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Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042

PubMed Central

2018-01-01

ABSTRACT Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae, cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli, out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue (hns2). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae. IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool. PMID
Evolution of Bacterial Global Modulators: Role of a Novel H-NS Paralogue in the Enteroaggregative Escherichia coli Strain 042.

PubMed

Prieto, A; Bernabeu, M; Aznar, S; Ruiz-Cruz, S; Bravo, A; Queiroz, M H; Juárez, A

2018-01-01

Bacterial genomes sometimes contain genes that code for homologues of global regulators, the function of which is unclear. In members of the family Enterobacteriaceae , cells express the global regulator H-NS and its paralogue StpA. In Escherichia coli , out of providing a molecular backup for H-NS, the role of StpA is poorly characterized. The enteroaggregative E. coli strain 042 carries, in addition to the hns and stpA genes, a third gene encoding an hns paralogue ( hns2 ). We present in this paper information about its biological function. Transcriptomic analysis has shown that the H-NS2 protein targets a subset of the genes targeted by H-NS. Genes targeted by H-NS2 correspond mainly with horizontally transferred (HGT) genes and are also targeted by the Hha protein, a fine-tuner of H-NS activity. Compared with H-NS, H-NS2 expression levels are lower. In addition, H-NS2 expression exhibits specific features: it is sensitive to the growth temperature and to the nature of the culture medium. This novel H-NS paralogue is widespread within the Enterobacteriaceae . IMPORTANCE Global regulators such as H-NS play key relevant roles enabling bacterial cells to adapt to a changing environment. H-NS modulates both core and horizontally transferred (HGT) genes, but the mechanism by which H-NS can differentially regulate these genes remains to be elucidated. There are several instances of bacterial cells carrying genes that encode homologues of the global regulators. The question is what the roles of these proteins are. We noticed that the enteroaggregative E. coli strain 042 carries a new hitherto uncharacterized copy of the hns gene. We decided to investigate why this pathogenic E. coli strain requires an extra H-NS paralogue, termed H-NS2. In our work, we show that H-NS2 displays specific expression and regulatory properties. H-NS2 targets a subset of H-NS-specific genes and may help to differentially modulate core and HGT genes by the H-NS cellular pool.
Interference of transcription across H-NS binding sites and repression by H-NS.

PubMed

Rangarajan, Aathmaja Anandhi; Schnetz, Karin

2018-05-01

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression. © 2018 John Wiley & Sons Ltd.
pH-Dependent Conformational Changes in the HCV NS3 Protein Modulate Its ATPase and Helicase Activities

PubMed Central

Ventura, Gustavo Tavares; da Costa, Emmerson Corrêa Brasil; Capaccia, Anne Miranda; Mohana-Borges, Ronaldo

2014-01-01

The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication. PMID:25551442
Interaction between the bacterial nucleoid associated proteins Hha and H-NS involves a conformational change of Hha.

PubMed

García, Jesús; Cordeiro, Tiago N; Nieto, José M; Pons, Ignacio; Juárez, Antonio; Pons, Miquel

2005-06-15

The H-NS family of proteins has been shown to participate in the regulation of a large number of genes in Gram-negative bacteria in response to environmental factors. In recent years, it has become apparent that proteins of the Hha family are essential elements for H-NS-regulated gene expression. Hha has been shown to bind H-NS, although the details for this interaction are still unknown. In the present paper, we report fluorescence anisotropy and NMR studies of the interaction between Hha and H-NS64, a truncated form of H-NS containing only its N-terminal dimerization domain. We demonstrate the initial formation of a complex between one Hha and two H-NS64 monomers in 150 mM NaCl. This complex seems to act as a nucleation unit for higher-molecular-mass complexes. NMR studies suggest that Hha is in equilibrium between two different conformations, one of which is stabilized by binding to H-NS64. A similar exchange is also observed for Hha in the absence of H-NS when temperature is increased to 37 degrees C, suggesting a key role for intrinsic conformational changes of Hha in modulating its interaction with H-NS.
Interaction between the bacterial nucleoid associated proteins Hha and H-NS involves a conformational change of Hha

PubMed Central

2005-01-01

The H-NS family of proteins has been shown to participate in the regulation of a large number of genes in Gram-negative bacteria in response to environmental factors. In recent years, it has become apparent that proteins of the Hha family are essential elements for H-NS-regulated gene expression. Hha has been shown to bind H-NS, although the details for this interaction are still unknown. In the present paper, we report fluorescence anisotropy and NMR studies of the interaction between Hha and H-NS64, a truncated form of H-NS containing only its N-terminal dimerization domain. We demonstrate the initial formation of a complex between one Hha and two H-NS64 monomers in 150 mM NaCl. This complex seems to act as a nucleation unit for higher-molecular-mass complexes. NMR studies suggest that Hha is in equilibrium between two different conformations, one of which is stabilized by binding to H-NS64. A similar exchange is also observed for Hha in the absence of H-NS when temperature is increased to 37 °C, suggesting a key role for intrinsic conformational changes of Hha in modulating its interaction with H-NS. PMID:15720293
StpA and Hha stimulate pausing by RNA polymerase by promoting DNA-DNA bridging of H-NS filaments.

PubMed

Boudreau, Beth A; Hron, Daniel R; Qin, Liang; van der Valk, Ramon A; Kotlajich, Matthew V; Dame, Remus T; Landick, Robert

2018-06-20

In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA-DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.
Dynamic Nucleolar Targeting of Dengue Virus Polymerase NS5 in Response to Extracellular pH

PubMed Central

Fraser, Johanna E.; Rawlinson, Stephen M.; Heaton, Steven M.

2016-01-01

ABSTRACT The nucleolar subcompartment of the nucleus is increasingly recognized as an important target of RNA viruses. Here we document for the first time the ability of dengue virus (DENV) polymerase, nonstructural protein 5 (NS5), to accumulate within the nucleolus of infected cells and to target green fluorescent protein (GFP) to the nucleolus of live transfected cells. Intriguingly, NS5 exchange between the nucleus and nucleolus is dynamically modulated by extracellular pH, responding rapidly and reversibly to pH change, in contrast to GFP alone or other nucleolar and non-nucleolar targeted protein controls. The minimal pH-sensitive nucleolar targeting region (pHNTR), sufficient to target GFP to the nucleolus in a pH-sensitive fashion, was mapped to NS5 residues 1 to 244, with mutation of key hydrophobic residues, Leu-165, Leu-167, and Val-168, abolishing pHNTR function in NS5-transfected cells, and severely attenuating DENV growth in infected cells. This is the first report of a viral protein whose nucleolar targeting ability is rapidly modulated by extracellular stimuli, suggesting that DENV has the ability to detect and respond dynamically to the extracellular environment. IMPORTANCE Infections by dengue virus (DENV) threaten 40% of the world's population yet there is no approved vaccine or antiviral therapeutic to treat infections. Understanding the molecular details that govern effective viral replication is key for the development of novel antiviral strategies. Here, we describe for the first time dynamic trafficking of DENV nonstructural protein 5 (NS5) to the subnuclear compartment, the nucleolus. We demonstrate that NS5's targeting to the nucleolus occurs in response to acidic pH, identify the key amino acid residues within NS5 that are responsible, and demonstrate that their mutation severely impairs production of infectious DENV. Overall, this study identifies a unique subcellular trafficking event and suggests that DENV is able to detect and respond
A single residue mutation in Hha preserving structure and binding to H-NS results in loss of H-NS mediated gene repression properties.

PubMed

Cordeiro, Tiago N; Garcia, Jesús; Pons, José-Ignacio; Aznar, Sonia; Juárez, Antonio; Pons, Miquel

2008-09-03

In this study, we report that a single mutation of cysteine 18 to isoleucine (C18I) in Escherichia coli Hha abolishes the repression of the hemolysin operon observed in the wild-type protein. The phenotype also includes a significant decrease in the growth rate of E. coli cells at low ionic strength. Other substitutions at this position (C18A, C18S) have no observable effects in E. coli growth or hemolysin repression. All mutants are stable and well folded and bind H-NS in vitro with similar affinities suggesting that Cys 18 is not directly involved in H-NS binding but this position is essential for the activity of the H-NS/Hha heterocomplexes in the regulation of gene expression.
Indirect DNA Readout by an H-NS Related Protein: Structure of the DNA Complex of the C-Terminal Domain of Ler

PubMed Central

Cordeiro, Tiago N.; Schmidt, Holger; Madrid, Cristina; Juárez, Antonio; Bernadó, Pau; Griesinger, Christian; García, Jesús; Pons, Miquel

2011-01-01

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family. PMID:22114557
Indirect DNA readout by an H-NS related protein: structure of the DNA complex of the C-terminal domain of Ler.

PubMed

Cordeiro, Tiago N; Schmidt, Holger; Madrid, Cristina; Juárez, Antonio; Bernadó, Pau; Griesinger, Christian; García, Jesús; Pons, Miquel

2011-11-01

Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.
H-NS Facilitates Sequence Diversification of Horizontally Transferred DNAs during Their Integration in Host Chromosomes

PubMed Central

Higashi, Koichi; Tobe, Toru; Kanai, Akinori; Uyar, Ebru; Ishikawa, Shu; Suzuki, Yutaka; Ogasawara, Naotake; Kurokawa, Ken; Oshima, Taku

2016-01-01

Bacteria can acquire new traits through horizontal gene transfer. Inappropriate expression of transferred genes, however, can disrupt the physiology of the host bacteria. To reduce this risk, Escherichia coli expresses the nucleoid-associated protein, H-NS, which preferentially binds to horizontally transferred genes to control their expression. Once expression is optimized, the horizontally transferred genes may actually contribute to E. coli survival in new habitats. Therefore, we investigated whether and how H-NS contributes to this optimization process. A comparison of H-NS binding profiles on common chromosomal segments of three E. coli strains belonging to different phylogenetic groups indicated that the positions of H-NS-bound regions have been conserved in E. coli strains. The sequences of the H-NS-bound regions appear to have diverged more so than H-NS-unbound regions only when H-NS-bound regions are located upstream or in coding regions of genes. Because these regions generally contain regulatory elements for gene expression, sequence divergence in these regions may be associated with alteration of gene expression. Indeed, nucleotide substitutions in H-NS-bound regions of the ybdO promoter and coding regions have diversified the potential for H-NS-independent negative regulation among E. coli strains. The ybdO expression in these strains was still negatively regulated by H-NS, which reduced the effect of H-NS-independent regulation under normal growth conditions. Hence, we propose that, during E. coli evolution, the conservation of H-NS binding sites resulted in the diversification of the regulation of horizontally transferred genes, which may have facilitated E. coli adaptation to new ecological niches. PMID:26789284
H-NS Facilitates Sequence Diversification of Horizontally Transferred DNAs during Their Integration in Host Chromosomes.

PubMed

Higashi, Koichi; Tobe, Toru; Kanai, Akinori; Uyar, Ebru; Ishikawa, Shu; Suzuki, Yutaka; Ogasawara, Naotake; Kurokawa, Ken; Oshima, Taku

2016-01-01

Bacteria can acquire new traits through horizontal gene transfer. Inappropriate expression of transferred genes, however, can disrupt the physiology of the host bacteria. To reduce this risk, Escherichia coli expresses the nucleoid-associated protein, H-NS, which preferentially binds to horizontally transferred genes to control their expression. Once expression is optimized, the horizontally transferred genes may actually contribute to E. coli survival in new habitats. Therefore, we investigated whether and how H-NS contributes to this optimization process. A comparison of H-NS binding profiles on common chromosomal segments of three E. coli strains belonging to different phylogenetic groups indicated that the positions of H-NS-bound regions have been conserved in E. coli strains. The sequences of the H-NS-bound regions appear to have diverged more so than H-NS-unbound regions only when H-NS-bound regions are located upstream or in coding regions of genes. Because these regions generally contain regulatory elements for gene expression, sequence divergence in these regions may be associated with alteration of gene expression. Indeed, nucleotide substitutions in H-NS-bound regions of the ybdO promoter and coding regions have diversified the potential for H-NS-independent negative regulation among E. coli strains. The ybdO expression in these strains was still negatively regulated by H-NS, which reduced the effect of H-NS-independent regulation under normal growth conditions. Hence, we propose that, during E. coli evolution, the conservation of H-NS binding sites resulted in the diversification of the regulation of horizontally transferred genes, which may have facilitated E. coli adaptation to new ecological niches.
Structural Insights into the Regulation of Foreign Genes in Salmonella by the Hha/H-NS Complex*

PubMed Central

Ali, Sabrina S.; Whitney, John C.; Stevenson, James; Robinson, Howard; Howell, P. Lynne; Navarre, William Wiley

2013-01-01

The bacterial nucleoid-associated proteins Hha and H-NS jointly repress horizontally acquired genes in Salmonella, including essential virulence loci encoded within Salmonella pathogenicity islands. Hha is known to interact with the N-terminal dimerization domain of H-NS; however, the manner in which this interaction enhances transcriptional silencing is not understood. To further understand this process, we solved the x-ray crystal structure of Hha in complex with the N-terminal dimerization domain of H-NS (H-NS(1–46)) to 3.2 Å resolution. Two monomers of Hha bind to symmetrical sites on either side of the H-NS(1–46) dimer. Disruption of the Hha/H-NS interaction by the H-NS site-specific mutation I11A results in increased expression of the Hha/H-NS co-regulated gene hilA without affecting the expression levels of proV, a target gene repressed by H-NS in an Hha-independent fashion. Examination of the structure revealed a cluster of conserved basic amino acids that protrude from the surface of Hha on the opposite side of the Hha/H-NS(1–46) interface. Hha mutants with a diminished positively charged surface maintain the ability to interact with H-NS but can no longer regulate hilA. Increased expression of the hilA locus did not correspond to significant depletion of H-NS at the promoter region in chromatin immunoprecipitation assays. However, in vitro, we find Hha improves H-NS binding to target DNA fragments. Taken together, our results show for the first time how Hha and H-NS interact to direct transcriptional repression and reveal that a positively charged surface of Hha enhances the silencing activity of H-NS nucleoprotein filaments. PMID:23515315
NS1-binding protein abrogates the elevation of cell viability by the influenza A virus NS1 protein in association with CRKL

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

Miyazaki, Masaya; Nishihara, Hiroshi, E-mail: hnishihara@med.hokudai.ac.jp; Hasegawa, Hideki

Highlights: •NS1 induced excessive phosphorylation of ERK and elevated cell viability. •NS1-BP expression and CRKL knockdown abolished survival effect of NS1. •NS1-BP and NS1 formed the complex through the interaction with CRKL-SH3(N). -- Abstract: The influenza A virus non-structural protein 1 (NS1) is a multifunctional virulence factor consisting of an RNA binding domain and several Src-homology (SH) 2 and SH3 binding motifs, which promotes virus replication in the host cell and helps to evade antiviral immunity. NS1 modulates general host cell physiology in association with various cellular molecules including NS1-binding protein (NS1-BP) and signaling adapter protein CRK-like (CRKL), while themore » physiological role of NS1-BP during influenza A virus infection especially in association with NS1 remains unclear. In this study, we analyzed the intracellular association of NS1-BP, NS1 and CRKL to elucidate the physiological roles of these molecules in the host cell. In HEK293T cells, enforced expression of NS1 of A/Beijing (H1N1) and A/Indonesia (H5N1) significantly induced excessive phosphorylation of ERK and elevated cell viability, while the over-expression of NS1-BP and the abrogation of CRKL using siRNA abolished such survival effect of NS1. The pull-down assay using GST-fusion CRKL revealed the formation of intracellular complexes of NS1-BP, NS1 and CRKL. In addition, we identified that the N-terminus SH3 domain of CRKL was essential for binding to NS1-BP using GST-fusion CRKL-truncate mutants. This is the first report to elucidate the novel function of NS1-BP collaborating with viral protein NS1 in modulation of host cell physiology. In addition, an alternative role of adaptor protein CRKL in association with NS1 and NS1-BP during influenza A virus infection is demonstrated.« less
Interactome Analysis of NS1 Protein Encoded by Influenza A H7N9 Virus Reveals an Inhibitory Role of NS1 in Host mRNA Maturation.

PubMed

Kuo, Rei-Lin; Chen, Chi-Jene; Tam, Ee-Hong; Huang, Chung-Guei; Li, Li-Hsin; Li, Zong-Hua; Su, Pei-Chia; Liu, Hao-Ping; Wu, Chih-Ching

2018-04-06

Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using coimmunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.
The non-structural (NS) gene segment of H9N2 influenza virus isolated from backyard poultry in Pakistan reveals strong genetic and functional similarities to the NS gene of highly pathogenic H5N1

PubMed Central

Munir, Muhammad; Zohari, Siamak; Iqbal, Munir; Abbas, Muhammad; Perez, Daniel Roberto; Berg, Mikael

2013-01-01

Apart from natural reassortment, co-circulation of different avian influenza virus strains in poultry populations can lead to generation of novel variants and reassortant viruses. In this report, we studied the genetics and functions of a reassorted non-structural gene (NS) of H9N2 influenza virus collected from back yard poultry (BYP) flock. Phylogenetic reconstruction based on hemagglutinin and neuraminidase genes indicates that an isolate from BYP belongs to H9N2. However, the NS gene-segment of this isolate cluster into genotype Z, clade 2.2 of the highly pathogenic H5N1. The NS gene plays essential roles in the host-adaptation, cell-tropism, and virulence of influenza viruses. However, such interpretations have not been investigated in naturally recombinant H9N2 viruses. Therefore, we compared the NS1 protein of H9N2 (H9N2/NS1) and highly pathogenic H5N1 (H5N1/NS1) in parallel for their abilities to regulate different signaling pathways, and investigated the molecular mechanisms of IFN-β production in human, avian, and mink lung cells. We found that H9N2/NS1 and H5N1/NS1 are comparably similar in inhibiting TNF-α induced nuclear factor κB and double stranded RNA induced activator protein 1 and interferon regulatory factor 3 transcription factors. Thus, the production of IFN-β was inhibited equally by both NS1s as demonstrated by IFN stimulatory response element and IFN-β promoter activation. Moreover, both NS1s predominantly localized in the nucleus when transfected to human A549 cells. This study therefore suggests the possible increased virulence of natural reassortant viruses for their efficient invasion of host immune responses, and proposes that these should not be overlooked for their epizootic and zoonotic potential. PMID:23959028
Identification of high-specificity H-NS binding site in LEE5 promoter of enteropathogenic Esherichia coli (EPEC).

PubMed

Bhat, Abhay Prasad; Shin, Minsang; Choy, Hyon E

2014-07-01

Histone-like nucleoid structuring protein (H-NS) is a small but abundant protein present in enteric bacteria and is involved in compaction of the DNA and regulation of the transcription. Recent reports have suggested that H-NS binds to a specific AT rich DNA sequence than to intrinsically curved DNA in sequence independent manner. We detected two high-specificity H-NS binding sites in LEE5 promoter of EPEC centered at -110 and -138, which were close to the proposed consensus H-NS binding motif. To identify H-NS binding sequence in LEE5 promoter, we took a random mutagenesis approach and found the mutations at around -138 were specifically defective in the regulation by H-NS. It was concluded that H-NS exerts maximum repression via the specific sequence at around -138 and subsequently contacts a subunit of RNAP through oligomerization.
RNA Modulates the Interaction between Influenza A Virus NS1 and Human PABP1.

PubMed

Arias-Mireles, Bryan H; de Rozieres, Cyrus M; Ly, Kevin; Joseph, Simpson

2018-05-25

Nonstructural protein 1 (NS1) is a multifunctional protein involved in preventing host-interferon response in influenza A virus (IAV). Previous studies have indicated that NS1 also stimulates the translation of viral mRNA by binding to conserved sequences in the viral 5'-UTR. Additionally, NS1 binds to poly(A) binding protein 1 (PABP1) and eukaryotic initiation factor 4G (eIF4G). The interaction of NS1 with the viral 5'-UTR, PABP1, and eIF4G has been suggested to specifically enhance the translation of viral mRNAs. In contrast, we report that NS1 does not directly bind to sequences in the viral 5'-UTR, indicating that NS1 is not responsible for providing the specificity to stimulate viral mRNA translation. We also monitored the interaction of NS1 with PABP1 using a new, quantitative FRET assay. Our data show that NS1 binds to PABP1 with high affinity; however, the binding of double-stranded RNA (dsRNA) to NS1 weakens the binding of NS1 to PABP1. Correspondingly, the binding of PABP1 to NS1 weakens the binding of NS1 to double-stranded RNA (dsRNA). In contrast, the affinity of PABP1 for binding to poly(A) RNA is not significantly changed by NS1. We propose that the modulation of NS1·PABP1 interaction by dsRNA may be important for the viral cycle.
H-NS represses transcription of the flagellin gene lafA of lateral flagella in Vibrio parahaemolyticus.

PubMed

Wang, Yan; Zhang, Yiquan; Yin, Zhe; Wang, Jie; Zhu, Yongzhe; Peng, Haoran; Zhou, Dongsheng; Qi, Zhongtian; Yang, Wenhui

2018-01-01

Swarming motility is ultimately mediated by the proton-powered lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf genes is tightly regulated by a number of environmental conditions and regulatory factors. The nucleoid-associated DNA-binding protein H-NS is a small and abundant protein that is widely distributed in bacteria, and H-NS-like protein-dependent expression of laf genes has been identified in Vibrio cholerae and V. parahaemolyticus. The data presented here show that H-NS acts as a repressor of the swarming motility in V. parahaemolyticus. A single σ 28 -dependent promoter was detected for lafA encoding the flagellin of the lateral flagella, and its activity was directly repressed by H-NS. Thus, H-NS represses swarming motility by directly acting on lafA. Briefly, this work revealed a novel function for H-NS as a repressor of the expression of lafA and swarming motility in V. parahaemolyticus.

Modulation of human multidrug-resistance MDR-1 gene by natural curcuminoids

PubMed Central

Limtrakul, Pornngarm; Anuchapreeda, Songyot; Buddhasukh, Duang

2004-01-01

Background Multidrug resistance (MDR) is a phenomenon that is often associated with decreased intracellular drug accumulation in patient's tumor cells resulting from enhanced drug efflux. It is related to the overexpression of a membrane protein, P-glycoprotein (Pgp-170), thereby reducing drug cytotoxicity. A variety of studies have tried to find MDR modulators which increase drug accumulation in cancer cells. Methods In this study, natural curcuminoids, pure curcumin, demethoxycurcumin and bisdemethoxycurcumin, isolated from turmeric (Curcuma longa Linn), were compared for their potential ability to modulate the human MDR-1 gene expression in multidrug resistant human cervical carcinoma cell line, KB-V1 by Western blot analysis and RT-PCR. Results Western blot analysis and RT-PCR showed that all the three curcuminoids inhibited MDR-1 gene expression, and bisdemethoxycurcumin produced maximum effect. In additional studies we found that commercial grade curcuminoid (approximately 77% curcumin, 17% demethoxycurcumin and 3% bisdemthoxycurcumin) decreased MDR-1 gene expression in a dose dependent manner and had about the same potent inhibitory effect on MDR-1 gene expression as our natural curcuminoid mixtures. Conclusion These results indicate that bisdemethoxycurcumin is the most active of the curcuminoids present in turmeric for modulation of MDR-1 gene. Treatment of drug resistant KB-V1 cells with curcumin increased their sensitivity to vinblastine, which was consistent with a decreased MDR-1 gene product, a P-glycoprotein, on the cell plasma membrane. Although many drugs that prevent the P-glycoprotein function have been reported, this report describes the inhibition of MDR-1 expression by a phytochemical. The modulation of MDR-1 expression may be an attractive target for new chemosensitizing agents. PMID:15090070
Alternate SlyA and H-NS nucleoprotein complexes control hlyE expression in Escherichia coli K-12

PubMed Central

Lithgow, James K; Haider, Fouzia; Roberts, Ian S; Green, Jeffrey

2007-01-01

Haemolysin E is a cytolytic pore-forming toxin found in several Escherichia coli and Salmonella enterica strains. Expression of hlyE is repressed by the global regulator H-NS (histone-like nucleoid structuring protein), but can be activated by the regulator SlyA. Expression of a chromosomal hlyE–lacZ fusion in an E. coli slyA mutant was reduced to 60% of the wild-type level confirming a positive role for SlyA. DNase I footprint analysis revealed the presence of two separate SlyA binding sites, one located upstream, the other downstream of the hlyE transcriptional start site. These sites overlap AT-rich H-NS binding sites. Footprint and gel shift data showed that whereas H-NS prevented binding of RNA polymerase (RNAP) at the hlyE promoter (PhlyE), SlyA allowed binding of RNAP, but inhibited binding of H-NS. Accordingly, in vitro transcription analyses showed that addition of SlyA protein relieved H-NS-mediated repression of hlyE. Based on these observations a model for SlyA/H-NS regulation of hlyE expression is proposed in which the relative concentrations of SlyA and H-NS govern the nature of the nucleoprotein complexes formed at PhlyE. When H-NS is dominant RNAP binding is inhibited and hlyE expression is silenced; when SlyA is dominant H-NS binding is inhibited allowing RNAP access to the promoter facilitating hlyE transcription. PMID:17892462
A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri.

PubMed

Tran, Chi Nhan; Giangrossi, Mara; Prosseda, Gianni; Brandi, Anna; Di Martino, Maria Letizia; Colonna, Bianca; Falconi, Maurizio

2011-10-01

The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5'-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense-antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem-loop motifs of the antisense region.
Efficacy of Live-Attenuated H9N2 Influenza Vaccine Candidates Containing NS1 Truncations against H9N2 Avian Influenza Viruses.

PubMed

Chen, Sujuan; Zhu, Yinbiao; Yang, Da; Yang, Yang; Shi, Shaohua; Qin, Tao; Peng, Daxin; Liu, Xiufan

2017-01-01

H9N2 avian influenza virus is a zoonotic agent with a broad host range that can contribute genetic information to H5 or H7N9 subtype viruses, which are significant threats to both humans and birds. Thus, there is a great need for a vaccine to control H9N2 avian influenza. Three mutant viruses of an H9N2 virus A/chicken/Taixing/10/2010 (rTX-NS1-73, rTX-NS1-100, and rTX-NS1-128) were constructed with different NS1 gene truncations and confirmed by western blot analysis. The genetic stability, pathogenicity, transmissibility, and host immune responses toward these mutants were evaluated. The mutant virus rTX-NS1-128 exhibited the most attenuated phenotype and lost transmissibility. The expression levels of interleukin 12 in the nasal and tracheal tissues from chickens immunized with rTX-NS1-128 were significantly upregulated on day 3 post-immunization and the IgA and IgG antibody levels were significantly increased on days 7, 14, and 21 post-immunization when compared to chickens that received an inactivated vaccine. rTX-NS1-128 also protected chickens from challenge by homologous and heterologous H9N2 avian influenza viruses. The results indicate that rTX-NS1-128 can be used as a potential live-attenuated vaccine against H9N2 avian influenza.
The NS segment of H5N1 avian influenza viruses (AIV) enhances the virulence of an H7N1 AIV in chickens.

PubMed

Vergara-Alert, Júlia; Busquets, Núria; Ballester, Maria; Chaves, Aida J; Rivas, Raquel; Dolz, Roser; Wang, Zhongfang; Pleschka, Stephan; Majó, Natàlia; Rodríguez, Fernando; Darji, Ayub

2014-01-25

Some outbreaks involving highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 were caused by avian-to-human transmissions. In nature, different influenza A viruses can reassort leading to new viruses with new characteristics. We decided to investigate the impact that the NS-segment of H5 HPAIV would have on viral pathogenicity of a classical avian H7 HPAIV in poultry, a natural host. We focussed this study based on our previous work that demonstrated that single reassortment of the NS-segment from an H5 HPAIV into an H7 HPAIV changes the ability of the virus to replicate in mammalian hosts. Our present data show that two different H7-viruses containing an NS-segment from H5-types (FPV NS GD or FPV NS VN) show an overall highly pathogenic phenotype compared with the wild type H7-virus (FPV), as characterized by higher viral shedding and earlier manifestation of clinical signs. Correlating with the latter, higher amounts of IFN-β mRNA were detected in the blood of NS-reassortant infected birds, 48 h post-infection (pi). Although lymphopenia was detected in chickens from all AIV-infected groups, also 48 h pi those animals challenged with NS-reassortant viruses showed an increase of peripheral monocyte/macrophage-like cells expressing high levels of IL-1β, as determined by flow cytometry. Taken together, these findings highlight the importance of the NS-segment in viral pathogenicity which is directly involved in triggering antiviral and pro-inflammatory cytokines found during HPAIV pathogenesis in chickens.
Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells.

PubMed

Gao, Yunfeng; Foo, Yong Hwee; Winardhi, Ricksen S; Tang, Qingnan; Yan, Jie; Kenney, Linda J

2017-11-21

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.
The 5.5 protein of phage T7 inhibits H-NS through interactions with the central oligomerization domain.

PubMed

Ali, Sabrina S; Beckett, Emily; Bae, Sandy Jeehoon; Navarre, William Wiley

2011-09-01

The 5.5 protein (T7p32) of coliphage T7 (5.5(T7)) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5(T7) protein is insoluble when expressed in Escherichia coli, but we find that 5.5(T7) can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5(T7) binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5(T7) can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se. Homologues of the 5.5(T7) protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5(T7) protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression. Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Development of a dual-protective live attenuated vaccine against H5N1 and H9N2 avian influenza viruses by modifying the NS1 gene.

PubMed

Choi, Eun-hye; Song, Min-Suk; Park, Su-Jin; Pascua, Philippe Noriel Q; Baek, Yun Hee; Kwon, Hyeok-il; Kim, Eun-Ha; Kim, Semi; Jang, Hyung-Kwan; Poo, Haryoung; Kim, Chul-Joong; Choi, Young Ki

2015-07-01

An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-β activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.
Structural characterization of the H-NS protein from Xylella fastidiosa and its interaction with DNA.

PubMed

Rosselli-Murai, Luciana K; Sforça, Maurício L; Sassonia, Rogério C; Azzoni, Adriano R; Murai, Marcelo J; de Souza, Anete P; Zeri, Ana C

2012-10-01

The nucleoid-associated protein H-NS is a major component of the bacterial nucleoid involved in DNA compaction and transcription regulation. The NMR solution structure of the Xylella fastidiosa H-NS C-terminal domain (residues 56-134) is presented here and consists of two beta-strands and two alpha helices, with one loop connecting the two beta-strands and a second loop connecting the second beta strand and the first helix. The amide (1)H and (15)N chemical shift signals for a sample of XfH-NS(56-134) were monitored in the course of a titration series with a 14-bp DNA duplex. Most of the residues involved in contacts to DNA are located around the first and second loops and in the first helix at a positively charged side of the protein surface. The overall structure of the Xylella H-NS C-terminal domain differ significantly from Escherichia coli and Salmonella enterica H-NS proteins, even though the DNA binding motif in loop 2 adopt similar conformation, as well as β-strand 2 and loop 1. Interestingly, we have also found that the DNA binding site is expanded to include helix 1, which is not seen in the other structures. Copyright © 2012 Elsevier Inc. All rights reserved.
Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity

PubMed Central

Allonso, Diego; Andrade, Iamara S.; Conde, Jonas N.; Coelho, Diego R.; Rocha, Daniele C. P.; da Silva, Manuela L.; Ventura, Gustavo T.

2015-01-01

ABSTRACT Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. IMPORTANCE Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the
Dengue Virus NS1 Protein Modulates Cellular Energy Metabolism by Increasing Glyceraldehyde-3-Phosphate Dehydrogenase Activity.

PubMed

Allonso, Diego; Andrade, Iamara S; Conde, Jonas N; Coelho, Diego R; Rocha, Daniele C P; da Silva, Manuela L; Ventura, Gustavo T; Silva, Emiliana M; Mohana-Borges, Ronaldo

2015-12-01

Dengue is one of the main public health concerns worldwide. Recent estimates indicate that over 390 million people are infected annually with the dengue virus (DENV), resulting in thousands of deaths. Among the DENV nonstructural proteins, the NS1 protein is the only one whose function during replication is still unknown. NS1 is a 46- to 55-kDa glycoprotein commonly found as both a membrane-associated homodimer and a soluble hexameric barrel-shaped lipoprotein. Despite its role in the pathogenic process, NS1 is essential for proper RNA accumulation and virus production. In the present study, we identified that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with intracellular NS1. Molecular docking revealed that this interaction occurs through the hydrophobic protrusion of NS1 and the hydrophobic residues located at the opposite side of the catalytic site. Moreover, addition of purified recombinant NS1 enhanced the glycolytic activity of GAPDH in vitro. Interestingly, we observed that DENV infection promoted the relocalization of GAPDH to the perinuclear region, where NS1 is commonly found. Both DENV infection and expression of NS1 itself resulted in increased GAPDH activity. Our findings indicate that the NS1 protein acts to increase glycolytic flux and, consequently, energy production, which is consistent with the recent finding that DENV induces and requires glycolysis for proper replication. This is the first report to propose that NS1 is an important modulator of cellular energy metabolism. The data presented here provide new insights that may be useful for further drug design and the development of alternative antiviral therapies against DENV. Dengue represents a serious public health problem worldwide and is caused by infection with dengue virus (DENV). Estimates indicate that half of the global population is at risk of infection, with almost 400 million cases occurring per year. The NS1 glycoprotein is found in both the intracellular and the
The NS segment of H5N1 avian influenza viruses (AIV) enhances the virulence of an H7N1 AIV in chickens

PubMed Central

2014-01-01

Some outbreaks involving highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 were caused by avian-to-human transmissions. In nature, different influenza A viruses can reassort leading to new viruses with new characteristics. We decided to investigate the impact that the NS-segment of H5 HPAIV would have on viral pathogenicity of a classical avian H7 HPAIV in poultry, a natural host. We focussed this study based on our previous work that demonstrated that single reassortment of the NS-segment from an H5 HPAIV into an H7 HPAIV changes the ability of the virus to replicate in mammalian hosts. Our present data show that two different H7-viruses containing an NS-segment from H5–types (FPV NS GD or FPV NS VN) show an overall highly pathogenic phenotype compared with the wild type H7–virus (FPV), as characterized by higher viral shedding and earlier manifestation of clinical signs. Correlating with the latter, higher amounts of IFN-β mRNA were detected in the blood of NS-reassortant infected birds, 48 h post-infection (pi). Although lymphopenia was detected in chickens from all AIV-infected groups, also 48 h pi those animals challenged with NS-reassortant viruses showed an increase of peripheral monocyte/macrophage-like cells expressing high levels of IL-1β, as determined by flow cytometry. Taken together, these findings highlight the importance of the NS-segment in viral pathogenicity which is directly involved in triggering antiviral and pro-inflammatory cytokines found during HPAIV pathogenesis in chickens. PMID:24460592
Probing the structure, function, and interactions of the Escherichia coli H-NS and StpA proteins by using dominant negative derivatives.

PubMed

Williams, R M; Rimsky, S; Buc, H

1996-08-01

Twelve different dominant negative mutants of the Escherichia coli nucleoid-associated protein, H-NS, have been selected and characterized in vivo. The mutants are all severely defective in promoter repression activity in a strain lacking H-NS, and they all disrupt the repression normally exerted by H-NS at two of its target promoters. From the locations of the alterations in these mutants, which result in both large truncations and amino acid substitutions, we propose that H-NAS contains at least two distinct domains. The in vitro protein-protein cross-linking data presented in this report indicate that the proposed N-terminal domain of H-NS has a role in H-NS multimerization. StpA is a protein with known structural and functional homologies to H-NS. We have analyzed the extent of these homologies by constructing and studying StpA mutants predicted to be dominant negative. Our data indicate that the substitutions and deletions found in dominant negative H-NS have similar effects in the context of StpA. We conclude that the domain organizations and functions in StpA and H-NS are closely related. Furthermore, dominant negative H-NS can disrupt the activity of native StpA, and reciprocally, dominant negative StpA can disrupt the activity of native H-NS. We demonstrate that the N-terminal domain of H-NS can be chemically cross-linked to both full-length H-NS and StpA. We account for these observations by proposing that H-NS and StpA have the ability to form hybrid species.
Hha has a defined regulatory role that is not dependent upon H-NS or StpA

PubMed Central

Solórzano, Carla; Srikumar, Shabarinath; Canals, Rocío; Juárez, Antonio; Paytubi, Sonia; Madrid, Cristina

2015-01-01

The Hha family of proteins is involved in the regulation of gene expression in enterobacteria by forming complexes with H-NS-like proteins. Whereas several amino acid residues of both proteins participate in the interaction, some of them play a key role. Residue D48 of Hha protein is essential for the interaction with H-NS, thus the D48N substitution in Hha protein abrogates H-NS/Hha interaction. Despite being a paralog of H-NS protein, StpA interacts with HhaD48N with higher affinity than with the wild type Hha protein. To analyze whether Hha is capable of acting independently of H-NS and StpA, we conducted transcriptomic analysis on the hha and stpA deletion strains and the hhaD48N substitution strain of Salmonella Typhimurium using a custom microarray. The results obtained allowed the identification of 120 genes regulated by Hha in an H-NS/StpA-independent manner, 38% of which are horizontally acquired genes. A significant number of the identified genes are involved in functions related to cell motility, iron uptake, and pathogenicity. Thus, motility assays, siderophore detection and intra-macrophage replication assays were performed to confirm the transcriptomic data. Our findings point out the importance of Hha protein as an independent regulator in S. Typhimurium, highlighting a regulatory role on virulence. PMID:26284052
X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus

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

Bornholdt, Zachary A.; Prasad, B.V. Venkataram

2009-04-08

The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains. NS1, which consists of two domains - a double-stranded RNA (dsRNA) binding domain and the effector domain, separated through a linker - is an antagonist of antiviral type-I interferon response in the host. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60%more » of human deaths in an outbreak in Vietnam. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-{angstrom}-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.« less
Ketoconazole and the modulation of multidrug resistance-mediated transport in Caco-2 and MDCKII-MDR1 drug transport models.

PubMed

Fan, Y; Rodriguez-Proteau, R

2008-02-01

The hypothesis tested was that ketoconazole can modulate P-glycoprotein, thereby altering cellular uptake and apparent permeability (P(app)) of multidrug-resistant substrates, such as cyclosporin A (CSA) and digoxin, across Caco-2, MDCKII-MDR1, and MDCKII wild-type cell transport models. (3)H-CSA/(3)H-digoxin transport experiments were performed with and without co-exposure to ketoconazole, and (3)H-ketoconzole transport experiments were performed with and without co-exposure to dietary flavonoids, epigallocatechin-3-gallate, and xanthohumol. Ketoconazole (3 microM) reduced the P(app) efflux of CSA and digoxin from 5.07 x 10(-6) to 2.91 x 10(-6) cm s(-1) and from 2.60 x 10(-6) to 1.41 x 10(-6) cm s(-1), respectively, in Caco-2 cells. In the MDCKII-MDR1 cells, ketoconazole reduced the P(app) efflux of CSA and increased the P(app) absorption of digoxin. Cellular uptake of ketoconazole in the Caco-2 cells was significantly inhibited by CSA and digoxin, whereas epigallocatechin-3-gallate and xanthohumol exhibited biphasic responses. In conclusion, ketoconazole modulates the P(app) of P-glycoprotein substrates by interacting with MDR1 protein. Epigallocatechin-3-gallate and xanthohumol modulate the transport and uptake of ketoconazole.
Disposition of Cremophor EL in humans limits the potential for modulation of the multidrug resistance phenotype in vivo.

