the end of cytokinesis. LEM-3 localization depends on factors needed for midbody assembly, and LEM-3 accumulation is increased and prolonged when chromatin bridges are trapped at the cleavage plane. LEM-3 locally processes chromatin bridges that arise from incomplete DNA replication, unresolved recombination intermediates, or the perturbance of chromosome structure. Proper LEM-3 midbody localization and function is regulated by AIR-2/Aurora B kinase. Strikingly, LEM-3 acts cooperatively with the BRC-1/BRCA1 homologous recombination factor to promote genome integrity. These findings provide a molecular basis for the suspected role of the LEM-3 orthologue Ankle1 in human breast cancer.

The SNM1B/APOLLO DNA nuclease functions in resolution of replication stress and maintenance of common fragile site stability.

PubMed

Mason, Jennifer M; Das, Ishita; Arlt, Martin; Patel, Neil; Kraftson, Stephanie; Glover, Thomas W; Sekiguchi, JoAnn M

2013-12-15

SNM1B/Apollo is a DNA nuclease that has important functions in telomere maintenance and repair of DNA interstrand crosslinks (ICLs) within the Fanconi anemia (FA) pathway. SNM1B is required for efficient localization of key repair proteins, such as the FA protein, FANCD2, to sites of ICL damage and functions epistatically to FANCD2 in cellular survival to ICLs and homology-directed repair. The FA pathway is also activated in response to replication fork stalling. Here, we sought to determine the importance of SNM1B in cellular responses to stalled forks in the absence of a blocking lesion, such as ICLs. We found that depletion of SNM1B results in hypersensitivity to aphidicolin, a DNA polymerase inhibitor that causes replication stress. We observed that the SNM1B nuclease is required for efficient localization of the DNA repair proteins, FANCD2 and BRCA1, to subnuclear foci upon aphidicolin treatment, thereby indicating SNM1B facilitates direct repair of stalled forks. Consistent with a role for SNM1B subsequent to recognition of the lesion, we found that SNM1B is dispensable for upstream events, including activation of ATR-dependent signaling and localization of RPA, γH2AX and the MRE11/RAD50/NBS1 complex to aphidicolin-induced foci. We determined that a major consequence of SNM1B depletion is a marked increase in spontaneous and aphidicolin-induced chromosomal gaps and breaks, including breakage at common fragile sites. Thus, this study provides evidence that SNM1B functions in resolving replication stress and preventing accumulation of genomic damage.

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.

Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis

PubMed Central

2011-01-01

Background Alkaline α-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline α-amylase gains increased industrial interest, the yield of alkaline α-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline α-amylase. Results The alkaline α-amylase gene from Bacillus alcalophilus JN21 (CCTCC NO. M 2011229) was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline α-amylase was stable at pH from 7.0 to 11.0 and temperature below 40°C. The optimum pH and temperature of alkaline α-amylase was 9.0 and 50°C, respectively. Using soluble starch as the substrate, the Km and Vmax of alkaline α-amylase were 9.64 g/L and 0.80 g/(L·min), respectively. The effects of medium compositions (starch, peptone, and soybean meal) and temperature on the recombinant production of alkaline α-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v), peptone concentration 1.45% (w/v), soybean meal concentration 1.3% (w/v), and temperature 37°C), the highest yield of alkaline α-amylase reached 415 U/mL. The yield of alkaline α-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline α-amylase from B. alcalophilus JN21. Conclusions This is the first report concerning the heterologous expression of alkaline α-amylase in B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline α-amylase with the recombinant B. subtilis. PMID:21978209

Electrocatalysts for Hydrogen Evolution in Alkaline Electrolytes: Mechanisms, Challenges, and Prospective Solutions.