PubMed

Sparreboom, A; Verweij, J; van der Burg, M E; Loos, W J; Brouwer, E; Viganò, L; Locatelli, A; de Vos, A I; Nooter, K; Stoter, G; Gianni, L

1998-08-01

The purpose of the present study was to characterize the distribution and elimination kinetics of the paclitaxel vehicle Cremophor EL (CrEL), a polyoxyethylated castor oil that can modulate P-glycoprotein-mediated multidrug resistance in vitro. The pharmacokinetics of CrEL were studied using noncompartmental models in 23 patients with histological proof of malignant solid tumors, receiving paclitaxel as a 3-h i.v. infusion at dose levels ranging from 100-225 mg/m2 (corresponding to CrEL doses of 8.33-18.8 ml/m2). Serial plasma samples were obtained before and up to 72 h after drug administration, and were analyzed for the presence of CrEL by a novel colorimetric dye-binding microassay. The area under the plasma concentration versus time curves and the peak plasma levels of CrEL increased from 253+/-36.8 (mean+/-SD) to 680+/- 180 microl.h/ml, and from 3.40+/-0.10 to 6.58+/-0.52 microl/ml, respectively, consistent with linear pharmacokinetics. Disappearance of CrEL from the central plasma compartment was characterized by a terminal elimination half-life of 84.1+/-20.4 h, resulting in extended persistence of substantial levels even at 1 week after paclitaxel treatment. The observed volume of distribution was extremely low and averaged 3.70+/-0.49 liters/m2, implying that the tumor delivery of CrEL is insignificant. Our results indicate that CrEL is a relatively slow clearance compound and that its distribution is limited to the central plasma compartment. Hence, CrEL is not likely to play a role in reversing P-glycoprotein-mediated multidrug resistance to paclitaxel in vivo.
Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

PubMed

Muñoz-Martínez, Francisco; Lu, Peihua; Cortés-Selva, Fernando; Pérez-Victoria, José María; Jiménez, Ignacio A; Ravelo, Angel G; Sharom, Frances J; Gamarro, Francisco; Castanys, Santiago

2004-10-01

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential
Dengue virus NS2 and NS4: Minor proteins, mammoth roles.

PubMed

Gopala Reddy, Sindhoora Bhargavi; Chin, Wei-Xin; Shivananju, Nanjunda Swamy

2018-04-17

Despite the ever-increasing global incidence of dengue fever, there are no specific chemotherapy regimens for its treatment. Structural studies on dengue virus (DENV) proteins have revealed potential drug targets. Major DENV proteins such as the envelope protein and non-structural (NS) proteins 3 and 5 have been extensively investigated in antiviral studies, but with limited success in vitro. However, the minor NS proteins NS2 and NS4 have remained relatively underreported. Emerging evidence indicating their indispensable roles in virus propagation and host immunomodulation should encourage us to target these proteins for drug discovery. This review covers current knowledge on DENV NS2 and NS4 proteins from structural and functional perspectives and assesses their potential as targets for antiviral design. Antiviral targets in NS2A include surface-exposed transmembrane regions involved in pathogenesis, while those in NS2B include protease-binding sites in a conserved hydrophilic domain. Ideal drug targets in NS4A include helix α4 and the PEPEKQR sequence, which are essential for NS4A-2K cleavage and NS4A-NS4B association, respectively. In NS4B, the cytoplasmic loop connecting helices α5 and α7 is an attractive target for antiviral design owing to its role in dimerization and NS4B-NS3 interaction. Findings implicating NS2A, NS2B, and NS4A in membrane-modulation and viroporin-like activities indicate an opportunity to target these proteins by disrupting their association with membrane lipids. Despite the lack of 3D structural data, recent topological findings and progress in structure-prediction methods should be sufficient impetus for targeting NS2 and NS4 for drug design. Copyright © 2018 Elsevier Inc. All rights reserved.
NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses

PubMed Central

Nogales, Aitor; Martinez-Sobrido, Luis

2016-01-01

ABSTRACT Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus

NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses.

PubMed

Nogales, Aitor; Martinez-Sobrido, Luis; Topham, David J; DeDiego, Marta L

2017-03-01

Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus
Green synthesised zinc oxide nanostructures through Periploca aphylla extract shows tremendous antibacterial potential against multidrug resistant pathogens.

PubMed

Abbas, Fazal; Maqbool, Qaisar; Nazar, Mudassar; Jabeen, Nyla; Hussain, Syed Zaheer; Anwaar, Sadaf; Mehmood, Nasir; Sheikh, Muhammad Saleem; Hussain, Talib; Iftikhar, Sidra

2017-12-01

To grapple with multidrug resistant bacterial infections, implementations of antibacterial nanomedicines have gained prime attention of the researchers across the globe. Nowadays, zinc oxide (ZnO) at nano-scale has emerged as a promising antibacterial therapeutic agent. Keeping this in view, ZnO nanostructures (ZnO-NS) have been synthesised through reduction by P. aphylla aqueous extract without the utilisation of any acid or base. Structural examinations via scanning electron microscopy (SEM) and X-ray diffraction have revealed pure phase morphology with highly homogenised average particle size of 18 nm. SEM findings were further supplemented by transmission electron microscopy examinations. The characteristic Zn-O peak has been observed around 363 nm using ultra-violet-visible spectroscopy. Fourier-transform infrared spectroscopy examination has also confirmed the formation of ZnO-NS through detection of Zn-O bond vibration frequencies. To check the superior antibacterial activity of ZnO-NS, the authors' team has performed disc diffusion assay and colony forming unit testing against multidrug resistant E. coli, S. marcescens and E. cloacae . Furthermore, protein kinase inhibition assay and cytotoxicity examinations have revealed that green fabricated ZnO-NS are non-hazardous, economical, environmental friendly and possess tremendous potential to treat lethal infections caused by multidrug resistant pathogens.
Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus.

PubMed

Nogales, Aitor; Rodriguez, Laura; DeDiego, Marta L; Topham, David J; Martínez-Sobrido, Luis

2017-09-01

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics, representing a serious public health concern. It has been described that one mechanism used by some IAV strains to escape the host innate immune responses and modulate virus pathogenicity involves the ability of the PA-X and NS1 proteins to inhibit the host protein synthesis in infected cells. It was reported that for the 2009 pandemic H1N1 IAV (pH1N1) only the PA-X protein had this inhibiting capability, while the NS1 protein did not. In this work, we have evaluated, for the first time, the combined effect of PA-X- and NS1-mediated inhibition of general gene expression on virus pathogenesis, using a temperature-sensitive, live-attenuated 2009 pandemic H1N1 IAV (pH1N1 LAIV). We found that viruses containing PA-X and NS1 proteins that simultaneously have (PA WT + /NS1 MUT + ) or do not have (PA MUT - /NS1 WT - ) the ability to block host gene expression showed reduced pathogenicity in vivo However, a virus where the ability to inhibit host protein expression was switched between PA-X and NS1 (PA MUT - /NS1 MUT + ) presented pathogenicity similar to that of a virus containing both wild-type proteins (PA WT + /NS1 WT - ). Our findings suggest that inhibition of host protein expression is subject to a strict balance, which can determine the successful progression of IAV infection. Importantly, knowledge obtained from our studies could be used for the development of new and more effective vaccine approaches against IAV. IMPORTANCE Influenza A viruses (IAVs) are one of the most common causes of respiratory infections in humans, resulting in thousands of deaths annually. Furthermore, IAVs can cause unpredictable pandemics of great consequence when viruses not previously circulating in humans are introduced into humans. The defense machinery provided by the host innate immune system limits IAV replication; however, to counteract host antiviral activities, IAVs have developed different inhibition
Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

PubMed

Yamanaka, Yuki; Winardhi, Ricksen S; Yamauchi, Erika; Nishiyama, So-Ichiro; Sowa, Yoshiyuki; Yan, Jie; Kawagishi, Ikuro; Ishihama, Akira; Yamamoto, Kaneyoshi

2018-06-15

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses.

PubMed

Clark, Amelia M; Nogales, Aitor; Martinez-Sobrido, Luis; Topham, David J; DeDiego, Marta L

2017-09-01

In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up
Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses

PubMed Central

Clark, Amelia M.; Nogales, Aitor; Martinez-Sobrido, Luis

2017-01-01

ABSTRACT In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people
Discriminative-stimulus effects of NS9283, a nicotinic α4β2* positive allosteric modulator, in nicotine-discriminating rats.

PubMed

Mohler, Eric G; Franklin, Stanley R; Rueter, Lynne E

2014-01-01

Neuronal α4β2* nicotinic acetylcholine receptors mediate cognition, pain, and the discriminative and reinforcing effects of nicotine. In addition to traditional orthosteric agonists, α4β2* positive allosteric modulators (PAMs) have recently been identified. With increased subtype selectivity relative to agonists, PAMs administered alone or in combination with low-dose α4β2* agonists may be used as powerful tools for increasing our understanding of α4β2* pharmacology. The present experiments tested the nicotine discriminative-stimulus effects of the α4β2* PAM NS9283 (A-969933) in the presence and absence of low-dose nicotine or nicotinic subtype-selective agonist. Rats were trained to discriminate 0.4 mg/kg nicotine from saline in a two-lever drug discrimination paradigm. In subsequent generalization tests, rats were administered nicotine, the α4β2*-preferring agonist ABT-594, and NS9283, alone or in two-drug combinations. Nicotine and ABT-594 showed dose-dependent nicotine generalization. NS9283 alone resulted in a non-significant increase in nicotine-appropriate lever selection. Combination of non-effective doses of nicotine or ABT-594 with escalating doses of NS9283 resulted in a complete conversion to 100 % nicotine-appropriate choice in the case of nicotine combination and incomplete, though significant, generalization for ABT-594. The α4β2* PAM NS9283 alone did not produce nicotine-like discriminative effects, but did demonstrate dose-related increases in nicotine lever choice when combined with a non-effective dose of nicotine or the α4β2* agonist ABT-594. This finding provides confirmation of the positive allosteric modulating effect of NS9283 in a functional in vivo paradigm. NS9283 is a potentially valuable tool for studying the role of α4β2* receptors in various nicotinic acetylcholine receptor-related functions.
DNA Unwinding Functions of Minute Virus of Mice NS1 Protein Are Modulated Specifically by the Lambda Isoform of Protein Kinase C

PubMed Central

Dettwiler, Sabine; Rommelaere, Jean; Nüesch, Jürg P. F.

1999-01-01

The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966–9977, 1998). The present study led to the identification of atypical PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKCλ phosphorylation at residue S473. PMID:10438831
The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development.

PubMed

Luo, Dahai; Vasudevan, Subhash G; Lescar, Julien

2015-06-01

The flavivirus NS3 protein is associated with the endoplasmic reticulum membrane via its close interaction with the central hydrophilic region of the NS2B integral membrane protein. The multiple roles played by the NS2B-NS3 protein in the virus life cycle makes it an attractive target for antiviral drug discovery. The N-terminal region of NS3 and its cofactor NS2B constitute the protease that cleaves the viral polyprotein. The NS3 C-terminal domain possesses RNA helicase, nucleoside and RNA triphosphatase activities and is involved both in viral RNA replication and virus particle formation. In addition, NS2B-NS3 serves as a hub for the assembly of the flavivirus replication complex and also modulates viral pathogenesis and the host immune response. Here, we review biochemical and structural advances on the NS2B-NS3 protein, including the network of interactions it forms with NS5 and NS4B and highlight recent drug development efforts targeting this protein. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery. Copyright © 2015 Elsevier B.V. All rights reserved.
DNA sequence-specific dimeric bisbenzimidazoles DBP(n) and DBPA(n) as inhibitors of H-NS silencing in bacterial cells.

PubMed

Melkina, Olga E; Koval, Vasilii S; Ivanov, Alexander A; Zhuze, Alexei L; Zavilgelsky, Gennadii B

2018-03-01

DNA sequence-specific fluorescent dimeric bisbenzimidazoles DBP(n) and DBPA(n), noncovalently interacting with A-T pairs in the minor groove of double-stranded DNA were used for studying and monitoring the expression of histone-like H-NS-dependent promoters. Histone-like H-NS selectively binds to AT-rich segments of DNA and silences a large number of genes in bacterial chromosomes. The H-NS-dependent promoters of Quorum Sensing (QS)-regulated lux operons of the marine bacteria mesophilic Aliivibrio fischeri, psychrophilic Aliivibrio logei were used. Escherichia coli lux biosensors were constructed by cloning fragments bearing QS-regulated promoters into the vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE genes. It was shown that the dimeric bisbenzimidazoles DBP(n) and DBPA(n) counteract the H-NS silencing activity. Thus, the presence of DBP(n) or DBPA(n) in the medium leads to an approximately 10-100-fold increase in the level of transcription of QS promoters in E. coli hns + . The largest decrease in the level of H-NS repression was observed using ligands containing a linker with a length of ca. 18Å, such as DBP(2) and DBPA(2). Ligands containing linkers with n=1 and 3 are an order of magnitude less active; ligands with n=4 are inactive. DBPA(2) exhibits activity starting with a concentration of 0.5μM; the minimum concentration of DBP(2) is 5-7 times higher. It is suggested that A-T pairs located at five nucleotide pair intervals, which correspond to the linker length in highly active ligands with n=2, play a key role in the structure of H-NS-binding sites in QS-regulated promoters. Copyright © 2017 Elsevier GmbH. All rights reserved.
Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

PubMed

Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.
Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

PubMed Central

Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272
Highly thermostable and pH-stable cellulases from Aspergillus niger NS-2: properties and application for cellulose hydrolysis.

PubMed

Bansal, Namita; Janveja, Chetna; Tewari, Rupinder; Soni, Raman; Soni, Sanjeev Kumar

2014-01-01

Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9 ± 20.1 U/g, FPase 101.1 ± 3.5 U/g and β-glucosidase 99 ± 4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0-9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92-98 %.
In-Depth Analysis of HA and NS1 Genes in A(H1N1)pdm09 Infected Patients.

PubMed

Caglioti, Claudia; Selleri, Marina; Rozera, Gabriella; Giombini, Emanuela; Zaccaro, Paola; Valli, Maria Beatrice; Capobianchi, Maria Rosaria

2016-01-01

In March/April 2009, a new pandemic influenza A virus (A(H1N1)pdm09) emerged and spread rapidly via human-to-human transmission, giving rise to the first pandemic of the 21th century. Influenza virus may be present in the infected host as a mixture of variants, referred to as quasi-species, on which natural and immune-driven selection operates. Since hemagglutinin (HA) and non-structural 1 (NS1) proteins are relevant in respect of adaptive and innate immune responses, the present study was aimed at establishing the intra-host genetic heterogeneity of HA and NS1 genes, applying ultra-deep pyrosequencing (UDPS) to nasopharyngeal swabs (NPS) from patients with confirmed influenza A(H1N1)pdm09 infection. The intra-patient nucleotide diversity of HA was significantly higher than that of NS1 (median (IQR): 37.9 (32.8-42.3) X 10-4 vs 30.6 (27.4-33.6) X 10-4 substitutions/site, p = 0.024); no significant correlation for nucleotide diversity of NS1 and HA was observed (r = 0.319, p = 0.29). Furthermore, a strong inverse correlation between nucleotide diversity of NS1 and viral load was observed (r = - 0.74, p = 0.004). For both HA and NS1, the variants appeared scattered along the genes, thus indicating no privileged mutation site. Known polymorphisms, S203T (HA) and I123V (NS1), were observed as dominant variants (>98%) in almost all patients; three HA and two NS1 further variants were observed at frequency >40%; a number of additional variants were detected at frequency <6% (minority variants), of which three HA and four NS1 variants were novel. In few patients multiple variants were observed at HA residues 203 and 222. According to the FLUSURVER tool, some of these variants may affect immune recognition and host range; however, these inferences are based on H5N1, and their extension to A(H1N1)pdm09 requires caution. More studies are necessary to address the significance of the composite nature of influenza virus quasi-species within infected patients.
H-NS Nucleoid Protein Controls Virulence Features of Klebsiella pneumoniae by Regulating the Expression of Type 3 Pili and the Capsule Polysaccharide.

PubMed

Ares, Miguel A; Fernández-Vázquez, José L; Rosales-Reyes, Roberto; Jarillo-Quijada, Ma Dolores; von Bargen, Kristine; Torres, Javier; González-y-Merchand, Jorge A; Alcántar-Curiel, María D; De la Cruz, Miguel A

2016-01-01

Klebsiella pneumoniae is an opportunistic pathogen causing nosocomial infections. Main virulence determinants of K. pneumoniae are pili, capsular polysaccharide, lipopolysaccharide, and siderophores. The histone-like nucleoid-structuring protein (H-NS) is a pleiotropic regulator found in several gram-negative pathogens. It has functions both as an architectural component of the nucleoid and as a global regulator of gene expression. We generated a Δhns mutant and evaluated the role of the H-NS nucleoid protein on the virulence features of K. pneumoniae. A Δhns mutant down-regulated the mrkA pilin gene and biofilm formation was affected. In contrast, capsule expression was derepressed in the absence of H-NS conferring a hypermucoviscous phenotype. Moreover, H-NS deficiency affected the K. pneumoniae adherence to epithelial cells such as A549 and HeLa cells. In infection experiments using RAW264.7 and THP-1 differentiated macrophages, the Δhns mutant was less phagocytized than the wild-type strain. This phenotype was likely due to the low adherence to these phagocytic cells. Taken together, our data indicate that H-NS nucleoid protein is a crucial regulator of both T3P and CPS of K. pneumoniae.
Progress on New Hepatitis C Virus Targets: NS2 and NS5A

NASA Astrophysics Data System (ADS)

Marcotrigiano, Joseph

Hepatitis C virus (HCV) is a major global health problem, affecting about 170 million people worldwide. Chronic infection can lead to cirrhosis and liver cancer. The replication machine of HCV is a multi-subunit membrane associated complex, consisting of nonstructural proteins (NS2-5B), which replicate the viral RNA genome. The structures of NS5A and NS2 were recently determined. NS5A is an essential replicase component that also modulates numerous cellular processes ranging from innate immunity to cell growth and survival. The structure reveals a novel protein fold, a new zinc coordination motif, a disulfide bond and a dimer interface. Analysis of molecular surfaces suggests the location of the membrane interaction surface of NS5A, as well as hypothetical protein and RNA binding sites. NS2 is one of two virally encoded proteases that are required for processing the viral polyprotein into the mature nonstructural proteins. NS2 is a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer and the nucleophilic cysteine by the other. The C-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. The structure also reveals possible sites of membrane interaction, a rare cis-proline residue, and highly conserved dimer contacts. The novel features of both structures have changed the current view of HCV polyprotein replication and present new opportunities for antiviral drug design.
Negative gating modulation by (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) depends on residues in the inner pore vestibule: pharmacological evidence of deep-pore gating of K(Ca)2 channels.

PubMed

Jenkins, David Paul; Strøbæk, Dorte; Hougaard, Charlotte; Jensen, Marianne L; Hummel, Rene; Sørensen, Ulrik S; Christophersen, Palle; Wulff, Heike

2011-06-01

Acting as a negative gating modulator, (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphthylamine (NS8593) shifts the apparent Ca(2+)-dependence of the small-conductance Ca(2+)-activated K(+) channels K(Ca)2.1-2.3 to higher Ca(2+) concentrations. Similar to the positive K(Ca) channel-gating modulators 1-ethyl-2-benzimidazolinone (1-EBIO) and cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methylpyrimidin-4-yl]-amine (CyPPA), the binding site for NS8593 has been assumed to be located in the C-terminal region, in which these channels interact with their Ca(2+) sensor calmodulin. However, by using a progressive chimeric approach, we were able to localize the site-of-action of NS8593 to the K(Ca)2 pore. For example, when we transferred the C terminus from the NS8593-insensitive intermediate-conductance K(Ca)3.1 channel to K(Ca)2.3, the chimeric channel remained as sensitive to NS8593 as wild-type K(Ca)2.3. In contrast, when we transferred the K(Ca)2.3 pore to K(Ca)3.1, the channel became sensitive to NS8593. Using site-directed mutagenesis, we subsequently identified two specific residues in the inner vestibule of K(Ca)2.3 (Ser507 and Ala532) that determined the effect of NS8593. Mutation of these residues to the corresponding residues in K(Ca)3.1 (Thr250 and Val275) made K(Ca)2.3 insensitive to NS8593, whereas introduction of serine and alanine into K(Ca)3.1 was sufficient to render this channel highly sensitive to NS8593. It is noteworthy that the same two residue positions have been found previously to mediate sensitivity of K(Ca)3.1 to clotrimazole and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). The location of Ser507 in the pore-loop near the selectivity filter and Ala532 in an adjacent position in S6 are within the region predicted to contain the K(Ca)2 channel gate. Hence, we propose that NS8593-mediated gating modulation occurs via interaction with gating structures at a position deep within the inner pore vestibule.
The T4 Phage DNA Mimic Protein Arn Inhibits the DNA Binding Activity of the Bacterial Histone-like Protein H-NS*

PubMed Central

Ho, Chun-Han; Wang, Hao-Ching; Ko, Tzu-Ping; Chang, Yuan-Chih; Wang, Andrew H.-J.

2014-01-01

The T4 phage protein Arn (Anti restriction nuclease) was identified as an inhibitor of the restriction enzyme McrBC. However, until now its molecular mechanism remained unclear. In the present study we used structural approaches to investigate biological properties of Arn. A structural analysis of Arn revealed that its shape and negative charge distribution are similar to dsDNA, suggesting that this protein could act as a DNA mimic. In a subsequent proteomic analysis, we found that the bacterial histone-like protein H-NS interacts with Arn, implying a new function. An electrophoretic mobility shift assay showed that Arn prevents H-NS from binding to the Escherichia coli hns and T4 p8.1 promoters. In vitro gene expression and electron microscopy analyses also indicated that Arn counteracts the gene-silencing effect of H-NS on a reporter gene. Because McrBC and H-NS both participate in the host defense system, our findings suggest that T4 Arn might knock down these mechanisms using its DNA mimicking properties. PMID:25118281
Structural basis for the blockade of MATE multidrug efflux pumps

DOE PAGES

Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; ...

2015-08-06

Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H + or Na + electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H +-coupled DinF transporter, as well as of an H +-coupled DinF and a Na +-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm themore » biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.« less
H-NS regulates the Vibrio parahaemolyticus type VI secretion system 1

PubMed Central

Salomon, Dor; Klimko, John A.

2014-01-01

The marine bacterium Vibrio parahaemolyticus, a major cause of food-borne gastroenteritis, employs a type VI secretion system 1 (T6SS1), a recently discovered protein secretion system, to combat competing bacteria. Environmental signals such as temperature, salinity, cell density and surface sensing, as well as the quorum-sensing master regulator OpaR, were previously reported to regulate T6SS1 activity and expression. In this work, we set out to identify additional transcription regulators that control the tightly regulated T6SS1 activity. To this end, we determined the effect of deletions in several known virulence regulators and in two regulators encoded within the T6SS1 gene cluster on expression and secretion of the core T6SS component Hcp1 and on T6SS1-mediated anti-bacterial activity. We report that VP1391 and VP1407, transcriptional regulators encoded within the T6SS1 gene cluster, are essential for T6SS1 activity. Moreover, we found that H-NS, a bacterial histone-like nucleoid structuring protein, which mediates transcription silencing of horizontally acquired genes, serves as a repressor of T6SS1. We also show that activation of surface sensing and high salt conditions alleviate the H-NS-mediated repression. Our results shed light on the complex network of environmental signals and transcription regulators that govern the tight regulation over T6SS1 activity. PMID:24987102

NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement on the Path to Activation

PubMed Central

Guo, Jiqing; Cheng, Yen May; Lees-Miller, James P.; Perissinotti, Laura L.; Claydon, Tom W.; Hull, Christina M.; Thouta, Samrat; Roach, Daniel E.; Durdagi, Serdar; Noskov, Sergei Y.; Duff, Henry J.

2015-01-01

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V1/2 of activation of L529I (−34 ± 4 mV) is similar to wild-type (WT) (−37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (−131 ± 4 mV for K525C and −120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed. PMID:25809253
Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation.

PubMed

Odermatt, Nina T; Sala, Claudia; Benjak, Andrej; Kolly, Gaëlle S; Vocat, Anthony; Lupien, Andréanne; Cole, Stewart T

2017-08-15

A handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the h istone-like n ucleoid- s tructuring protein, is one of these NAPs and protects Escherichia coli from foreign gene expression. Though lacking any sequence similarity with E. coli H-NS, Rv3852 was annotated as the H-NS ortholog in Mycobacterium tuberculosis , as it resembles human histone H1. The role of Rv3852 was thoroughly investigated by immunoblotting, subcellular localization, construction of an unmarked rv3852 deletion in the M. tuberculosis genome, and subsequent analysis of the resulting Δ rv3852 strain. We found that Rv3852 was predominantly present in the logarithmic growth phase with a decrease in protein abundance in stationary phase. Furthermore, it was strongly associated with the cell membrane and not detected in the cytosolic fraction, nor was it secreted. The Δ rv3852 strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in the Δ rv3852 mutant strain compared to that in the parental H37Rv strain showed no difference in nucleoid position or spread. Infection of macrophages as well as severe combined immunodeficient (SCID) mice demonstrated that loss of Rv3852 had no detected influence on the virulence of M. tuberculosis We thus conclude that M. tuberculosis Rv3852 is not involved in pathogenesis and is not a typical NAP. The existence of an as yet undiscovered Rv3852 ortholog cannot be excluded, although this role is likely played by the well-characterized Lsr2 protein. IMPORTANCE Mycobacterium tuberculosis is the causative agent of the lung infection tuberculosis, claiming more than 1.5 million lives each year. To understand the mechanisms of latent infection, where M. tuberculosis can stay dormant inside the human host, we require deeper knowledge of the basic biology and of the regulatory networks. In our work, we show that Rv3852, previously annotated as H-NS, is not
Exploring the Lead Compounds for Zika Virus NS2B-NS3 Protein: an e-Pharmacophore-Based Approach.

PubMed

Rohini, K; Agarwal, Pratika; Preethi, B; Shanthi, V; Ramanathan, K

2018-06-18

The rapid spread of the Zika virus and its association with the abnormal brain development constitute a global health emergency. With a continuing spread of the mosquito vector, the exposure is expected to accelerate in the coming years. Despite number of efforts, there is still no proper vaccine or medicine to combat this virus. Of note, the NS2B-NS3 protein is proven to be the potential target for the Zika virus therapeutics. Hence, e-pharmacophore-based drug design strategy was employed to identify potent inhibitors of NS2B-NS3 protein from ASINEX database consisting of 467,802 molecules. A 3D e-pharmacophore model was generated using PHASE module of Schrödinger Suite. The generated model consists of one hydrogen bond acceptor (A), two hydrogen bond donors (D), and two aromatic rings (R), ADDRR. The model was further evaluated for its ability to screen actives using enrichment analysis. Subsequently, high-throughput virtual screening protocol was employed, and the resultant hit molecules were also examined for its binding free energies and ADME properties using Prime MM-GBSA and Qikprop module of Schrodinger packages, respectively. Finally, the screened hit molecule was subjected to molecular dynamics simulation to examine its stability. Overall, the results from our analysis suggest that compound BAS 19192837 could be a potent inhibitor for the NS2B-NS3 protein of the Zika virus. It is also noteworthy to mention that our results are in good agreement with literature evidences. We hope that this result is of immense importance in designing potential drug molecules to combat the spread of Zika virus in the near future.
Magnetic Resonance Imaging-Guided Multi-Drug Chemotherapy and Photothermal Synergistic Therapy with pH and NIR-Stimulation Release.

PubMed

Yang, Ji-Chun; Chen, Yang; Li, Yu-Hao; Yin, Xue-Bo

2017-07-12

The combination of multidrug chemotherapy and photothermal therapy (PTT) enhances cancer therapeutic efficacy. Herein, we develop a simple and smart pH/NIR dual-stimulus-responsive degradable mesoporous CoFe 2 O 4 @PDA@ZIF-8 sandwich nanocomposite. The mesoporous CoFe 2 O 4 core acts as T 2 -weighted magnetic resonance (MR) imaging probe, PTT agent, and loading platform of hydrophilic doxorubicin (DOX). A polydopamine (PDA) layer is used to avoid the premature leakage of DOX before arriving at tumor site, enhance PTT efficiency, and facilitate the integration of ZIF-8 (a kind of metal-organic framework). The ZIF-8 shell serves to encapsulate hydrophobic camptothecin (CPT) and as the switch for the pH and NIR stimulation-responsive release of the two drugs. Therefore, T 2 -weighted MR imaging-guided multidrug chemotherapy and PTT synergistic treatment is achieved. Two kinds of anticancer drugs, hydrophilic DOX and hydrophobic CPT, are successfully loaded in CoFe 2 O 4 and ZIF-8, respectively, so no mutual interference between the two drugs exists. A unique two-stage stepwise release process is exhibited for CPT and DOX with an interval of 12 h to improve the anticancer efficacy under the acidic microenvironment of tumor tissue. NIR irradiation achieves the burst drug-release and PTT after laser stimulation, simultaneously. With this smart design, high drug concentration is achieved at the tumor site by quick release, especially for the therapeutic drugs that show nonlinear pharmacokinetics, and PTT is integrated efficiently. Furthermore, negligible biotoxicity and a remarkable synergic antitumor effect of the hybrid nanocomposites are validated by HepG2 cells and tumor-bearing mice as models. Our multidrug delivery-releasing composite improves tumor therapeutic efficiency significantly compared with a single-drug chemotherapy system. The simple multifunctional composite system can be applied as an effective platform for personal nanomedicine with diagnosis, smart
The DNA-mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription.

PubMed

Melkina, Olga E; Goryanin, Ignatiy I; Zavilgelsky, Gennadii B

2016-11-01

The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.
Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs

USDA-ARS?s Scientific Manuscript database

The diversity of contemporary swine influenza virus (SIV) strains impedes effective immunization of swine herds. Mucosally delivered, attenuated virus vaccines are one approach with potential to provide broad cross-protection. Reverse genetics-derived H3N2 SIV virus with truncated NS1 (NS1delta126 T...
H-NS-like nucleoid-associated proteins, mobile genetic elements and horizontal gene transfer in bacteria.

PubMed

Dorman, Charles J

2014-09-01

Horizontal gene transfer plays an important role in the evolution of bacterial species, conferring new genetic traits on the recipient bacterium that extend its range of phenotypes and plasmids make important contributions to this process. However, the inappropriate expression of newly acquired genes may lead to a loss of competitive fitness, resulting in the elimination of the new gene-bacterium combination. It is thought that transcriptional silencing of horizontally acquired genes offers a route out of this dilemma and that nucleoid-associated proteins, especially those related to the H-NS protein, play a particularly important role in the silencing process. The discovery that many plasmids express orthologues of nucleoid-associated proteins adds an interesting dimension to current models of regulatory integration following lateral transfer of DNA. Other horizontally acquired genetic elements, such as genomic islands, also express nucleoid-associated proteins of their own. Here the interactions of H-NS-like nucleoid-associated proteins encoded by the core genome, genomic islands and plasmids are described. Copyright © 2014 Elsevier Inc. All rights reserved.
PARP12 suppresses Zika virus infection through PARP-dependent degradation of NS1 and NS3 viral proteins.

PubMed

Li, Lili; Zhao, Hui; Liu, Ping; Li, Chunfeng; Quanquin, Natalie; Ji, Xue; Sun, Nina; Du, Peishuang; Qin, Cheng-Feng; Lu, Ning; Cheng, Genhong

2018-06-19

Zika virus infection stimulates a type I interferon (IFN) response in host cells, which suppresses viral replication. Type I IFNs exert antiviral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). To screen for antiviral ISGs that restricted Zika virus replication, we individually knocked out 21 ISGs in A549 lung cancer cells and identified PARP12 as a strong inhibitor of Zika virus replication. Our findings suggest that PARP12 mediated the ADP-ribosylation of NS1 and NS3, nonstructural viral proteins that are involved in viral replication and modulating host defense responses. This modification of NS1 and NS3 triggered their proteasome-mediated degradation. These data increase our understanding of the antiviral activity of PARP12 and suggest a molecular basis for the potential development of therapeutics against Zika virus. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
NS Segment of a 1918 Influenza A Virus-Descendent Enhances Replication of H1N1pdm09 and Virus-Induced Cellular Immune Response in Mammalian and Avian Systems

PubMed Central

Petersen, Henning; Mostafa, Ahmed; Tantawy, Mohamed A.; Iqbal, Azeem A.; Hoffmann, Donata; Tallam, Aravind; Selvakumar, Balachandar; Pessler, Frank; Beer, Martin; Rautenschlein, Silke; Pleschka, Stephan

2018-01-01

The 2009 pandemic influenza A virus (IAV) H1N1 strain (H1N1pdm09) has widely spread and is circulating in humans and swine together with other human and avian IAVs. This fact raises the concern that reassortment between H1N1pdm09 and co-circulating viruses might lead to an increase of H1N1pdm09 pathogenicity in different susceptible host species. Herein, we explored the potential of different NS segments to enhance the replication dynamics, pathogenicity and host range of H1N1pdm09 strain A/Giessen/06/09 (Gi-wt). The NS segments were derived from (i) human H1N1- and H3N2 IAVs, (ii) highly pathogenic- (H5- or H7-subtypes) or (iii) low pathogenic avian influenza viruses (H7- or H9-subtypes). A significant increase of growth kinetics in A549 (human lung epithelia) and NPTr (porcine tracheal epithelia) cells was only noticed in vitro for the reassortant Gi-NS-PR8 carrying the NS segment of the 1918-descendent A/Puerto Rico/8/34 (PR8-wt, H1N1), whereas all other reassortants showed either reduced or comparable replication efficiencies. Analysis using ex vivo tracheal organ cultures of turkeys (TOC-Tu), a species susceptible to IAV H1N1 infection, demonstrated increased replication of Gi-NS-PR8 compared to Gi-wt. Also, Gi-NS-PR8 induced a markedly higher expression of immunoregulatory and pro-inflammatory cytokines, chemokines and interferon-stimulated genes in A549 cells, THP-1-derived macrophages (dHTP) and TOC-Tu. In vivo, Gi-NS-PR8 induced an earlier onset of mortality than Gi-wt in mice, whereas, 6-week-old chickens were found to be resistant to both viruses. These data suggest that the specific characteristics of the PR8 NS segments can impact on replication, virus induced cellular immune responses and pathogenicity of the H1N1pdm09 in different avian and mammalian host species. PMID:29623073
Uncommon and Emissive {[Au2(C3H6NS2)2][Au(C3H6NS2)2]2(PF6)2} Mixed Au+ and Au3+ Pseudotetranuclear Crystalline Compound: Synthesis, Structural Characterization, and Optical Properties.

PubMed

Langaro, Ana P; Souza, Ana K R; Morassuti, Claudio Y; Lima, Sandro M; Casagrande, Gleison A; Deflon, Victor M; Nunes, Luiz A O; Da Cunha Andrade, Luis H

2016-11-23

An uncommon emissive pseudotetranuclear compound, {[Au 2 (C 3 H 6 NS 2 ) 2 ][Au(C 3 H 6 NS 2 ) 2 ] 2 (PF 6 ) 2 }, was synthesized and characterized in terms of its structure and optical properties. The synthesis produced a crystalline compound composed of four gold atoms with two different oxidation states (Au + and Au 3+ ) in the same crystalline structure. The title complex belonged to a triclinic crystalline system involving the centrosymmetric P1̅ space group. X-ray diffractometry and vibrational spectroscopy (infrared, Raman, and SERS) were used for structural characterization of the new crystal. The vibrational spectroscopy techniques supported the X-ray diffraction results and confirmed the presence of bonds including Au-Au and Au-S. Optical characterization performed using UV-vis spectroscopy showed that under ultraviolet excitation, the emissive crystalline complex presented characteristic broad luminescent bands centered at 420 and 670 nm.
Regulation of the yjjQ-bglJ Operon, Encoding LuxR-Type Transcription Factors, and the Divergent yjjP Gene by H-NS and LeuO▿ †

PubMed Central

Stratmann, Thomas; Madhusudan, S.; Schnetz, Karin

2008-01-01

The yjjQ and bglJ genes encode LuxR-type transcription factors conserved in several enterobacterial species. YjjQ is a potential virulence factor in avian pathogenic Escherichia coli. BglJ counteracts the silencing of the bgl (β-glucoside) operon by H-NS in E. coli K-12. Here we show that yjjQ and bglJ form an operon carried by E. coli K-12, whose expression is repressed by the histone-like nucleoid structuring (H-NS) protein. The LysR-type transcription factor LeuO counteracts this repression. Furthermore, the yjjP gene, encoding a membrane protein of unknown function and located upstream in divergent orientation to the yjjQ-bglJ operon, is likewise repressed by H-NS. Mapping of the promoters as well as the H-NS and LeuO binding sites within the 555-bp intergenic region revealed that H-NS binds to the center of the AT-rich regulatory region and distal to the divergent promoters. LeuO sites map to the center and to positions distal to the yjjQ promoters, while one LeuO binding site overlaps with the divergent yjjP promoter. This latter LeuO site is required for full derepression of the yjjQ promoters. The arrangement of regulatory sites suggests that LeuO restructures the nucleoprotein complex formed by H-NS. Furthermore, the data support the conclusion that LeuO, whose expression is likewise repressed by H-NS and which is a virulence factor in Salmonella enterica, is a master regulator that among other loci, also controls the yjjQ-bglJ operon and thus indirectly the presumptive targets of YjjQ and BglJ. PMID:18055596
H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease.

PubMed

Yang, Li-Yuan; Lin, Jun; Zhou, Bin; Liu, Yan-Gang; Zhu, Bao-Quan

2016-04-01

The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3β, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.
Conformational flexibility of DENV NS2B/NS3pro: from the inhibitor effect to the serotype influence

NASA Astrophysics Data System (ADS)

Piccirillo, Erika; Merget, Benjamin; Sotriffer, Christoph A.; do Amaral, Antonia T.

2016-03-01

The dengue virus (DENV) has four well-known serotypes, namely DENV1 to DENV4, which together cause 50-100 million infections worldwide each year. DENV NS2B/NS3pro is a protease recognized as a valid target for DENV antiviral drug discovery. However, NS2B/NS3pro conformational flexibility, involving in particular the NS2B region, is not yet completely understood and, hence, a big challenge for any virtual screening (VS) campaign. Molecular dynamics (MD) simulations were performed in this study to explore the DENV3 NS2B/NS3pro binding-site flexibility and obtain guidelines for further VS studies. MD simulations were done with and without the Bz-nKRR-H inhibitor, showing that the NS2B region stays close to the NS3pro core even in the ligand-free structure. Binding-site conformational states obtained from the simulations were clustered and further analysed using GRID/PCA, identifying four conformations of potential importance for VS studies. A virtual screening applied to a set of 31 peptide-based DENV NS2B/NS3pro inhibitors, taken from literature, illustrated that selective alternative pharmacophore models can be constructed based on conformations derived from MD simulations. For the first time, the NS2B/NS3pro binding-site flexibility was evaluated for all DENV serotypes using homology models followed by MD simulations. Interestingly, the number of NS2B/NS3pro conformational states differed depending on the serotype. Binding-site differences could be identified that may be crucial to subsequent VS studies.
Inhibitor Bound Dengue NS2B-NS3pro Reveals Multiple Dynamic Binding Modes.

PubMed

Gibbs, Alan C; Steele, Ruth; Liu, Gaohua; Tounge, Brett A; Montelione, Gaetano T

2018-03-13

Dengue virus poses a significant global health threat as the source of increasingly deleterious dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. As no specific antiviral treatment exists for dengue infection, considerable effort is being applied to discover therapies and drugs for maintenance and prevention of these afflictions. The virus is primarily transmitted by mosquitoes, and infection occurs following viral endocytosis by host cells. Upon entering the cell, viral RNA is translated into a large multisubunit polyprotein which is post-translationally cleaved into mature, structural and nonstructural (NS) proteins. The viral genome encodes the enzyme to carry out cleavage of the large polyprotein, specifically the NS2B-NS3pro cofactor-protease complex-a target of high interest for drug design. One class of recently discovered NS2B-NS3pro inhibitors is the substrate-based trifluoromethyl ketone containing peptides. These compounds interact covalently with the active site Ser135 via a hemiketal adduct. A detailed picture of the intermolecular protease/inhibitor interactions of the hemiketal adduct is crucial for rational drug design. We demonstrate, through the use of protein- and ligand-detected solution-state 19 F and 1 H NMR methods, an unanticipated multibinding mode behavior of a representative of this class of inhibitors to dengue NS2B-NS3pro. Our results illustrate the highly dynamic nature of both the covalently bound ligand and protease protein structure, and the need to consider these dynamics when designing future inhibitors in this class.
Implementation of quantum key distribution network simulation module in the network simulator NS-3

NASA Astrophysics Data System (ADS)

Mehic, Miralem; Maurhart, Oliver; Rass, Stefan; Voznak, Miroslav

2017-10-01

As the research in quantum key distribution (QKD) technology grows larger and becomes more complex, the need for highly accurate and scalable simulation technologies becomes important to assess the practical feasibility and foresee difficulties in the practical implementation of theoretical achievements. Due to the specificity of the QKD link which requires optical and Internet connection between the network nodes, to deploy a complete testbed containing multiple network hosts and links to validate and verify a certain network algorithm or protocol would be very costly. Network simulators in these circumstances save vast amounts of money and time in accomplishing such a task. The simulation environment offers the creation of complex network topologies, a high degree of control and repeatable experiments, which in turn allows researchers to conduct experiments and confirm their results. In this paper, we described the design of the QKD network simulation module which was developed in the network simulator of version 3 (NS-3). The module supports simulation of the QKD network in an overlay mode or in a single TCP/IP mode. Therefore, it can be used to simulate other network technologies regardless of QKD.
Imipenem represses CRISPR-Cas interference of DNA acquisition through H-NS stimulation in Klebsiella pneumoniae.

PubMed

Lin, Tzu-Lung; Pan, Yi-Jiun; Hsieh, Pei-Fang; Hsu, Chun-Ru; Wu, Meng-Chuan; Wang, Jin-Town

2016-08-17

Analysis of the genome of Klebsiella pneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5'-TTN-3' was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem.
Imipenem represses CRISPR-Cas interference of DNA acquisition through H-NS stimulation in Klebsiella pneumoniae

PubMed Central

Lin, Tzu-Lung; Pan, Yi-Jiun; Hsieh, Pei-Fang; Hsu, Chun-Ru; Wu, Meng-Chuan; Wang, Jin-Town

2016-01-01

Analysis of the genome of Klebsiella pneumoniae NTUH-K2044 strain revealed the presence of two clustered regularly interspaced short palindromic repeats (CRISPR) arrays separated with CRISPR-associated (cas) genes. Carbapenem-resistant K. pneumoniae isolates were observed to be less likely to have CRISPR-Cas than sensitive strains (5/85 vs. 22/132). Removal of the transcriptional repressor, H-NS, was shown to prevent the transformation of plasmids carrying a spacer and putative proto-spacer adjacent motif (PAM). The CRISPR-Cas system also decreased pUC-4K plasmid stability, resulting in plasmid loss from the bacteria with acquisition of new spacers. Analysis of the acquired proto-spacers in pUC-4K indicated that 5′-TTN-3′ was the preferred PAM in K. pneumoniae. Treatment of cells by imipenem induced hns expression, thereby decreasing cas3 expression and consequently repressed CRISPR-Cas activity resulted in increase of plasmid stability. In conclusion, NTUH-K2044 CRISPR-Cas contributes to decrease of plasmid transformation and stability. Through repression of CRISPR-Cas activity by induced H-NS, bacteria might be more able to acquire DNA to confront the challenge of imipenem. PMID:27531594
NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses

PubMed Central

DeDiego, Marta L.; Nogales, Aitor; Lambert-Emo, Kris; Martinez-Sobrido, Luis

2016-01-01

ABSTRACT Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus. IMPORTANCE Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence
H-NS Mutation-Mediated CRISPR-Cas Activation Inhibits Phage Release and Toxin Production of Escherichia coli Stx2 Phage Lysogen.

PubMed

Fu, Qiang; Li, Shiyu; Wang, Zhaofei; Shan, Wenya; Ma, Jingjiao; Cheng, Yuqiang; Wang, Hengan; Yan, Yaxian; Sun, Jianhe

2017-01-01

Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. There is limited understanding of the effect that an Escherichia coli ( E. coli ) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune system has on Stx phage lysogen. We investigated heat-stable nucleoid-structuring (H-NS) mutation-mediated CRISPR-Cas activation and its effect on E. coli Stx2 phage lysogen. The Δ hns mutant (MG1655Δ hns ) of the E. coli K-12 strain MG1655 was obtained. The Δ hns mutant lysogen that was generated after Stx phage lysogenic infection had a repressed growth status and showed subdued group behavior, including biofilm formation and swarming motility, in comparison to the wild-type strain. The de-repression effect of the H-NS mutation on CRISPR-Cas activity was then verified. The results showed that cas gene expression was upregulated and the transformation efficiency of the wild-type CRISPR plasmids was decreased, which may indicate activation of the CRISPR-Cas system. Furthermore, the function of CRISPR-Cas on Stx2 phage lysogen was investigated by activating the CRISPR-Cas system, which contains an insertion of the protospacer regions of the Stx2 phage Min27. The phage release and toxin production of four lysogens harboring the engineered CRISPRs were investigated. Notably, in the supernatant of the Δ hns mutant lysogen harboring the Min27 spacer, both the progeny phage release and the toxin production were inhibited after mitomycin C induction. These observations demonstrate that the H-NS mutation-activated CRISPR-Cas system plays a role in modifying the effects of the Stx2 phage lysogen. Our findings indicated that H-NS mutation-mediated CRISPR-Cas activation in E. coli protects bacteria against Stx2 phage lysogeny by inhibiting the phage release and toxin production of the lysogen.
Functional interplay among the flavivirus NS3 protease, helicase, and cofactors.

PubMed

Li, Kuohan; Phoo, Wint Wint; Luo, Dahai

2014-04-01

Flaviviruses are positive-sense RNA viruses, and many are important human pathogens. Nonstructural protein 2B and 3 of the flaviviruses (NS2BNS3) form an endoplasmic reticulum (ER) membrane-associated hetero-dimeric complex through the NS2B transmembrane region. The NS2BNS3 complex is multifunctional. The N-terminal region of NS3, and its cofactor NS2B fold into a protease that is responsible for viral polyprotein processing, and the C-terminal domain of NS3 possesses NTPase/RNA helicase activities and is involved in viral RNA replication and virus particle formation. In addition, NS2BNS3 complex has also been shown to modulate viral pathogenesis and the host immune response. Because of the essential functions that the NS2BNS3 complex plays in the flavivirus life cycle, it is an attractive target for antiviral development. This review focuses on the recent biochemical and structural advances of NS2BNS3 and provides a brief update on the current status of drug development targeting this viral protein complex.

Python Cathelicidin CATHPb1 Protects against Multidrug-Resistant Staphylococcal Infections by Antimicrobial-Immunomodulatory Duality.

PubMed

Cai, Shasha; Qiao, Xue; Feng, Lan; Shi, Nannan; Wang, Hui; Yang, Huaixin; Guo, Zhilai; Wang, Mengke; Chen, Yan; Wang, Yipeng; Yu, Haining

2018-03-08

Multidrug-resistant Staphylococcus aureus, including MRSA (methicillin-resistant) and VRSA (vancomycin-resistant), causes serious healthcare-associated infections, even sepsis and death. Here, we identified six novel cathelicidins (CATHPb1-6) from Python bivittatu, and CATHPb1 displayed the best in vitro pharmacological and toxicological profile. We further show that CATHPb1 exhibited evident protection in mice MRSA/VRSA infection models, given either 24 h before or 4 h after infection. The protection was all effective through different administration routes, but was blocked by in vivo depletion of monocyte/macrophages or neutrophils. CATHPb1 can rapidly and massively modulate macrophages/monocytes and neutrophils trafficking to the infection site, and potentiate their bactericidal functions. Meanwhile, CATHPb1 remarkably augmented neutrophil-mediated bacteria killing by facilitating neutrophil extracellular traps (NETs) formation and preventing its degradation. Acting through MAPKs and NF-κB pathways, CATHPb1 selectively enhanced the levels of chemokines while reducing the production of pro-inflammatory cytokines without undesirable toxicities. The much improved serum half-life and stabilities confer CATHPb1 an excellent prospect to become a novel therapeutic agent against multidrug-resistant staphylococcal infections.
Multidrug Resistance: Physiological Principles and Nanomedical Solutions

PubMed Central

Storm, Gert; Kiessling, Fabian; Lammers, Twan

2014-01-01

Multidrug (MDR) resistance is a pathophysiological phenomenon employed by cancer cells which limits the prolonged and effective use of chemotherapeutic agents. MDR is primarily based on the over-expression of drug efflux pumps in the cellular membrane. Prominent examples of such efflux pumps, which belong to the ATP-binding cassette (ABC) superfamily of proteins, are Pgp (P-glycoprotein) and MRP (multidrug resistance-associated protein), nowadays officially known as ABCB1 and ABCC1. Over the years, several strategies have been evaluated to overcome MDR, based not only on the use of low-molecular-weight MDR modulators, but also on the implementation of 1-100(0) nm-sized drug delivery systems. In the present manuscript, after introducing the most important physiological principles of MDR, we summarize prototypic nanomedical strategies to overcome multidrug resistance, including the use of carrier materials with intrinsic anti-MDR properties, the use of nanomedicines to modify the mode of cellular uptake, and the co-formulation of chemotherapeutic drugs together with low- and high-molecular-weight MDR inhibitors within a single drug delivery system. While certain challenges still need to be overcome before such constructs and concepts can be widely applied in the clinic, the insights obtained and the progress made strongly suggest that nanomedicine formulations hold significant potential for improving the treatment of multidrug-resistant malignancies. PMID:24120954
PGLa-H tandem-repeat peptides active against multidrug resistant clinical bacterial isolates.

PubMed

Rončević, Tomislav; Gajski, Goran; Ilić, Nada; Goić-Barišić, Ivana; Tonkić, Marija; Zoranić, Larisa; Simunić, Juraj; Benincasa, Monica; Mijaković, Marijana; Tossi, Alessandro; Juretić, Davor

2017-02-01

Antimicrobial peptides (AMPs) are promising candidates for new antibiotic classes but often display an unacceptably high toxicity towards human cells. A naturally produced C-terminal fragment of PGLa, named PGLa-H, has been reported to have a very low haemolytic activity while maintaining a moderate antibacterial activity. A sequential tandem repeat of this fragment, diPGLa-H, was designed, as well as an analogue with a Val to Gly substitution at a key position. These peptides showed markedly improved in vitro bacteriostatic and bactericidal activity against both reference strains and multidrug resistant clinical isolates of Gram-negative and Gram-positive pathogens, with generally low toxicity for human cells as assessed by haemolysis, cell viability, and DNA damage assays. The glycine substitution analogue, kiadin, had a slightly better antibacterial activity and reduced haemolytic activity, which may correlate with an increased flexibility of its helical structure, as deduced using molecular dynamics simulations. These peptides may serve as useful lead compounds for developing anti-infective agents against resistant Gram-negative and Gram-positive species. Copyright © 2016 Elsevier B.V. All rights reserved.
A folate modified pH sensitive targeted polymeric micelle alleviated systemic toxicity of doxorubicin (DOX) in multi-drug resistant tumor bearing mice.

PubMed

Li, Xinru; Yang, Xiucong; Lin, Zhiqiang; Wang, Dan; Mei, Dong; He, Bing; Wang, Xiaoyou; Wang, Xueqing; Zhang, Qiang; Gao, Wei

2015-08-30

The purpose of this work was to demonstrate the advantages of a folate modified pH sensitive micelle system (HPPF) on reducing the systemic toxicity of antitumor drug doxorubicin (DOX) as well as increasing the antitumor efficacy on multi-drug resistant tumor. The micelle HPPF was fabricated by PHIS-PEG and Fol-PEG-PLA using dialysis method. Multi-drug resistant human breast-cancer cell (MCF-7Adr) was used to test the therapeutic effect of DOX loaded HPPF micelles (HPPF/DOX). Nude mice bearing MCF-7Adr tumor was used to evaluate the systemic toxicity of HPPF/DOX. The micelle HPPF was successfully prepared with good size uniformity and pH sensitivity. The in vitro experiments showed that HPPF significantly increased the intracellular level and cytotoxicity of DOX. The in vivo experiments demonstrated that HPPF had largely reduced the mortality and body weight loss, improved the animal status and decreased damages on heart and lung tissues comparing to free DOX. The HPPF/DOX could significantly increase the antitumor efficacy of DOX and largely alleviate the systemic side effects induced by high dose DOX in the treatment of multi-drug resistant tumor. Copyright © 2015 Elsevier B.V. All rights reserved.
NS1 Protein Mutation I64T Affects Interferon Responses and Virulence of Circulating H3N2 Human Influenza A Viruses.

PubMed

DeDiego, Marta L; Nogales, Aitor; Lambert-Emo, Kris; Martinez-Sobrido, Luis; Topham, David J

2016-11-01

Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus. Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence, the pathogenicity of
Dilatonic parallelizable NS-NS backgrounds

NASA Astrophysics Data System (ADS)

Kawano, Teruhiko; Yamaguchi, Satoshi

2003-08-01

We complete the classification of parallelizable NS-NS backgrounds in type II supergravity by adding the dilatonic case to the result of Figueroa-O'Farrill on the non-dilatonic case. We also study the supersymmetry of these parallelizable backgrounds. It is shown that all the dilatonic parallelizable backgrounds have sixteen supersymmetries.
STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

PubMed

Schöne, Tobias; Grimm, Lena Lisbeth; Sakai, Naoki; Zhang, Linlin; Hilgenfeld, Rolf; Peters, Thomas

2017-10-01

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3 pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3 pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3 pro , we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3 pro . Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC 50 ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3 pro . To identify the matching protein binding site, chemical shift perturbation studies employing 1 H, 15 N-TROSY-HSQC experiments with uniformly 2 H, 15 N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3 pro inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.
A Salmonella nanoparticle mimic overcomes multidrug resistance in tumours.

PubMed

Mercado-Lubo, Regino; Zhang, Yuanwei; Zhao, Liang; Rossi, Kyle; Wu, Xiang; Zou, Yekui; Castillo, Antonio; Leonard, Jack; Bortell, Rita; Greiner, Dale L; Shultz, Leonard D; Han, Gang; McCormick, Beth A

2016-07-25

Salmonella enterica serotype Typhimurium is a food-borne pathogen that also selectively grows in tumours and functionally decreases P-glycoprotein (P-gp), a multidrug resistance transporter. Here we report that the Salmonella type III secretion effector, SipA, is responsible for P-gp modulation through a pathway involving caspase-3. Mimicking the ability of Salmonella to reverse multidrug resistance, we constructed a gold nanoparticle system packaged with a SipA corona, and found this bacterial mimic not only accumulates in tumours but also reduces P-gp at a SipA dose significantly lower than free SipA. Moreover, the Salmonella nanoparticle mimic suppresses tumour growth with a concomitant reduction in P-gp when used with an existing chemotherapeutic drug (that is, doxorubicin). On the basis of our finding that the SipA Salmonella effector is fundamental for functionally decreasing P-gp, we engineered a nanoparticle mimic that both overcomes multidrug resistance in cancer cells and increases tumour sensitivity to conventional chemotherapeutics.
The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

PubMed Central

URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

2015-01-01

The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820
Attenuation of nicotine taking and seeking in rats by the stoichiometry-selective alpha4beta2 nicotinic acetylcholine receptor positive allosteric modulator NS9283.

PubMed

Maurer, John J; Sandager-Nielsen, Karin; Schmidt, Heath D

2017-02-01

The rewarding and reinforcing effects of nicotine are produced, in large part, by activation of neuronal α4β2* nicotinic acetylcholine receptors (nAChRs), pentameric protein complexes comprised of different stoichiometries of α4 and β2 subunits. However, little is known about the functional role of distinct subtypes of α4β2* nAChRs in nicotine addiction. NS9283 represents a new class of stoichiometry-selective positive allosteric modulators (PAMs) that selectively bind to α4β2 nAChRs containing three α4 and two β2 subunits (3(α4)2(β2) nAChRs). The present experiments were designed to determine the effects of NS9283 on nicotine self-administration and the reinstatement of nicotine-seeking behavior, an animal model of smoking relapse. Parallel studies of sucrose self-administration and reinstatement were conducted in separate cohorts of rats to determine if the effects of NS9283 generalized to other reinforced behaviors. Acute and repeated administration of NS9283 dose-dependently reduced nicotine self-administration and reinstatement in male Sprague Dawley rats. These effects were reinforcer specific as no effects of NS9283 on sucrose self-administration and reinstatement were noted. NS9283 also failed to substitute for nicotine in supporting self-administration behavior suggesting that, at the doses tested, NS9283 alone is not reinforcing. Taken together, these results provide compelling evidence that stoichiometry-selective PAMs of 3(α4)2(β2) nAChRs attenuate nicotine taking and seeking in rats and suggest that targeting 3(α4)2(β2) nAChRs may represent a promising therapeutic strategy for preventing smoking relapse.
Characterization of NS5A and NS5B Resistance-Associated Substitutions from Genotype 1 Hepatitis C Virus Infected Patients in a Portuguese Cohort.

PubMed

Brandão, Ruben; Marcelino, Rute; Gonçalves, Fátima; Diogo, Isabel; Carvalho, Ana; Cabanas, Joaquim; Costa, Inês; Brogueira, Pedro; Ventura, Fernando; Miranda, Ana; Mansinho, Kamal; Gomes, Perpétua

2018-04-26

This study is focused on the prevalent NS5 coding region resistance-associated substitutions (RASs) in DAA-naive genotype (GT)1 HCV-infected patients and their potential impact on success rates. Plasma RNA from 81 GT1 HCV-infected patients was extracted prior to an in-house nested RT-PCR of the NS5 coding region, which is followed by Sanger population sequencing. NS5A RASs were present in 28.4% (23/81) of all GT1-infected patients with 9.9% (8/81) having the Y93C/H mutation. NS5B RASs showed a prevalence of 14.8% (12/81) and were only detected in GT1b. Overall 38.3% (31/81) of all GT1 HCV-infected patients presented baseline RASs. The obtained data supports the usefulness of resistance testing prior to treatment since a statistically significant association was found between treatment failure and the baseline presence of specific NS5 RASs known as Y93C/H ( p = 0.04).
The CRISPR/Cas Immune System Is an Operon Regulated by LeuO, H-NS, and Leucine-Responsive Regulatory Protein in Salmonella enterica Serovar Typhi ▿ †

PubMed Central

Medina-Aparicio, L.; Rebollar-Flores, J. E.; Gallego-Hernández, A. L.; Vázquez, A.; Olvera, L.; Gutiérrez-Ríos, R. M.; Calva, E.; Hernández-Lucas, I.

2011-01-01

Prokaryotes have developed multiple strategies to survive phage attack and invasive DNA. Recently, a novel genetic program denominated the CRISPR/Cas system was demonstrated to have a role in these biological processes providing genetic immunity. This defense mechanism is widespread in the Archaea and Bacteria, suggesting an ancient origin. In the last few years, progress has been made regarding the functionality of the CRISPR/Cas system; however, many basic aspects of the system remain unknown. For instance, there are few studies about the conditions and regulators involved in its transcriptional control. In this work, we analyzed the transcriptional organization of the CRISPR/Cas system as well as the positive and negative regulators involved in its genetic expression in Salmonella enterica serovar Typhi. The results obtained show that in S. Typhi the CRISPR/Cas system is a LeuO-dependent operon silenced by the global regulator LRP, in addition to the previously known nucleoid-associated protein H-NS; both LRP and H-NS bind upstream and downstream of the transcriptional start site of casA. In this study, relevant nucleotides of the casA regulatory region that mediate its LeuO transcriptional activation were identified. Interestingly, specific growth conditions (N-minimal medium) were found for the LeuO-independent expression of the CRISPR/Cas system in S. Typhi. Thus, our work provides evidence that there are multiple modulators involved in the genetic expression of this immune system in S. Typhi IMSS-1. PMID:21398529
pH. Training Module 5.305.2.77.

ERIC Educational Resources Information Center

Kirkwood Community Coll., Cedar Rapids, IA.

This document is an instructional module package prepared in objective form for use by an instructor familiar with pH, measurement of pH with a pH meter and maintenance of pH meter electrodes. Included are objectives, instructor guides, student handouts and transparency masters. This module considers the definition of pH, types of electrodes and…
Modulation of expression and activity of intestinal multidrug resistance-associated protein 2 by xenobiotics

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

Tocchetti, Guillermo Nicolás

The multidrug resistance-associated protein 2 (MRP2/ABCC2) is a transporter that belongs to the ATP-binding cassette (ABC) superfamily. In the intestine, it is localized to the apical membrane of the enterocyte and plays a key role in limiting the absorption of xenobiotics incorporated orally. MRP2 may also play a role in systemic clearance of xenobiotics available from the serosal side of the intestine. MRP2 transports a wide range of substrates, mainly organic anions conjugated with glucuronic acid, glutathione and sulfate and its expression can be modulated by xenobiotics at transcriptional- and post-transcriptional levels. Transcriptional regulation is usually mediated by a groupmore » of nuclear receptors. The pregnane X receptor (PXR) is a major member of this group. Relevant drugs described to up-regulate intestinal MRP2 via PXR are rifampicin, spironolactone and carbamazepine, among others. The constitutive androstane receptor (CAR, NR1I3) was also reported to modulate MRP2 expression, phenobarbital being a typical activator. Dietary compounds, including micronutrients and other natural products, are also capable of regulating intestinal MRP2 expression transcriptionally. We have given them particular attention since the composition of the food ingested daily is not necessarily supervised and may result in interactions with therapeutic drugs. Post-transcriptional regulation of MRP2 activity by xenobiotics, e.g. as a consequence of inhibitory actions, is also described in this review. Unfortunately, only few studies report on drug-drug or nutrient-drug interactions as a consequence of modulation of intestinal MRP2 activity by xenobiotics. Future clinical studies are expected to identify additional interactions resulting in changes in efficacy or safety of therapeutic drugs. - Highlights: • Intestinal MRP2 (ABCC2) expression and activity can be regulated by xenobiotics. • PXR and CAR are major MRP2 modulators through a transcriptional mechanism. �
Extended Surface for Membrane Association in Zika Virus NS1 Structure

PubMed Central

Brown, W. Clay; Akey, David L.; Konwerski, Jamie; Tarrasch, Jeffrey T.; Skiniotis, Georgios; Kuhn, Richard J.; Smith, Janet L.

2018-01-01

The Zika virus, which is implicated in an increase in neonatal microcephaly and Guillain-Barré syndrome, has spread rapidly through tropical regions of the world. The virulence protein NS1 functions in genome replication and host immune system modulation. Here we report the crystal structure of full-length Zika virus NS1, revealing an elongated hydrophobic surface for membrane association and a polar surface that varies substantially among flaviviruses. PMID:27455458
Hippocampal A-type current and Kv4.2 channel modulation by the sulfonylurea compound NS5806.

PubMed

Witzel, Katrin; Fischer, Paul; Bähring, Robert

2012-12-01

We examined the effects of the sulfonylurea compound NS5806 on neuronal A-type channel function. Using whole-cell patch-clamp we studied the effects of NS5806 on the somatodendritic A-type current (I(SA)) in cultured hippocampal neurons and the currents mediated by Kv4.2 channels coexpressed with different auxiliary β-subunits, including both Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-related proteins (DPPs), in HEK 293 cells. The amplitude of the I(SA) component in hippocampal neurons was reduced in the presence of 20 μM NS5806. I(SA) decay kinetics were slowed and the recovery kinetics accelerated, but the voltage dependence of steady-state inactivation was shifted to more negative potentials by NS5806. The peak amplitudes of currents mediated by ternary Kv4.2 channel complexes, associated with DPP6-S (short splice-variant) and either KChIP2, KChIP3 or KChIP4, were potentiated and their macroscopic inactivation slowed by NS5806, whereas the currents mediated by binary Kv4.2 channels, associated only with DPP6-S, were suppressed, and the NS5806-mediated slowing of macroscopic inactivation was less pronounced. Neither potentiation nor suppression and no effect on current decay kinetics in the presence of NS5806 were observed for Kv4.2 channels associated with KChIP3 and the N-type inactivation-conferring DPP6a splice-variant. For all recombinant channel complexes, NS5806 slowed the recovery from inactivation and shifted the voltage dependence of steady-state inactivation to more negative potentials. Our results demonstrate the activity of NS5806 on native I(SA) and possible molecular correlates in the form of recombinant Kv4.2 channels complexed with different KChIPs and DPPs, and they shed some light on the mechanism of NS5806 action. Copyright © 2012 Elsevier Ltd. All rights reserved.
The possible existence of Pop III NS-BH binary and its detectability

NASA Astrophysics Data System (ADS)

Kinugawa, Tomoya; Nakamura, Takashi; Nakano, Hiroyuki

2017-02-01

In the population synthesis simulations of Pop III stars, many BH (black hole)-BH binaries with merger time less than the age of the Universe (τH) are formed, while NS (neutron star)-BH binaries are not. The reason is that Pop III stars have no metal so that no mass loss is expected. Then, in the final supernova explosion to NS, much mass is lost so that the semimajor axis becomes too large for Pop III NS-BH binaries to merge within τH . However it is almost established that the kick velocity of the order of 200 ‑500 km s‑1 exists for NS from the observation of the proper motion of the pulsar. Therefore, the semimajor axis of the half of NS-BH binaries can be smaller than that of the previous argument for Pop III NS-BH binaries to decrease the merging time. We perform population synthesis Monte Carlo simulations of Pop III NS-BH binaries including the kick of NS and find that the event rate of Pop III NS-BH merger rate is 1 Gpc‑3 yr‑1 . This suggests that there is a good chance of detecting Pop III NS-BH mergers in O2 (Observation run 2) of Advanced LIGO and Advanced Virgo from this autumn.
Modulation of the multidrug efflux pump EmrD-3 from Vibrio cholerae by Allium sativum extract and the bioactive agent allyl sulfide plus synergistic enhancement of antimicrobial susceptibility by A. sativum extract.

PubMed

Bruns, Merissa M; Kakarla, Prathusha; Floyd, Jared T; Mukherjee, Mun Mun; Ponce, Robert C; Garcia, John A; Ranaweera, Indrika; Sanford, Leslie M; Hernandez, Alberto J; Willmon, T Mark; Tolson, Grace L; Varela, Manuel F

2017-10-01

The causative agent of cholera, Vibrio cholerae, is a public health concern. Multidrug-resistant V. cholerae variants may reduce chemotherapeutic efficacies of severe cholera. We previously reported that the multidrug efflux pump EmrD-3 from V. cholerae confers resistance to multiple structurally distinct antimicrobials. Medicinal plant compounds are potential candidates for EmrD-3 efflux pump modulation. The antibacterial activities of garlic Allium sativum, although poorly understood, predicts that a main bioactive component, allyl sulfide, modulates EmrD-3 efflux. Thus, we tested whether A. sativum extract acts in synergy with antimicrobials and that a main bioactive component allyl sulfide inhibits EmrD-3 efflux. We found that A. sativum extract and allyl sulfide inhibited ethidium bromide efflux in cells harboring EmrD-3 and that A. sativum lowered the MICs of multiple antibacterials. We conclude that A. sativum and allyl sulfide inhibit EmrD-3 and that A. sativum extract synergistically enhances antibacterial agents.
4H-Chromene-based anticancer agents towards multi-drug resistant HL60/MX2 human leukemia: SAR at the 4th and 6th positions.

PubMed

Puppala, Manohar; Zhao, Xinghua; Casemore, Denise; Zhou, Bo; Aridoss, Gopalakrishnan; Narayanapillai, Sreekanth; Xing, Chengguo

2016-03-15

4H-Chromene-based compounds, for example, CXL017, CXL035, and CXL055, have a unique anticancer potential that they selectively kill multi-drug resistant cancer cells. Reported herein is the extended structure-activity relationship (SAR) study, focusing on the ester functional group at the 4th position and the conformation at the 6th position. Sharp SARs were observed at both positions with respect to cellular cytotoxic potency and selectivity between the parental HL60 and the multi-drug resistant HL60/MX2 cells. These results provide critical guidance for future medicinal optimization. Copyright © 2016. Published by Elsevier Ltd.
Influenza A H3N2 subtype virus NS1 protein targets into the nucleus and binds primarily via its C-terminal NLS2/NoLS to nucleolin and fibrillarin

PubMed Central

2012-01-01

Background Influenza A virus non-structural protein 1 (NS1) is a virulence factor, which is targeted into the cell cytoplasm, nucleus and nucleolus. NS1 is a multi-functional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. Previously, we have shown that the NS1 protein of the H3N2 subtype influenza viruses possesses a C-terminal nuclear localization signal (NLS) that also functions as a nucleolar localization signal (NoLS) and targets the protein into the nucleolus. Results Here, we show that the NS1 protein of the human H3N2 virus subtype interacts in vitro primarily via its C-terminal NLS2/NoLS and to a minor extent via its N-terminal NLS1 with the nucleolar proteins, nucleolin and fibrillarin. Using chimeric green fluorescence protein (GFP)-NS1 fusion constructs, we show that the nucleolar retention of the NS1 protein is determined by its C-terminal NLS2/NoLS in vivo. Confocal laser microscopy analysis shows that the NS1 protein colocalizes with nucleolin in nucleoplasm and nucleolus and with B23 and fibrillarin in the nucleolus of influenza A/Udorn/72 virus-infected A549 cells. Since some viral proteins contain NoLSs, it is likely that viruses have evolved specific nucleolar functions. Conclusion NS1 protein of the human H3N2 virus interacts primarily via the C-terminal NLS2/NoLS and to a minor extent via the N-terminal NLS1 with the main nucleolar proteins, nucleolin, B23 and fibrillarin. PMID:22909121

The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

PubMed

Tay, Moon Y F; Saw, Wuan Geok; Zhao, Yongqian; Chan, Kitti W K; Singh, Daljit; Chong, Yuwen; Forwood, Jade K; Ooi, Eng Eong; Grüber, Gerhard; Lescar, Julien; Luo, Dahai; Vasudevan, Subhash G

2015-01-23

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Sus scrofa miR-204 and miR-4331 Negatively Regulate Swine H1N1/2009 Influenza A Virus Replication by Targeting Viral HA and NS, Respectively.

PubMed

Zhang, Shishuo; Wang, Ruifang; Su, Huijuan; Wang, Biaoxiong; Sizhu, Suolang; Lei, Zhixin; Jin, Meilin; Chen, Huanchun; Cao, Jiyue; Zhou, Hongbo

2017-04-03

The prevalence of swine pandemic H1N1/2009 influenza A virus (SIV-H1N1/2009) in pigs has the potential to generate novel reassortant viruses, posing a great threat to human health. Cellular microRNAs (miRNAs) have been proven as promising small molecules for regulating influenza A virus replication by directly targeting viral genomic RNA. In this study, we predicted potential Sus scrofa (ssc-, swine) miRNAs targeting the genomic RNA of SIV-H1N1/2009 by RegRNA 2.0, and identified ssc-miR-204 and ssc-miR-4331 to target viral HA and NS respectively through dual-luciferase reporter assays. The messenger RNA (mRNA) levels of viral HA and NS were significantly suppressed when newborn pig trachea (NPTr) cells respectively overexpressed ssc-miR-204 and ssc-miR-4331 and were infected with SIV-H1N1/2009, whereas the suppression effect could be restored when respectively decreasing endogenous ssc-miR-204 and ssc-miR-4331 with inhibitors. Because of the importance of viral HA and NS in the life cycle of influenza A virus, ssc-miR-204 and ssc-miR-4331 exhibited an inhibition effect on SIV-H1N1/2009 replication. The antiviral effect was sequence-specific of SIV-H1N1/2009, for the target sites in HA and NS of H5N1 or H9N2 influenza A virus were not conserved. Furthermore, SIV-H1N1/2009 infection reversely downregulated the expression of ssc-miR-204 and ssc-miR-4331, which might facilitate the virus replication in the host. In summary, this work will provide us some important clues for controlling the prevalence of SIV-H1N1/2009 in pig populations.
West Nile Virus Temperature Sensitivity and Avian Virulence Are Modulated by NS1-2B Polymorphisms.

PubMed

Dietrich, Elizabeth A; Langevin, Stanley A; Huang, Claire Y-H; Maharaj, Payal D; Delorey, Mark J; Bowen, Richard A; Kinney, Richard M; Brault, Aaron C

2016-08-01

West Nile virus (WNV) replicates in a wide variety of avian species, which serve as reservoir and amplification hosts. WNV strains isolated in North America, such as the prototype strain NY99, elicit a highly pathogenic response in certain avian species, notably American crows (AMCRs; Corvus brachyrhynchos). In contrast, a closely related strain, KN3829, isolated in Kenya, exhibits a low viremic response with limited mortality in AMCRs. Previous work has associated the difference in pathogenicity primarily with a single amino acid mutation at position 249 in the helicase domain of the NS3 protein. The NY99 strain encodes a proline residue at this position, while KN3829 encodes a threonine. Introduction of an NS3-T249P mutation in the KN3829 genetic background significantly increased virulence and mortality; however, peak viremia and mortality were lower than those of NY99. In order to elucidate the viral genetic basis for phenotype variations exclusive of the NS3-249 polymorphism, chimeric NY99/KN3829 viruses were created. We show herein that differences in the NS1-2B region contribute to avian pathogenicity in a manner that is independent of and additive with the NS3-249 mutation. Additionally, NS1-2B residues were found to alter temperature sensitivity when grown in avian cells.
Escherichia coli Sequence Type 131 (ST131) Subclone H30 as an Emergent Multidrug-Resistant Pathogen Among US Veterans

PubMed Central

Colpan, Aylin; Johnston, Brian; Porter, Stephen; Clabots, Connie; Anway, Ruth; Thao, Lao; Kuskowski, Michael A.; Tchesnokova, Veronika; Sokurenko, Evgeni V.; Johnson, James R.; Allen, Bradley L.; Baracco, Gio J.; Bedimo, Roger; Bessesen, Mary; Bonomo, Robert A.; Brecher, Stephen M.; Brown, Sheldon T.; Castellino, Laila; Desai, Arundhati S.; Fernau, Fletcher; Fisher, Mark A.; Fleckenstein, James; Fleming, Carol S.; Fries, Narla J.; Kan, Virginia L.; Kauffman, Carol A.; Klutts, Stacey; Ohl, Michael; Russo, Thomas; Swiatlo, Andrea; Swiatlo, Edwin

2013-01-01

Background. Escherichia coli sequence type 131 (ST131), typically fluoroquinolone-resistant (FQ-R) and/or extended-spectrum β-lactamase (ESBL)–producing, has emerged globally. We assessed its prevalence and characteristics among US veterans. Methods. In 2011, 595 de-identified E. coli clinical isolates were collected systematically within 3 resistance groups (FQ-susceptible [FQ-S], FQ-R, and ESBL-producing) from 24 nationally distributed Veterans Affairs Medical Centers (VAMCs). ST131 and its H30 subclone were detected by polymerase chain reaction and compared with other E. coli for molecular traits, source, and resistance profiles. Results. ST131 accounted for 78% (184/236) of FQ-R and 64.2% (79/123) of ESBL-producing isolates, but only 7.2% (17/236) of FQ-S isolates (P < .001). The H30 subclone accounted for ≥95% of FQ-R and ESBL-producing, but only 12.5% of FQ-S, ST131 isolates (P < .001). By back-calculation, 28% of VAMC E. coli isolates nationally represented ST131. Overall, ST131 varied minimally in prevalence by specimen type, inpatient/outpatient source, or locale; was the most prevalent ST, followed distantly by ST95 and ST12 (13% each); and accounted for ≥40% (β-lactams), >50% (trimethoprim-sulfamethoxazole , multidrug), or >70% (ciprofloxacin, gentamicin) of total antimicrobial resistance. FQ-R and ESBL-producing ST131 isolates had higher virulence scores than corresponding non-ST131 isolates. ST131 pulsotypes overlapped extensively among VAMCs. Conclusions. Among US veterans, ST131, primarily its H30 subclone, accounts for most antimicrobial-resistant E. coli and is the dominant E. coli strain overall. Possible contributors include multidrug resistance, extensive virulence gene content, and ongoing transmission. Focused attention to ST131, especially its H30 subclone, could reduce infection-related morbidity, mortality, and costs among veterans. PMID:23926176
Molecular mechanism of influenza A NS1-mediated TRIM25 recognition and inhibition.

PubMed

Koliopoulos, Marios G; Lethier, Mathilde; van der Veen, Annemarthe G; Haubrich, Kevin; Hennig, Janosch; Kowalinski, Eva; Stevens, Rebecca V; Martin, Stephen R; Reis E Sousa, Caetano; Cusack, Stephen; Rittinger, Katrin

2018-05-08

RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1.
MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells.

PubMed

Klappe, Karin; Hinrichs, John W J; Kroesen, Bart-Jan; Sietsma, Hannie; Kok, Jan Willem

2004-07-01

Previously we have described a novel multidrug-resistant cell line, HT29(col), which displayed over expression of the multidrug-resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu C, Muller M. Int J Cancer 2000;87:172-8). In our study, long-term screening revealed that, during colchicine-induced acquisition of multidrug resistance in a new HT29(col) cell line, increases in GlcCer occurred concomitantly with upregulation of MRP1 expression. Both MRP1 and GlcCer were found enriched in Lubrol-insoluble membrane domains. The expression of MRP1 and GlcCer were tightly correlated, as indicated also by a reversal of both at the later stage of colchicine consolidation. Resistance to colchicine was determined by MRP1, while glucosylceramide synthase (GCS) did not contribute: 1). Resistance was fully inhibited by MK571. 2). GCS expression and activity were not upregulated in HT29(col) cells. 3). Inhibition of GCS did not affect MRP1-mediated efflux function or sensitivity to colchicine. Instead, overall sphingolipid metabolism was upregulated through an increased rate of ceramide biosynthesis. In conclusion, upregulation of MRP1 occurs in concert with upregulation of GlcCer during multidrug-resistance acquisition, and both are enriched in rafts. The increased GlcCer pool does not directly modulate MRP1 function and cell survival. Copyright 2004 Wiley-Liss, Inc.
Transferrin receptor-targeted pH-sensitive micellar system for diminution of drug resistance and targetable delivery in multidrug-resistant breast cancer

PubMed Central

Gao, Wei; Ye, Guihua; Duan, Xiaochuan; Yang, Xiaoying; Yang, Victor C

2017-01-01

The emergence of drug resistance is partially associated with overproduction of transferrin receptor (TfR). To overcome multidrug resistance (MDR) and achieve tumor target delivery, we designed a novel biodegradable pH-sensitive micellar system modified with HAIYPRH, a TfR ligand (7pep). First, the polymers poly(l-histidine)-coupled polyethylene glycol-2000 (PHIS-PEG2000) and 7pep-modified 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (7pep-DSPE-PEG2000) were synthesized, and the mixed micelles were prepared by blending of PHIS-PEG2000 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG2000) or 7pep-DSPE-PEG2000 (7-pep HD micelles). The micelles exhibited good size uniformity, high encapsulation efficiency, and a low critical micelle concentration. By changing the polymer ratio in the micellar formulation, the pH response range was specially tailored to pH ~6.0. When loaded with antitumor drug doxorubicin (DOX), the micelle showed an acid pH-triggering drug release profile. The cellular uptake and cytotoxicity study demonstrated that 7-pep HD micelles could significantly enhance the intracellular level and antitumor efficacy of DOX in multidrug-resistant cells (MCF-7/Adr), which attributed to the synergistic effect of poly(l-histidine)-triggered endolysosom escape and TfR-mediated endocytosis. Most importantly, the in vivo imaging study confirmed the target-ability of 7-pep HD micelles to MDR tumor. These findings indicated that 7-pep HD micelles would be a promising drug delivery system in the treatment of drug-resistant tumors. PMID:28223798
Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events.