PubMed

Mahmood, Nasir; Yao, Yunduo; Zhang, Jing-Wen; Pan, Lun; Zhang, Xiangwen; Zou, Ji-Jun

2018-02-01

Hydrogen evolution reaction (HER) in alkaline medium is currently a point of focus for sustainable development of hydrogen as an alternative clean fuel for various energy systems, but suffers from sluggish reaction kinetics due to additional water dissociation step. So, the state-of-the-art catalysts performing well in acidic media lose considerable catalytic performance in alkaline media. This review summarizes the recent developments to overcome the kinetics issues of alkaline HER, synthesis of materials with modified morphologies, and electronic structures to tune the active sites and their applications as efficient catalysts for HER. It first explains the fundamentals and electrochemistry of HER and then outlines the requirements for an efficient and stable catalyst in alkaline medium. The challenges with alkaline HER and limitation with the electrocatalysts along with prospective solutions are then highlighted. It further describes the synthesis methods of advanced nanostructures based on carbon, noble, and inexpensive metals and their heterogeneous structures. These heterogeneous structures provide some ideal systems for analyzing the role of structure and synergy on alkaline HER catalysis. At the end, it provides the concluding remarks and future perspectives that can be helpful for tuning the catalysts active-sites with improved electrochemical efficiencies in future.

Electrocatalysts for Hydrogen Evolution in Alkaline Electrolytes: Mechanisms, Challenges, and Prospective Solutions

PubMed Central

Mahmood, Nasir; Yao, Yunduo; Zhang, Jing‐Wen; Pan, Lun; Zhang, Xiangwen

2017-01-01

Abstract Hydrogen evolution reaction (HER) in alkaline medium is currently a point of focus for sustainable development of hydrogen as an alternative clean fuel for various energy systems, but suffers from sluggish reaction kinetics due to additional water dissociation step. So, the state‐of‐the‐art catalysts performing well in acidic media lose considerable catalytic performance in alkaline media. This review summarizes the recent developments to overcome the kinetics issues of alkaline HER, synthesis of materials with modified morphologies, and electronic structures to tune the active sites and their applications as efficient catalysts for HER. It first explains the fundamentals and electrochemistry of HER and then outlines the requirements for an efficient and stable catalyst in alkaline medium. The challenges with alkaline HER and limitation with the electrocatalysts along with prospective solutions are then highlighted. It further describes the synthesis methods of advanced nanostructures based on carbon, noble, and inexpensive metals and their heterogeneous structures. These heterogeneous structures provide some ideal systems for analyzing the role of structure and synergy on alkaline HER catalysis. At the end, it provides the concluding remarks and future perspectives that can be helpful for tuning the catalysts active‐sites with improved electrochemical efficiencies in future. PMID:29610722

Interfacial behavior of alkaline protease at the air-water and oil-water interfaces

NASA Astrophysics Data System (ADS)

Zhang, Jian; Li, Yanyan; Wang, Jing; Zhang, Yue

2018-03-01

The interfacial behavior of alkaline protease at the air-water and n-hexane-water interfaces was investigated using interfacial tension, dilatational rheology and dynamic light scattering. Additionally, different adsorption models which are Langmuir, Frumkin, Reorientation-A and Reorientation-R were used to fitting the data of equilibrium interfacial tension for further understanding the interfacial behavior of alkaline protease. Data fitting of the equilibrium interfacial tension was achieved by IsoFit software. The results show that the molecules arrangement of the alkaline protease at the n-hexane-water interface is more tightly than at the air-water interface. The data were further analyzed to indicate that the hydrophobic chains of alkaline protease penetrate into oil phase deeper than the air phase. Also data indicate that the electrostatic interactions and hydrophobic interactions at the n-hexane-water interface are stronger than at the air-water interface within molecules of the alkaline protease. Based on comprehensive analysis of the adsorption kinetics and interfacial rheological properties, interfacial structures mechanism of alkaline protease at n-hexane-water and air-water interfaces was proposed.

Systemic and local effects of long-term exposure to alkaline drinking water in rats