PubMed

Wong, Vanessa K; Baker, Stephen; Pickard, Derek J; Parkhill, Julian; Page, Andrew J; Feasey, Nicholas A; Kingsley, Robert A; Thomson, Nicholas R; Keane, Jacqueline A; Weill, François-Xavier; Edwards, David J; Hawkey, Jane; Harris, Simon R; Mather, Alison E; Cain, Amy K; Hadfield, James; Hart, Peter J; Thieu, Nga Tran Vu; Klemm, Elizabeth J; Glinos, Dafni A; Breiman, Robert F; Watson, Conall H; Kariuki, Samuel; Gordon, Melita A; Heyderman, Robert S; Okoro, Chinyere; Jacobs, Jan; Lunguya, Octavie; Edmunds, W John; Msefula, Chisomo; Chabalgoity, Jose A; Kama, Mike; Jenkins, Kylie; Dutta, Shanta; Marks, Florian; Campos, Josefina; Thompson, Corinne; Obaro, Stephen; MacLennan, Calman A; Dolecek, Christiane; Keddy, Karen H; Smith, Anthony M; Parry, Christopher M; Karkey, Abhilasha; Mulholland, E Kim; Campbell, James I; Dongol, Sabina; Basnyat, Buddha; Dufour, Muriel; Bandaranayake, Don; Naseri, Take Toleafoa; Singh, Shalini Pravin; Hatta, Mochammad; Newton, Paul; Onsare, Robert S; Isaia, Lupeoletalalei; Dance, David; Davong, Viengmon; Thwaites, Guy; Wijedoru, Lalith; Crump, John A; De Pinna, Elizabeth; Nair, Satheesh; Nilles, Eric J; Thanh, Duy Pham; Turner, Paul; Soeng, Sona; Valcanis, Mary; Powling, Joan; Dimovski, Karolina; Hogg, Geoff; Farrar, Jeremy; Holt, Kathryn E; Dougan, Gordon

2015-06-01

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.
Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events

PubMed Central

Wong, Vanessa K; Baker, Stephen; Pickard, Derek J; Parkhill, Julian; Page, Andrew J; Feasey, Nicholas A; Kingsley, Robert A; Thomson, Nicholas R; Keane, Jacqueline A; Weill, François-Xavier; Edwards, David J; Hawkey, Jane; Harris, Simon R; Mather, Alison E; Cain, Amy K; Hadfield, James; Hart, Peter J; Thieu, Nga Tran Vu; Klemm, Elizabeth J; Glinos, Dafni A; Breiman, Robert F; Watson, Conall H; Kariuki, Samuel; Gordon, Melita A; Heyderman, Robert S; Okoro, Chinyere; Jacobs, Jan; Lunguya, Octavie; Edmunds, W John; Msefula, Chisomo; Chabalgoity, Jose A; Kama, Mike; Jenkins, Kylie; Dutta, Shanta; Marks, Florian; Campos, Josefina; Thompson, Corinne; Obaro, Stephen; MacLennan, Calman A; Dolecek, Christiane; Keddy, Karen H; Smith, Anthony M; Parry, Christopher M; Karkey, Abhilasha; Mulholland, E Kim; Campbell, James I; Dongol, Sabina; Basnyat, Buddha; Dufour, Muriel; Bandaranayake, Don; Naseri, Take Toleafoa; Singh, Shalini Pravin; Hatta, Mochammad; Newton, Paul; Onsare, Robert S; Isaia, Lupeoletalalei; Dance, David; Davong, Viengmon; Thwaites, Guy; Wijedoru, Lalith; Crump, John A; De Pinna, Elizabeth; Nair, Satheesh; Nilles, Eric J; Thanh, Duy Pham; Turner, Paul; Soeng, Sona; Valcanis, Mary; Powling, Joan; Dimovski, Karolina; Hogg, Geoff; Farrar, Jeremy; Holt, Kathryn E; Dougan, Gordon

2016-01-01

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species. PMID:25961941
Could GRB170817A be really correlated to an NS-NS merging?

NASA Astrophysics Data System (ADS)

Fargion, D.; Khlopov, M. Yu.; Oliva, P.

The exciting development of gravitational wave (GW) astronomy in the correlation of LIGO and VIRGO detection of GW signals makes possible to expect registration of effects of not only binary black hole (BH) coalescence but also binary neutron star (NS) merging accompanied by electromagnetic (gamma ray burst; GRB) signal. Here we consider the possibility that an NS, merging in an NS-NS or NS-BH system might be (soon) observed in correlation with any LIGO-VIRGO GWs detection. We analyze as an example the recent case of the short GRB170817A observed by Fermi and integral. The associated optical transient (OT) source in NGC4993 implies a rare near source, a consequent averaged large rate of such events (almost) compatible with expected NS-NS merging rate. However the expected beamed GRB (or short GRB) may be mostly aligned to a different direction than ours. Therefore, even soft GRB photons, spread more than hard ones, might be hardly able to shower to us. Nevertheless, a prompt spiraling electron turbine jet in largest magnetic fields, at the base of the NS-NS collapse, might shine by its tangential synchrotron radiation in spread way with its skimming photons shining in large open disk. The consequent solid angle for such soft disk gamma radiation may be large enough to be nevertheless often observed.
Linezolid susceptibility in Helicobacter pylori, including strains with multidrug resistance.

PubMed

Boyanova, Lyudmila; Evstatiev, Ivailo; Gergova, Galina; Yaneva, Penka; Mitov, Ivan

2015-12-01

Only a few studies have evaluated Helicobacter pylori susceptibility to linezolid. The aim of the present study was to assess linezolid susceptibility in H. pylori, including strains with double/multidrug resistance. The susceptibility of 53 H. pylori strains was evaluated by Etest and a breakpoint susceptibility testing method. Helicobacter pylori resistance rates were as follows: amoxicillin, 1.9%; metronidazole, 37.7%; clarithromycin, 17.0%; tetracycline, 1.9%; levofloxacin, 24.5%; and linezolid (>4 mg/L), 39.6%. The linezolid MIC50 value was 31.2-fold higher than that of clarithromycin and 10.5-fold higher than that of levofloxacin; however, 4 of 11 strains with double/multidrug resistance were linezolid-susceptible. The MIC range of the oxazolidinone agent was larger (0.125-64 mg/L) compared with those in the previous two reports. The linezolid resistance rate was 2.2-fold higher in metronidazole-resistant strains and in strains resistant to at least one antibiotic compared with the remaining strains. Briefly, linezolid was less active against H. pylori compared with clarithromycin and levofloxacin, and linezolid resistance was linked to resistance to metronidazole as well as to resistance to at least one antibiotic. However, linezolid activity against some strains with double/multidrug resistance may render the agent appropriate to treat some associated H. pylori infections following in vitro susceptibility testing of the strains. Clinical trials are required to confirm this suggestion. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Differential responses of rabbit ventricular and atrial transient outward current (Ito) to the Ito modulator NS5806.

PubMed

Cheng, Hongwei; Cannell, Mark B; Hancox, Jules C

2017-03-01

Transient outward potassium current (I to ) in the heart underlies phase 1 repolarization of cardiac action potentials and thereby affects excitation-contraction coupling. Small molecule activators of I to may therefore offer novel treatments for cardiac dysfunction, including heart failure and atrial fibrillation. NS5806 has been identified as a prototypic activator of canine I to This study investigated, for the first time, actions of NS5806 on rabbit atrial and ventricular I to Whole cell patch-clamp recordings of I to and action potentials were made at physiological temperature from rabbit ventricular and atrial myocytes. 10 μ mol/L NS5806 increased ventricular I to with a leftward shift in I to activation and accelerated restitution. At higher concentrations, stimulation of I to was followed by inhibition. The EC 50 for stimulation was 1.6 μ mol/L and inhibition had an IC 50 of 40.7 μ mol/L. NS5806 only inhibited atrial I to (IC 50 of 18 μ mol/L) and produced a modest leftward shifts in I to activation and inactivation, without an effect on restitution. 10 μ mol/L NS5806 shortened ventricular action potential duration (APD) at APD 20 -APD 90 but prolonged atrial APD NS5806 also reduced atrial AP upstroke and amplitude, consistent with an additional atrio-selective effect on Na + channels. In contrast to NS5806, flecainide, which discriminates between Kv1.4 and 4.x channels, produced similar levels of inhibition of ventricular and atrial I to NS5806 discriminates between rabbit ventricular and atrial I to, with mixed activator and inhibitor actions on the former and inhibitor actions against the later. NS5806 may be of significant value for pharmacological interrogation of regional differences in native cardiac I to . © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.
Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication.

PubMed

Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R; Li, Melody M H; Rice, Charles M; MacDonald, Margaret R

2016-01-06

DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is
Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

PubMed Central

Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R.; Li, Melody M. H.; Rice, Charles M.

2016-01-01

ABSTRACT DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCE Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single
Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

PubMed

Lin, Kuan-Hung; Ali, Akbar; Rusere, Linah; Soumana, Djade I; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-15

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors
Three Conformational Snapshots of the Hepatitis Virus NS3 Helicase Reveal a Ratchet Translocation Mechanism

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

Gu, M.; Rice, C

2010-01-01

A virally encoded superfamily-2 (SF2) helicase (NS3h) is essential for the replication of hepatitis C virus, a leading cause of liver disease worldwide. Efforts to elucidate the function of NS3h and to develop inhibitors against it, however, have been hampered by limited understanding of its molecular mechanism. Here we show x-ray crystal structures for a set of NS3h complexes, including ground-state and transition-state ternary complexes captured with ATP mimics (ADP {center_dot} BeF{sub 3} and ADP {center_dot} AlF{sub 4}{sup -}). These structures provide, for the first time, three conformational snapshots demonstrating the molecular basis of action for a SF2 helicase. Uponmore » nucleotide binding, overall domain rotation along with structural transitions in motif V and the bound DNA leads to the release of one base from the substrate base-stacking row and the loss of several interactions between NS3h and the 3{prime} DNA segment. As nucleotide hydrolysis proceeds into the transition state, stretching of a 'spring' helix and another overall conformational change couples rearrangement of the (d)NTPase active site to additional hydrogen-bonding between NS3h and DNA. Together with biochemistry, these results demonstrate a 'ratchet' mechanism involved in the unidirectional translocation and define the step size of NS3h as one base per nucleotide hydrolysis cycle. These findings suggest feasible strategies for developing specific inhibitors to block the action of this attractive, yet largely unexplored drug target.« less
Inhibition of vincristine binding to plasma membrane vesicles from daunorubicin-resistant Ehrlich ascites cells by multidrug resistance modulators.

PubMed Central

Sehested, M.; Jensen, P. B.; Skovsgaard, T.; Bindslev, N.; Demant, E. J.; Friche, E.; VindelÃ¸v, L.

1989-01-01

The multidrug resistance (MDR) phenotype is presumed to be mostly dependent on changes in the resistant cell plasma membrane, notably the emergence of a 170 kDa glycoprotein called P-glycoprotein, which facilitate increased drug efflux. We have previously demonstrated that ATP-enhanced binding of vincristine (VCR) to plasma membrane vesicles is much greater in MDR than in wild type cells. The present study has shown that VCR binding to MDR Ehrlich ascites tumour cell plasma membrane vesicles is inhibited 50% most efficiently by quinidine (0.5 microM) followed by verapamil (4.1 microM) and trifluoperazine (23.2 microM). This is the reverse order of the effect on whole cells where a ranking of efficiency in terms of enhancement of VCR accumulation, inhibition of VCR efflux, DNA perturbation and modulation of resistance in a clonogenic assay, was trifluoperazine greater than or equal to verapamil much greater than quinidine. The detergent Tween 80 inhibited VCR binding to plasma membrane vesicles at 0.001% v/v which agreed with the level which modulated resistance and increased VCR accumulation in whole cells. No effect was observed on daunorubicin binding to MDR plasma membrane vesicles after incubation with either Tween 80 (up to 0.1% v/v) or verapamil (up to 25 microM). We conclude that the effect of a modulating drug in reversing resistance to VCR correlates with its ability to raise intracellular VCR levels but not with its capability to inhibit VCR binding to the plasma membrane. Thus, enhancement of VCR accumulation in MDR cells is hardly solely due to competition for a drug binding site on P-glycoprotein. Furthermore, the lack of a demonstrable effect on daunorubicin binding to the plasma membrane by modulators points to transport mechanisms which do not utilise specific drug binding to the plasma membrane. PMID:2605092
Cloacael Carriage and Multidrug Resistance Escherichia coli O157:H7 from Poultry Farms, Eastern Ethiopia

PubMed Central

Shecho, Mude; Muktar, Yimer

2017-01-01

A cross-sectional study was carried out to determine antimicrobial drug resistance patterns of E. coli O157:H7 isolates and estimate the level of the pathogen. A total of 194 cloacae swab samples were collected randomly in two poultry farms. Standard cultural, biochemical, and serological (latex agglutination) methods were used to isolate E. coli O157:H7. The isolates were subjected to antimicrobial susceptibility testing using disc diffusion method. Out of 194 cloacae samples examined, 13.4% (n = 26) were found to be positive for E. coli O157:H7. The finding indicated differences in E. coli O157:H7 infection among the different risk factors. Chicken from Adele Poultry Farm showed higher E. coli O157:H7 infection (OR = 3.89) than Haramaya University poultry farm and young birds had more infection (OR = 4.62) than adult birds. Of the total 14 antimicrobials included in the panel of study, the susceptibility results were varied with 96.15% and 0% E. coli O157:H7 isolates expressing resistance to erythromycin, clindamycin, spectinomycin, and ciprofloxacin, respectively. Multidrug resistance to more than two antimicrobial agents was detected in 24 (92.30%) of the isolates. The study showed high presence of antimicrobial resistant isolates of E. coli O157:H7. Further study is required to better understand the ecology and evolution of bacterial resistance to antimicrobial agents. PMID:28349121
Cloacael Carriage and Multidrug Resistance Escherichia coli O157:H7 from Poultry Farms, Eastern Ethiopia.

PubMed

Shecho, Mude; Thomas, Naod; Kemal, Jelalu; Muktar, Yimer

2017-01-01

A cross-sectional study was carried out to determine antimicrobial drug resistance patterns of E. coli O157:H7 isolates and estimate the level of the pathogen. A total of 194 cloacae swab samples were collected randomly in two poultry farms. Standard cultural, biochemical, and serological (latex agglutination) methods were used to isolate E. coli O157:H7. The isolates were subjected to antimicrobial susceptibility testing using disc diffusion method. Out of 194 cloacae samples examined, 13.4% ( n = 26) were found to be positive for E. coli O157:H7. The finding indicated differences in E. coli O157:H7 infection among the different risk factors. Chicken from Adele Poultry Farm showed higher E. coli O157:H7 infection (OR = 3.89) than Haramaya University poultry farm and young birds had more infection (OR = 4.62) than adult birds. Of the total 14 antimicrobials included in the panel of study, the susceptibility results were varied with 96.15% and 0% E. coli O157:H7 isolates expressing resistance to erythromycin, clindamycin, spectinomycin, and ciprofloxacin, respectively. Multidrug resistance to more than two antimicrobial agents was detected in 24 (92.30%) of the isolates. The study showed high presence of antimicrobial resistant isolates of E. coli O157:H7. Further study is required to better understand the ecology and evolution of bacterial resistance to antimicrobial agents.
pH/NIR Light-Controlled Multidrug Release via a Mussel-Inspired Nanocomposite Hydrogel for Chemo-Photothermal Cancer Therapy

NASA Astrophysics Data System (ADS)

Ghavaminejad, Amin; Samarikhalaj, Melisa; Aguilar, Ludwig Erik; Park, Chan Hee; Kim, Cheol Sang

2016-09-01

This study reports on an intelligent composite hydrogel with both pH-dependent drug release in a cancer environment and heat generation based on NIR laser exposure, for the combined application of photothermal therapy (PTT) and multidrug chemotherapy. For the first time in the literature, Dopamine nanoparticle (DP) was incorporated as a highly effective photothermal agent as well as anticancer drug, bortezomib (BTZ) carrier inside a stimuli responsive pNIPAAm-co-pAAm hydrogel. When light is applied to the composite hydrogel, DP nanoparticle absorbs the light, which is dissipated locally as heat to impact cancer cells via hyperthermia. On the other hand, facile release of the anticancer drug BTZ from the surface of DP encapsulated hydrogel could be achieved due to the dissociation between catechol groups of DP and the boronic acid functionality of BTZ in typical acidic cancer environment. In order to increase the synergistic effect by dual drug delivery, Doxorubicin (DOXO) were also loaded to pNIPAAm-co-pAAm/DP-BTZ hydrogel and the effect of monotherapy as well as combined therapy were detailed by a complete characterization. Our results suggest that these mussel inspired nanocomposite with excellent heating property and controllable multidrug release can be considered as a potential material for cancer therapy.

Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus.

PubMed

Li, Xiao-Dan; Deng, Cheng-Lin; Ye, Han-Qing; Zhang, Hong-Lei; Zhang, Qiu-Yan; Chen, Dong-Dong; Zhang, Pan-Tao; Shi, Pei-Yong; Yuan, Zhi-Ming; Zhang, Bo

2016-06-15

Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis
NS309 decreases rat detrusor smooth muscle membrane potential and phasic contractions by activating SK3 channels

PubMed Central

Parajuli, Shankar P; Hristov, Kiril L; Soder, Rupal P; Kellett, Whitney F; Petkov, Georgi V

2013-01-01

Background and Purpose Overactive bladder (OAB) is often associated with abnormally increased detrusor smooth muscle (DSM) contractions. We used NS309, a selective and potent opener of the small or intermediate conductance Ca2+-activated K+ (SK or IK, respectively) channels, to evaluate how SK/IK channel activation modulates DSM function. Experimental Approach We employed single-cell RT-PCR, immunocytochemistry, whole cell patch-clamp in freshly isolated rat DSM cells and isometric tension recordings of isolated DSM strips to explore how the pharmacological activation of SK/IK channels with NS309 modulates DSM function. Key Results We detected SK3 but not SK1, SK2 or IK channels expression at both mRNA and protein levels by RT-PCR and immunocytochemistry in DSM single cells. NS309 (10 μM) significantly increased the whole cell SK currents and hyperpolarized DSM cell resting membrane potential. The NS309 hyperpolarizing effect was blocked by apamin, a selective SK channel inhibitor. NS309 inhibited the spontaneous phasic contraction amplitude, force, frequency, duration and tone of isolated DSM strips in a concentration-dependent manner. The inhibitory effect of NS309 on spontaneous phasic contractions was blocked by apamin but not by TRAM-34, indicating no functional role of the IK channels in rat DSM. NS309 also significantly inhibited the pharmacologically and electrical field stimulation-induced DSM contractions. Conclusions and Implications Our data reveal that SK3 channel is the main SK/IK subtype in rat DSM. Pharmacological activation of SK3 channels with NS309 decreases rat DSM cell excitability and contractility, suggesting that SK3 channels might be potential therapeutic targets to control OAB associated with detrusor overactivity. PMID:23145946
Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses.

PubMed

Strauss, E G; Levinson, R; Rice, C M; Dalrymple, J; Strauss, J H

1988-05-01

We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function.
Hospital costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition.

PubMed

Morales, Eva; Cots, Francesc; Sala, Maria; Comas, Mercè; Belvis, Francesc; Riu, Marta; Salvadó, Margarita; Grau, Santiago; Horcajada, Juan P; Montero, Maria Milagro; Castells, Xavier

2012-05-23

We aimed to assess the hospital economic costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition. A retrospective study of all hospital admissions between January 1, 2005, and December 31, 2006 was carried out in a 420-bed, urban, tertiary-care teaching hospital in Barcelona (Spain). All patients with a first positive clinical culture for P. aeruginosa more than 48 h after admission were included. Patient and hospitalization characteristics were collected from hospital and microbiology laboratory computerized records. According to antibiotic susceptibility, isolates were classified as non-resistant, resistant and multi-drug resistant. Cost estimation was based on a full-costing cost accounting system and on the criteria of clinical Activity-Based Costing methods. Multivariate analyses were performed using generalized linear models of log-transformed costs. Cost estimations were available for 402 nosocomial incident P. aeruginosa positive cultures. Their distribution by antibiotic susceptibility pattern was 37.1% non-resistant, 29.6% resistant and 33.3% multi-drug resistant. The total mean economic cost per admission of patients with multi-drug resistant P. aeruginosa strains was higher than that for non-resistant strains (15,265 vs. 4,933 Euros). In multivariate analysis, resistant and multi-drug resistant strains were independently predictive of an increased hospital total cost in compared with non-resistant strains (the incremental increase in total hospital cost was more than 1.37-fold and 1.77-fold that for non-resistant strains, respectively). P. aeruginosa multi-drug resistance independently predicted higher hospital costs with a more than 70% increase per admission compared with non-resistant strains. Prevention of the nosocomial emergence and spread of antimicrobial resistant microorganisms is essential to limit the strong economic impact.
Structures of a Na+-coupled, substrate-bound MATE multidrug transporter

PubMed Central

Lu, Min; Symersky, Jindrich; Radchenko, Martha; Koide, Akiko; Guo, Yi; Nie, Rongxin; Koide, Shohei

2013-01-01

Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na+ or H+ gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na+-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs+ (a Na+ congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation–π interaction in the Na+-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na+-induced drug export. Based on our structural and functional analyses, we suggest that Na+ triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport. PMID:23341609
Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient
Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient
Multiple molecular mechanisms for multidrug resistance transporters.

PubMed

Higgins, Christopher F

2007-04-12

The acquisition of multidrug resistance is a serious impediment to improved healthcare. Multidrug resistance is most frequently due to active transporters that pump a broad spectrum of chemically distinct, cytotoxic molecules out of cells, including antibiotics, antimalarials, herbicides and cancer chemotherapeutics in humans. The paradigm multidrug transporter, mammalian P-glycoprotein, was identified 30 years ago. Nonetheless, success in overcoming or circumventing multidrug resistance in a clinical setting has been modest. Recent structural and biochemical data for several multidrug transporters now provide mechanistic insights into how they work. Organisms have evolved several elegant solutions to ridding the cell of such cytotoxic compounds. Answers are emerging to questions such as how multispecificity for different drugs is achieved, why multidrug resistance arises so readily, and what chance there is of devising a clinical solution.
The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus▿

PubMed Central

Jones, Daniel M.; Patel, Arvind H.; Targett-Adams, Paul; McLauchlan, John

2009-01-01

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release. PMID:19073716
Implications of PSR J0737-3039B for the Galactic NS-NS binary merger rate

NASA Astrophysics Data System (ADS)

Kim, Chunglee; Perera, Benetge Bhakthi Pranama; McLaughlin, Maura A.

2015-03-01

The Double Pulsar (PSR J0737-3039) is the only neutron star-neutron star (NS-NS) binary in which both NSs have been detectable as radio pulsars. The Double Pulsar has been assumed to dominate the Galactic NS-NS binary merger rate R_g among all known systems, solely based on the properties of the first-born, recycled pulsar (PSR J0737-3039A, or A) with an assumption for the beaming correction factor of 6. In this work, we carefully correct observational biases for the second-born, non-recycled pulsar (PSR J0737-0737B, or B) and estimate the contribution from the Double Pulsar on R_g using constraints available from both A and B. Observational constraints from the B pulsar favour a small beaming correction factor for A (˜2), which is consistent with a bipolar model. Considering known NS-NS binaries with the best observational constraints, including both A and B, we obtain R_g=21_{-14}^{+28} Myr-1 at 95 per cent confidence from our reference model. We expect the detection rate of gravitational waves from NS-NS inspirals for the advanced ground-based gravitational-wave detectors is to be 8^{+10}_{-5} yr-1 at 95 per cent confidence. Within several years, gravitational-wave detections relevant to NS-NS inspirals will provide us useful information to improve pulsar population models.
Hospital costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition

PubMed Central

2012-01-01

Background We aimed to assess the hospital economic costs of nosocomial multi-drug resistant Pseudomonas aeruginosa acquisition. Methods A retrospective study of all hospital admissions between January 1, 2005, and December 31, 2006 was carried out in a 420-bed, urban, tertiary-care teaching hospital in Barcelona (Spain). All patients with a first positive clinical culture for P. aeruginosa more than 48 h after admission were included. Patient and hospitalization characteristics were collected from hospital and microbiology laboratory computerized records. According to antibiotic susceptibility, isolates were classified as non-resistant, resistant and multi-drug resistant. Cost estimation was based on a full-costing cost accounting system and on the criteria of clinical Activity-Based Costing methods. Multivariate analyses were performed using generalized linear models of log-transformed costs. Results Cost estimations were available for 402 nosocomial incident P. aeruginosa positive cultures. Their distribution by antibiotic susceptibility pattern was 37.1% non-resistant, 29.6% resistant and 33.3% multi-drug resistant. The total mean economic cost per admission of patients with multi-drug resistant P. aeruginosa strains was higher than that for non-resistant strains (15,265 vs. 4,933 Euros). In multivariate analysis, resistant and multi-drug resistant strains were independently predictive of an increased hospital total cost in compared with non-resistant strains (the incremental increase in total hospital cost was more than 1.37-fold and 1.77-fold that for non-resistant strains, respectively). Conclusions P. aeruginosa multi-drug resistance independently predicted higher hospital costs with a more than 70% increase per admission compared with non-resistant strains. Prevention of the nosocomial emergence and spread of antimicrobial resistant microorganisms is essential to limit the strong economic impact. PMID:22621745
Process Performances of 2 ns Pulsed Discharge Plasma

NASA Astrophysics Data System (ADS)

Matsumoto, Takao; Wang, Douyan; Namihira, Takao; Akiyama, Hidenori

2011-08-01

Pulsed discharge plasmas have been used to treat exhaust gases. Since pulse duration and the rise time of applied voltage to the discharge electrode has a strong influence on the energy efficiency of pollutant removal, the development of a short-pulse generator is of paramount importance for practical applications. In this work, it is demonstrated that the non thermal plasma produced by the 2 ns pulsed discharge has a higher energy efficiency than the 5 ns pulsed discharge plasma for NO removal and ozone generation. Typically, the NO removal efficiency was 1.0 mol kW-1 h-1 for 70% NO removal (initial NO concentration = 200 ppm, gas flow = 10 L/min). Meanwhile, the ozone yield was 500 g kW-1 h-1 for 20 g/m3 ozone concentration in the case of oxygen feeding. These energy efficiencies are the highest in the literature.
Microfluidic assembly of monodisperse multistage pH-responsive polymer/porous silicon composites for precisely controlled multi-drug delivery.

PubMed

Liu, Dongfei; Zhang, Hongbo; Herranz-Blanco, Bárbara; Mäkilä, Ermei; Lehto, Vesa-Pekka; Salonen, Jarno; Hirvonen, Jouni; Santos, Hélder A

2014-05-28

We report an advanced drug delivery platform for combination chemotherapy by concurrently incorporating two different drugs into microcompoistes with ratiometric control over the loading degree. Atorvastatin and celecoxib were selected as model drugs due to their different physicochemical properties and synergetic effect on colorectal cancer prevention and inhibition. To be effective in colorectal cancer prevention and inhibition, the produced microcomposite contained hypromellose acetate succinate, which is insoluble in acidic conditions but highly dissolving at neutral or alkaline pH conditions. Taking advantage of the large pore volume of porous silicon (PSi), atorvastatin was firstly loaded into the PSi matrix, and then encapsulated into the pH-responsive polymer microparticles containing celecoxib by microfluidics in order to obtain multi-drug loaded polymer/PSi microcomposites. The prepared microcomposites showed monodisperse size distribution, multistage pH-response, precise ratiometric controlled loading degree towards the simultaneously loaded drug molecules, and tailored release kinetics of the loaded cargos. This attractive microcomposite platform protects the payloads from being released at low pH-values, and enhances their release at higher pH-values, which can be further used for colon cancer prevention and treatment. Overall, the pH-responsive polymer/PSi-based microcomposite can be used as a universal platform for the delivery of different drug molecules for combination therapy. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3

PubMed Central

Zheng, Fengwei; Lu, Guoliang; Li, Ling

2017-01-01

ABSTRACT The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a “closed” global conformation related to the NS3-NS4A cis-cleavage event. Although this conformation is incompatible with protease trans-cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo. Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis-cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through
ABC multidrug transporters: target for modulation of drug pharmacokinetics and drug-drug interactions.

PubMed

Marquez, Béatrice; Van Bambeke, Françoise

2011-05-01

Nine proteins of the ABC superfamily (P-glycoprotein, 7 MRPs and BCRP) are involved in multidrug transport. Being localised at the surface of endothelial or epithelial cells, they expel drugs back to the external medium (if located at the apical side [P-glycoprotein, BCRP, MRP2, MRP4 in the kidney]) or to the blood (if located at the basolateral side [MRP1, MRP3, MRP4, MRP5]), modulating thereby their absorption, distribution, and elimination. In the CNS, most transporters are oriented to expel drugs to the blood. Transporters also cooperate with Phase I/Phase II metabolism enzymes by eliminating drug metabolites. Their major features are (i) their capacity to recognize drugs belonging to unrelated pharmacological classes, and (ii) their redundancy, a single molecule being possibly substrate for different transporters. This ensures an efficient protection of the body against invasion by xenobiotics. Competition for transport is now characterized as a mechanism of interaction between co-administered drugs, one molecule limiting the transport of the other, potentially affecting bioavailability, distribution, and/or elimination. Again, this mechanism reinforces drug interactions mediated by cytochrome P450 inhibition, as many substrates of P-glycoprotein and CYP3A4 are common. Induction of the expression of genes coding for MDR transporters is another mechanism of drug interaction, which could affect all drug substrates of the up-regulated transporter. Overexpression of MDR transporters confers resistance to anticancer agents and other therapies. All together, these data justify why studying drug active transport should be part of the evaluation of new drugs, as recently recommended by the FDA.
Phytochemical Prospection and Modulation of Antibiotic Activity In Vitro by Lippia origanoides H.B.K. in Methicillin Resistant Staphylococcus aureus

PubMed Central

Medeiros Barreto, Humberto; Cerqueira Fontinele, Filipe; Pereira de Oliveira, Aldeídia; Arcanjo, Daniel Dias Rufino; Cavalcanti dos Santos, Bernadete Helena; de Abreu, Aislan Pereira Lira; Douglas Melo Coutinho, Henrique; Alves Carvalho da Silva, Romezio; Oliveira de Sousa, Taciana; Freire de Medeiros, Maria das Graças; Lopes Citó, Antonia Maria das Graças; Dantas Lopes, José Arimateia

2014-01-01

The Lippia origanoides H.B.K. ethanol extract (LOEE) and hexane (LOHEX), dichloromethane (LODCM), and ethyl acetate (LOEA) fractions were tested for their antimicrobial activity alone or in combination with antibiotics against a methicillin resistant Staphylococcus aureus (MRSA) strain. The natural products did not show antimicrobial activity against multidrug resistant strain at the clinically significant concentrations tested. However, a modulatory effect in the antibacterial activity of the neomycin and amikacin was verified when LOEE, LOHEX and LODCM were added to the growth medium at subinhibitory concentrations. A similar modulation was found when the natural products were changed for chlorpromazine, an inhibitor of bacterial efflux pumps, suggesting the involvement of resistance mediated by efflux system in the MRSA tested. The fractions LOHEX and LODCM showed a modulatory activity bigger than their majority compounds (carvacrol, thymol, and naringenin), indicating that this activity is not due to their majority compounds only, but it is probably due to a synergism between their chemical components. These results indicate that L. origanoides H.B.K. can be a source of phytochemicals able to modify the phenotype of resistance to aminoglycosides in MRSA. PMID:24683545
[MOLECULAR CHARACTERISTICS OF THE MULTIDRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS IN THE NORTHWEST RUSSIA].

PubMed

Vyazovaya, A A; Mokrousov, I V; Zhuravlev, V Yu; Solovieva, N S; Otten, T F; Manicheva, O A; Vishnevsky, B I; Narvskaya, O V

2016-01-01

The goal of this work was to study the genotypic characteristics of the multidrug-resistant (MDR, i.e., resistant to at least rifampicine and isoniazid) Mycobacterium tuberculosis strains isolated in 2011-2012 from tuberculosis (TB) patients in the Northwest Russia. Spoligotyping of 195 M. tuberculosis isolates identified 14 different spoligotypes and assigned isolates to the genetic families Beijing (n = 162, 83%), LAM (n = 15), H3/URAL (n = 14), as well as T, Haarlem and X. Spoligotypes SIT1 (Beijing), SIT42 (LAM) and SIT262 (H3/URAL) were the most prevalent. Irrespective to the genotype, all the isolates were resistant to streptomycin. The multidrug resistance was accompanied by the resistance to ethionamide (56%), amikacin (31%), kanamycin (40%), and capreomycin (33%). The ethambutol resistance was found in 71% (n = 115) and 42% (n = 14) of the Beijing and non-Beijing strains, respectively (p < 0.05). In conclusion, the multidrug resistant M. tuberculosis population circulating in the Northwest Russia continues to be dominated by the Beijing family strains.
Jatrophane diterpenoids from Euphorbia sororia as potent modulators against P-glycoprotein-based multidrug resistance.

PubMed

Hu, Rui; Gao, Jie; Rozimamat, Rushangul; Aisa, Haji Akber

2018-02-25

Five new (1-5) and ten known (6-15) jatrophane diterpenoids were isolated from the fructus of Euphorbia sororia and their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds 1 and 4 were confirmed by X-ray crystallographic analysis. Cytotoxicity and anti-multidrug resistance effects of these jatrophane diterpenoids were evaluated in multidrug-resistant MCF-7/ADR breast cancer cells with an overexpression of P-glycoprotein (P-gp). Eight compounds (1, 2, 4, 6, 8, 10, 11, and 15) showed promising chemoreversal abilities compared to verapamil (VRP). The most potent compound, Euphosorophane A (1), possessed many advantages, including (1) high potency (EC 50  = 92.68 ± 18.28 nM) in reversing P-gp-mediated resistance to doxorubicin (DOX), low cytotoxicity, and a high therapeutic index, (2) potency in reversing resistance to other cytotoxic agents associated with MDR, and (3) inhibition of P-gp-mediated Rhodamine123 (Rh123) efflux function in MCF-7/ADR cells. The results of the Western blot analysis indicated that the multidrug resistance (MDR) reversal induced by 1 was not due to the inhibiton of P-gp expression. Compound 1 stimulated P-gp-ATPase activity and caused the dose-dependent inhibition of DOX transport activity. Lineweaver-Burk and Dixon plots implied that 1 was a competitive inhibitor to DOX in the binding site of P-gp with a Ki of 0.49-0.50 μM. Our data suggested that 1 had a high binding affinity toward the DOX recognition site of P-gp. This resulted in inhibiting DOX transport, increasing intracellular DOX concentration, and finally resensitizing MCF-7/ADR to DOX. In addition, we discussed some added contents in the structure-activity relationship (SAR) of jatrophane diterpenoids. Copyright © 2018 Elsevier Masson SAS. All rights reserved.
In vivo effects of the IKr agonist NS3623 on cardiac electrophysiology of the guinea pig.

PubMed

Hansen, Rie Schultz; Olesen, Søren-Peter; Rønn, Lars Christian B; Grunnet, Morten

2008-07-01

The long QT syndrome is characterized by a prolongation of the QT interval measured on the surface electrocardiogram. Prolonging the QT interval increases the risk of dangerous ventricular fibrillations, eventually leading to sudden cardiac death. Pharmacologically induced QT interval prolongations are most often caused by antagonizing effects on the repolarizing cardiac current called IKr. In humans IKr is mediated by the human ether-a-go-go related gene (hERG) potassium channel. We recently presented NS3623, a compound that selectively activates this channel. The present study was dedicated to examining the in vivo effects of NS3623. Injection of 30 mg/kg NS3623 shortened the corrected QT interval by 25 +/- 4% in anaesthetized guinea pigs. Accordingly, 50 mg/kg of NS3623 shortened the QT interval by 30 +/- 6% in conscious guinea pigs. Finally, pharmacologically induced QT prolongation by a hERG channel antagonist (0.15 mg/kg E-4031) could be reverted by injection of NS3623 (50 mg/kg) in conscious guinea pigs. In conclusion, the present in vivo study demonstrates that injection of the hERG channel agonist NS3623 results in shortening of the QTc interval as well as reversal of a pharmacologically induced QT prolongation in both anaesthetized and conscious guinea pigs.
miR-297 modulates multidrug resistance in human colorectal carcinoma by down-regulating MRP-2.

PubMed

Xu, Ke; Liang, Xin; Shen, Ke; Cui, Daling; Zheng, Yuanhong; Xu, Jianhua; Fan, Zhongze; Qiu, Yanyan; Li, Qi; Ni, Lei; Liu, Jianwen

2012-09-01

Colorectal carcinoma is a frequent cause of cancer-related death in men and women. miRNAs (microRNAs) are endogenous small non-coding RNAs that regulate gene expression negatively at the post-transcriptional level. In the present study we investigated the possible role of microRNAs in the development of MDR (multidrug resistance) in colorectal carcinoma cells. We analysed miRNA expression levels between MDR colorectal carcinoma cell line HCT116/L-OHP cells and their parent cell line HCT116 using a miRNA microarray. miR-297 showed lower expression in HCT116/L-OHP cells compared with its parental cells. MRP-2 (MDR-associated protein 2) is an important MDR protein in platinum-drug-resistance cells and is a predicted target of miR-297. Additionally miR-297 was down-regulated in a panel of human colorectal carcinoma tissues and negatively correlated with expression levels of MRP-2. Furthermore, we found that ectopic expression of miR-297 in MDR colorectal carcinoma cells reduced MRP-2 protein level and sensitized these cells to anti-cancer drugs in vitro and in vivo. Taken together, our findings suggest that miR-297 could play a role in the development of MDR in colorectal carcinoma cells, at least in part by modulation of MRP-2.

Zirconium catalyzed synthesis of 2-arylidene Indan-1,3-diones and evaluation of their inhibitory activity against NS2B-NS3 WNV protease.

PubMed

Oliveira, Ana Flávia C da S; de Souza, Ana Paula M; de Oliveira, André S; da Silva, Milene L; de Oliveira, Fabrício M; Santos, Edjon G; da Silva, Ítalo Esposti P; Ferreira, Rafaela S; Villela, Filipe S; Martins, Felipe T; Leal, Daniel H S; Vaz, Boniek G; Teixeira, Róbson R; de Paula, Sergio O

2018-04-10

A simple and efficient Knoevenagel procedure for the synthesis of 2-arylidene indan-1,3-diones is herein reported. These compounds were prepared via ZrOCl2·8H2O catalyzed reactions of indan-1,3-dione with several aromatic aldehydes and using water as the solvent. The 2-arylidene indan-1,3-diones were obtained with 53%-95% yield within 10-45 min. The synthesized compounds were evaluated as inhibitors of the NS2B-NS3 protease of West Nile Virus (WNV). It was found that hydroxylated derivatives impaired enzyme activity with varying degrees of effectiveness. The most active hydroxylated derivatives, namely 2-(4-hydroxybenzylidene)-1H-indene-1,3(2H)-dione (14) and 2-(3,4-dihydroxybenzylidene)-1H-indene-1,3(2H)-dione (17), were characterized as noncompetitive enzymes inhibitors, with IC 50 values of 11 μmol L -1 and 3 μmol L -1 , respectively. Docking and electrostatic potential surfaces investigations provided insight on the possible binding mode of the most active compounds within an allosteric site. Copyright © 2018 Elsevier Masson SAS. All rights reserved.
Evolution of multi-drug resistant HCV clones from pre-existing resistant-associated variants during direct-acting antiviral therapy determined by third-generation sequencing

NASA Astrophysics Data System (ADS)

Takeda, Haruhiko; Ueda, Yoshihide; Inuzuka, Tadashi; Yamashita, Yukitaka; Osaki, Yukio; Nasu, Akihiro; Umeda, Makoto; Takemura, Ryo; Seno, Hiroshi; Sekine, Akihiro; Marusawa, Hiroyuki

2017-03-01

Resistance-associated variant (RAV) is one of the most significant clinical challenges in treating HCV-infected patients with direct-acting antivirals (DAAs). We investigated the viral dynamics in patients receiving DAAs using third-generation sequencing technology. Among 283 patients with genotype-1b HCV receiving daclatasvir + asunaprevir (DCV/ASV), 32 (11.3%) failed to achieve sustained virological response (SVR). Conventional ultra-deep sequencing of HCV genome was performed in 104 patients (32 non-SVR, 72 SVR), and detected representative RAVs in all non-SVR patients at baseline, including Y93H in 28 (87.5%). Long contiguous sequences spanning NS3 to NS5A regions of each viral clone in 12 sera from 6 representative non-SVR patients were determined by third-generation sequencing, and showed the concurrent presence of several synonymous mutations linked to resistance-associated substitutions in a subpopulation of pre-existing RAVs and dominant isolates at treatment failure. Phylogenetic analyses revealed close genetic distances between pre-existing RAVs and dominant RAVs at treatment failure. In addition, multiple drug-resistant mutations developed on pre-existing RAVs after DCV/ASV in all non-SVR cases. In conclusion, multi-drug resistant viral clones at treatment failure certainly originated from a subpopulation of pre-existing RAVs in HCV-infected patients. Those RAVs were selected for and became dominant with the acquisition of multiple resistance-associated substitutions under DAA treatment pressure.
NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains

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

Benzine, Tiffany; Brandt, Ryan; Lovell, William C.