PubMed Central

Merne, Marina ET; Syrjänen, Kari J; Syrjänen, Stina M

2001-01-01

Alkaline conditions in the oral cavity may be caused by a variety of stimuli, including tobacco products, antacids, alkaline drinking water or bicarbonate toothpaste. The effects of alkaline pH on oral mucosa have not been systematically studied. To assess the systemic (organ) and local (oral mucosal) effects of alkalinity, drinking water supplemented with Ca(OH)2 or NaOH, with pH 11.2 or 12 was administered to rats (n = 36) for 52 weeks. Tissues were subjected to histopathological examination; oral mucosal biopsy samples were also subjected to immunohistochemical (IHC) analyses for pankeratin, CK19, CK5, CK4, PCNA, ICAM-1, CD44, CD68, S-100, HSP 60, HSP70, and HSP90. At completion of the study, animals in the study groups had lower body weights (up to 29% less) than controls despite equal food and water intake, suggesting a systemic response to the alkaline treatment. The lowest body weight was found in rats exposed to water with the highest pH value and starting the experiment when young (6 weeks). No histological changes attributable to alkaline exposure occurred in the oral mucosa or other tissues studied. Alkaline exposure did not affect cell proliferation in the oral epithelium, as shown by the equal expression of PCNA in groups. The up-regulation of HSP70 protein expression in the oral mucosa of rats exposed to alkaline water, especially Ca(OH)2 treated rats, may indicate a protective response. Intercellular adhesion molecule-1 (ICAM-1) positivity was lost in 6/12 rats treated with Ca(OH)2 with pH 11.2, and loss of CD44 expression was seen in 3/6 rats in both study groups exposed to alkaline water with pH 12. The results suggest that the oral mucosa in rats is resistant to the effects of highly alkaline drinking water. However, high alkalinity may have some unknown systemic effects leading to growth retardation, the cause of which remains to be determined. PMID:11493345

Systemic and local effects of long-term exposure to alkaline drinking water in rats.

PubMed

Merne, M E; Syrjänen, K J; Syrjänen, S M

2001-08-01

Alkaline conditions in the oral cavity may be caused by a variety of stimuli, including tobacco products, antacids, alkaline drinking water or bicarbonate toothpaste. The effects of alkaline pH on oral mucosa have not been systematically studied. To assess the systemic (organ) and local (oral mucosal) effects of alkalinity, drinking water supplemented with Ca(OH)2 or NaOH, with pH 11.2 or 12 was administered to rats (n = 36) for 52 weeks. Tissues were subjected to histopathological examination; oral mucosal biopsy samples were also subjected to immunohistochemical (IHC) analyses for pankeratin, CK19, CK5, CK4, PCNA, ICAM-1, CD44, CD68, S-100, HSP 60, HSP70, and HSP90. At completion of the study, animals in the study groups had lower body weights (up to 29% less) than controls despite equal food and water intake, suggesting a systemic response to the alkaline treatment. The lowest body weight was found in rats exposed to water with the highest pH value and starting the experiment when young (6 weeks). No histological changes attributable to alkaline exposure occurred in the oral mucosa or other tissues studied. Alkaline exposure did not affect cell proliferation in the oral epithelium, as shown by the equal expression of PCNA in groups. The up-regulation of HSP70 protein expression in the oral mucosa of rats exposed to alkaline water, especially Ca(OH)2 treated rats, may indicate a protective response. Intercellular adhesion molecule-1 (ICAM-1) positivity was lost in 6/12 rats treated with Ca(OH)2 with pH 11.2, and loss of CD44 expression was seen in 3/6 rats in both study groups exposed to alkaline water with pH 12. The results suggest that the oral mucosa in rats is resistant to the effects of highly alkaline drinking water. However, high alkalinity may have some unknown systemic effects leading to growth retardation, the cause of which remains to be determined.

Sequential eruption of alkaline and sub-alkaline magmas from a small monogenetic volcano in the Auckland Volcanic Field, New Zealand

NASA Astrophysics Data System (ADS)

Needham, A. J.; Lindsay, J. M.; Smith, I. E. M.; Augustinus, P.; Shane, P. A.

2011-04-01

Rangitoto Volcano is the youngest and largest eruptive centre in the monogenetic intraplate Auckland Volcanic Field (AVF). The stratigraphy of Rangitoto pyroclastic deposits that have been preserved in swamps on nearby Motutapu Island and in Lake Pupuke on the mainland reveals that the volcano erupted twice; radiocarbon dating of 10 samples from the two tephra units in the swamps indicates eruption ages of 553 ± 7 and 504 ± 5 Cal years BP, for the lower and upper tephra layers, respectively. Geochemistry of the lava field and various scoria cones on Rangitoto Island itself reveals two distinct compositional groups: an alkaline olivine basalt group (that correlates geochemically with the lower tephra layer) and a group that is sub-alkaline and transitional to tholeiite (that correlates geochemically with the upper tephra layer). Based on this data, we infer that, following a phreatomagmatic vent-clearing phase, the early magmatic eruption of Rangitoto Volcano was Strombolian in character and produced an alkaline olivine basalt scoria cone and an associated thick ash deposit on nearby Motutapu Island. This was followed by a time gap of up to several decades, after which a second eruptive phase built the current summit scoria cone together with an encircling lava field. We suggest this later, sub-alkaline eruptive period was associated with the deposition of the thin upper tephra layer on Motutapu Island. The two suites of Rangitoto samples are chemically quite distinct, and each is associated with a distinct parental composition. Trace element modelling indicates the alkaline and sub-alkaline parental melts could have been derived by ~ 1 and 6 wt.% partial melting of an anhydrous garnet peridotite source at ~ 80 and 65 km depth, respectively. The compositional range within each suite is similar, and can be explained by mainly olivine together with minor clinopyroxene fractionation within a relatively simple conduit system in which mixing and mingling were not

Alkaline Earth Core Level Photoemission Spectroscopy of High-Temperature Superconductors

NASA Technical Reports Server (NTRS)

Vasquez, R.

1993-01-01

This paper examines photoemission measurements of the alkaline Earth core levels of high-temperature superconductors and related materials, models that seek to explain the large negative shifts observed relative to the corresponding alkaline Earth metals, and the effect of lattice site disorder on the core level spectra and the presence or absence of intrinsic surface peaks.

CARINA alkalinity data in the Atlantic Ocean

NASA Astrophysics Data System (ADS)

Velo, A.; Perez, F. F.; Brown, P.; Tanhua, T.; Schuster, U.; Key, R. M.

2009-08-01

Data on carbon and carbon-relevant hydrographic and hydrochemical parameters from previously non-publicly available cruise data sets in the Arctic, Atlantic and Southern Ocean have been retrieved and merged to a new database: CARINA (CARbon IN the Atlantic). These data have gone through rigorous quality control (QC) procedures to assure the highest possible quality and consistency. The data for most of the measured parameters in the CARINA data base were objectively examined in order to quantify systematic differences in the reported values, i.e. secondary quality control. Systematic biases found in the data have been corrected in the data products, i.e. three merged data files with measured, calculated and interpolated data for each of the three CARINA regions; Arctic, Atlantic and Southern Ocean. Out of a total of 188 cruise entries in the CARINA database, 98 were conducted in the Atlantic Ocean and of these, 75 cruises report alkalinity values. Here we present details of the secondary QC on alkalinity for the Atlantic Ocean part of CARINA. Procedures of quality control, including crossover analysis between cruises and inversion analysis of all crossover data are briefly described. Adjustments were applied to the alkalinity values for 16 of the cruises in the Atlantic Ocean region. With these adjustments the CARINA database is consistent both internally as well as with GLODAP data, an oceanographic data set based on the World Hydrographic Program in the 1990s. Based on our analysis we estimate the internal accuracy of the CARINA-ATL alkalinity data to be 3.3 μmol kg-1. The CARINA data are now suitable for accurate assessments of, for example, oceanic carbon inventories and uptake rates and for model validation.

CARINA alkalinity data in the Atlantic Ocean

NASA Astrophysics Data System (ADS)

Velo, A.; Perez, F. F.; Brown, P.; Tanhua, T.; Schuster, U.; Key, R. M.

2009-11-01

Data on carbon and carbon-relevant hydrographic and hydrochemical parameters from previously non-publicly available cruise data sets in the Arctic, Atlantic and Southern Ocean have been retrieved and merged to a new database: CARINA (CARbon IN the Atlantic). These data have gone through rigorous quality control (QC) procedures to assure the highest possible quality and consistency. The data for most of the measured parameters in the CARINA data base were objectively examined in order to quantify systematic differences in the reported values, i.e. secondary quality control. Systematic biases found in the data have been corrected in the data products, i.e. three merged data files with measured, calculated and interpolated data for each of the three CARINA regions; Arctic, Atlantic and Southern Ocean. Out of a total of 188 cruise entries in the CARINA database, 98 were conducted in the Atlantic Ocean and of these, 75 cruises report alkalinity values. Here we present details of the secondary QC on alkalinity for the Atlantic Ocean part of CARINA. Procedures of quality control, including crossover analysis between cruises and inversion analysis of all crossover data are briefly described. Adjustments were applied to the alkalinity values for 16 of the cruises in the Atlantic Ocean region. With these adjustments the CARINA database is consistent both internally as well as with GLODAP data, an oceanographic data set based on the World Hydrographic Program in the 1990s. Based on our analysis we estimate the internal accuracy of the CARINA-ATL alkalinity data to be 3.3 μmol kg-1. The CARINA data are now suitable for accurate assessments of, for example, oceanic carbon inventories and uptake rates and for model validation.