We synthesized the Hepatitis C virus (HCV) RNA by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by preformed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOmore » R) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPO S) viruses (e.g. H77S.3 and N.2). Furthermore, in luciferase assays, LPO S HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPO R HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNAdependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPO S H77S.3 and the LPO R H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. The mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPO S and LPO R viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.« less
NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains

DOE PAGES

Benzine, Tiffany; Brandt, Ryan; Lovell, William C.; ...

2017-06-08

We synthesized the Hepatitis C virus (HCV) RNA by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by preformed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOmore » R) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPO S) viruses (e.g. H77S.3 and N.2). Furthermore, in luciferase assays, LPO S HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPO R HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNAdependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPO S H77S.3 and the LPO R H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. The mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPO S and LPO R viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.« less
Comparison of gene expression profiles between pansensitive and multidrug-resistant strains of Mycobacterium tuberculosis.

PubMed

Peñuelas-Urquides, K; González-Escalante, L; Villarreal-Treviño, L; Silva-Ramírez, B; Gutiérrez-Fuentes, D J; Mojica-Espinosa, R; Rangel-Escareño, C; Uribe-Figueroa, L; Molina-Salinas, G M; Dávila-Velderrain, J; Castorena-Torres, F; Bermúdez de León, M; Said-Fernández, S

2013-09-01

Mycobacterium tuberculosis has developed resistance to anti-tuberculosis first-line drugs. Multidrug-resistant strains complicate the control of tuberculosis and have converted it into a worldwide public health problem. Mutational studies of target genes have tried to envisage the resistance in clinical isolates; however, detection of these mutations in some cases is not sufficient to identify drug resistance, suggesting that other mechanisms are involved. Therefore, the identification of new markers of susceptibility or resistance to first-line drugs could contribute (1) to specifically diagnose the type of M. tuberculosis strain and prescribe an appropriate therapy, and (2) to elucidate the mechanisms of resistance in multidrug-resistant strains. In order to identify specific genes related to resistance in M. tuberculosis, we compared the gene expression profiles between the pansensitive H37Rv strain and a clinical CIBIN:UMF:15:99 multidrug-resistant isolate using microarray analysis. Quantitative real-time PCR confirmed that in the clinical multidrug-resistant isolate, the esxG, esxH, rpsA, esxI, and rpmI genes were upregulated, while the lipF, groES, and narG genes were downregulated. The modified genes could be involved in the mechanisms of resistance to first-line drugs in M. tuberculosis and could contribute to increased efficiency in molecular diagnosis approaches of infections with drug-resistant strains.
Effects of indomethacin, NS-398 (a selective prostaglandin H synthase-2 inhibitor) and protein synthesis inhibitors on prostaglandin production by the guinea-pig placenta.

PubMed

Aitken, H; Poyser, N L

2001-01-01

The outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)were similar from the day 22 guinea-pig placenta and sub-placenta in culture, except for PGE2 output from the sub-placenta which was lower. Between days 22 and 29 of pregnancy, the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)during the initial 2 h culture period increased 6.9-, 1.1- and 3.2-fold, respectively, from the placenta, and 2.1-, 1.4- and 2.2-fold, respectively, from the sub-placenta. Therefore, there was a relatively specific increase in PGF(2 alpha)production by the guinea-pig placenta between days 22 and 29 of pregnancy. The output of PGFM from the cultured placenta also increased between days 22 and 29, indicating that the increase in PGF(2 alpha)output was due to increased synthesis rather than to decreased metabolism. By comparing the amounts of prostaglandins produced by tissue homogenates during a 1 h incubation period, it appears that there is approximately a 2-fold increase in the amount of prostaglandin H synthase (PGHS) present in the guinea-pig placenta between days 22 and 29. NS-398 (a specific inhibitor of PGHS-2) and indomethacin (an inhibitor of both PGHS-1 and PGHS-2) both inhibited prostaglandin production by homogenates of day 22 and day 29 placenta. Indomethacin was more effective than NS-398, except for their actions on PGF(2 alpha)production by the day 29 placenta where indomethacin and NS-398 were equiactive. Indomethacin and NS-398 were both very effective at inhibiting the outputs of PGF(2 alpha), PGE2 and 6-keto-PGF(1 alpha)from the day 22 and day 29 placenta and sub-placenta in culture, indicating that prostaglandin production by the guinea-pig placenta and sub-placenta in culture is largely dependent upon the activity of PGHS-2. The high production of PGF(2 alpha)by the day 29 placenta is not dependent on the continual synthesis of fresh protein(s), as inhibitors of protein synthesis did not reduce PGF(2 alpha)output from the day 29 guinea-pig placenta in culture
[System of ns time-resolved spectroscopy diagnosis and radioprotection].

PubMed

Yao, Wei-Bo; Guo, Jian-Ming; Zhang, Yong-min; Tang, Jun-Ping; Cheng, Liang; Xu, Qi-fuo

2014-06-01

Cathode plasma of high current electron beam diode is an important research on high power microwave and strong pulsed radio accelerator. It is a reliable method to study cathode plasma by diagnosing the cathode plasma parameters with non-contact spectroscopy measurement system. The present paper introduced the work principle, system composition and performance of the nanosecond (ns) time-resolved spectroscopy diagnosis system. Furthermore, it introduced the implementing method and the temporal relation of lower jitter synchronous trigger system. Simultaneously, the authors designed electromagnetic and radio shield room to protect the diagnosis system due to the high electromagnetic and high X-ray and γ-ray radiation, which seriously interferes with the system. Time-resolved spectroscopy experiment on brass (H62) cathode shows that, the element and matter composition of cathode plasma is clearly increase with the increase in the diode pulsed voltage and current magnitude. The spectroscopy diagnosis system could be of up to 10 ns time resolve capability. It's least is 2 ns. Synchronous trigger system's jitter is less than 4 ns. The spectroscopy diagnosis system will open a new way to study the cathode emission mechanism in depth.
Unexpected Functional Divergence of Bat Influenza Virus NS1 Proteins.

PubMed

Turkington, Hannah L; Juozapaitis, Mindaugas; Tsolakos, Nikos; Corrales-Aguilar, Eugenia; Schwemmle, Martin; Hale, Benjamin G

2018-03-01

Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85β interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85β-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs. IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The
Unexpected Functional Divergence of Bat Influenza Virus NS1 Proteins

PubMed Central

Turkington, Hannah L.; Juozapaitis, Mindaugas; Tsolakos, Nikos; Corrales-Aguilar, Eugenia; Schwemmle, Martin

2017-01-01

ABSTRACT Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85β interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85β-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs. IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or
[Construction and expression of six deletion mutants of human astrovirus C-terminal nsP1a/4 protein].

PubMed

Zhao, Wei; Niu, Ke; Zhao, Jian; Jin, Yi-ming; Sui, Ting-ting; Wang, Wen

2013-09-01

Human astrovirus (HAstV) is one of the leading causes of actue virual diarrhea in infants. HAstV-induced epithdlial cell apoptosis plays an important role in the pathogenesis of HAstV infection. Our previous study indicated that HAstV non-structural protein nsPla C-terminal protein nsPla/4 was the major apoptosis functional protein and probably contained the main apoptosis domains. In order to screen for astrovirus encoded apoptotic protien, nsPla/4 and six turncated proteins, which possessed nsPla/4 protein different function domain ,were cloned into green fluorescent protein (GFP) vector pEG-FP-N3. After 24-72 h transfection, the fusion protein expression in BHK21 cells, was analysis by fluorescence microscope and Western blot. The results indicated seven fusion proteins were observed successfully in BHK21 cell after transfected for 24 h. Western blot analysis showed that the level of fusion protein expressed in BHK21 cells was increased significantly at 72h compared to 48h in transfected cells. The successful expression of deletion mutants of nsPla/4 protein was an important foundation to gain further insights into the function of apoptosis domains of nsPla/4 protein and it would also provide research platform to further confirm the molecule pathogenic mechanism of human astrovirus.
Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide

PubMed Central

Nüesch, Jürg P. F.; Corbau, Romuald; Tattersall, Peter; Rommelaere, Jean

1998-01-01

NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1O) and the native (NS1P) polypeptides. Biochemical analyses of replicative functions of NS1O revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2–3 was enhanced compared to that of NS1P. In the presence of endogenous protein kinases found in replication extracts, NS1O showed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1O by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1O was incubated with protein kinase C. PMID:9733839
Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.

PubMed

Takahashi, Hirotaka; Takahashi, Chikako; Moreland, Nicole J; Chang, Young-Tae; Sawasaki, Tatsuya; Ryo, Akihide; Vasudevan, Subhash G; Suzuki, Youichi; Yamamoto, Naoki

2012-12-01

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. Copyright © 2012 Elsevier B.V. All rights reserved.
Diffraction-assisted micropatterning of silicon surfaces by ns-laser irradiation

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

Haro-Poniatowski, E., E-mail: haro@xanum.uam.mx; Acosta-Zepeda, C.; Mecalco, G.

2014-06-14

Single-pulse (532 nm, 8 ns) micropatterning of silicon with nanometric surface modulation is demonstrated by irradiating through a diffracting pinhole. The irradiation results obtained at fluences above the melting threshold are characterized by scanning electron and scanning force microscopy and reveal a good agreement with Fresnel diffraction theory. The physical mechanism is identified and discussed on basis of both thermocapillary and chemicapillary induced material transport during the molten state of the surface.
Discovery and SAR studies of methionine-proline anilides as dengue virus NS2B-NS3 protease inhibitors.

PubMed

Zhou, Guo-Chun; Weng, Zhibing; Shao, Xiaoxia; Liu, Fang; Nie, Xin; Liu, Jinsong; Wang, Decai; Wang, Chunguang; Guo, Kai

2013-12-15

A series of methionine-proline dipeptide derivatives and their analogues were designed, synthesized and assayed against the serotype 2 dengue virus NS2B-NS3 protease, and methionine-proline anilides 1 and 2 were found to be the most active DENV 2 NS2B-NS3 competitive inhibitors with Ki values of 4.9 and 10.5 μM. The structure and activity relationship and the molecular docking revealed that L-proline, L-methionine and p-nitroaniline in 1 and 2 are the important characters in blocking the active site of NS2B-NS3 protease. Our current results suggest that the title dipeptidic scaffold represents a promising structural core to discover a new class of active NS2B-NS3 competitive inhibitors. Copyright © 2013 Elsevier Ltd. All rights reserved.
Structural basis for histone H2B deubiquitination by the SAGA DUB module

DOE PAGES

Morgan, Michael T.; Haj-Yahya, Mahmood; Ringel, Alison E.; ...

2016-02-12

Monoubiquitinated histone H2B plays multiple roles in transcription activation. H2B is deubiquitinated by the Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator, which contains a four-protein subcomplex known as the deubiquitinating (DUB) module. In this paper, the crystal structure of the Ubp8/Sgf11/Sus1/Sgf73 DUB module bound to a ubiquitinated nucleosome reveals that the DUB module primarily contacts H2A/H2B, with an arginine cluster on the Sgf11 zinc finger domain docking on the conserved H2A/H2B acidic patch. The Ubp8 catalytic domain mediates additional contacts with H2B, as well as with the conjugated ubiquitin. Finally, we find that the DUB module deubiquitinates H2B both in the context ofmore » the nucleosome and in H2A/H2B dimers complexed with the histone chaperone, FACT, suggesting that SAGA could target H2B at multiple stages of nucleosome disassembly and reassembly during transcription.« less
Interplay Between Antibiotic Resistance and Virulence During Disease Promoted by Multidrug-Resistant Bacteria

PubMed Central

Geisinger, Edward

2017-01-01

Abstract Diseases caused by antibiotic-resistant bacteria in hospitals are the outcome of complex relationships between several dynamic factors, including bacterial pathogenicity, the fitness costs of resistance in the human host, and selective forces resulting from interventions such as antibiotic therapy. The emergence and fate of mutations that drive antibiotic resistance are governed by these interactions. In this review, we will examine how different forms of antibiotic resistance modulate bacterial fitness and virulence potential, thus influencing the ability of pathogens to evolve in the context of nosocomial infections. We will focus on 3 important multidrug-resistant pathogens that are notoriously problematic in hospitals: Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus. An understanding of how antibiotic resistance mutations shape the pathobiology of multidrug-resistant infections has the potential to drive novel strategies that can control the development and spread of drug resistance. PMID:28375515
The expression of Longus type 4 pilus of enterotoxigenic Escherichia coli is regulated by LngR and LngS and by H-NS, CpxR and CRP global regulators.

PubMed

De la Cruz, Miguel A; Ruiz-Tagle, Alejandro; Ares, Miguel A; Pacheco, Sabino; Yáñez, Jorge A; Cedillo, Lilia; Torres, Javier; Girón, Jorge A

2017-05-01

Enterotoxigenic Escherichia coli produces a long type 4 pilus called Longus. The regulatory elements and the environmental signals controlling the expression of Longus-encoding genes are unknown. We identified two genes lngR and lngS in the Longus operon, whose predicted products share homology with transcriptional regulators. Isogenic lngR and lngS mutants were considerably affected in transcription of lngA pilin gene. The expression of lngA, lngR and lngS genes was optimally expressed at 37°C at pH 7.5. The presence of glucose and sodium chloride had a positive effect on Longus expression. The presence of divalent ions, particularly calcium, appears to be an important stimulus for Longus production. In addition, we studied H-NS, CpxR and CRP global regulators, on Longus expression. The response regulator CpxR appears to function as a positive regulator of lng genes as the cpxR mutant showed reduced levels of lngRSA expression. In contrast, H-NS and CRP function as negative regulators since expression of lngA was up-regulated in isogenic hns and crp mutants. H-NS and CRP were required for salt- and glucose-mediated regulation of Longus. Our data suggest the existence of a complex regulatory network controlling Longus expression, involving both local and global regulators in response to different environmental signals. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.
Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

PubMed Central

2011-01-01

Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy. PMID:22014111
Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen.

PubMed

Le Breton, Marc; Meyniel-Schicklin, Laurène; Deloire, Alexandre; Coutard, Bruno; Canard, Bruno; de Lamballerie, Xavier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent; Davoust, Nathalie

2011-10-20

The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.
A translational study of resistance emergence using sequential direct-acting antiviral agents for hepatitis C using ultra-deep sequencing.

PubMed

Abe, Hiromi; Hayes, C Nelson; Hiraga, Nobuhiko; Imamura, Michio; Tsuge, Masataka; Miki, Daiki; Takahashi, Shoichi; Ochi, Hidenori; Chayama, Kazuaki

2013-09-01

Direct-acting antiviral agents (DAAs) against hepatitis C virus (HCV) have recently been developed and are ultimately hoped to replace interferon-based therapy. However, DAA monotherapy results in rapid emergence of resistant strains and DAAs must be used in combinations that present a high genetic barrier to resistance, although viral kinetics of multidrug-resistant strains remain poorly characterized. The aim of this study is to track the emergence and fitness of resistance using combinations of telaprevir and NS5A or NS5B inhibitors with genotype 1b clones. HCV-infected chimeric mice were treated with DAAs, and resistance was monitored using direct and ultra-deep sequencing. Combination therapy with telaprevir and BMS-788329 (NS5A inhibitor) reduced serum HCV RNA to undetectable levels. The presence of an NS3-V36A telaprevir resistance mutation resulted in poor response to telaprevir monotherapy but showed significant HCV reduction when telaprevir was combined with BMS-788329. However, a BMS-788329-resistant strain emerged at low frequency. Infection with a BMS-788329-resistant NS5A-L31V mutation rapidly resulted in gain of an additional NS5A-Y93A mutation that conferred telaprevir resistance during combination therapy. Infection with dual NS5AL31V/NS5AY93H mutations resulted in poor response to combination therapy and development of telaprevir resistance. Although HCV RNA became undetectable soon after the beginning of combination therapy with BMS-788329 and BMS-821095 (NS5B inhibitor), rebound with emergence of resistance against all three drugs occurred. Triple resistance also occurred following infection with the NS3V36A/NS5AL31V/NS5AY93H triple mutation. Resistant strains easily develop from cloned virus strains. Sequential use of DAAs should be avoided to prevent emergence of multidrug-resistant strains.

Indolcarboxamide is a preclinical candidate for treating multidrug-resistant tuberculosis.

PubMed

Rao, Srinivasa P S; Lakshminarayana, Suresh B; Kondreddi, Ravinder R; Herve, Maxime; Camacho, Luis R; Bifani, Pablo; Kalapala, Sarath K; Jiricek, Jan; Ma, Ng L; Tan, Bee H; Ng, Seow H; Nanjundappa, Mahesh; Ravindran, Sindhu; Seah, Peck G; Thayalan, Pamela; Lim, Siao H; Lee, Boon H; Goh, Anne; Barnes, Whitney S; Chen, Zhong; Gagaring, Kerstin; Chatterjee, Arnab K; Pethe, Kevin; Kuhen, Kelli; Walker, John; Feng, Gu; Babu, Sreehari; Zhang, Lijun; Blasco, Francesca; Beer, David; Weaver, Margaret; Dartois, Veronique; Glynne, Richard; Dick, Thomas; Smith, Paul W; Diagana, Thierry T; Manjunatha, Ujjini H

2013-12-04

New chemotherapeutic compounds against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed to combat drug resistance in tuberculosis (TB). We have identified and characterized the indolcarboxamides as a new class of antitubercular bactericidal agent. Genetic and lipid profiling studies identified the likely molecular target of indolcarboxamides as MmpL3, a transporter of trehalose monomycolate that is essential for mycobacterial cell wall biosynthesis. Two lead candidates, NITD-304 and NITD-349, showed potent activity against both drug-sensitive and multidrug-resistant clinical isolates of Mtb. Promising pharmacokinetic profiles of both compounds after oral dosing in several species enabled further evaluation for efficacy and safety. NITD-304 and NITD-349 were efficacious in treating both acute and chronic Mtb infections in mouse efficacy models. Furthermore, dosing of NITD-304 and NITD-349 for 2 weeks in exploratory rat toxicology studies revealed a promising safety margin. Finally, neither compound inhibited the activity of major cytochrome P-450 enzymes or the hERG (human ether-a-go-go related gene) channel. These results suggest that NITD-304 and NITD-349 should undergo further development as a potential treatment for multidrug-resistant TB.
Differential roles for the C-terminal hexapeptide domains of NS2 splice variants during MVM infection of murine cells.

PubMed

Ruiz, Zandra; D'Abramo, Anthony; Tattersall, Peter

2006-06-05

The MVM NS2 proteins are required for viral replication in cells of its normal murine host, but are dispensable in transformed human 324K cells. Alternate splicing at the minor intron controls synthesis of three forms of this protein, which differ in their C-terminal hexapeptides and in their relative abundance, with NS2P and NS2Y, the predominant isoforms, being expressed at a 5:1 ratio. Mutant genomes were constructed with premature termination codons in the C-terminal exons of either NS2P or NS2Y, which resulted in their failure to accumulate in vivo. To modulate their expression levels, we also introduced a mutation at the putative splice branch point of the large intron, dubbed NS2(lo), that reduced total NS2 expression in murine A9 cells such that NS2P accumulated to approximately half the level normally seen for NS2Y. All mutants replicated productively in human 324K cells. In A9 cells, NS2Y(-) mutants replicated like wildtype, and the NS2(lo) mutants expressed NS1 and replicated duplex viral DNA like wildtype, although their progeny single-strand DNA synthesis was reduced. However, while NS2P(-) and NS2-null viruses initiated infection efficiently in A9 cells, they gave diminished NS1 levels, and viral macromolecular synthesis appeared to become paralyzed shortly after the onset of viral duplex DNA amplification, such that no progeny single-strand DNA could be detected. Thus, the NS2P isoform, even when expressed at a level lower than that of NS2Y, performs a critical role in infection of A9 cells that cannot be accomplished by the NS2Y isoform alone.
Computer Aided Screening of Phytochemicals from Garcinia against the Dengue NS2B/NS3 Protease.

PubMed

Qamar, Tahir Ul; Mumtaz, Arooj; Ashfaq, Usman Ali; Azhar, Samia; Fatima, Tabeer; Hassan, Muhammad; Hussain, Syed Sajid; Akram, Waheed; Idrees, Sobia

2014-01-01

Dengue virus NS2/NS3 protease because of its ability to cleave viral proteins is considered as an attractive target to screen antiviral agents. Medicinal plants contain a variety of phytochemicals that can be used as drug against different diseases and infections. Therefore, this study was designed to uncover possible phytochemical of different classes (Aromatic, Carbohydrates, Lignin, Saponins, Steroids, Tannins, Terpenoids, Xanthones) that could be used as inhibitors against the NS2B/NS3 protease of DENV. With the help of molecular docking, Garcinia phytochemicals found to be bound deeply inside the active site of DENV NS2B/NS3 protease among all tested phytochemicals and had interactions with catalytic triad (His51, Asp75, Ser135). Thus, it can be concluded from the study that these Gracinia phytochemicals could serve as important inhibitors to inhibit the viral replication inside the host cell. Further in-vitro investigations require confirming their efficacy.
Computer Aided Screening of Phytochemicals from Garcinia against the Dengue NS2B/NS3 Protease

PubMed Central

Qamar, Tahir ul; Mumtaz, Arooj; Ashfaq, Usman Ali; Azhar, Samia; Fatima, Tabeer; Hassan, Muhammad; Hussain, Syed Sajid; Akram, Waheed; Idrees, Sobia

2014-01-01

Dengue virus NS2/NS3 protease because of its ability to cleave viral proteins is considered as an attractive target to screen antiviral agents. Medicinal plants contain a variety of phytochemicals that can be used as drug against different diseases and infections. Therefore, this study was designed to uncover possible phytochemical of different classes (Aromatic, Carbohydrates, Lignin, Saponins, Steroids, Tannins, Terpenoids, Xanthones) that could be used as inhibitors against the NS2B/NS3 protease of DENV. With the help of molecular docking, Garcinia phytochemicals found to be bound deeply inside the active site of DENV NS2B/NS3 protease among all tested phytochemicals and had interactions with catalytic triad (His51, Asp75, Ser135). Thus, it can be concluded from the study that these Gracinia phytochemicals could serve as important inhibitors to inhibit the viral replication inside the host cell. Further in-vitro investigations require confirming their efficacy. PMID:24748749
Highly potent non-peptidic inhibitors of the HCV NS3/NS4A serine protease.

PubMed

Sperandio, David; Gangloff, Anthony R; Litvak, Joane; Goldsmith, Richard; Hataye, Jason M; Wang, Vivian R; Shelton, Emma J; Elrod, Kyle; Janc, James W; Clark, James M; Rice, Ken; Weinheimer, Steve; Yeung, Kap-Sun; Meanwell, Nicholas A; Hernandez, Dennis; Staab, Andrew J; Venables, Brian L; Spencer, Jeffrey R

2002-11-04

Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A led to the identification of a potent Zn(2+)-dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn(2+) conditions (K(i)=27nM). This compound (46) binds also to NS3/NS4A in a Zn(2+) independent fashion (K(i)=1microM). The SAR of this class of compounds under Zn(2+) conditions is highly divergent compared to the SAR in the absence of Zn(2+), suggesting two distinct binding modes.
Replacement of the respiratory syncytial virus nonstructural proteins NS1 and NS2 by the V protein of parainfluenza virus 5

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

Tran, Kim C.; He, Biao; Teng, Michael N.

2007-11-10

Paramyxoviruses have been shown to produce proteins that inhibit interferon production and signaling. For human respiratory syncytial virus (RSV), the nonstructural NS1 and NS2 proteins have been shown to have interferon antagonist activity through an unknown mechanism. To understand further the functions of NS1 and NS2, we generated recombinant RSV in which both NS1 and NS2 were replaced by the PIV5 V protein, which has well-characterized IFN antagonist activities ({delta}NS1/2-V). Expression of V was able to partially inhibit IFN responses in {delta}NS1/2-V-infected cells. In addition, the replication kinetics of {delta}NS1/2-V were intermediate between {delta}NS1/2 and wild-type (rA2) in A549 cells.more » However, expression of V did not affect the ability of {delta}NS1/2-V to activate IRF3 nuclear translocation and IFN{beta} transcription. These data indicate that V was able to replace some of the IFN inhibitory functions of the RSV NS1 and NS2 proteins, but also that NS1 and NS2 have functions in viral replication beyond IFN antagonism.« less
A Proteomic Analysis Reveals Differential Regulation of the σS-Dependent yciGFE(katN) Locus by YncC and H-NS in Salmonella and Escherichia coli K-12

PubMed Central

Beraud, Mélanie; Kolb, Annie; Monteil, Véronique; D'Alayer, Jacques; Norel, Françoise

2010-01-01

The stationary phase sigma factor σS (RpoS) controls a regulon required for general stress resistance of the closely related enterobacteria Salmonella and Escherichia coli. The σS-dependent yncC gene encodes a putative DNA binding regulatory protein. Application of the surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) ProteinChip technology for proteome profiling of wild-type and mutant strains of Salmonella enterica serovar Typhimurium revealed potential protein targets for YncC regulation, which were identified by mass spectrometry, and subsequently validated. These proteins are encoded by the σS-dependent operon yciGFEkatN and regulation of their expression by YncC operates at the transcriptional level, as demonstrated by gene fusion analyses and by in vitro transcription and DNase I footprinting experiments with purified YncC. The yciGFE genes are present (without katN) in E. coli K-12 but are poorly expressed, compared with the situation in Salmonella. We report that the yciGFE(katN) locus is silenced by the histone-like protein H-NS in both species, but that σS efficiently relieves silencing in Salmonella but not in E. coli K-12. In Salmonella, YncC acts in concert with σS to activate transcription at the yciG promoter (pyciG). When overproduced, YncC also activated σS-dependent transcription at pyciG in E. coli K-12, but solely by countering the negative effect of H-NS. Our results indicate that differences between Salmonella and E. coli K-12, in the architecture of cis-acting regulatory sequences upstream of pyciG, contribute to the differential regulation of the yciGFE(katN) genes by H-NS and YncC in these two enterobacteria. In E. coli, this locus is subject to gene rearrangements and also likely to horizontal gene transfer, consistent with its repression by the xenogeneic silencer H-NS. PMID:20713450
Synthetic Analogs of Curcumin Modulate the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCG2.

PubMed

Murakami, Megumi; Ohnuma, Shinobu; Fukuda, Michihiro; Chufan, Eduardo E; Kudoh, Katsuyoshi; Kanehara, Keigo; Sugisawa, Norihiko; Ishida, Masaharu; Naitoh, Takeshi; Shibata, Hiroyuki; Iwabuchi, Yoshiharu; Ambudkar, Suresh V; Unno, Michiaki

2017-11-01

Multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) transporters in cancer cells is a major obstacle in cancer chemotherapy. Previous studies have shown that curcumin, a natural product and a dietary constituent of turmeric, inhibits the function of MDR-related ABC transporters, including ABCB1, ABCC1, and especially ABCG2. However, the limited bioavailability of curcumin prevents its use for modulation of the function of these transporters in the clinical setting. In this study, we investigated the effects of 24 synthetic curcumin analogs with increased bioavailability on the transport function of ABCG2. The screening of the 24 synthetic analogs by means of flow cytometry revealed that four of the curcumin analogs (GO-Y030, GO-Y078, GO-Y168, and GO-Y172) significantly inhibited the efflux of the ABCG2 substrates, mitoxantrone and pheophorbide A, from ABCG2-overexpressing K562/breast cancer resistance protein (BCRP) cells. Biochemical analyses showed that GO-Y030, GO-Y078, and GO-Y172 stimulated the ATPase activity of ABCG2 at nanomolar concentrations and inhibited the photolabeling of ABCG2 with iodoarylazidoprazosin, suggesting that these analogs interact with the substrate-binding sites of ABCG2. In addition, when used in cytotoxicity assays, GO-Y030 and GO-Y078 were found to improve the sensitivity of the anticancer drug, SN-38, in K562/BCRP cells. Taken together, these results suggest that nontoxic synthetic curcumin analogs with increased bioavailability, especially GO-Y030 and GO-Y078, inhibit the function of ABCG2 by directly interacting at the substrate-binding site. These synthetic curcumin analogs could therefore be developed as potent modulators to overcome ABCG2-mediated MDR in cancer cells. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.
Flavonoids as noncompetitive inhibitors of Dengue virus NS2B-NS3 protease: inhibition kinetics and docking studies.

PubMed

de Sousa, Lorena Ramos Freitas; Wu, Hongmei; Nebo, Liliane; Fernandes, João Batista; da Silva, Maria Fátima das Graças Fernandes; Kiefer, Werner; Kanitz, Manuel; Bodem, Jochen; Diederich, Wibke E; Schirmeister, Tanja; Vieira, Paulo Cezar

2015-02-01

NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44 μM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7 μM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis experiments, yielded a dissociation constant KD of 20 μM. Our results help to understand the mechanism of inhibition of the Dengue virus serine protease by flavonoids, which is essential for the development of improved inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.
H-reflex modulation in the human medial and lateral gastrocnemii during standing and walking

PubMed Central

Makihara, Yukiko; Segal, Richard L.; Wolpaw, Jonathan R.; Thompson, Aiko K.

2011-01-01

Introduction The soleus H-reflex is dynamically modulated during walking. However, modulation of the gastrocnemii H-reflexes has not been studied systematically. Methods The medial and lateral gastrocnemii (MG and LG) and soleus H-reflexes were measured during standing and walking in humans. Results Maximum H-reflex amplitude was significantly smaller in MG (mean 1.1 mV) or LG (1.1 mV) than in soleus (3.3 mV). Despite these size differences, the reflex amplitudes of the three muscles were positively correlated. The MG and LG H-reflexes were phase- and task-dependently modulated in ways similar to the soleus H-reflex. Discussion Although there are anatomical and physiological differences between the soleus and gastrocnemii muscles, the reflexes of the three muscles are similarly modulated during walking and between standing and walking. The findings support the hypothesis that these reflexes are synergistically modulated during walking to facilitate ongoing movement. PMID:22190317
15. VIEW OF MODULE H, THE HIGH PRESSURE ASSEMBLY AREA. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

15. VIEW OF MODULE H, THE HIGH PRESSURE ASSEMBLY AREA. PROCESSES IN THIS MODULE OCCURRED UNDER HIGH PRESSURES AND TEMPERATURES. (5/70) - Rocky Flats Plant, Plutonium Manufacturing Facility, North-central section of Plant, just south of Building 776/777, Golden, Jefferson County, CO
Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments.

PubMed

Wu, Ren-Huang; Tsai, Ming-Han; Tsai, Kuen-Nan; Tian, Jia Ni; Wu, Jian-Sung; Wu, Su-Ying; Chern, Jyh-Haur; Chen, Chun-Hong; Yueh, Andrew

2017-06-15

The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMS1 to -8) and participates in RNA replication, virion assembly, and host antiviral response. However, the roles of specific amino acid residues within the pTMS regions of NS2A during the viral life cycle are not clear. Here, we explore the function of DENV NS2A by introducing a series of alanine substitutions into the N-terminal half (pTMS1 to -4) of the protein in the context of a DENV infectious clone or subgenomic replicon. Six NS2A mutants (NM5, -7, -9, and -17 to -19) around pTMS1 and -2 displayed a novel phenotype showing a >1,000-fold reduction in virus yield, an absence of plaque formation despite wild-type-like replicon activity, and infectious-virus-like particle yields. HEK-293 cells infected with the six NS2A mutant viruses failed to cause a virus-induced cytopathic effect (CPE) by MitoCapture staining, cell proliferation, and lactate dehydrogenase release assays. Sequencing analyses of pseudorevertant viruses derived from lethal-mutant viruses revealed two consensus reversion mutations, leucine to phenylalanine at codon 181 (L181F) within pTMS7 of NS2A and isoleucine to threonine at codon 114 (I114T) within NS2B. The introduction of an NS2A-L181F mutation into the lethal (NM15, -16, -25, and -33) and CPE-defective (NM7, -9, and -19) mutants substantially rescued virus infectivity and virus-induced CPE, respectively, whereas the NS2B-L114T mutation rescued the NM16, -25, and -33 mutants. In conclusion, the results revealed the essential roles of the N-terminal half of NS2A in RNA replication and virus-induced CPE. Intramolecular interactions between pTMSs of NS2A and intermolecular interactions between the NS2A and NS2B proteins were also implicated. IMPORTANCE The characterization of the N-terminal (current study) and C-terminal halves of DENV NS2A is the most comprehensive mutagenesis study to date to investigate the function of NS2A during the flaviviral life cycle
Genetic diversity of drug and multidrug-resistant Mycobacterium tuberculosis circulating in Veracruz, Mexico

PubMed Central

Munro-Rojas, Daniela; Fernandez-Morales, Esdras; Zarrabal-Meza, José; Martínez-Cazares, Ma. Teresa; Parissi-Crivelli, Aurora; Fuentes-Domínguez, Javier; Séraphin, Marie Nancy; Lauzardo, Michael; González-y-Merchand, Jorge Alberto; Rivera-Gutierrez, Sandra

2018-01-01

Background Mexico is one of the most important contributors of drug and multidrug-resistant tuberculosis in Latin America; however, knowledge of the genetic diversity of drug-resistant tuberculosis isolates is limited. Methods In this study, the genetic structure of 112 Mycobacterium tuberculosis strains from the southeastern Mexico was determined by spoligotyping and 24-loci MIRU-VNTRs. Findings The results show eight major lineages, the most of which was T1 (24%), followed by LAM (16%) and H (15%). A total of 29 (25%) isolates were identified as orphan. The most abundant SITs were SIT53/T1 and SIT42/LAM9 with 10 isolates each and SIT50/H3 with eight isolates. Fifty-two spoligotype patterns, twenty-seven clusters and ten clonal complexes were observed, demonstrating an important genetic diversity of drug and multidrug-resistant tuberculosis isolates in circulation and transmission level of these aggravated forms of tuberculosis. Being defined as orphan or as part of an orphan cluster, was a risk factor for multidrug resistant-tuberculosis (OR 2.5, IC 1.05–5.86 and OR 3.3, IC 1–11.03, respectively). Multiple correspondence analyses showed association of some clusters and SITs with specific geographical locations. Conclusions Our study provides one of the most detailed description of the genetic structure of drug and multidrug-resistant tuberculosis strains in southeast Mexico, establishing for the first time a baseline of the genotypes observed in resistant isolates circulating, however further studies are required to better elucidate the genetic structure of tuberculosis in region and the factors that could be participating in their dispersion. PMID:29543819
Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus

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

Lee, Hyun; Ren, Jinhong; Nocadello, Salvatore

Zika flavivirus infection during pregnancy appears to produce higher risk of microcephaly, and also causes multiple neurological problems such as Guillain–Barré syndrome. The Zika virus is now widespread in Central and South America, and is anticipated to become an increasing risk in the southern United States. With continuing global travel and the spread of the mosquito vector, the exposure is expected to accelerate, but there are no currently approved treatments against the Zika virus. The Zika NS2B/NS3 protease is an attractive drug target due to its essential role in viral replication. Our studies have identified several compounds with inhibitory activitymore » (IC50) and binding affinity (KD) of ~5–10 μM against the Zika NS2B-NS3 protease from testing 71 HCV NS3/NS4A inhibitors that were initially discovered by high-throughput screening of 40,967 compounds. Competition surface plasmon resonance studies and mechanism of inhibition analyses by enzyme kinetics subsequently determined the best compound to be a competitive inhibitor with a Ki value of 9.5 μM. We also determined the X-ray structure of the Zika NS2B-NS3 protease in a “pre-open conformation”, a conformation never observed before for any flavivirus proteases. This provides the foundation for new structure-based inhibitor design.« less
The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

PubMed Central

2013-01-01

Background Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L
The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication.

PubMed

Morais, Ana Ts; Terzian, Ana Cb; Duarte, Danilo Vb; Bronzoni, Roberta Vm; Madrid, Maria Cfs; Gavioli, Arieli F; Gil, Laura Hvg; Oliveira, Amanda G; Zanelli, Cleslei F; Valentini, Sandro R; Rahal, Paula; Nogueira, Mauricio L

2013-06-22

Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role
Characterisation of divergent flavivirus NS3 and NS5 protein sequences detected in Rhipicephalus microplus ticks from Brazil

PubMed Central

Maruyama, Sandra Regina; Castro-Jorge, Luiza Antunes; Ribeiro, José Marcos Chaves; Gardinassi, Luiz Gustavo; Garcia, Gustavo Rocha; Brandão, Lucinda Giampietro; Rodrigues, Aline Rezende; Okada, Marcos Ituo; Abrão, Emiliana Pereira; Ferreira, Beatriz Rossetti; da Fonseca, Benedito Antonio Lopes; de Miranda-Santos, Isabel Kinney Ferreira

2013-01-01

Transcripts similar to those that encode the nonstructural (NS) proteins NS3 and NS5 from flaviviruses were found in a salivary gland (SG) complementary DNA (cDNA) library from the cattle tick Rhipicephalus microplus. Tick extracts were cultured with cells to enable the isolation of viruses capable of replicating in cultured invertebrate and vertebrate cells. Deep sequencing of the viral RNA isolated from culture supernatants provided the complete coding sequences for the NS3 and NS5 proteins and their molecular characterisation confirmed similarity with the NS3 and NS5 sequences from other flaviviruses. Despite this similarity, phylogenetic analyses revealed that this potentially novel virus may be a highly divergent member of the genus Flavivirus. Interestingly, we detected the divergent NS3 and NS5 sequences in ticks collected from several dairy farms widely distributed throughout three regions of Brazil. This is the first report of flavivirus-like transcripts in R. microplus ticks. This novel virus is a potential arbovirus because it replicated in arthropod and mammalian cells; furthermore, it was detected in a cDNA library from tick SGs and therefore may be present in tick saliva. It is important to determine whether and by what means this potential virus is transmissible and to monitor the virus as a potential emerging tick-borne zoonotic pathogen. PMID:24626302
Assessment of Drug Binding Potential of Pockets in the NS2B/NS3 Dengue Virus Protein

NASA Astrophysics Data System (ADS)

Amelia, F.; Iryani; Sari, P. Y.; Parikesit, A. A.; Bakri, R.; Toepak, E. P.; Tambunan, U. S. F.

2018-04-01

Every year an endemic dengue fever estimated to affect over 390 million cases in over 128 countries occurs. However, the antigen types which stimulate the human immune response are variable, as a result, neither effective vaccines nor antiviral treatments have been successfully developed for this disease. The NS2B/NS3 protease of the dengue virus (DENV) responsible for viral replication is a potential drug target. The ligand-enzyme binding site determination is a key role in the success of virtual screening of new inhibitors. The NS2B/NS3 protease of DENV (PDB ID: 2FOM) has two pockets consisting of 37 (Pocket 1) and 27 (Pocket 2) amino acid residues in each pocket. In this research, we characterized the amino acid residues for binding sites in NS3/NS2B based on the hydrophobicity, the percentage of charged residues, volume, depth, ΔGbinding, hydrogen bonding and bond length. The hydrophobic percentages of both pockets are high, 59 % (Pocket 1) and 41% (Pocket 2) and the percentage of charged residues in Pocket 1 and 2 are 22% and 48%, and the pocket volume is less than 700 Å3. An interaction analysis using molecular docking showed that interaction between the ligand complex and protein in Pocket 1 is more negative than Pocket 2. As a result, Pocket 1 is the better potential target for a ligand to inhibit the action of NS2B/NS3 DENV.
hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

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

Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to themore » cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.« less
Targeting multidrug resistance protein 1 (MRP1, ABCC1): past, present, and future.

PubMed

Cole, Susan P C

2014-01-01

The human ATP-binding cassette transporter multidrug resistance protein 1 (MRP1), encoded by ABCC1, was initially identified because of its ability to confer multidrug resistance in lung cancer cells. It is now established that MRP1 plays a role in protecting certain tissues from xenobiotic insults and that it mediates the cellular efflux of the proinflammatory cysteinyl leukotriene C4 as well as a vast array of other endo- and xenobiotic organic anions. Many of these are glutathione (GSH) or glucuronide conjugates, the products of Phase II drug metabolism. MRP1 also plays a role in the cellular efflux of the reduced and oxidized forms of GSH and thus contributes to the many physiological and pathophysiological processes influenced by these small peptides, including oxidative stress. In this review, the pharmacological and physiological aspects of MRP1 are considered in the context of the current status and future prospects of pharmacological and genetic modulation of MRP1 activity.