Reduction of nitrobenzene with alkaline ascorbic acid: Kinetics and pathways.

PubMed

Liang, Chenju; Lin, Ya-Ting; Shiu, Jia-Wei

2016-01-25

Alkaline ascorbic acid (AA) exhibits the potential to reductively degrade nitrobenzene (NB), which is the simplest of the nitroaromatic compounds. The nitro group (NO2(-)) of NB has a +III oxidation state of the N atom and tends to gain electrons. The effect of alkaline pH ranging from 9 to 13 was initially assessed and the results demonstrated that the solution pH, when approaching or above the pKa2 of AA (11.79), would increase reductive electron transfer to NB. The rate equation for the reactions between NB and AA at pH 12 can be described as r=((0.89±0.11)×10(-4) mM(1-(a+b))h(-1))×[NB](a=1.35±0.10)[AA](b=0.89±0.01). The GC/MS analytical method identified nitrosobenzene, azoxybenzene, and azobenzene as NB reduction intermediates, and aniline (AN) as a final product. These experimental results indicate that the alkaline AA reduction of NB to AN mainly proceeds via the direct route, consisting of a series of two-electron or four-electron transfers, and the condensation reaction plays a minor route. Preliminary evaluation of the remediation of spiked NB contaminated soils revealed that maintenance of alkaline pH and a higher water to soil ratio are essential for a successful alkaline AA application. Copyright © 2015 Elsevier B.V. All rights reserved.

Ethylene Inhibits Root Elongation during Alkaline Stress through AUXIN1 and Associated Changes in Auxin Accumulation.

PubMed

Li, Juan; Xu, Heng-Hao; Liu, Wen-Cheng; Zhang, Xiao-Wei; Lu, Ying-Tang

2015-08-01

Soil alkalinity causes major reductions in yield and quality of crops worldwide. The plant root is the first organ sensing soil alkalinity, which results in shorter primary roots. However, the mechanism underlying alkaline stress-mediated inhibition of root elongation remains to be further elucidated. Here, we report that alkaline conditions inhibit primary root elongation of Arabidopsis (Arabidopsis thaliana) seedlings by reducing cell division potential in the meristem zones and that ethylene signaling affects this process. The ethylene perception antagonist silver (Ag(+)) alleviated the inhibition of root elongation by alkaline stress. Moreover, the ethylene signaling mutants ethylene response1-3 (etr1-3), ethylene insensitive2 (ein2), and ein3-1 showed less reduction in root length under alkaline conditions, indicating a reduced sensitivity to alkalinity. Ethylene biosynthesis also was found to play a role in alkaline stress-mediated root inhibition; the ethylene overproducer1-1 mutant, which overproduces ethylene because of increased stability of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE5, was hypersensitive to alkaline stress. In addition, the ethylene biosynthesis inhibitor cobalt (Co(2+)) suppressed alkaline stress-mediated inhibition of root elongation. We further found that alkaline stress caused an increase in auxin levels by promoting expression of auxin biosynthesis-related genes, but the increase in auxin levels was reduced in the roots of the etr1-3 and ein3-1 mutants and in Ag(+)/Co(2+)-treated wild-type plants. Additional genetic and physiological data showed that AUXIN1 (AUX1) was involved in alkaline stress-mediated inhibition of root elongation. Taken together, our results reveal that ethylene modulates alkaline stress-mediated inhibition of root growth by increasing auxin accumulation by stimulating the expression of AUX1 and auxin biosynthesis-related genes. © 2015 American Society of Plant Biologists. All Rights Reserved.

Survival and stress responses of E. coli exposed to alkaline cleaners

USDA-ARS?s Scientific Manuscript database

Studies were undertaken to evaluate the effects of alkaline cleaners commonly used in food processing environments on survival and stress responses of the foodborne pathogen Escherichia coli O157:H7. Alkaline cleaners containing either sodium hydroxide or potassium hydroxide and hypochlorite had gre...

Alternative alkaline conditioning of amidoxime based adsorbent for uranium extraction from seawater

DOE PAGES

Das, Sadananda; Liao, Wei -Po; Byers, Maggie Flicker; ...