Structure-based design of NS2 mutants for attenuated influenza A virus vaccines.

PubMed

Akarsu, Hatice; Iwatsuki-Horimoto, Kiyoko; Noda, Takeshi; Kawakami, Eiryo; Katsura, Hiroaki; Baudin, Florence; Horimoto, Taisuke; Kawaoka, Yoshihiro

2011-01-01

We previously characterised the matrix 1 (M1)-binding domain of the influenza A virus NS2/nuclear export protein (NEP), reporting a critical role for the tryptophan (W78) residue that is surrounded by a cluster of glutamate residues in the C-terminal region that interacts with the M1 protein (Akarsu et al., 2003). To gain further insight into the functional role of this interaction, here we used reverse genetics to generate a series of A/WSN/33 (H1N1)-based NS2/NEP mutants for W78 or the C-terminal glutamate residues and assessed their effect on virus growth. We found that simultaneous mutations at three positions (E67S/E74S/E75S) of NS2/NEP were important for inhibition of influenza viral polymerase activity, although the W78S mutant and other glutamate mutants with single substitutions were not. In addition, double and triple substitutions in the NS2/NEP glutamine residues, which resulted in the addition of seven amino acids to the C-terminus of NS1 due to gene overlapping, resulted in virus attenuation in mice. Animal studies with this mutant suggest a potential benefit to incorporating these NS mutations into live vaccines. Copyright © 2010 Elsevier B.V. All rights reserved.
Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in Escherichia coli

PubMed Central

2013-01-01

Background In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis. Results Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress. Conclusions Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli. PMID:23701827
Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in Escherichia coli.

PubMed

Holdsworth, Scarlett R; Law, Christopher J

2013-05-23

In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis. Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress. Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli.
Multidrug resistance reversal and apoptosis induction in human colon cancer cells by some flavonoids present in citrus plants.

PubMed

Wesołowska, Olga; Wiśniewski, Jerzy; Sroda-Pomianek, Kamila; Bielawska-Pohl, Aleksandra; Paprocka, Maria; Duś, Danuta; Duarte, Noélia; Ferreira, Maria-José U; Michalak, Krystyna

2012-11-26

Multidrug resistance (MDR) of cancer cells constitutes one of the main reasons for chemotherapy failure. The search for nontoxic modulators that reduce MDR is a task of great importance. An ability to enhance apoptosis of resistant cells would also be beneficial. In the present study, the MDR reversal and apoptosis-inducing potency of three flavonoids produced by Citrus plants, namely, naringenin (1a), aromadendrin (2), and tangeretin (3), and the methylated naringenin derivatives (1b, 1c), have been studied in sensitive (LoVo) and multidrug-resistant (LoVo/Dx) human colon adenocarcinoma cells. Cytotoxicity of methoxylated flavonoids was higher as compared to hydroxylated analogues. Only 3 turned out to inhibit P-glycoprotein, as demonstrated by a rhodamine 123 accumulation assay. It also increased doxorubicin accumulation in LoVo/Dx cells and enabled doxorubicin to enter cellular nuclei. In addition, 3 was found to be an effective MDR modulator in resistant cells by sensitizing them to doxorubicin. Tangeretin-induced caspase-3 activation and elevated surface phosphatidylserine exposure demonstrated its apoptosis-inducing activity in LoVo/Dx cells, while the other flavonoids evaluated were not active. Additionally, 3 was more toxic to resistant rather than to sensitive cancer cells. Its apoptosis-inducing activity was also higher in LoVo/Dx than in LoVo cells. It was concluded that the activity of 3 against multidrug-resistant cancer cells may be enhanced by its apoptosis-inducing activity.
Antibacterial activity of exogenous glutathione and its synergism on antibiotics sensitize carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii.

PubMed

Alharbe, Roaa; Almansour, Ayidh; Kwon, Dong H

2017-10-01

A major clinical impact of A. baumannii is hospital-acquired infections including ventilator-associated pneumonia. The treatment of this pathogen is often difficult due to its innate and acquired resistance to almost all commercially available antibiotics. Infections with carbapenem-associated multidrug resistant A. baumannii is the most problematic. Glutathione is a tripeptide thiol-antioxidant and antibacterial activity of exogenous glutathione was reported in some bacteria. However, clinical relevance and molecular details of the antibacterial activity of glutathione are currently unclear. Seventy clinical isolates of A. baumannii including 63 carbapenem-associated multidrug resistant isolates and a type strain A. baumannii ATCC 19606 were used to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Fractional inhibitory concentration (FIC) and time-killing activity with meropenem and/or glutathione were also determined in the carbapenem-associated multidrug resistant isolates. In addition, the roles of exogenous glutathione in multidrug efflux pumps and β-lactamase production were examined. Levels of MIC and MBC were ranged from 10 to 15mM of exogenous glutathione. All tested carbapenem-associated multidrug resistant isolates were sensitized by all tested antibiotics in combination with subinhibitory concentrations of glutathione. FIC levels of glutathione with carbapenem (meropenem) were all<0.5 and the carbapenem-associated multidrug resistant isolates were killed by subinhibitory concentrations of both glutathione and meropenem at>2log10 within 12h, suggesting glutathione synergistically interacts with meropenem. The roles of multidrug efflux pumps and β-lactamase production were excluded for the glutathione-mediated antibiotic susceptibility. Overall results demonstrate that the antibacterial activity of glutathione is clinically relevant and its synergism on antibiotics sensitizes clinical isolates of A
Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

PubMed

Asante-Appiah, Ernest; Curry, Stephanie; McMonagle, Patricia; Ingravallo, Paul; Chase, Robert; Nickle, David; Qiu, Ping; Howe, Anita; Lahser, Frederick C

2017-07-01

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC 50 ) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC 50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC 50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC 50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir
Spin Complicates Eccentric BH-NS Mergers

NASA Astrophysics Data System (ADS)

Kohler, Susanna

2015-08-01

When a neutron star (NS) has a glancing encounter with a black hole (BH), its spin has a significant effect on the outcome, according to new simulations run by William East of Stanford University and his collaborators. Spotting an Eccentric Merger. In a traditional BH-NS merger, the two objects orbit each other quasi-circularly as they spiral in. But there's another kind of merger that's possible in high-density environments like galactic nuclei or globular clusters: a dynamical capture merger, in which a NS and BH pass each other just close enough that the gravity of the black hole "catches" the NS, leading the two objects to merge with very eccentric orbits. During an eccentric merger, the NS can be torn apart -- at which point some fraction of the tidally-disrupted material will escape the system, while some fraction instead accretes back onto the BH. Knowing these fractions is important for being able to model the expected electromagnetic signatures for the merger: the unbound material can power transients like kilonovae, whereas the accreting material may be the cause of short gamma-ray bursts. The amount of material available for events like these would change their observable strengths. Testing the Effects of Spin. To see whether NS spin has an impact on the behavior of the merger, East and collaborators use a general-relativistic hydrodynamic code to simulate the glancing encounter of a BH and a NS with dimensionless spin between a=0 (non-spinning) and a=0.756 (rotation period of 1 ms). They also vary the separation of the first encounter. The group finds that changing the NS's spin can change a number of outcomes of the merger. To start with, it can affect whether the NS is captured by the BH, or if the encounter is glancing and then both objects carry on their merry way. And if the NS is trapped by the BH and torn apart, then the higher the NS's spin, the more matter outside of the BH ends up unbound, instead of getting trapped into an accretion disk
Physiological characterisation of the efflux pump system of antibiotic-susceptible and multidrug-resistant Enterobacter aerogenes.

PubMed

Martins, A; Spengler, G; Martins, M; Rodrigues, L; Viveiros, M; Davin-Regli, A; Chevalier, J; Couto, I; Pagès, J M; Amaral, L

2010-10-01

Enterobacter aerogenes predominates amongst Enterobacteriaceae species that are increasingly reported as producers of extended-spectrum beta-lactamases. Although this mechanism of resistance to beta-lactams is important, other mechanisms bestowing a multidrug-resistant (MDR) phenotype in this species are now well documented. Amongst these mechanisms is the overexpression of efflux pumps that extrude structurally unrelated antibiotics prior to their reaching their targets. Interestingly, although knowledge of the genetic background behind efflux pumps is rapidly advancing, few studies assess the physiological nature of the overall efflux pump system of this, or for that matter any other, bacterium. The study reported here evaluates physiologically the efflux pump system of an E. aerogenes ATCC reference as well as two strains whose MDR phenotypes are mediated by overexpressed efflux pumps. The activities of the efflux pumps in these strains are modulated by pH and glucose, although the effects of the latter are essentially restricted to pH 8, suggesting the presence of two general efflux pump systems, i.e. proton-motive force-dependent and ABC transporter types, respectively. Copyright 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Regulation of the Production of Infectious Genotype 1a Hepatitis C Virus by NS5A Domain III▿

PubMed Central

Kim, Seungtaek; Welsch, Christoph; Yi, MinKyung; Lemon, Stanley M.

2011-01-01

Although hepatitis C virus (HCV) assembly remains incompletely understood, recent studies with the genotype 2a JFH-1 strain suggest that it is dependent upon the phosphorylation of Ser residues near the C terminus of NS5A, a multifunctional nonstructural protein. Since genotype 1 viruses account for most HCV disease yet differ substantially in sequence from that of JFH-1, we studied the role of NS5A in the production of the H77S virus. While less efficient than JFH-1, genotype 1a H77S RNA produces infectious virus when transfected into permissive Huh-7 cells. The exchange of complete NS5A sequences between these viruses was highly detrimental to replication, while exchanges of the C-terminal domain III sequence (46% amino acid sequence identity) were well tolerated, with little effect on RNA synthesis. Surprisingly, the placement of the H77S domain III sequence into JFH-1 resulted in increased virus yields; conversely, H77S yields were reduced by the introduction of domain III from JFH-1. These changes in infectious virus yield correlated well with changes in the abundance of NS5A in RNA-transfected cells but not with RNA replication or core protein expression levels. Alanine replacement mutagenesis of selected Ser and Thr residues in the C-terminal domain III sequence revealed no single residue to be essential for infectious H77S virus production. However, virus production was eliminated by Ala substitutions at multiple residues and could be restored by phosphomimetic Asp substitutions at these sites. Thus, despite low overall sequence homology, the production of infectious virus is regulated similarly in JFH-1 and H77S viruses by a conserved function associated with a C-terminal Ser/Thr cluster in domain III of NS5A. PMID:21525356
Quantitative Proteomic Analysis of the Influenza A Virus Nonstructural Proteins NS1 and NS2 during Natural Cell Infection Identifies PACT as an NS1 Target Protein and Antiviral Host Factor

PubMed Central

Tawaratsumida, Kazuki; Phan, Van; Hrincius, Eike R.; High, Anthony A.; Webby, Richard; Redecke, Vanessa

2014-01-01

ABSTRACT Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production. IMPORTANCE Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host
Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C

PubMed Central

Nüesch, Jürg P. F.; Dettwiler, Sabine; Corbau, Romuald; Rommelaere, Jean

1998-01-01

NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro. PMID:9811734
Purification and crystallization of dengue and West Nile virus NS2B–NS3 complexes

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

D’Arcy, Allan, E-mail: allan.darcy@novartis.com; Chaillet, Maxime; Schiering, Nikolaus

Crystals of dengue serotype 2 and West Nile virus NS2B–NS3 protease complexes have been obtained and the crystals of both diffract to useful resolution. Sample homogeneity was essential for obtaining X-ray-quality crystals of the dengue protease. Controlled proteolysis produced a crystallizable fragment of the apo West Nile virus NS2B–NS3 and crystals were also obtained in the presence of a peptidic inhibitor. Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but maymore » also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B–NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.« less
Influenza A virus strains that circulate in humans differ in the ability of their NS1 proteins to block the activation of IRF3 and interferon-β transcription.

PubMed

Kuo, Rei-Lin; Zhao, Chen; Malur, Meghana; Krug, Robert M

2010-12-20

We demonstrate that influenza A virus strains that circulate in humans differ markedly in the ability of their NS1 proteins to block the activation of IRF3 and interferon-β transcription. Strong activation occurs in cells infected with viruses expressing NS1 proteins of seasonal H3N2 and H2N2 viruses, whereas activation is blocked in cells infected with viruses expressing NS1 proteins of some, but not all seasonal H1N1 viruses. The NS1 proteins of the 2009 H1N1 and H5N1 viruses also block these activations. The difference in this NS1 function is mediated largely by the C-terminal region of the effector domain, which contains the only amino acid (K or E at position 196) that covaries with the functional difference. Further, we show that TRIM25 binds the NS1 protein whether or not IRF3 activation is blocked, demonstrating that binding of TRIM25 by the NS1 protein does not necessarily lead to the blocking of IRF3 activation. Copyright © 2010 Elsevier Inc. All rights reserved.
Magnetoelectric gradiometer with enhanced vibration rejection efficiency under H-field modulation

NASA Astrophysics Data System (ADS)

Xu, Junran; Zhuang, Xin; Leung, Chung Ming; Staruch, Margo; Finkel, Peter; Li, Jiefang; Viehland, D.

2018-03-01

A magnetoelectric (ME) gradiometer consisting of two Metglas/Pb(Zr,Ti)O3 fiber-based sensors has been developed. The equivalent magnetic noise of both sensors was first determined to be about 60 pT/√Hz while using an H-field modulation technique. The common mode rejection ratio of a gradiometer based on these two sensors was determined to be 74. The gradiometer response curve was then measured, which provided the dependence of the gradiometer output as a function of the source-gradiometer-normalized distance. Investigations in the presence of vibration noise revealed that a ME gradiometer consisting of two ME magnetometers working under H-field modulation was capable of significant vibration rejection. The results were compared to similar studies of ME gradiometers operated in a passive working mode. Our findings demonstrate that this active gradiometer has a good vibration rejection capability in the presence of both magnetic signals and vibration noise/interferences by using two magnetoelectric sensors operated under H-field modulation.
Performance of commercial dengue NS1 ELISA and molecular analysis of NS1 gene of dengue viruses obtained during surveillance in Indonesia.

PubMed

Aryati, Aryati; Trimarsanto, Hidayat; Yohan, Benediktus; Wardhani, Puspa; Fahri, Sukmal; Sasmono, R Tedjo

2013-12-29

Early diagnosis of dengue infection is crucial for better management of the disease. Diagnostic tests based on the detection of dengue virus (DENV) Non Structural Protein 1 (NS1) antigen are commercially available with different sensitivities and specificities observed in various settings. Dengue is endemic in Indonesia and clinicians are increasingly using the NS1 detection for dengue confirmation. This study described the performance of Panbio Dengue Early NS1 and IgM Capture ELISA assays for dengue detection during our surveillance in eight cities in Indonesia as well as the genetic diversity of DENV NS1 genes and its relationship with the NS1 detection. The NS1 and IgM/IgG ELISA assays were used for screening and confirmation of dengue infection during surveillance in 2010-2012. Collected serum samples (n = 440) were subjected to RT-PCR and virus isolation, in which 188 samples were confirmed for dengue infection. The positivity of the ELISA assays were correlated with the RT-PCR results to obtain the sensitivity of the assays. The NS1 genes of 48 Indonesian virus isolates were sequenced and their genetic characteristics were studied. Using molecular data as gold standard, the sensitivity of NS1 ELISA assay for samples from Indonesia was 56.4% while IgM ELISA was 73.7%. When both NS1 and IgM results were combined, the sensitivity increased to 89.4%. The NS1 sensitivity varied when correlated with city/geographical origins and DENV serotype, in which the lowest sensitivity was observed for DENV-4 (19.0%). NS1 sensitivity was higher in primary (67.6%) compared to secondary infection (48.2%). The specificity of NS1 assay for non-dengue samples were 100%. The NS1 gene sequence analysis of 48 isolates revealed the presence of polymorphisms of the NS1 genes which apparently did not influence the NS1 sensitivity. We observed a relatively low sensitivity of NS1 ELISA for dengue detection on RT-PCR-positive dengue samples. The detection rate increased significantly
20(S)-Protopanaxadiol (PPD) analogues chemosensitize multidrug-resistant cancer cells to clinical anticancer drugs.

PubMed

Liu, Junhua; Wang, Xu; Liu, Peng; Deng, Rongxin; Lei, Min; Chen, Wantao; Hu, Lihong

2013-07-15

Novel 20(S)-protopanoxadiol (PPD) analogues were designed, synthesized, and evaluated for the chemosensitizing activity against a multidrug resistant (MDR) cell line (KBvcr) overexpressing P-glycoprotein (P-gp). Structure-activity relationship analysis showed that aromatic substituted aliphatic amine at the 24-positions (groups V) effectively and significantly sensitized P-gp overexpressing multidrug resistant (MDR) cells to anticancer drugs, such as docetaxel (DOC), vincristine (VCR), and adriamycin (ADM). PPD derivatives 12 and 18 showed 1.3-2.6 times more effective reversal ability than verapamil (VER) for DOC and VCR. Importantly, no cytotoxicity was observed by the active PPD analogues (5μM) against both non-MDR and MDR cells, suggesting that PPD analogues serve as novel lead compounds toward a potent and safe resistance modulator. Moreover, a preliminary mechanism study demonstrated that the chemosensitizing activity of PPD analogues results from inhibition of P-glycoprotein (P-gp) overexpressed in MDR cancer cells. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.
Protective immunity to Japanese encephalitis virus associated with anti-NS1 antibodies in a mouse model.

PubMed

Li, Yize; Counor, Dorian; Lu, Peng; Duong, Veasna; Yu, Yongxin; Deubel, Vincent

2012-07-24

Japanese encephalitis virus (JEV) is a major mosquito-borne pathogen that causes viral encephalitis throughout Asia. Vaccination with an inactive JEV particle or attenuated virus is an efficient preventative measure for controlling infection. Flavivirus NS1 protein is a glycoprotein secreted during viral replication that plays multiple roles in the viral life cycle and pathogenesis. Utilizing JEV NS1 as an antigen in viral vectors induces a limited protective immune response against infection. Previous studies using E. coli-expressed JEV NS1 to immunize mice induced protection against lethal challenge; however, the protection mechanism through cellular and humoral immune responses was not described. JEV NS1 was expressed in and purified from Drosophila S2 cells in a native glycosylated multimeric form, which induced T-cell and antibody responses in immunized C3H/HeN mice. Mice vaccinated with 1 μg NS1 with or without water-in-oil adjuvant were partially protected against viral challenge and higher protection was observed in mice with higher antibody titers. IgG1 was preferentially elicited by an adjuvanted NS1 protein, whereas a larger load of IFN-γ was produced in splenocytes from mice immunized with aqueous NS1. Mice that passively received anti-NS1 mouse polyclonal immune sera were protected, and this phenomenon was dose-dependent, whereas protection was low or delayed after the passive transfer of anti-NS1 MAbs. The purified NS1 subunit induced protective immunity in relation with anti-NS1 IgG1 antibodies. NS1 protein efficiently stimulated Th1-cell proliferation and IFN-γ production. Protection against lethal challenge was elicited by passive transfer of anti-NS1 antisera, suggesting that anti-NS1 antibodies play a substantial role in anti-viral immunity.
Naturally occurring mutations associated with resistance to HCV NS5B polymerase and NS3 protease inhibitors in treatment-naïve patients with chronic hepatitis C.

PubMed

Costantino, Angela; Spada, Enea; Equestre, Michele; Bruni, Roberto; Tritarelli, Elena; Coppola, Nicola; Sagnelli, Caterina; Sagnelli, Evangelista; Ciccaglione, Anna Rita

2015-11-14

The detection of baseline resistance mutations to new direct-acting antivirals (DAAs) in HCV chronically infected treatment-naïve patients could be important for their management and outcome prevision. In this study, we investigated the presence of mutations, which have been previously reported to be associated with resistance to DAAs in HCV polymerase (NS5B) and HCV protease (NS3) regions, in sera of treatment-naïve patients. HCV RNA from 152 naïve patients (84 % Italian and 16 % immigrants from various countries) infected with different HCV genotypes (21,1a; 21, 1b; 2, 2a; 60, 2c; 22, 3a; 25, 4d and 1, 4k) was evaluated for sequence analysis. Amplification and sequencing of fragments in the NS5B (nt 8256-8640) and NS3 (nt 3420-3960) regions of HCV genome were carried out for 152 and 28 patients, respectively. The polymorphism C316N/H in NS5B region, associated with resistance to sofosbuvir, was detected in 9 of the 21 (43 %) analysed sequences from genotype 1b-infected patients. Naturally occurring mutations V36L, and M175L in the NS3 protease region were observed in 100 % of patients infected with subtype 2c and 4. A relevant proportion of treatment naïve genotype 1b infected patients evaluated in this study harboured N316 polymorphism and might poorly respond to sofosbuvir treatment. As sofosbuvir has been approved for treatment of HCV chronic infection in USA and Europe including Italy, pre-treatment testing for N316 polymorphism on genotype 1b naïve patients should be considered for this drug.
Multidrug Efflux Systems in Microaerobic and Anaerobic Bacteria

PubMed Central

Xu, Zeling; Yan, Aixin

2015-01-01

Active drug efflux constitutes an important mechanism of antibiotic and multidrug resistance in bacteria. Understanding the distribution, expression, and physiological functions of multidrug efflux pumps, especially under physiologically and clinically relevant conditions of the pathogens, is the key to combat drug resistance. In animal hosts, most wounded, infected and inflamed tissues display low oxygen tensions. In this article, we summarize research development on multidrug efflux pumps in the medicinally relevant microaerobic and anaerobic pathogens and their implications in the effort to combat drug-resistant infections. PMID:27025630
Isolation and evaluation of cocktail phages for the control of multidrug-resistant Escherichia coli serotype O104: H4 and E. coli O157: H7 isolates causing diarrhea.

PubMed

Safwat Mohamed, Doaa; Farouk Ahmed, Eman; Mohamed Mahmoud, Abobakr; Abd El-Baky, Rehab Mahmoud; John, James

2018-02-01

Escherichia coli serotype O157: H7 and E. coli O104: H4 are well known foodborne pathogens causing sever enteric illness. Using bacteriophages as biocontrol agents of some foodborne pathogens and multidrug-resistant (MDR) bacteria has a great attention nowadays. This study aims to test the effect of cocktail phages on the growth of some foodborne pathogens and MDR E. coli. Routine conventional PCR was used to confirm the identification of E. coli isolates. Double-layered culture technique was used to isolate phages from sewage water. Morphology of bacteriophage was described using transmission electron microscopy, and spot test was performed to determine host range of the phage cocktail. Phage cocktail of Siphoviridae and Podoviridae family infecting E. coli O157: H7, E. coli O104: H4 and untypeable E. coli (neither O157 nor O104) has been isolated from sewage water. Phage cocktail showed both lytic and lysogenic activity. Lytic activity was observed against E. coli O157: H7, E. coli O104: H4 isolates, Staphylococcus. aureus ATCC6538 and Pseudomonas aeruginosa ATCC 10145, while the lysogenic activity was observed against the untypeable strain. The tested phage cocktail showed a promising inhibitory action on E. coli O157: H7 and O104: H4, S. aureus ATCC6538 and P. aeruginosa ATCC 10145, suggesting the possibility of its use as a biocontrol tool or as natural food preservatives for many food products. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Rationalizing meat consumption. The 4Ns.

PubMed

Piazza, Jared; Ruby, Matthew B; Loughnan, Steve; Luong, Mischel; Kulik, Juliana; Watkins, Hanne M; Seigerman, Mirra

2015-08-01

Recent theorizing suggests that the 4Ns - that is, the belief that eating meat is natural, normal, necessary, and nice - are common rationalizations people use to defend their choice of eating meat. However, such theorizing has yet to be subjected to empirical testing. Six studies were conducted on the 4Ns. Studies 1a and 1b demonstrated that the 4N classification captures the vast majority (83%-91%) of justifications people naturally offer in defense of eating meat. In Study 2, individuals who endorsed the 4Ns tended also to objectify (dementalize) animals and included fewer animals in their circle of moral concern, and this was true independent of social dominance orientation. Subsequent studies (Studies 3-5) showed that individuals who endorsed the 4Ns tend not to be motivated by ethical concerns when making food choices, are less involved in animal-welfare advocacy, less driven to restrict animal products from their diet, less proud of their animal-product decisions, tend to endorse Speciesist attitudes, tend to consume meat and animal products more frequently, and are highly committed to eating meat. Furthermore, omnivores who strongly endorsed the 4Ns tended to experience less guilt about their animal-product decisions, highlighting the guilt-alleviating function of the 4Ns. Copyright © 2015 Elsevier Ltd. All rights reserved.
GESPA: classifying nsSNPs to predict disease association.

PubMed

Khurana, Jay K; Reeder, Jay E; Shrimpton, Antony E; Thakar, Juilee

2015-07-25

Non-synonymous single nucleotide polymorphisms (nsSNPs) are the most common DNA sequence variation associated with disease in humans. Thus determining the clinical significance of each nsSNP is of great importance. Potential detrimental nsSNPs may be identified by genetic association studies or by functional analysis in the laboratory, both of which are expensive and time consuming. Existing computational methods lack accuracy and features to facilitate nsSNP classification for clinical use. We developed the GESPA (GEnomic Single nucleotide Polymorphism Analyzer) program to predict the pathogenicity and disease phenotype of nsSNPs. GESPA is a user-friendly software package for classifying disease association of nsSNPs. It allows flexibility in acceptable input formats and predicts the pathogenicity of a given nsSNP by assessing the conservation of amino acids in orthologs and paralogs and supplementing this information with data from medical literature. The development and testing of GESPA was performed using the humsavar, ClinVar and humvar datasets. Additionally, GESPA also predicts the disease phenotype associated with a nsSNP with high accuracy, a feature unavailable in existing software. GESPA's overall accuracy exceeds existing computational methods for predicting nsSNP pathogenicity. The usability of GESPA is enhanced by fast SQL-based cloud storage and retrieval of data. GESPA is a novel bioinformatics tool to determine the pathogenicity and phenotypes of nsSNPs. We anticipate that GESPA will become a useful clinical framework for predicting the disease association of nsSNPs. The program, executable jar file, source code, GPL 3.0 license, user guide, and test data with instructions are available at http://sourceforge.net/projects/gespa.
The multidrug transporter ABCG2 (BCRP) is inhibited by plant-derived cannabinoids.

PubMed

Holland, M L; Lau, D T T; Allen, J D; Arnold, J C

2007-11-01

Cannabinoids are used therapeutically for the palliation of the adverse side effects associated with cancer chemotherapy. However, cannabinoids also inhibit both the activity and expression of the multidrug transporter, P-glycoprotein in vitro. Here we address the interaction of cannabinol (CBN), cannabidiol (CBD) and delta 9-tetrahydrocannabinol (THC) with the related multidrug transporter, ABCG2. Cannabinoid inhibition of Abcg2/ABCG2 was assessed using flow cytometric analysis of substrate accumulation and ATPase activity assays. The cytotoxicity and chemosensitization by cannabinoids was determined with cell viability assays. Expression of cannabinoid and vanilloid receptors was assessed using reverse transcriptase polymerase chain reaction, and cannabinoid modulation of ABCG2 expression was examined using immunoblotting. CBN, CBD and THC increased the intracellular accumulation of the Abcg2/ABCG2 substrate, mitoxantrone, in an over-expressing cell line. The THC metabolite, (-)-11-nor-9-carboxy-delta 9-THC was much less potent. The plant cannabinoids inhibited both basal and substrate stimulated ATPase activity of human ABCG2. Cannabinoid cytotoxicity occurred in the absence of known cannabinoid cell surface receptors, and only at concentrations higher than those required for Abcg2/ABCG2 inhibition. Sub-toxic concentrations of the cannabinoids resensitized the overexpressing cell line to the cytotoxic effect of Abcg2/ABCG2 substrates, mitoxantrone and topotecan. This occurred in the absence of any effect on ABCG2 expression. Cannabinoids are novel Abcg2/ABCG2 inhibitors, reversing the Abcg2-mediated multidrug-resistant phenotype in vitro. This finding may have implications for the co-administration of cannabinoids with pharmaceuticals that are ABCG2 substrates.
Suitable technological conditions for enzymatic hydrolysis of waste paper by Novozymes® enzymes NS50013 and NS50010.

PubMed

Brummer, Vladimir; Skryja, Pavel; Jurena, Tomas; Hlavacek, Viliam; Stehlik, Petr

2014-10-01

Waste paper belongs to a group of quantitatively the most produced waste types. Enzymatic hydrolysis is becoming a suitable way to treat this type of waste and at the same time, to produce a valuable liquid biofuel, because reducing sugars solutions that are formed during the process of saccharification can be a precursor for following or simultaneous fermentation. If it will be possible to make the enzymatic hydrolysis of the waste paper economically viable, it could serve as one of the new ways to lower the dependence of the transport sector on oil in the future. Only several studies comparing the enzymatic hydrolysis of different waste papers were performed in the past; they are summarized in this manuscript. In our experimental trials, suitable technological conditions for waste paper enzymatic hydrolysis using enzymes from Novozymes® biomass kit: enzymes NS50013 and NS50010 were investigated. The following enzymatic hydrolysis parameters in laboratory scale trials were verified on high cellulose content substrates-filter paper and cellulose pulp: type of buffer, pH, temperature, concentration of the substrate, loading of the enzyme and rate of stirring.
Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by RIG-I

PubMed Central

Gack, Michaela Ulrike; Albrecht, Randy Allen; Urano, Tomohiko; Inn, Kyung-Soo; Huang, I-Chueh; Carnero, Elena; Farzan, Michael; Inoue, Satoshi; Jung, Jae Ung; García-Sastre, Adolfo

2009-01-01

SUMMARY TRIM25 mediates Lys 63-linked ubiquitination of the N-terminal CARDs of the viral RNA sensor RIG-I, leading to type I interferon (IFN) production. Here, we report that the influenza A virus non-structural protein 1 (NS1) specifically inhibits TRIM25-mediated RIG-I CARD ubiquitination, thereby suppressing RIG-I signal transduction. A novel domain in NS1 comprising E96/E97 residues mediates its interaction with the coiled-coil domain of TRIM25, thus blocking TRIM25 multimerization and RIG-I CARD ubiquitination. Furthermore, a recombinant influenza A virus expressing an E96A/E97A NS1 mutant is defective in blocking TRIM25-mediated anti-viral IFN response and loses virulence in mice. Our findings reveal a novel mechanism of influenza virus to inhibit host IFN response and also emphasize the vital role of TRIM25 in modulating viral infections. PMID:19454348
TFaNS-Tone Fan Noise Design/Prediction System: Users' Manual TFaNS Version 1.5

NASA Technical Reports Server (NTRS)

Topol, David A.; Huff, Dennis L. (Technical Monitor)

2003-01-01

TFaNS is the Tone Fan Noise Design/Prediction System developed by Pratt & Whitney under contract to NASA Glenn. The purpose of this system is to predict tone noise emanating from a fan stage including the effects of reflection and transmission by the rotor and stator and by the duct inlet and nozzle. The first version of this design system was developed under a previous NASA contract. Several improvements have been made to TFaNS. This users' manual shows how to run this new system. TFaNS consists of the codes that compute the acoustic properties (reflection and transmission coefficients) of the various elements and writes them to files, CUP3D Fan Noise Coupling Code that reads these files, solves the coupling problem, and outputs the desired noise predictions, and AWAKEN CFD/Measured Wake Postprocessor which reformats CFD wake predictions and/or measured wake data so they can be used by the system. This report provides information on code input and file structure essential for potential users of TFaNS.
Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries.

PubMed

Flachbartova, Z; Pulzova, L; Bencurova, E; Potocnakova, L; Comor, L; Bednarikova, Z; Bhide, M

2016-01-01

The aim of the study was to isolate and characterize novel antimicrobial peptides from peptide phage library with antimicrobial activity against multidrug resistant Listeria monocytogenes. Combinatorial phage-display library was used to affinity select peptides binding to the cell surface of multidrug resistant L. monocytogenes. After several rounds of affinity selection followed by sequencing, three peptides were revealed as the most promising candidates. Peptide L2 exhibited features common to antimicrobial peptides (AMPs), and was rich in Asp, His and Lys residues. Peptide L3 (NSWIQAPDTKSI), like peptide L2, inhibited bacterial growth in vitro, without any hemolytic or cytotoxic effects on eukaryotic cells. L1 peptide showed no inhibitory effect on Listeria. Structurally, peptides L2 and L3 formed random coils composed of α-helix and β-sheet units. Peptides L2 and L3 exhibited antimicrobial activity against multidrug resistant isolates of L. monocytogenes with no haemolytic or toxic effects. Both peptides identified in this study have the potential to be beneficial in human and veterinary medicine. Copyright © 2016 Elsevier GmbH. All rights reserved.
Effects of Zuccagnia punctata extracts and their flavonoids on the function and expression of ABCB1/P-glycoprotein multidrug transporter.

PubMed

Chieli, Elisabetta; Romiti, Nadia; Catiana Zampini, Iris; Garrido, Gabino; Inés Isla, María

2012-12-18

Zuccagnia punctata extracts (ZpE) are used in ethnomedicine as antimicrobial and anti-inflammatory drugs. The pharmacological properties of ZpE and their polyphenolic components suggest that they may be used as potential modulators on the P-glycoprotein (P-gp) multidrug transporter. P-gp is well known for its role in the acquired drug resistance by tumors following chemotherapy, causing a low drug bioavailability by extruding them out of the cells. To evaluate the effects of ZpE and three of their phenolic components: 7-hydroxyflavanone (HF), 3,7-dihydroxyflavone (DHF) and 2',4'-dihydroxychalcone (DHC) on P-gp activity and expression. The effects of natural products on ABCB1/P-gp function and expression were evaluated by R-123 accumulation assay and western blot analysis using HK-2 cells as experimental model. The ABCB1 mRNA content was determined by SQRT-PCR. The accumulation of R-123 in HK-2 cells was significantly increased by ZpE and DHF, and to a lesser extent by DHC, indicating their roles on the efflux transporter activity. However, HF did not show any effect. HK-2 cells maintained in the presence of ZpE or DHF for 72 h, showed an increase in P-gp expression whereas activity was unchanged or decreased. No changes were observed in ABCB1 mRNA content. Furthermore, in these assay conditions, more sensibility of HK-2 cells to the cytotoxic action of cyclosporine A (P-gp substrate) was observed. These results may suggest an impact of Zuccagnia punctata and some of its components on the pharmacokinetics of drugs that are P-gp substrates, as well as a potential role on multidrug resistance modulation. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.
Feeding a sub-ns-risetime rectangular pulse onto a rod-shaped resistive high-voltage divider in risetime <2 ns

NASA Astrophysics Data System (ADS)

Zeng, Zhengzhong; Ma, Lianying

2004-01-01

A simple and effective bridge-type feeding network consisting only of ordinary resistors and conductive wires is designed and tested which launches a 0.8 ns risetime, 40 ns width, and kV-level rectangular pulse from a coaxial cable onto a rod-shaped resistive high-voltage divider with risetime <2 ns with no significant distortion.
Two distinct sets of NS2A molecules are responsible for dengue virus RNA synthesis and virion assembly.

PubMed

Xie, Xuping; Zou, Jing; Puttikhunt, Chunya; Yuan, Zhiming; Shi, Pei-Yong

2015-01-15

Flavivirus nonstructural protein 2A (NS2A) plays important roles in both viral RNA synthesis and virion assembly. The molecular details of how the NS2A protein modulates the two distinct events have not been defined. To address this question, we have performed a systematic mutagenesis of NS2A using dengue virus (DENV) serotype 2 (DENV-2) as a model. We identified two sets of NS2A mutations with distinct defects during a viral infection cycle. One set of NS2A mutations (D125A and G200A) selectively abolished viral RNA synthesis. Mechanistically, the D125A mutation abolished viral RNA synthesis through blocking the N-terminal cleavage of the NS2A protein, leading to an unprocessed NS1-NS2A protein; this result suggests that amino acid D125 (far downstream of the N terminus of NS2A) may contribute to the recognition of host protease at the NS1-NS2A junction. The other set of NS2A mutations (G11A, E20A, E100A, Q187A, and K188A) specifically impaired virion assembly without significantly affecting viral RNA synthesis. Remarkably, mutants defective in virion assembly could be rescued by supplying in trans wild-type NS2A molecules expressed from a replicative replicon, by wild-type NS2A protein expressed alone, by a mutant NS2A (G200A) that is lethal for viral RNA synthesis, or by a different mutant NS2A that is defective in virion assembly. In contrast, none of the mutants defective in viral RNA synthesis could be rescued by trans-complementation. Collectively, the results indicate that two distinct sets of NS2A molecules are responsible for DENV RNA synthesis and virion assembly. Dengue virus (DENV) represents the most prevalent mosquito-borne human pathogen. Understanding the replication of DENV is essential for development of vaccines and therapeutics. Here we characterized the function of DENV-2 NS2A using a systematic mutagenesis approach. The mutagenesis results revealed two distinct sets of NS2A mutations: one set of mutations that result in defects in viral RNA
Identification of drug resistance and immune-driven variations in hepatitis C virus (HCV) NS3/4A, NS5A and NS5B regions reveals a new approach toward personalized medicine.

PubMed

Ikram, Aqsa; Obaid, Ayesha; Awan, Faryal Mehwish; Hanif, Rumeza; Naz, Anam; Paracha, Rehan Zafar; Ali, Amjad; Janjua, Hussnain Ahmed

2017-01-01

Cellular immune responses (T cell responses) during hepatitis C virus (HCV) infection are significant factors for determining the outcome of infection. HCV adapts to host immune responses by inducing mutations in its genome at specific sites that are important for HLA processing/presentation. Moreover, HCV also adapts to resist potential drugs that are used to restrict its replication, such as direct-acting antivirals (DAAs). Although DAAs have significantly reduced disease burden, resistance to these drugs is still a challenge for the treatment of HCV infection. Recently, drug resistance mutations (DRMs) observed in HCV proteins (NS3/4A, NS5A and NS5B) have heightened concern that the emergence of drug resistance may compromise the effectiveness of DAAs. Therefore, the NS3/4A, NS5A and NS5B drug resistance variations were investigated in this study, and their prevalence was examined in a large number of protein sequences from all HCV genotypes. Furthermore, potential CD4 + and CD8 + T cell epitopes were predicted and their overlap with genetic variations was explored. The findings revealed that many reported DRMs within NS3/4A, NS5A and NS5B are not drug-induced; rather, they are already present in HCV strains, as they were also detected in HCV-naïve patients. This study highlights several hot spots in which HLA and drug selective pressure overlap. Interestingly, these overlapping mutations were frequently observed among many HCV genotypes. This study implicates that knowledge of the host HLA type and HCV subtype/genotype can provide important information in defining personalized therapy. Copyright © 2016 Elsevier B.V. All rights reserved.
Multidrug-Resistant Salmonella enterica Serotype Typhi, Gulf of Guinea Region, Africa

PubMed Central

Baltazar, Murielle; Ngandjio, Antoinette; Holt, Kathryn Elizabeth; Lepillet, Elodie; Pardos de la Gandara, Maria; Collard, Jean-Marc; Bercion, Raymond; Nzouankeu, Ariane; Le Hello, Simon; Dougan, Gordon; Fonkoua, Marie-Christine

2015-01-01

We identified 3 lineages among multidrug-resistant (MDR) Salmonella enterica serotype Typhi isolates in the Gulf of Guinea region in Africa during the 2000s. However, the MDR H58 haplotype, which predominates in southern Asia and Kenya, was not identified. MDR quinolone-susceptible isolates contained a 190-kb incHI1 pST2 plasmid or a 50-kb incN pST3 plasmid. PMID:25811307
The interaction between NOLC1 and IAV NS1 protein promotes host cell apoptosis and reduces virus replication.

PubMed

Zhu, Chunyu; Zheng, Fangliang; Zhu, Junfeng; Liu, Meichen; Liu, Na; Li, Xue; Zhang, Li; Deng, Zaidong; Zhao, Qi; Liu, Hongsheng

2017-11-07

NS1 of the influenza virus plays an important role in the infection ability of the influenza virus. Our previous research found that NS1 protein interacts with the NOLC1 protein of host cells, however, the function of the interaction is unknown. In the present study, the role of the interaction between the two proteins in infection was further studied. Several analyses, including the use of a pull-down assay, Co-IP, western blot analysis, overexpression, RNAi, flow cytometry, etc., were used to demonstrate that the NS1 protein of H3N2 influenza virus interacts with host protein NOLC1 and reduces the quantity of NOLC1. The interaction also promotes apoptosis in A549 host cells, while the suppression of NOLC1 protein reduces the proliferation of the H3N2 virus. Based on these data, it was concluded that during the process of infection, NS1 protein interacts with NOLC1 protein, reducing the level of NOLC1, and that the interaction between the two proteins promotes apoptosis of host cells, thus reducing the proliferation of the virus. These findings provide new information on the biological function of the interaction between NS1 and NOLC1.
Influenza Viral Vectors Expressing Two Kinds of HA Proteins as Bivalent Vaccine Against Highly Pathogenic Avian Influenza Viruses of Clade 2.3.4.4 H5 and H7N9

PubMed Central

Li, Jinping; Hou, Guangyu; Wang, Yan; Wang, Suchun; Peng, Cheng; Yu, Xiaohui; Jiang, Wenming

2018-01-01

The H5 and H7N9 subtypes of highly pathogenic avian influenza viruses (HPAIVs) in China pose a serious challenge to public health and the poultry industry. In this study, a replication competent recombinant influenza A virus of the Í5N1 subtype expressing the H7 HA1 protein from a tri-cistronic NS segment was constructed. A heterologous dimerization domain was used to combine with the truncated NS1 protein of 73 amino acids to increase protein stability. H7 HA1, nuclear export protein coding region, and the truncated NS1 were fused in-frame into a single open reading frame via 2A self-cleaving peptides. The resulting PR8-H5-NS1(73)H7 stably expressed the H5 HA and H7 HA1 proteins, and exhibited similar growth kinetics as the parental PR8-H5 virus in vitro. PR8-H5-NS1(73)H7 induced specific hemagglutination inhibition (HI) antibody against H5, which was comparable to that of the combination vaccine of PR8-H5 and PR8-H7. The HI antibody titers against H7 virus were significantly lower than that by the combination vaccine. PR8-H5-NS1(73)H7 completely protected chickens from challenge with both H5 and H7 HPAIVs. These results suggest that PR8-H5-NS1(73)H7 is highly immunogenic and efficacious against both H5 and H7N9 HPAIVs in chickens. Highlights: - PR8-H5-NS1(73)H7 simultaneously expressed two HA proteins of different avian influenza virus subtypes. - PR8-H5-NS1(73)H7 was highly immunogenic in chickens. - PR8-H5-NS1(73)H7 provided complete protection against challenge with both H5 and H7N9 HPAIVs. PMID:29670587
Combined 3D-QSAR, molecular docking, molecular dynamics simulation, and binding free energy calculation studies on the 5-hydroxy-2H-pyridazin-3-one derivatives as HCV NS5B polymerase inhibitors.

PubMed

Yu, Haijing; Fang, Yu; Lu, Xia; Liu, Yongjuan; Zhang, Huabei

2014-01-01

The NS5B RNA-dependent RNA polymerase (RdRP) is a promising therapeutic target for developing novel anti-hepatitis C virus (HCV) drugs. In this work, a combined molecular modeling study was performed on a series of 193 5-hydroxy-2H-pyridazin-3-one derivatives as inhibitors of HCV NS5B Polymerase. The best 3D-QSAR models, including CoMFA and CoMSIA, are based on receptor (or docking). Furthermore, a 40-ns molecular dynamics (MD) simulation and binding free energy calculations using docked structures of NS5B with ten compounds, which have diverse structures and pIC50 values, were employed to determine the detailed binding process and to compare the binding modes of the inhibitors with different activities. On one side, the stability and rationality of molecular docking and 3D-QSAR results were validated by MD simulation. The binding free energies calculated by the MM-PBSA method gave a good correlation with the experimental biological activity. On the other side, by analyzing some differences between the molecular docking and the MD simulation results, we can find that the MD simulation could also remedy the defects of molecular docking. The analyses of the combined molecular modeling results have identified that Tyr448, Ser556, and Asp318 are the key amino acid residues in the NS5B binding pocket. The results from this study can provide some insights into the development of novel potent NS5B inhibitors. © 2013 John Wiley & Sons A/S.
Baseline NS5A resistance associated substitutions may impair DAA response in real-world hepatitis C patients.

PubMed

Carrasco, Itzíar; Arias, Ana; Benítez-Gutiérrez, Laura; Lledó, Gemma; Requena, Silvia; Cuesta, Miriam; Cuervas-Mons, Valentín; de Mendoza, Carmen

2018-03-01

Oral DAA have demonstrated high efficacy as treatment of hepatitis C. However, the presence of resistance-associated substitutions (RAS) at baseline has occasionally been associated with impaired treatment response. Herein, we examined the impact of baseline RAS at the HCV NS5A gene region on treatment response in a real-life setting. All hepatitis C patients treated with DAA including NS5A inhibitors at our institution were retrospectively examined. The virus NS5A gene was analyzed using population sequencing at baseline and after 24 weeks of completing therapy in all patients that failed. All changes recorded at positions 28, 29, 30, 31, 32, 58, 62, 92, and 93 were considered. A total of 166 patients were analyzed. HCV genotypes were as follows: G1a (31.9%), G1b (48.2%), G3 (10.2%), and G4 (9.6%). Overall, 69 (41.6%) patients were coinfected with HIV and 46.7% had advanced liver fibrosis (Metavir F3-F4). Sixty (36.1%) patients had at least one RAS at baseline, including M28A/G/T (5), Q30X (12), L31I/F/M/V (6), T58P/S (25), Q/E62D (1), A92 K (7), and Y93C/H (15). Overall, 4.8% had two or more RAS, being more frequent in G4 (12.5%) followed by G1b (6.3%) and G1a (1.9%). Of 10 (6%) patients that failed DAA therapy, five had baseline NS5A RAS. No association was found for specific baseline RAS, although changes at position 30 were more frequent in failures than cures (22.2% vs 6.4%, P = 0.074). Moreover, the presence of two or more RAS at baseline was more frequent in failures (HR: 7.2; P = 0.029). Upon failure, six patients showed emerging RAS, including Q30C/H/R (3), L31M (1), and Y93C/H (2). Baseline NS5A RAS are frequently seen in DAA-naïve HCV patients. Two or more baseline NS5A RAS were found in nearly 5% and were significantly associated to DAA failure. Therefore, baseline NS5A testing should be considered when HCV treatment is planned with NS5A inhibitors. © 2017 Wiley Periodicals, Inc.
Quantitative Assessment of Combination Antimicrobial Therapy against Multidrug-Resistant Acinetobacter baumannii▿

PubMed Central

Lim, Tze-Peng; Ledesma, Kimberly R.; Chang, Kai-Tai; Hou, Jing-Guo; Kwa, Andrea L.; Nikolaou, Michael; Quinn, John P.; Prince, Randall A.; Tam, Vincent H.

2008-01-01

Treatment of multidrug-resistant bacterial infections poses a therapeutic challenge to clinicians; combination therapy is often the only viable option for multidrug-resistant infections. A quantitative method was developed to assess the combined killing abilities of antimicrobial agents. Time-kill studies (TKS) were performed using a multidrug-resistant clinical isolate of Acinetobacter baumannii with escalating concentrations of cefepime (0 to 512 mg/liter), amikacin (0 to 256 mg/liter), and levofloxacin (0 to 64 mg/liter). The bacterial burden data in single and combined (two of the three agents with clinically achievable concentrations in serum) TKS at 24 h were mathematically modeled to provide an objective basis for comparing various antimicrobial agent combinations. Synergy and antagonism were defined as interaction indices of <1 and >1, respectively. A hollow-fiber infection model (HFIM) simulating various clinical (fluctuating concentrations over time) dosing exposures was used to selectively validate our quantitative assessment of the combined killing effect. Model fits in all single-agent TKS were satisfactory (r2 > 0.97). An enhanced combined overall killing effect was seen in the cefepime-amikacin combination (interactive index, 0.698; 95% confidence interval [CI], 0.675 to 0.722) and the cefepime-levofloxacin combination (interactive index, 0.929; 95% CI, 0.903 to 0.956), but no significant difference in the combined overall killing effect for the levofloxacin-amikacin combination was observed (interactive index, 0.994; 95% CI, 0.982 to 1.005). These assessments were consistent with observations in HFIM validation studies. Our method could be used to objectively rank the combined killing activities of two antimicrobial agents when used together against a multidrug-resistant A. baumannii isolate. It may offer better insights into the effectiveness of various antimicrobial combinations and warrants further investigations. PMID:18505848
Natural lignans from Arctium lappa modulate P-glycoprotein efflux function in multidrug resistant cancer cells.

PubMed

Su, Shan; Cheng, Xinlai; Wink, Michael

2015-02-15

Arctium lappa is a well-known traditional medicinal plant in China (TCM) and Europe that has been used for thousands of years to treat arthritis, baldness or cancer. The plant produces lignans as secondary metabolites which have a wide range of bioactivities. Yet, their ability to reverse multidrug resistance (MDR) in cancer cells has not been explored. In this study, we isolated six lignans from A. lappa seeds, namely arctigenin, matairesinol, arctiin, (iso)lappaol A, lappaol C, and lappaol F. The MDR reversal potential of the isolated lignans and the underlying mechanism of action were studied using two MDR cancer cell lines, CaCo2 and CEM/ADR 5000 which overexpress P-gp and other ABC transporters. In two-drug combinations of lignans with the cytotoxic doxorubicin, all lignans exhibited synergistic effects in CaCo2 cells and matairesinol, arctiin, lappaol C and lappaol F display synergistic activity in CEM/ADR 5000 cells. Additionally, in three-drug combinations of lignans with the saponin digitonin and doxorubicin MDR reversal activity was even stronger enhanced. The lignans can increase the retention of the P-gp substrate rhodamine 123 in CEM/ADR 5000 cells, indicating that lignans can inhibit the activity of P-gp. Our study provides a first insight into the potential chemosensitizing activity of a series of natural lignans, which might be candidates for developing novel adjuvant anticancer agents. Copyright © 2015 Elsevier GmbH. All rights reserved.
Regulation of MVM NS1 by protein kinase C: impact of mutagenesis at consensus phosphorylation sites on replicative functions and cytopathic effects.

PubMed

Corbau, R; Duverger, V; Rommelaere, J; Nüesch, J P

2000-12-05

Minute virus of mice NS1, an 83-kDa mainly nuclear phosphoprotein, is the only viral nonstructural protein required in all cell types and it is involved in multiple processes necessary for virus propagation. The diversity of functions assigned to NS1, together with the variation of its complex phosphorylation pattern during infection, suggested that the various activities of NS1 could be regulated by distinct phosphorylation events. So far, it has been demonstrated that NS1 replicative functions, in particular, DNA-unwinding activities, are regulated by protein kinase C (PKC), as exemplified by the modulation of NS1 helicase activity by PKClambda phosphorylation. In order to determine further impact of phosphorylation on NS1 functions, including the induction of cytopathic effects, a mutational approach was pursued in order to produce NS1 variants harboring amino acid substitutions at candidate PKC target residues. Besides the determination of two additional in vivo phosphorylation sites in NS1, this mutagenesis allowed the segregation of distinct NS1 functions from one another, generating NS1 variants with a distinct activity profile. Thus, we obtained NS1 mutants that were fully proficient for trans activation of the viral P38 promoter, while being impaired in their replicative functions. Moreover, the alterations of specific PKC phosphorylation sites gave rise to NS1 polypeptides that exerted reduced cytotoxicity, leading to sustained gene expression, while keeping functions necessary for progeny virus production, i.e., viral DNA replication and activation of the capsid gene promoter. These data suggested that in the course of a viral infection, NS1 may undergo a shift from productive to cytotoxic functions as a result of a phosphorylation-dependent regulation. Copyright 2000 Academic Press.
Multidrug Transport Protein NorM from Vibrio cholerae Simultaneously Couples to Sodium- and Proton-Motive Force*

PubMed Central

Jin, Yoonhee; Nair, Asha; van Veen, Hendrik W.

2014-01-01

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na+ or H+. We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na+ or H+ binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures. PMID:24711447

Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor.

PubMed

Owens, Christopher M; Brasher, Bradley B; Polemeropoulos, Alex; Rhodin, Michael H J; McAllister, Nicole; Wong, Kelly A; Jones, Christopher T; Jiang, Lijuan; Lin, Kai; Or, Yat Sun

2016-10-01

EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.). Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication.

PubMed

Naik, Nenavath Gopal; Wu, Huey-Nan

2015-07-01

machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
The multidrug transporter ABCG2 (BCRP) is inhibited by plant-derived cannabinoids

PubMed Central

Holland, M L; Lau, D T T; Allen, J D; Arnold, J C

2007-01-01

Background and purpose: Cannabinoids are used therapeutically for the palliation of the adverse side effects associated with cancer chemotherapy. However, cannabinoids also inhibit both the activity and expression of the multidrug transporter, P-glycoprotein in vitro. Here we address the interaction of cannabinol (CBN), cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) with the related multidrug transporter, ABCG2. Experimental approach: Cannabinoid inhibition of Abcg2/ABCG2 was assessed using flow cytometric analysis of substrate accumulation and ATPase activity assays. The cytotoxicity and chemosensitization by cannabinoids was determined with cell viability assays. Expression of cannabinoid and vanilloid receptors was assessed using reverse transcriptase polymerase chain reaction, and cannabinoid modulation of ABCG2 expression was examined using immunoblotting. Key results: CBN, CBD and THC increased the intracellular accumulation of the Abcg2/ABCG2 substrate, mitoxantrone, in an over-expressing cell line. The THC metabolite, (−)-11-nor-9-carboxy-Δ9-THC was much less potent. The plant cannabinoids inhibited both basal and substrate stimulated ATPase activity of human ABCG2. Cannabinoid cytotoxicity occurred in the absence of known cannabinoid cell surface receptors, and only at concentrations higher than those required for Abcg2/ABCG2 inhibition. Sub-toxic concentrations of the cannabinoids resensitized the overexpressing cell line to the cytotoxic effect of Abcg2/ABCG2 substrates, mitoxantrone and topotecan. This occurred in the absence of any effect on ABCG2 expression. Conclusions and implications: Cannabinoids are novel Abcg2/ABCG2 inhibitors, reversing the Abcg2-mediated multidrug-resistant phenotype in vitro. This finding may have implications for the co-administration of cannabinoids with pharmaceuticals that are ABCG2 substrates. PMID:17906686
Heat-Storage Modules Containing LiNO3-3H2O and Graphite Foam

NASA Technical Reports Server (NTRS)

Bootle, John

2008-01-01

A heat-storage module based on a commercial open-cell graphite foam (Poco-Foam or equivalent) imbued with lithium nitrate trihydrate (LiNO3-3H2O) has been developed as a prototype of other such modules for use as short-term heat sources or heat sinks in the temperature range of approximately 28 to 30 C. In this module, the LiNO3-3H2O serves as a phase-change heat-storage material and the graphite foam as thermally conductive filler for transferring heat to or from the phase-change material. In comparison with typical prior heat-storage modules in which paraffins are the phase-change materials and aluminum fins are the thermally conductive fillers, this module has more than twice the heat-storage capacity per unit volume.
NS-398, a selective COX-2 inhibitor, inhibits proliferation of IL-1{beta}-stimulated vascular smooth muscle cells by induction of {eta}{omicron}-1

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

Choi, Hyoung Chul; Kim, Hee Sun; Lee, Kwang Youn

2008-11-28

We investigated whether NS-398, a selective inhibitor of COX-2, induces HO-1 in IL-1{beta}-stimulated vascular smooth muscle cells (VSMC). NS-398 reduced the production of PGE{sub 2} without modulation of expression of COX-2 in IL-1{beta}-stimulated VSMC. NS-398 increased HO-1 mRNA and protein in a dose-dependent manner, but inhibited proliferation of IL-1{beta}-stimulated VSMC. Furthermore, SnPPIX, a HO-1 inhibitor, reversed the effects of NS-398 on PGE{sub 2} production, suggesting that COX-2 activity can be affected by HO-1. Hemin, a HO-1 inducer, also reduced the production of PGE{sub 2} and proliferation of IL-1{beta}-stimulated VSMC. CORM-2, a CO-releasing molecule, but not bilirubin inhibited proliferation of IL-1{beta}-stimulatedmore » VSMC. NS-398 inhibited proliferation of IL-1{beta}-stimulated VSMC in a HbO{sub 2}-sensitive manner. In conclusion, NS-398 inhibits proliferation of IL-1{beta}-stimulated VSMC by HO-1-derived CO. Thus, NS-398 may facilitate the healing process of vessels in vascular inflammatory disorders such as atherosclerosis.« less
Redox Control of Multidrug Resistance and Its Possible Modulation by Antioxidants

PubMed Central

Cort, Aysegul; Ozben, Tomris; Saso, Luciano; De Luca, Chiara

2016-01-01

Clinical efficacy of anticancer chemotherapies is dramatically hampered by multidrug resistance (MDR) dependent on inherited traits, acquired defence against toxins, and adaptive mechanisms mounting in tumours. There is overwhelming evidence that molecular events leading to MDR are regulated by redox mechanisms. For example, chemotherapeutics which overrun the first obstacle of redox-regulated cellular uptake channels (MDR1, MDR2, and MDR3) induce a concerted action of phase I/II metabolic enzymes with a temporal redox-regulated axis. This results in rapid metabolic transformation and elimination of a toxin. This metabolic axis is tightly interconnected with the inducible Nrf2-linked pathway, a key switch-on mechanism for upregulation of endogenous antioxidant enzymes and detoxifying systems. As a result, chemotherapeutics and cytotoxic by-products of their metabolism (ROS, hydroperoxides, and aldehydes) are inactivated and MDR occurs. On the other hand, tumour cells are capable of mounting an adaptive antioxidant response against ROS produced by chemotherapeutics and host immune cells. The multiple redox-dependent mechanisms involved in MDR prompted suggesting redox-active drugs (antioxidants and prooxidants) or inhibitors of inducible antioxidant defence as a novel approach to diminish MDR. Pitfalls and progress in this direction are discussed. PMID:26881027
Activation of c-jun N-terminal kinase upon influenza A virus (IAV) infection is independent of pathogen-related receptors but dependent on amino acid sequence variations of IAV NS1.

PubMed

Nacken, Wolfgang; Anhlan, Darisuren; Hrincius, Eike R; Mostafa, Ahmed; Wolff, Thorsten; Sadewasser, Anne; Pleschka, Stephan; Ehrhardt, Christina; Ludwig, Stephan

2014-08-01

A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation
Activation of c-jun N-Terminal Kinase upon Influenza A Virus (IAV) Infection Is Independent of Pathogen-Related Receptors but Dependent on Amino Acid Sequence Variations of IAV NS1

PubMed Central

Nacken, Wolfgang; Anhlan, Darisuren; Hrincius, Eike R.; Mostafa, Ahmed; Wolff, Thorsten; Sadewasser, Anne; Pleschka, Stephan; Ehrhardt, Christina

2014-01-01

ABSTRACT A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. IMPORTANCE Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that
Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP)

PubMed Central

Glickman, Randolph D.; Tolstykh, Gleb P.; Estlack, Larry E.; Moen, Erick K.; Echchgadda, Ibtissam; Beier, Hope T.; Barnes, Ronald A.; Ibey, Bennett L.

2016-01-01

Nanosecond electrical pulse (nsEP) exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s) between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress. PMID:27135944
Multidrug resistance in enteric and other gram-negative bacteria.

PubMed

George, A M

1996-05-15

In Gram-negative bacteria, multidrug resistance is a term that is used to describe mechanisms of resistance by chromosomal genes that are activated by induction or mutation caused by the stress of exposure to antibiotics in natural and clinical environments. Unlike plasmid-borne resistance genes, there is no alteration or degradation of drugs or need for genetic transfer. Exposure to a single drug leads to cross-resistance to many other structurally and functionally unrelated drugs. The only mechanism identified for multidrug resistance in bacteria is drug efflux by membrane transporters, even though many of these transporters remain to be identified. The enteric bacteria exhibit mostly complex multidrug resistance systems which are often regulated by operons or regulons. The purpose of this review is to survey molecular mechanisms of multidrug resistance in enteric and other Gram-negative bacteria, and to speculate on the origins and natural physiological functions of the genes involved.
Substrate inhibition kinetic model for West Nile virus NS2B-NS3 protease.

PubMed

Tomlinson, Suzanne M; Watowich, Stanley J

2008-11-11

West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested that peptide substrates could bind to two binding sites on the protease. Reaction product analogues also showed inhibition of the protease, demonstrating product inhibition in addition to and distinct from substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1'-P3' interaction surface (i.e., the S1'-S3' sites). Optimization of substrate analogue inhibitors that target these two independent sites may lead to novel anti-WNV drugs.
Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly.

PubMed

Senthilvel, Padmanaban; Lavanya, Pandian; Kumar, Kalavathi Murugan; Swetha, Rayapadi; Anitha, Parimelzaghan; Bag, Susmita; Sarveswari, Sundaramoorthy; Vijayakumar, Vijayaparthasarathi; Ramaiah, Sudha; Anbarasu, Anand

2013-01-01

Dengue virus belongs to the virus family Flaviviridae. Dengue hemorrhagic disease caused by dengue virus is a public health problem worldwide. The viral non structural 2B and 3 (NS2B-NS3) protease complex is crucial for virus replication and hence, it is considered to be a good anti-viral target. Leaf extracts from Carica papaya is generally prescribed for patients with dengue fever, but there are no scientific evidences for its anti-dengue activity; hence we intended to investigate the anti-viral activity of compounds present in the leaves of Carica papaya against dengue 2 virus (DENV-2). We analysed the anti-dengue activities of the extracts from Carica papaya by using bioinformatics tools. Interestingly, we find the flavonoid quercetin with highest binding energy against NS2B-NS3 protease which is evident by the formation of six hydrogen bonds with the amino acid residues at the binding site of the receptor. Our results suggest that the flavonoids from Carica papaya have significant anti-dengue activities. ADME - Absorption, distribution, metabolism and excretion, BBB - Blood brain barrier, CYP - Cytochrome P450, DENV - - Dengue virus, DHF - Dengue hemorrhagic fever, DSS - Dengue shock syndrome, GCMS - - Gas chromatography- Mass spectrometry, MOLCAD - Molecular Computer Aided Design, NS - Non structural, PDB - Protein data bank, PMF - Potential Mean Force.
Multidrug transport protein norM from vibrio cholerae simultaneously couples to sodium- and proton-motive force.

PubMed

Jin, Yoonhee; Nair, Asha; van Veen, Hendrik W

2014-05-23

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na(+) or H(+). We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na(+) or H(+) binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Extended substrate specificity and first potent irreversible inhibitor/activity-based probe design for Zika virus NS2B-NS3 protease.

PubMed

Rut, Wioletta; Zhang, Linlin; Kasperkiewicz, Paulina; Poreba, Marcin; Hilgenfeld, Rolf; Drąg, Marcin

2017-03-01

Zika virus is spread by Aedes mosquitoes and is linked to acute neurological disorders, especially to microcephaly in newborn children and Guillan-Barré Syndrome. The NS2B-NS3 protease of this virus is responsible for polyprotein processing and therefore considered an attractive drug target. In this study, we have used the Hybrid Combinatorial Substrate Library (HyCoSuL) approach to determine the substrate specificity of ZIKV NS2B-NS3 protease in the P4-P1 positions using natural and a large spectrum of unnatural amino acids. Obtained data demonstrate a high level of specificity of the S3-S1 subsites, especially for basic amino acids. However, the S4 site exhibits a very broad preference toward natural and unnatural amino acids with selected D-amino acids being favored over L enantiomers. This information was used for the design of a very potent phosphonate inhibitor/activity-based probe of ZIKV NS2B-NS3 protease. Copyright © 2016 Elsevier B.V. All rights reserved.
Green Tea Catechin-Based Complex Micelles Combined with Doxorubicin to Overcome Cardiotoxicity and Multidrug Resistance

PubMed Central

Cheng, Tangjian; Liu, Jinjian; Ren, Jie; Huang, Fan; Ou, Hanlin; Ding, Yuxun; Zhang, Yumin; Ma, Rujiang; An, Yingli; Liu, Jianfeng; Shi, Linqi

2016-01-01

Chemotherapy for cancer treatment has been demonstrated to cause some side effects on healthy tissues and multidrug resistance of the tumor cells, which greatly limits therapeutic efficacy. To address these limitations and achieve better therapeutic efficacy, combination therapy based on nanoparticle platforms provides a promising approach through delivering different agents simultaneously to the same destination with synergistic effect. In this study, a novel green tea catechin-based polyion complex (PIC) micelle loaded with doxorubicin (DOX) and (-)-Epigallocatechin-3-O-gallate (EGCG) was constructed through electrostatic interaction and phenylboronic acid-catechol interaction between poly(ethylene glycol)-block-poly(lysine-co-lysine-phenylboronic acid) (PEG-PLys/PBA) and EGCG. DOX was co-loaded in the PIC micelles through π-π stacking interaction with EGCG. The phenylboronic acid-catechol interaction endowed the PIC micelles with high stability under physiological condition. Moreover, acid cleavability of phenylboronic acid-catechol interaction in the micelle core has significant benefits for delivering EGCG and DOX to same destination with synergistic effects. In addition, benefiting from the oxygen free radicals scavenging activity of EGCG, combination therapy with EGCG and DOX in the micelle core could protect the cardiomyocytes from DOX-mediated cardiotoxicity according to the histopathologic analysis of hearts. Attributed to modulation of EGCG on P-glycoprotein (P-gp) activity, this kind of PIC micelles could effectively reverse multidrug resistance of cancer cells. These results suggested that EGCG based PIC micelles could effectively overcome DOX induced cardiotoxicity and multidrug resistance. PMID:27375779
Green Tea Catechin-Based Complex Micelles Combined with Doxorubicin to Overcome Cardiotoxicity and Multidrug Resistance.

PubMed

Cheng, Tangjian; Liu, Jinjian; Ren, Jie; Huang, Fan; Ou, Hanlin; Ding, Yuxun; Zhang, Yumin; Ma, Rujiang; An, Yingli; Liu, Jianfeng; Shi, Linqi

2016-01-01

Chemotherapy for cancer treatment has been demonstrated to cause some side effects on healthy tissues and multidrug resistance of the tumor cells, which greatly limits therapeutic efficacy. To address these limitations and achieve better therapeutic efficacy, combination therapy based on nanoparticle platforms provides a promising approach through delivering different agents simultaneously to the same destination with synergistic effect. In this study, a novel green tea catechin-based polyion complex (PIC) micelle loaded with doxorubicin (DOX) and (-)-Epigallocatechin-3-O-gallate (EGCG) was constructed through electrostatic interaction and phenylboronic acid-catechol interaction between poly(ethylene glycol)-block-poly(lysine-co-lysine-phenylboronic acid) (PEG-PLys/PBA) and EGCG. DOX was co-loaded in the PIC micelles through π-π stacking interaction with EGCG. The phenylboronic acid-catechol interaction endowed the PIC micelles with high stability under physiological condition. Moreover, acid cleavability of phenylboronic acid-catechol interaction in the micelle core has significant benefits for delivering EGCG and DOX to same destination with synergistic effects. In addition, benefiting from the oxygen free radicals scavenging activity of EGCG, combination therapy with EGCG and DOX in the micelle core could protect the cardiomyocytes from DOX-mediated cardiotoxicity according to the histopathologic analysis of hearts. Attributed to modulation of EGCG on P-glycoprotein (P-gp) activity, this kind of PIC micelles could effectively reverse multidrug resistance of cancer cells. These results suggested that EGCG based PIC micelles could effectively overcome DOX induced cardiotoxicity and multidrug resistance.
Pathogenic and multidrug-resistant Escherichia fergusonii from broiler chicken.

PubMed

Forgetta, V; Rempel, H; Malouin, F; Vaillancourt, R; Topp, E; Dewar, K; Diarra, M S

2012-02-01

An Escherichia spp. isolate, ECD-227, was previously identified from the broiler chicken as a phylogenetically divergent and multidrug-resistant Escherichia coli possessing numerous virulence genes. In this study, whole genome sequencing and comparative genome analysis was used to further characterize this isolate. The presence of known and putative antibiotic resistance and virulence open reading frames were determined by comparison to pathogenic (E. coli O157:H7 TW14359, APEC O1:K1:H7, and UPEC UTI89) and nonpathogenic species (E. coli K-12 MG1655 and Escherichia fergusonii ATCC 35469). The assembled genome size of 4.87 Mb was sequenced to 18-fold depth of coverage and predicted to contain 4,376 open reading frames. Phylogenetic analysis of 537 open reading frames present across 110 enteric bacterial species identifies ECD-227 to be E. fergusonii. The genome of ECD-227 contains 5 plasmids showing similarity to known E. coli and Salmonella enterica plasmids. The presence of virulence and antibiotic resistance genes were identified and localized to the chromosome and plasmids. The mutation in gyrA (S83L) involved in fluoroquinolone resistance was identified. The Salmonella-like plasmids harbor antibiotic resistance genes on a class I integron (aadA, qacEΔ-sul1, aac3-VI, and sulI) as well as numerous virulence genes (iucABCD, sitABCD, cib, traT). In addition to the genome analysis, the virulence of ECD-227 was evaluated in a 1-d-old chick model. In the virulence assay, ECD-227 was found to induce 18 to 30% mortality in 1-d-old chicks after 24 h and 48 h of infection, respectively. This study documents an avian multidrug-resistant and virulent E. fergusonii. The existence of several resistance genes to multiple classes of antibiotics indicates that infection caused by ECD-227 would be difficult to treat using antimicrobials currently available for poultry.
Anthracene-based Inhibitors of Dengue Virus NS2B-NS3 Protease†

PubMed Central

Tomlinson, Suzanne M.; Watowich, Stanley J.

2010-01-01

Summary Dengue virus (DENV) is a mosquito-borne flavivirus that has strained global healthcare systems throughout tropical and subtropical regions of the world. In addition to plaguing developing nations, it has re-emerged in several developed countries with recent outbreaks in the USA (CDC, 2010), Australia (Hanna et al., 2009), Taiwan (Kuan et al., 2010) and France (La Ruche et al., 2010). DENV infection can cause significant disease, including dengue fever, dengue hemorrhagic fever, dengue shock syndrome, and death. There are no approved vaccines or antiviral therapies to prevent or treat dengue-related illnesses. However, the viral NS2B-NS3 protease complex provides a strategic target for antiviral drug development since NS3 protease activity is required for virus replication. Recently, we reported two compounds with inhibitory activity against the DENV protease in vitro and antiviral activity against dengue 2 (DEN2V) in cell culture (Tomlinson et al., 2009a). Analogs of one of the lead compounds were purchased, tested in protease inhibition assays, and the data evaluated with detailed kinetic analyses. A structure activity relationship (SAR) identified key atomic determinants (i.e. functional groups) important for inhibitory activity. Four “second series” analogs were selected and tested to validate our SAR and structural models. Here, we report improvements to inhibitory activity ranging between ~2- and 60-fold, resulting in selective low micromolar dengue protease inhibitors. PMID:21185332
NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.

PubMed

Su, Xun-Cheng; Ozawa, Kiyoshi; Yagi, Hiromasa; Lim, Siew P; Wen, Daying; Ekonomiuk, Dariusz; Huang, Danzhi; Keller, Thomas H; Sonntag, Sebastian; Caflisch, Amedeo; Vasudevan, Subhash G; Otting, Gottfried

2009-08-01

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.
Nedocromil sodium modulates nonadrenergic, noncholinergic bronchoconstrictor nerves in guinea pig airways in vitro.

PubMed

Verleden, G M; Belvisi, M G; Stretton, C D; Barnes, P J

1991-01-01

Nonadrenergic, noncholinergic (NANC) neural bronchoconstrictor responses in guinea pig airways are due to the release of tachykinins from sensory nerves. We have performed an in vitro study using electrical field stimulation (EFS; 40 V, 0.5 ms, 8 Hz for 20 s) in guinea pig bronchi to investigate the effect of nedocromil sodium (NS) on NANC bronchoconstrictor responses. NS inhibited NANC bronchoconstriction in bronchi in a concentration-dependent manner, with a maximum inhibition of 40 +/- 4% (p less than 0.001, n = 6) at 100 microM. Cromolyn sodium, however, produced only 9 +/- 8% inhibition at the same molar concentration (p less than 0.05). NS did not affect the contractile response to substance P, nor did it modulate the cholinergic bronchoconstrictor response to EFS in tracheal smooth muscle. These results indicate that NS may modulate the release of tachykinins from airway sensory nerves.

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities.

PubMed

Aydin, Cihan; Mukherjee, Sourav; Hanson, Alicia M; Frick, David N; Schiffer, Celia A

2013-12-01

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full-length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain-domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild-type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full-length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an "extended" catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo. © 2013 The Protein Society.
Multidrug Resistance Proteins (MRPs/ABCCs) in Cancer Chemotherapy and Genetic Diseases

PubMed Central

Chen, Zhe-Sheng; Tiwari, Amit K.

2011-01-01

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins that are best known for their ability to transport a wide variety of exogenous and endogenous substances across membranes against a concentration gradient via ATP hydrolysis. There are seven subfamilies of human ABC transporters, one of the largest being the ‘C’ subfamily (gene symbol ABCC). Nine ABCC subfamily members, the so-called Multidrug Resistance Proteins (MRPs) 1-9, have been implicated in mediating multidrug resistance in tumor cells to varying degrees as the efflux extrude chemotherapeutic compounds (or their metabolites) from malignant cells. Some of the MRPs are also known to either influence drug disposition in normal tissues or modulate the elimination of drugs (or their metabolites) via hepatobiliary or renal excretory pathways. In addition, the cellular efflux of physiologically important organic anions such as leukotriene C4 and cAMP is mediated by one or more of the MRPs. Finally, mutations in several MRPs are associated with human genetic disorders. In this review article, the current biochemical and physiological knowledge of MRP1-MRP9 in cancer chemotherapy and human genetic disease is summarized. The mutations in MRP2/ABCC2 leading to conjugated hyperbilirubinemia (Dubin-Johnson syndrome) and in MRP6/ABCC6 leading to the connective tissue disorder Pseudoxanthoma elasticum are also discussed. PMID:21740521
Nanomedicine to Deal With Cancer Cell Biology in Multi-Drug Resistance.

PubMed

Tekchandani, Pawan; Kurmi, Balak Das; Paliwal, Shivani Rai

2017-01-01

Today Cancer still remains a major cause of mortality and death worldwide, in humans. Chemotherapy, a key treatment strategy in cancer, has significant hurdles such as the occurrence of chemoresistance in cancer, which is inherent unresponsiveness or acquired upon exposure to chemotherapeutics. The resistance of cancer cells to an antineoplastic agent accompanied to other chemotherapeutic drugs with different structures and mechanisms of action called multi-drug resistance (MDR) plays an important role in the failure of chemo- therapeutics. MDR is primarily based on the overexpression of drug efflux pumps in the cellular membrane, which belongs to the ATP-binding cassette (ABC) superfamily of proteins, are P-gp (P-glycoprotein) and multidrug resistance-associated protein (MRP). Over the years, various strategies have been evaluated to overcome MDR, based not only on the use of MDR modulators but also on the implementation an innovative approach and advanced nanosized drug delivery systems. Nanomedicine is an emerging tool of chemotherapy that focuses on alternative drug delivery for improvement of the treatment efficacy and reducing side effects to normal tissues. This review aims to focus on the details biology, reversal strategies option with the limitation of MDR and various advantages of the present medical science nanotechnology with intracellular delivery aspects for overcoming the significant potential for improving the treatment of MDR malignancies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
Coarse-grained Simulations of Conformational Changes in Multidrug Resistance Transporters

NASA Astrophysics Data System (ADS)

Jewel, S. M. Yead; Dutta, Prashanta; Liu, Jin

2016-11-01

The overexpression of multidrug resistance (MDR) systems on the gram negative bacteria causes serious problems for treatment of bacterial infectious diseases. The system effectively pumps the antibiotic drugs out of the bacterial cells. During the pumping process one of the MDR components, AcrB undergoes a series of large-scale conformational changes which are responsible for drug recognition, binding and expelling. All-atom simulations are unable to capture those conformational changes because of computational cost. Here, we implement a hybrid coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid, to investigate the proton-dependent conformational changes of AcrB. The simulation results in early stage ( 100 ns) of proton-dependent conformational changes agree with all-atom simulations, validating the coarse-grained model. The coarse-grained force field allows us to explore the process in microsecond simulations. Starting from the crystal structures of Access(A)/Binding(B)/Extrusion(E) monomers in AcrB, we find that deprotonation of Asp407 and Asp408 in monomer E causes a series of large-scale conformational changes from ABE to AAA in absence of drug molecules, which is consistent with experimental findings. This work is supported by NIH Grant: 1R01GM122081-01.
Genomic Analysis of Multidrug-Resistant Escherichia coli from North Carolina Community Hospitals: Ongoing Circulation of CTX-M-Producing ST131-H30Rx and ST131-H30R1 Strains.