2015-10-18

Alkaline conditioning of the amidoxime based adsorbents is a significant step in the preparation of the adsorbent for uranium uptake from seawater. The effects of various alkaline conditioning parameters such as the type of alkaline reagent, reaction temperature, and reaction time were investigated with respect to uranium adsorption capacity from simulated seawater (spiked with 8 ppm uranium) and natural seawater (from Sequim Bay, WA). An adsorbent (AF1) was prepared at the Oak Ridge National Laboratory by radiation-induced graft polymerization (RIGP) with acrylonitrile and itaconic acid onto high-surface-area polyethylene fibers. For the AF1 adsorbent, sodium hydroxide emerged as a better reagentmore » for alkaline conditioning over potassium hydroxide, which has typically been used in previous studies, because of higher uranium uptake capacity and lower cost over the other candidate alkaline reagents investigated in this study. Furthermore, the use of sodium hydroxide in place of potassium hydroxide is shown to result in a 21–30% decrease in the cost of uranium recovery.« less

Alternative alkaline conditioning of amidoxime based adsorbent for uranium extraction from seawater

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

Das, Sadananda; Liao, Wei -Po; Byers, Maggie Flicker

Alkaline conditioning of the amidoxime based adsorbents is a significant step in the preparation of the adsorbent for uranium uptake from seawater. The effects of various alkaline conditioning parameters such as the type of alkaline reagent, reaction temperature, and reaction time were investigated with respect to uranium adsorption capacity from simulated seawater (spiked with 8 ppm uranium) and natural seawater (from Sequim Bay, WA). An adsorbent (AF1) was prepared at the Oak Ridge National Laboratory by radiation-induced graft polymerization (RIGP) with acrylonitrile and itaconic acid onto high-surface-area polyethylene fibers. For the AF1 adsorbent, sodium hydroxide emerged as a better reagentmore » for alkaline conditioning over potassium hydroxide, which has typically been used in previous studies, because of higher uranium uptake capacity and lower cost over the other candidate alkaline reagents investigated in this study. Furthermore, the use of sodium hydroxide in place of potassium hydroxide is shown to result in a 21–30% decrease in the cost of uranium recovery.« less

The viability of MCM-41 as separator in secondary alkaline cells

NASA Astrophysics Data System (ADS)

Meskon, S. R.; Othman, R.; Ani, M. H.

2018-01-01

The viability of MCM-41 membrane as a separator material in secondary alkaline cell is investigated. The inorganic membrane was employed in an alkaline nickel-zinc system. MCM-41 mesoporous material consists of arrays of hexagonal nano-pore channels. The membrane was synthesized using sol-gel route from parent solution comprising of quarternary ammonium surfactant, cethyltrimethylammonium bromide C16H33(CH3)3NBr (CTAB), hydrochloric acid (HCl), deionized water (H2O), ethanol (C2H5OH), and tetraethylortosilicate (TEOS). Both the anodic zinc/zinc oxide and cathodic nickel hydroxide electrodeposited film were coated with MCM-41 membrane. The Ni/MCM-41/Zn alkaline cell was then subjected to 100-cycle durability test and the structural stability of MCM-41 separator throughout the progression of the charge-discharge cycles is studied. X-ray diffraction (XRD) analysis on the dismantled cell shows that MCM-41 began to transform to lamellar MCM-50 on the 5th cycle and transformed almost completely on the 25th cycle. The phase transformation of MCM-41 hexagonal structure into gel-like MCM-50 prevents the mesoporous cell separator from diminished in the caustic alkaline surround. This work has hence demonstrated MCM-41 membrane is viable to be employed in secondary alkaline cells.

Root Damage under Alkaline Stress Is Associated with Reactive Oxygen Species Accumulation in Rice (Oryza sativa L.).

PubMed

Zhang, Hui; Liu, Xiao-Long; Zhang, Rui-Xue; Yuan, Hai-Yan; Wang, Ming-Ming; Yang, Hao-Yu; Ma, Hong-Yuan; Liu, Duo; Jiang, Chang-Jie; Liang, Zheng-Wei

2017-01-01

Alkaline stress (high pH) severely damages root cells, and consequently, inhibits rice ( Oryza sativa L.) seedling growth. In this study, we demonstrate the accumulation of reactive oxygen species (ROS) in root cells under alkaline stress. Seedlings of two rice cultivars with different alkaline tolerances, 'Dongdao-4' (moderately alkaline-tolerant) and 'Jiudao-51' (alkaline-sensitive), were subjected to alkaline stress simulated by 15 mM sodium carbonate (Na 2 CO 3 ). Alkaline stress greatly reduced seedling survival rate, shoot and root growth, and root vigor. Moreover, severe root cell damage was observed under alkaline stress, as shown by increased membrane injury, malondialdehyde accumulation, and Evan's Blue staining. The expression of the cell death-related genes OsKOD1 , OsHsr203j , OsCP1 , and OsNAC4 was consistently upregulated, while that of a cell death-suppressor gene, OsBI1 , was downregulated. Analysis of the ROS contents revealed that alkaline stress induced a marked accumulation of superoxide anions ([Formula: see text]) and hydrogen peroxide (H 2 O 2 ) in rice roots. The application of procyanidins (a potent antioxidant) to rice seedlings 24 h prior to alkaline treatment significantly alleviated alkalinity-induced root damage and promoted seedling growth inhibition, which were concomitant with reduced ROS accumulation. These results suggest that root cell damage, and consequently growth inhibition, of rice seedlings under alkaline stress is closely associated with ROS accumulation. The antioxidant activity of superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase increased under alkaline stress in the roots, probably in response to the cellular damage induced by oxidative stress. However, this response mechanism may be overwhelmed by the excess ROS accumulation observed under stress, resulting in oxidative damage to root cells. Our findings provide physiological insights into the molecular mechanisms of alkalinity-induced damage to

Priming effect of abscisic acid on alkaline stress tolerance in rice (Oryza sativa L.) seedlings.

PubMed

Wei, Li-Xing; Lv, Bing-Sheng; Wang, Ming-Ming; Ma, Hong-Yuan; Yang, Hao-Yu; Liu, Xiao-Long; Jiang, Chang-Jie; Liang, Zheng-Wei

2015-05-01

Saline-alkaline stress is characterized by high salinity and high alkalinity (high pH); alkaline stress has been shown to be the primary factor inhibiting rice seedling growth. In this study, we investigated the potential priming effect of abscisic acid (ABA) on tolerance of rice seedlings to alkaline stress simulated by Na2CO3. Seedlings were pretreated with ABA at concentrations of 0 (control), 10, and 50 μM by root-drench for 24 h and then transferred to a Na2CO3 solution that did not contain ABA. Compared to control treatment, pretreatment with ABA substantially improved the survival rate of rice seedlings and increased biomass accumulation after 7 days under the alkaline condition. ABA application at 10 μM also alleviated the inhibitory effects of alkaline stress on the total root length and root surface area. Physiologically, ABA increased relative water content (RWC) and decreased cell membrane injury degree (MI) and Na(+)/K(+) ratios. In contrast, fluridone (an ABA biosynthesis inhibitor) decreased the RWC and increased MI in shoots under the alkaline conditions. These data suggest that ABA has a potent priming effect on the adaptive response to alkaline stress in rice and may be useful for improving rice growth in saline-alkaline paddy fields. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Grace DAKASEP alkaline battery separator

NASA Technical Reports Server (NTRS)

Giovannoni, R. T.; Lundquist, J. T.; Choi, W. M.

1987-01-01

The Grace DAKASEP separator was originally developed as a wicking layer for nickel-zinc alkaline batteries. The DAKASEP is a filled non-woven separator which is flexible and heat sealable. Through modification of formulation and processing variables, products with a variety of properties can be produced. Variations of DAKASEP were tested in Ni-H2, Ni-Zn, Ni-Cd, and primary alkaline batteries with good results. The properties of DAKASEP which are optimized for Hg-Zn primary batteries are shown in tabular form. This separator has high tensile strength, 12 micron average pore size, relatively low porosity at 46-48 percent, and consequently moderately high resistivity. Versions were produced with greater than 70 percent porosity and resistivities in 33 wt percent KOH as low as 3 ohm cm. Performance data for Hg-Zn E-1 size cells containing DAKASEP with the properties shown in tabular form, are more reproducible than data obtained with a competitive polypropylene non-woven separator. In addition, utilization of active material is in general considerably improved.