PubMed

Kanamori, Hajime; Parobek, Christian M; Juliano, Jonathan J; Johnson, James R; Johnston, Brian D; Johnson, Timothy J; Weber, David J; Rutala, William A; Anderson, Deverick J

2017-08-01

Escherichia coli sequence type 131 (ST131) predominates globally among multidrug-resistant (MDR) E. coli strains. We used whole-genome sequencing (WGS) to investigate 63 MDR E. coli isolates from 7 North Carolina community hospitals (2010 to 2015). Of these, 39 (62%) represented ST131, including 37 (95%) from the ST131- H 30R subclone: 10 (27%) from its H 30R1 subset and 27 (69%) from its H 30Rx subset. ST131 core genomes differed by a median of 15 (range, 0 to 490) single-nucleotide variants (SNVs) overall versus only 7 within H 30R1 (range, 3 to 12 SNVs) and 11 within H 30Rx (range, 0 to 21). The four isolates with identical core genomes were all H 30Rx. Epidemiological and clinical characteristics did not vary significantly by strain type, but many patients with MDR E. coli or H 30Rx infection were critically ill and had poor outcomes. H 30Rx isolates characteristically exhibited fluoroquinolone resistance and CTX-M-15 production, had a high prevalence of trimethoprim-sulfamethoxazole resistance (89%), sul1 (89%), and dfrA17 (85%), and were enriched for specific virulence traits, and all qualified as extraintestinal pathogenic E. coli The high overall prevalence of CTX-M-15 appeared to be possibly attributable to its association with the ST131- H 30Rx subclone and IncF[F2:A1:B-] plasmids. Some phylogenetically clustered non-ST131 MDR E. coli isolates also had distinctive serotypes/ fimH types, fluoroquinolone mutations, CTX-M variants, and IncF types. Thus, WGS analysis of our community hospital source MDR E. coli isolates suggested ongoing circulation and differentiation of E. coli ST131 subclones, with clonal segregation of CTX-M variants, other resistance genes, Inc-type plasmids, and virulence genes. Copyright © 2017 American Society for Microbiology.
Identification and subcellular localization of porcine deltacoronavirus accessory protein NS6

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

Fang, Puxian; Fang, Liurong; Liu, Xiaorong

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus. Accessory proteins are genus-specific for coronavirus, and two putative accessory proteins, NS6 and NS7, are predicted to be encoded by PDCoV; however, this remains to be confirmed experimentally. Here, we identified the leader-body junction sites of NS6 subgenomic RNA (sgRNA) and found that the actual transcription regulatory sequence (TRS) utilized by NS6 is non-canonical and is located upstream of the predicted TRS. Using the purified NS6 from an Escherichia coli expression system, we obtained two anti-NS6 monoclonal antibodies that could detect the predicted NS6 in cells infected with PDCoV or transfectedmore » with NS6-expressing plasmids. Further studies revealed that NS6 is always localized in the cytoplasm of PDCoV-infected cells, mainly co-localizing with the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments, as well as partially with the Golgi apparatus. Together, our results identify the NS6 sgRNA and demonstrate its expression in PDCoV-infected cells. -- Highlights: •The leader-body fusion site of NS6 sgRNA is identified. •NS6 sgRNA uses a non-canonical transcription regulatory sequence (TRS). •NS6 can be expressed in PDCoV-infected cell. •NS6 predominantly localize to the ER complex and ER-Golgi intermediate compartment.« less
Multidrug resistance in fungi: regulation of transporter-encoding gene expression

PubMed Central

Paul, Sanjoy; Moye-Rowley, W. Scott

2014-01-01

A critical risk to the continued success of antifungal chemotherapy is the acquisition of resistance; a risk exacerbated by the few classes of effective antifungal drugs. Predictably, as the use of these drugs increases in the clinic, more resistant organisms can be isolated from patients. A particularly problematic form of drug resistance that routinely emerges in the major fungal pathogens is known as multidrug resistance. Multidrug resistance refers to the simultaneous acquisition of tolerance to a range of drugs via a limited or even single genetic change. This review will focus on recent progress in understanding pathways of multidrug resistance in fungi including those of most medical relevance. Analyses of multidrug resistance in Saccharomyces cerevisiae have provided the most detailed outline of multidrug resistance in a eukaryotic microorganism. Multidrug resistant isolates of S. cerevisiae typically result from changes in the activity of a pair of related transcription factors that in turn elicit overproduction of several target genes. Chief among these is the ATP-binding cassette (ABC)-encoding gene PDR5. Interestingly, in the medically important Candida species, very similar pathways are involved in acquisition of multidrug resistance. In both C. albicans and C. glabrata, changes in the activity of transcriptional activator proteins elicits overproduction of a protein closely related to S. cerevisiae Pdr5 called Cdr1. The major filamentous fungal pathogen, Aspergillus fumigatus, was previously thought to acquire resistance to azole compounds (the principal antifungal drug class) via alterations in the azole drug target-encoding gene cyp51A. More recent data indicate that pathways in addition to changes in the cyp51A gene are important determinants in A. fumigatus azole resistance. We will discuss findings that suggest azole resistance in A. fumigatus and Candida species may share more mechanistic similarities than previously thought. PMID:24795641
Alkyl-Lysophospholipid Resistance in Multidrug-Resistant Leishmania tropica and Chemosensitization by a Novel P-Glycoprotein-Like Transporter Modulator

PubMed Central

Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Parodi-Talice, Adriana; Jiménez, Ignacio A.; Ravelo, Angel G.; Castanys, Santiago; Gamarro, Francisco

2001-01-01

Drug resistance has emerged as a major impediment in the treatment of leishmaniasis. Alkyl-lysophospholipids (ALP), originally developed as anticancer drugs, are considered to be the most promising antileishmanial agents. In order to anticipate probable clinical failure in the near future, we have investigated possible mechanisms of resistance to these drugs in Leishmania spp. The results presented here support the involvement of a member of the ATP-binding cassette (ABC) superfamily, the Leishmania P-glycoprotein-like transporter, in the resistance to ALP. (i) First, a multidrug resistance (MDR) Leishmania tropica line overexpressing a P-glycoprotein-like transporter displays significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1-fold, respectively. (ii) Reduced expression of P-glycoprotein in the MDR line correlates with a significant decrease in ALP resistance. (iii) The ALP were able to modulate the P-glycoprotein-mediated resistance to daunomycin in the MDR line. (iv) We have found a new inhibitor of this transporter, the sesquiterpene C-3, that completely sensitizes MDR parasites to ALP. (v) Finally, the MDR line exhibits a lower accumulation than the wild-type line of bodipy-C5-PC, a fluorescent analogue of phosphatidylcholine that has a structure resembling that of edelfosine. Also, C-3 significantly increases the accumulation of the fluorescent analogue to levels similar to those of wild-type parasites. The involvement of the Leishmania P-glycoprotein-like transporter in resistance to drugs used in the treatment of leishmaniasis also supports the importance of developing new specific inhibitors of this ABC transporter. PMID:11502516
Proton mediated control of biochemical reactions with bioelectronic pH modulation

NASA Astrophysics Data System (ADS)

Deng, Yingxin; Miyake, Takeo; Keene, Scott; Josberger, Erik E.; Rolandi, Marco

2016-04-01

In Nature, protons (H+) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H+ channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H+ currents and H+ concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H+ between PdHx contacts and solution. The present transducer records bistable pH modulation from an “enzymatic flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. The transducer also controls bioluminescence from firefly luciferase by affecting solution pH.
Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity

PubMed Central

Mendez, Ernesto; Ruggli, Nicolas; Collett, Marc S.; Rice, Charles M.

1998-01-01

Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>105 PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity. PMID:9573238
Strategies to overcome or circumvent P-glycoprotein mediated multidrug resistance.

PubMed

Yuan, Hongyu; Li, Xun; Wu, Jifeng; Li, Jinpei; Qu, Xianjun; Xu, Wenfang; Tang, Wei

2008-01-01

Cancer patients who receive chemotherapy often experience intrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents. The phenomenon, termed multidrug resistance (MDR), is often associated with the over-expression of P-glycoprotein, a transmembrane protein pump, which can enhance efflux of a various chemicals structurally unrelated at the expense of ATP depletion, resulting in decrease of the intracellular cytotoxic drug accumulation. The MDR has been a big threaten to the human health and the war fight for it continues. Although several other mechanisms for MDR are elucidated in recent years, considerable efforts attempting to inverse MDR are involved in exploring P-glycoprotein modulators and suppressing P-glycoprotein expression. In this review, we will report on the recent advances in various strategies for overcoming or circumventing MDR mediated by P-glycoprotein.
pH-induced conformational changes of AcrA, the membrane fusion protein of Escherichia coli multidrug efflux system.

PubMed

Ip, Hermia; Stratton, Kelly; Zgurskaya, Helen; Liu, Jun

2003-12-12

The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.
Modulatory effects of plant phenols on human multidrug-resistance proteins 1, 4 and 5 (ABCC1, 4 and 5).

PubMed

Wu, Chung-Pu; Calcagno, Anna Maria; Hladky, Stephen B; Ambudkar, Suresh V; Barrand, Margery A

2005-09-01

Plant flavonoids are polyphenolic compounds, commonly found in vegetables, fruits and many food sources that form a significant portion of our diet. These compounds have been shown to interact with several ATP-binding cassette transporters that are linked with anticancer and antiviral drug resistance and, as such, may be beneficial in modulating drug resistance. This study investigates the interactions of six common polyphenols; quercetin, silymarin, resveratrol, naringenin, daidzein and hesperetin with the multidrug-resistance-associated proteins, MRP1, MRP4 and MRP5. At nontoxic concentrations, several of the polyphenols were able to modulate MRP1-, MRP4- and MRP5-mediated drug resistance, though to varying extents. The polyphenols also reversed resistance to NSC251820, a compound that appears to be a good substrate for MRP4, as predicted by data-mining studies. Furthermore, most of the polyphenols showed direct inhibition of MRP1-mediated [3H]dinitrophenyl S-glutathione and MRP4-mediated [3H]cGMP transport in inside-out vesicles prepared from human erythrocytes. Also, both quercetin and silymarin were found to inhibit MRP1-, MRP4- and MRP5-mediated transport from intact cells with high affinity. They also had significant effects on the ATPase activity of MRP1 and MRP4 without having any effect on [32P]8-azidoATP[alphaP] binding to these proteins. This suggests that these flavonoids most likely interact at the transporter's substrate-binding sites. Collectively, these results suggest that dietary flavonoids such as quercetin and silymarin can modulate transport activities of MRP1, -4 and -5. Such interactions could influence bioavailability of anticancer and antiviral drugs in vivo and thus, should be considered for increasing efficacy in drug therapies.
A Naturally Occurring Deletion in the Effector Domain of H5N1 Swine Influenza Virus Nonstructural Protein 1 Regulates Viral Fitness and Host Innate Immunity.

PubMed

Wang, Junyong; Zeng, Yan; Xu, Shuai; Yang, Jiayun; Wang, Wanbing; Zhong, Bo; Ge, Jinying; Yin, Lei; Bu, Zhigao; Shu, Hong-Bing; Chen, Hualan; Lei, Cao-Qi; Zhu, Qiyun

2018-06-01

Nonstructural protein 1 (NS1) of influenza A virus regulates innate immune responses via various mechanisms. We previously showed that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus impairs the inhibition of type I interferon (IFN) in chicken fibroblasts and attenuates virulence in chickens. Here we found that the virus bearing this deletion in its NS1 effector domain showed diminished inhibition of IFN-related cytokine expression and attenuated virulence in mice. We further showed that deletion of the EALQR motif disrupted NS1 dimerization, impairing double-stranded RNA (dsRNA) sequestration and competitive binding with RIG-I. In addition, the EALQR-deleted NS1 protein could not bind to TRIM25, unlike full-length NS1, and was less able to block TRIM25 oligomerization and self-ubiquitination, further impairing the inhibition of TRIM25-mediated RIG-I ubiquitination compared to that with full-length NS1. Our data demonstrate that the EALQR deletion prevents NS1 from blocking RIG-I-mediated IFN induction via a novel mechanism to attenuate viral replication and virulence in mammalian cells and animals. IMPORTANCE H5 highly pathogenic avian influenza viruses have infected more than 800 individuals across 16 countries, with an overall case fatality rate of 53%. Among viral proteins, nonstructural protein 1 (NS1) of influenza virus is considered a key determinant for type I interferon (IFN) antagonism, pathogenicity, and host range. However, precisely how NS1 modulates virus-host interaction, facilitating virus survival, is not fully understood. Here we report that a naturally occurring deletion (of the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus disrupted NS1 dimerization, which diminished the blockade of IFN induction via the RIG-I signaling pathway, thereby impairing virus replication and virulence in the host. Our study demonstrates that the EALQR motif
A Three-protein Charge Zipper Stabilizes a Complex Modulating Bacterial Gene Silencing*

PubMed Central

Cordeiro, Tiago N.; García, Jesús; Bernadó, Pau; Millet, Oscar; Pons, Miquel

2015-01-01

The Hha/YmoA nucleoid-associated proteins help selectively silence horizontally acquired genetic material, including pathogenicity and antibiotic resistance genes and their maintenance in the absence of selective pressure. Members of the Hha family contribute to gene silencing by binding to the N-terminal dimerization domain of H-NS and modifying its selectivity. Hha-like proteins and the H-NS N-terminal domain are unusually rich in charged residues, and their interaction is mostly electrostatic-driven but, nonetheless, highly selective. The NMR-based structural model of the complex between Hha/YmoA and the H-NS N-terminal dimerization domain reveals that the origin of the selectivity is the formation of a three-protein charge zipper with interdigitated complementary charged residues from Hha and the two units of the H-NS dimer. The free form of YmoA shows collective microsecond-millisecond dynamics that can by measured by NMR relaxation dispersion experiments and shows a linear dependence with the salt concentration. The number of residues sensing the collective dynamics and the population of the minor form increased in the presence of H-NS. Additionally, a single residue mutation in YmoA (D43N) abolished H-NS binding and the dynamics of the apo-form, suggesting the dynamics and binding are functionally related. PMID:26085102
Delayed and highly specific antibody response to nonstructural protein 1 (NS1) revealed during natural human ZIKV infection by NS1-based capture ELISA.

PubMed

Gao, Xiujie; Wen, Yingfen; Wang, Jian; Hong, Wenxin; Li, Chunlin; Zhao, Lingzhai; Yin, Chibiao; Jin, Xia; Zhang, Fuchun; Yu, Lei

2018-06-14

Zika virus (ZIKV) had spread rapidly in the past few years in southern hemisphere where dengue virus (DENV) had caused epidemic problems for over half a century. The high degree of cross-reactivity of Envelope (E) protein specific antibody responses between ZIKV and DENV made it challenging to perform differential diagnosis between the two infections using standard ELISA method for E protein. Using an IgG capture ELISA, we investigated the kinetics of nonstructural protein 1 (NS1) antibody response during natural ZIKV infection and the cross-reactivity to NS1 proteins using convalescent sera obtained from patients infected by either DENV or ZIKV. The analyses of the sequential serum samples from ZIKV infected individuals showed NS1 specific Abs appeared 2 weeks later than E specific Abs. Notably, human sera from ZIKV infected individuals did not contain cross-reactivity to NS1 proteins of any of the four DENV serotypes. Furthermore, four out of five NS1-specific monoclonal antibodies (mAbs) isolated from ZIKV infected individuals did not bind to DENV NS1 proteins. Only limited amount of cross-reactivity to ZIKV NS1 was displayed in 108 DENV1 immune sera at 1:100 dilution. The high degree of NS1-specific Abs in both ZIKV and DENV infection revealed here suggest that NS1-based diagnostics would significantly improve the differential diagnosis between DENV and ZIKV infections.
A Solid-State Modulator for High Speed Kickers

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

Watson, J A; Cook, E G; Chen, Y J

2001-06-11

An all solid-state modulator with multi-pulse burst capability, very fast rise and fall times, pulse width agility, and amplitude modulation capability for use with high-speed beam kickers has been designed and tested at LLNL. The modulator uses multiple solid-state modules stacked in an inductive-adder configuration. It provides a nominal 18kV pulse with {+-} 10% amplitude modulation on the order of several MHz, rise times on the order of 10nS, and can be configured for either positive or negative polarity. The presentation will include measured performance data.
Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and Jurkat cells

NASA Astrophysics Data System (ADS)

Mahlke, Megan A.; Navara, Christopher; Ibey, Bennett L.

2014-03-01

Exposure to nano-second pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to
Proton mediated control of biochemical reactions with bioelectronic pH modulation

DOE PAGES

Deng, Yingxin; Miyake, Takeo; Keene, Scott; ...

2016-04-07

In Nature, protons (H +) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H + channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H + currents and H + concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H + between PdH x contacts and solution. The present transducer records bistable pH modulation from an “enzymaticmore » flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. Furthermore, the transducer also controls bioluminescence from firefly luciferase by affecting solution pH.« less
Proton mediated control of biochemical reactions with bioelectronic pH modulation

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

Deng, Yingxin; Miyake, Takeo; Keene, Scott

In Nature, protons (H +) can mediate metabolic process through enzymatic reactions. Examples include glucose oxidation with glucose dehydrogenase to regulate blood glucose level, alcohol dissolution into carboxylic acid through alcohol dehydrogenase, and voltage-regulated H + channels activating bioluminescence in firefly and jellyfish. Artificial devices that control H + currents and H + concentration (pH) are able to actively influence biochemical processes. Here, we demonstrate a biotransducer that monitors and actively regulates pH-responsive enzymatic reactions by monitoring and controlling the flow of H + between PdH x contacts and solution. The present transducer records bistable pH modulation from an “enzymaticmore » flip-flop” circuit that comprises glucose dehydrogenase and alcohol dehydrogenase. Furthermore, the transducer also controls bioluminescence from firefly luciferase by affecting solution pH.« less

Expression of multidrug resistance proteins in retinoblastoma

PubMed Central

Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

2017-01-01

AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307
Monodispersed calcium carbonate nanoparticles modulate local pH and inhibit tumor growth in vivo

NASA Astrophysics Data System (ADS)

Som, Avik; Raliya, Ramesh; Tian, Limei; Akers, Walter; Ippolito, Joseph E.; Singamaneni, Srikanth; Biswas, Pratim; Achilefu, Samuel

2016-06-01

The acidic extracellular environment of tumors potentiates their aggressiveness and metastasis, but few methods exist to selectively modulate the extracellular pH (pHe) environment of tumors. Transient flushing of biological systems with alkaline fluids or proton pump inhibitors is impractical and nonselective. Here we report a nanoparticles-based strategy to intentionally modulate the pHe in tumors. Biochemical simulations indicate that the dissolution of calcium carbonate nanoparticles (nano-CaCO3) in vivo increases pH asymptotically to 7.4. We developed two independent facile methods to synthesize monodisperse non-doped vaterite nano-CaCO3 with distinct size range between 20 and 300 nm. Using murine models of cancer, we demonstrate that the selective accumulation of nano-CaCO3 in tumors increases tumor pH over time. The associated induction of tumor growth stasis is putatively interpreted as a pHe increase. This study establishes an approach to prepare nano-CaCO3 over a wide particle size range, a formulation that stabilizes the nanomaterials in aqueous solutions, and a pH-sensitive nano-platform capable of modulating the acidic environment of cancer for potential therapeutic benefits.The acidic extracellular environment of tumors potentiates their aggressiveness and metastasis, but few methods exist to selectively modulate the extracellular pH (pHe) environment of tumors. Transient flushing of biological systems with alkaline fluids or proton pump inhibitors is impractical and nonselective. Here we report a nanoparticles-based strategy to intentionally modulate the pHe in tumors. Biochemical simulations indicate that the dissolution of calcium carbonate nanoparticles (nano-CaCO3) in vivo increases pH asymptotically to 7.4. We developed two independent facile methods to synthesize monodisperse non-doped vaterite nano-CaCO3 with distinct size range between 20 and 300 nm. Using murine models of cancer, we demonstrate that the selective accumulation of nano-CaCO3
Further theoretical insight into the reaction mechanism of the hepatitis C NS3/NS4A serine protease

NASA Astrophysics Data System (ADS)

Martínez-González, José Ángel; Rodríguez, Alex; Puyuelo, María Pilar; González, Miguel; Martínez, Rodrigo

2015-01-01

The main reactions of the hepatitis C virus NS3/NS4A serine protease are studied using the second-order Møller-Plesset ab initio method and rather large basis sets to correct the previously reported AM1/CHARMM22 potential energy surfaces. The reaction efficiencies measured for the different substrates are explained in terms of the tetrahedral intermediate formation step (the rate-limiting process). The energies of the barrier and the corresponding intermediate are so close that the possibility of a concerted mechanism is open (especially for the NS5A/5B substrate). This is in contrast to the suggested general reaction mechanism of serine proteases, where a two-step mechanism is postulated.
Tolerance response of multidrug-resistant Salmonella enterica strains to habituation to Origanum vulgare L. essential oil

PubMed Central

Monte, Daniel F. M.; Tavares, Adassa G.; Albuquerque, Allan R.; Sampaio, Fábio C.; Oliveira, Tereza C. R. M.; Franco, Octavio L.; Souza, Evandro L.; Magnani, Marciane

2014-01-01

Multidrug-resistant Salmonella enterica isolates from human outbreaks or from poultry origin were investigated for their ability to develop direct-tolerance or cross-tolerance to sodium chloride, potassium chloride, lactic acid, acetic acid, and ciprofloxacin after habituation in subinhibitory amounts ( of the minimum inhibitory concentration – (MIC) and of the minimum inhibitory concentration – MIC) of Origanum vulgare L. essential oil (OVEO) at different time intervals. The habituation of S. enterica to OVEO did not induce direct-tolerance or cross-tolerance in the tested strains, as assessed by the modulation of MIC values. However, cells habituated to OVEO maintained or increased susceptibility to the tested antimicrobials agents, with up to fourfold double dilution decrease from previously determined MIC values. This study reports for the first time the non-inductive effect of OVEO on the acquisition of direct-tolerance or cross-tolerance in multidrug-resistant S. enterica strains to antimicrobial agents that are largely used in food preservation, as well as to CIP, the therapeutic drug of salmonellosis. PMID:25566231
Regulation of the Expression of Bacterial Multidrug Exporters by Two-Component Signal Transduction Systems.

PubMed

Nishino, Kunihiko

2018-01-01

Bacterial multidrug exporters confer resistance to a wide range of antibiotics, dyes, and biocides. Recent studies have shown that there are many multidrug exporters encoded in bacterial genome. For example, it was experimentally identified that E. coli has at least 20 multidrug exporters. Because many of these multidrug exporters have overlapping substrate spectra, it is intriguing that bacteria, with their economically organized genomes, harbor such large sets of multidrug exporter genes. The key to understanding how bacteria utilize these multiple exporters lies in the regulation of exporter expression. Bacteria have developed signaling systems for eliciting a variety of adaptive responses to their environments. These adaptive responses are often mediated by two-component regulatory systems. In this chapter, the method to identify response regulators that affect expression of multidrug exporters is described.
Burden of Multidrug Resistant Mycobacterium tuberculosis Among New Cases in Al-Madinah Al-Monawarah, Saudi Arabia.

PubMed

Elhassan, Mogahid M; Hemeg, Hassan A; Elmekki, Miskelyemen A; Turkistani, Khalid A; Abdul-Aziz, Ahmed A

2017-01-01

The pattern of Mycobacterium tuberculosis susceptibility to first line drugs and multidrug resistance in Al-Madinah Al-Munawarah, a seasonally overcrowded are during Hajj and Omrah, is not well studied. This study aimed to investigate anti-tuberculosis drug resistance and its distribution among new cases in Al-Madinah Al-Monawarah. Study subjects included 622 patients with first time confirmed TB referred to the central tuberculosis laboratory in Al-Madinah between January 2012 and December 2014. Out of the 622 isolates, 99 (15.9%) were Mycobacteria Other Than Tuberculosis (MOTTS) and 25 (4.0%), three of which (12%) were children under five years of age, revealed multidrug resistance (MDR). Monoresistance to isoniazid (H) was (1.8%), to rifampin (R) was (1.4%), to streptomycin (S) was (1.9 %) to ethambutol (E) was (1.1 %) and to pyrazinamide (Z) was (2.1%). Being among the new cases, multidrug resistant tuberculosis (MDR TB) is supposed to be caused by strains which are originally multidrug resistant. Neither nationality nor gender was found to be associated with MDR TB. Since 12% of MDR cases were among children, a probability of primary infection with MDR strains is to be considered. Moreover, mass gathering during Hajj and Omrah seasons does not seem to increase the burden of MDR in the region. However, further investigation is needed to molecularly characterize MDR isolates and their phylogenetics and geographical origin. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses.

PubMed Central

Ahlquist, P; Strauss, E G; Rice, C M; Strauss, J H; Haseloff, J; Zimmern, D

1985-01-01

Although the genetic organization of tobacco mosaic virus (TMV) differs considerably from that of the tripartite viruses (alfalfa mosaic virus [AlMV] and brome mosaic virus [BMV]), all of these RNA plant viruses share three domains of homology among their nonstructural proteins. One such domain, common to the AlMV and BMV 2a proteins and the readthrough portion of TMV p183, is also homologous to the readthrough protein nsP4 of Sindbis virus (Haseloff et al., Proc. Natl. Acad. Sci. U.S.A. 81:4358-4362, 1984). Two more domains are conserved among the AlMV and BMV 1a proteins and TMV p126. We show here that these domains have homology with portions of the Sindbis proteins nsP1 and nsP2, respectively. These results strengthen the view that the four viruses share mechanistic similarities in their replication strategies and may be evolutionarily related. These results also suggest that either the AlMV 1a, BMV 1a, and TMV p126 proteins are multifunctional or Sindbis proteins nsP1 and nsP2 function together as subunits in a single complex. PMID:3968720
The PB2, PA, HA, NP, and NS genes of a highly pathogenic avian influenza virus A/whooper swan/Mongolia/3/2005 (H5N1) are responsible for pathogenicity in ducks

PubMed Central

2013-01-01

Background Wild ducks are the natural hosts of influenza A viruses. Duck influenza, therefore, has been believed inapparent infection with influenza A viruses, including highly pathogenic avian influenza viruses (HPAIVs) in chickens. In fact, ducks experimentally infected with an HPAIV strain, A/Hong Kong/483/1997 (H5N1) (HK483), did not show any clinical signs. Another HPAIV strain, A/whooper swan/Mongolia/3/2005 (H5N1) (MON3) isolated from a dead swan, however, caused neurological dysfunction and death in ducks. Method To understand the mechanism whereby MON3 shows high pathogenicity in ducks, HK483, MON3, and twenty-four reassortants generated between these two H5N1 viruses were compared for their pathogenicity in domestic ducks. Results None of the ducks infected with MON3-based single-gene reassortants bearing the PB2, NP, or NS gene segment of HK483 died, and HK483-based single-gene reassortants bearing PB2, NP, or NS genes of MON3 were not pathogenic in ducks, suggesting that multiple gene segments contribute to the pathogenicity of MON3 in ducks. All the ducks infected with the reassortant bearing PB2, PA, HA, NP, and NS gene segments of MON3 died within five days post-inoculation, as did those infected with MON3. Each of the viruses was assessed for replication in ducks three days post-inoculation. MON3 and multi-gene reassortants pathogenic in ducks were recovered from all of the tissues examined and replicated with high titers in the brains and lungs. Conclusion The present results indicate that multigenic factors are responsible for efficient replication of MON3 in ducks. In particular, virus growth in the brain might correlate with neurological dysfunction and the disease severity. PMID:23374292
Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1).

PubMed

Bragança, Caio Roberto Soares; Colombo, Lívia Tavares; Roberti, Alvaro Soares; Alvim, Mariana Caroline Tocantins; Cardoso, Silvia Almeida; Reis, Kledna Constancio Portes; de Paula, Sérgio Oliveira; da Silveira, Wendel Batista; Passos, Flavia Maria Lopes

2015-02-01

The yeast Kluyveromyces marxianus is a convenient host for industrial synthesis of biomolecules. However, despite its potential, there are few studies reporting the expression of heterologous proteins using this yeast. Here, we report expression of a dengue virus protein in K. marxianus for the first time. The dengue virus type 1 nonstructural protein 1 (NS1) was integrated into the K. marxianus UFV-3 genome at the LAC4 locus using an adapted integrative vector designed for high-level expression of recombinant protein in Kluyveromyces lactis. The NS1 gene sequence was codon-optimized to increase the level of protein expression in yeast. The synthetic gene was cloned in frame with K. lactis α-mating factor signal peptide, and the recombinant plasmid obtained was used to transform K. marxianus UFV-3 by electroporation. The transformed cells, selected in yeast extract peptone dextrose containing 200 μg mL(-1) Geneticin, were mitotically stable. Analysis of recombinant strains by RT-PCR and protein detection using blot analysis confirmed both transcription and expression of extracellular NS1 polypeptide. After induction with galactose, the NS1 protein was analyzed by sodium dodecyl sulfate-PAGE and immunogenic detection. Protein production was investigated under two conditions: with galactose and biotin pulses at 24-h intervals during 96 h of induction and without galactose and biotin supplementation. Protease activity was not detected in post-growth medium. Our results indicate that recombinant K. marxianus is a good host for the production of dengue virus NS1 protein, which has potential for diagnostic applications.
Novel dengue virus NS2B/NS3 protease inhibitors.

PubMed

Wu, Hongmei; Bock, Stefanie; Snitko, Mariya; Berger, Thilo; Weidner, Thomas; Holloway, Steven; Kanitz, Manuel; Diederich, Wibke E; Steuber, Holger; Walter, Christof; Hofmann, Daniela; Weißbrich, Benedikt; Spannaus, Ralf; Acosta, Eliana G; Bartenschlager, Ralf; Engels, Bernd; Schirmeister, Tanja; Bodem, Jochen

2015-02-01

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against cervical and pancreatic carcinomas

PubMed Central

Li, Junwei; Bonifati, Serena; Hristov, Georgi; Marttila, Tiina; Valmary-Degano, Séverine; Stanzel, Sven; Schnölzer, Martina; Mougin, Christiane; Aprahamian, Marc; Grekova, Svitlana P; Raykov, Zahari; Rommelaere, Jean; Marchini, Antonio

2013-01-01

The rat parvovirus H-1PV has oncolytic and tumour-suppressive properties potentially exploitable in cancer therapy. This possibility is being explored and results are encouraging, but it is necessary to improve the oncotoxicity of the virus. Here we show that this can be achieved by co-treating cancer cells with H-1PV and histone deacetylase inhibitors (HDACIs) such as valproic acid (VPA). We demonstrate that these agents act synergistically to kill a range of human cervical carcinoma and pancreatic carcinoma cell lines by inducing oxidative stress, DNA damage and apoptosis. Strikingly, in rat and mouse xenograft models, H-1PV/VPA co-treatment strongly inhibits tumour growth promoting complete tumour remission in all co-treated animals. At the molecular level, we found acetylation of the parvovirus nonstructural protein NS1 at residues K85 and K257 to modulate NS1-mediated transcription and cytotoxicity, both of which are enhanced by VPA treatment. These results warrant clinical evaluation of H-1PV/VPA co-treatment against cervical and pancreatic ductal carcinomas. PMID:24092664
2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

PubMed Central

Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee-Mei; Dedon, Peter C.; Shi, Pei-Yong

2012-01-01

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of
Morning glory resin glycosides as modulators of antibiotic activity in multidrug-resistant gram-negative bacteria.

PubMed

Corona-Castañeda, Berenice; Pereda-Miranda, Rogelio

2012-01-01

Twenty-six microbiologically inactive (MIC > 512 µg/mL) convolvulaceous resin glycosides ( 1- 26) were tested for resistance modulatory activity in vitro against Escherichia coli Rosetta-gami and two nosocomial pathogens, Salmonella typhi and Shigella flexneri. These compounds exerted a potentiation effect of the clinically useful antibiotics tetracycline, kanamycin, and chloramphenicol against the tested gram-negative bacteria by increasing antibiotic susceptibility up to 32-fold at concentrations of 25 µg/mL. Therefore, the oligosaccharides from the morning glory family (Convolvulaceae) represent metabolites that reverse microbial resistance mechanisms, favoring an increase in the strength and effectiveness of current antibiotics that are not effective in the treatment of refractive infections caused by multidrug-resistant strains. © Georg Thieme Verlag KG Stuttgart · New York.
Complement factor H family proteins in their non-canonical role as modulators of cellular functions.

PubMed

Józsi, Mihály; Schneider, Andrea E; Kárpáti, Éva; Sándor, Noémi

2018-01-04

Complement factor H is a major regulator of the alternative pathway of the complement system. The factor H-related proteins are less characterized, but recent data indicate that they rather promote complement activation. These proteins have some common ligands with factor H and have both overlapping and distinct functions depending on domain composition and the degree of conservation of amino acid sequence. Factor H and some of the factor H-related proteins also appear in a non-canonical function that is beyond their role in the modulation of complement activation. This review covers our current understanding on this emerging role of factor H family proteins in modulating the activation and function of various cells by binding to receptors or receptor ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.
Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

PubMed

Shiryaev, Sergey A; Kozlov, Igor A; Ratnikov, Boris I; Smith, Jeffrey W; Lebl, Michal; Strongin, Alex Y

2007-02-01

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.
Metal-graphene heterojunction modulation via H{sub 2} interaction

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

Cadore, A. R., E-mail: alissoncadore@gmail.com, E-mail: lccampos@fisica.ufmg.br; Mania, E.; Lacerda, R. G.

2016-07-18

Combining experiment and theory, we investigate how a naturally created heterojunction (pn junction) at a graphene and metallic contact interface is modulated via interaction with molecular hydrogen (H{sub 2}). Due to an electrostatic interaction, metallic electrodes induce pn junctions in graphene, leading to an asymmetrical resistance in electronic transport for electrons and holes. We report that the asymmetry in the resistance can be tuned in a reversible manner by exposing graphene devices to H{sub 2}. The interaction between the H{sub 2} and graphene occurs solely at the graphene-contact pn junction and induces a modification on the electrostatic interaction between graphenemore » and metallic contacts. We explain the experimental data with theory providing information concerning the length of the heterojunction and how it changes as a function of H{sub 2} adsorption. Our results are valuable for understanding the nature of the metal-graphene interfaces and have potential application for selective sensors of molecular hydrogen.« less
Modulation of P-glycoprotein activity by novel synthetic curcumin derivatives in sensitive and multidrug-resistant T-cell acute lymphoblastic leukemia cell lines

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

Ooko, Edna

Background: Multidrug resistance (MDR) and drug transporter P-glycoprotein (P-gp) represent major obstacles in cancer chemotherapy. We investigated 19 synthetic curcumin derivatives in drug-sensitive acute lymphoblastic CCRF–CEM leukemia cells and their multidrug-resistant P-gp-overexpressing subline, CEM/ADR5000. Material and methods: Cytotoxicity was tested by resazurin assays. Doxorubicin uptake was assessed by flow cytometry. Binding modes of compounds to P-gp were analyzed by molecular docking. Chemical features responsible for bioactivity were studied by quantitative structure activity relationship (QSAR) analyses. A 7-descriptor QSAR model was correlated with doxorubicin uptake values, IC{sub 50} values and binding energies. Results: The compounds displayed IC{sub 50} values between 0.7more » ± 0.03 and 20.2 ± 0.25 μM. CEM/ADR5000 cells exhibited cross-resistance to 10 compounds, collateral sensitivity to three compounds and regular sensitivity to the remaining six curcumins. Molecular docking studies at the intra-channel transmembrane domain of human P-gp resulted in lowest binding energies ranging from − 9.00 ± 0.10 to − 6.20 ± 0.02 kcal/mol and pKi values from 0.24 ± 0.04 to 29.17 ± 0.88 μM. At the ATP-binding site of P-gp, lowest binding energies ranged from − 9.78 ± 0.17 to − 6.79 ± 0.01 kcal/mol and pKi values from 0.07 ± 0.02 to 0.03 ± 0.03 μM. CEM/ADR5000 cells accumulated approximately 4-fold less doxorubicin than CCRF–CEM cells. The control P-gp inhibitor, verapamil, partially increased doxorubicin uptake in CEM/ADR5000 cells. Six curcumins increased doxorubicin uptake in resistant cells or even exceeded uptake levels compared to sensitive one. QSAR yielded good activity prediction (R = 0.797 and R = 0.794 for training and test sets). Conclusion: Selected derivatives may serve to guide future design of novel P-gp inhibitors and collateral sensitive drugs to combat MDR. - Highlights: • Novel derivatives of curcumin in
Synthesis and disulfide bond connectivity-activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B-NS3 protease.

PubMed

Gao, Yaojun; Cui, Taian; Lam, Yulin

2010-02-01

Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B-NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B-NS3 protease. Both oxidized forms showed potent inhibition with K(i) of 1.39+/-0.35 and 3.03+/-0.75 microM, respectively. Copyright (c) 2009 Elsevier Ltd. All rights reserved.
Modulation of sheep ruminal urea transport by ammonia and pH.

PubMed

Lu, Zhongyan; Stumpff, Friederike; Deiner, Carolin; Rosendahl, Julia; Braun, Hannah; Abdoun, Khalid; Aschenbach, Jörg R; Martens, Holger

2014-09-01

Ruminal fermentation products such as short-chain fatty acids (SCFA) and CO2 acutely stimulate urea transport across the ruminal epithelium in vivo, whereas ammonia has inhibitory effects. Uptake and signaling pathways remain obscure. The ruminal expression of SLC14a1 (UT-B) was studied using polymerase chain reaction (PCR). The functional short-term effects of ammonia on cytosolic pH (pHi) and ruminal urea transport across native epithelia were investigated using pH-sensitive microelectrodes and via flux measurements in Ussing chambers. Two variants (UT-B1 and UT-B2) could be fully sequenced from ovine ruminal cDNA. Functionally, transport was passive and modulated by luminal pH in the presence of SCFA and CO2, rising in response to luminal acidification to a peak value at pH 5.8 and dropping with further acidification, resulting in a bell-shaped curve. Presence of ammonia reduced the amplitude, but not the shape of the relationship between urea flux and pH, so that urea flux remained maximal at pH 5.8. Effects of ammonia were concentration dependent, with saturation at 5 mmol/l. Clamping the transepithelial potential altered the inhibitory potential of ammonia on urea flux. Ammonia depolarized the apical membrane and acidified pHi, suggesting that, at physiological pH (< 7), uptake of NH4 (+) into the cytosol may be a key signaling event regulating ruminal urea transport. We conclude that transport of urea across the ruminal epithelium involves proteins subject to rapid modulation by manipulations that alter pHi and the cytosolic concentration of NH4 (+). Implications for epithelial and ruminal homeostasis are discussed. Copyright © 2014 the American Physiological Society.
Research Progress in Reversal of Tumor Multi-drug Resistance via Natural Products.

PubMed

Guo, Qi; Cao, Hongyan; Qi, Xianghui; Li, Huikai; Ye, Peizhi; Wang, Zhiguo; Wang, Danqiao; Sun, Mingyu

2017-11-24

Multidrug resistance occurs when a tumor develops resistance to multiple chemotherapeutic drugs, which may include antitumor drugs with different chemical structures and mechanisms. Multidrug resistance limits the treatment effects of antitumor drugs, and is the main cause of chemotherapy failure. Multidrug resistance is caused by numerous factors including changes in ATP-binding cassette transporters, target proteins, detoxification, deoxyribonucleic acid repair, drug metabolic enzymes, and signal pathways of apoptosis. Clinical research indicates that natural products have great potential to treat tumors and reverse multidrug resistance. Natural products, which often have multiple targets, could play an important role in tumor treatment, have beneficial effects on tumor inhibition, improve symptoms, reduce radiotherapy and chemotherapy side effects, enhance immunity, and prolong survival. Because natural products often have few adverse reactions and less drug resistance, the antitumor activities of natural products have attracted extensive research. We aimed to review the basic research and clinical application of natural products in the reversal of multidrug resistance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A host YB-1 ribonucleoprotein complex is hijacked by hepatitis C virus for the control of NS3-dependent particle production.

PubMed

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin; Lamarre, Daniel

2013-11-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.
A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

PubMed Central

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin

2013-01-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production. PMID:23986595
Note: A rectangular pulse generator for 50 kV voltage, 0.8 ns rise time, and 10 ns pulse width based on polymer-film switch.

PubMed

Wu, Hanyu; Zhang, Xinjun; Sun, Tieping; Zeng, Zhengzhong; Cong, Peitian; Zhang, Shaoguo

2015-10-01

In this article, we describe a rectangular pulse generator, consisting of a polymer-film switch, a tri-plate transmission line, and parallel post-shaped ceramic resistor load, for 50-kV voltage, 0.8-ns rise time, and 10-ns width. The switch and resistors are arranged in atmospheric air and the transmission line can work in atmospheric air or in transformer oil to change the pulse width from 6.7 ns to 10 ns. The fast switching and low-inductance characteristics of the polymer-film switch ensure the fast rising wavefront of <1 ns. This generator can be applied in the calibration of nanosecond voltage dividers and used for electromagnetic pulse tests as a fast-rising current injection source.
Hybrid Simulation of Duty Cycle Influences on Pulse Modulated RF SiH4/Ar Discharge

NASA Astrophysics Data System (ADS)

Wang, Xifeng; Song, Yuanhong; Zhao, Shuxia; Dai, Zhongling; Wang, Younian

2016-04-01

A one-dimensional fluid/Monte-Carlo (MC) hybrid model is developed to describe capacitively coupled SiH4/Ar discharge, in which the lower electrode is applied by a RF source and pulse modulated by a square-wave, to investigate the modulation effects of the pulse duty cycle on the discharge mechanism. An electron Monte Carlo simulation is used to calculate the electron energy distribution as a function of position and time phase. Rate coefficients in chemical reactions can then be obtained and transferred to the fluid model for the calculation of electron temperature and densities of different species, such as electrons, ions, and radicals. The simulation results show that, the electron energy distribution f(ɛ) is modulated evidently within a pulse cycle, with its tail extending to higher energies during the power-on period, while shrinking back promptly in the afterglow period. Thus, the rate coefficients could be controlled during the discharge, resulting in modulation of the species composition on the substrate compared with continuous excitation. Meanwhile, more negative ions, like SiH-3 and SiH-2, may escape to the electrodes owing to the collapse of ambipolar electric fields, which is beneficial to films deposition. Pulse modulation is thus expected to provide additional methods to customize the plasma densities and components. supported by National Natural Science Foundation of China (No. 11275038)
Kinetic control of TolC recruitment by multidrug efflux complexes.

PubMed

Tikhonova, Elena B; Dastidar, Vishakha; Rybenkov, Valentin V; Zgurskaya, Helen I

2009-09-22

In Gram-negative pathogens, multidrug efflux pumps that provide clinically significant levels of antibiotic resistance function as three-component complexes. They are composed of the inner membrane transporters belonging to one of three superfamilies of proteins, RND, ABC, or MF; periplasmic proteins belonging to the membrane fusion protein (MFP) family; and outer membrane channels exemplified by the Escherichia coli TolC. The three-component complexes span the entire two-membrane envelope of Gram-negative bacteria and expel toxic molecules from the cytoplasmic membrane to the medium. The architecture of these complexes is expected to vary significantly because of the structural diversity of the inner membrane transporters. How the three-component pumps are assembled, their architecture, and their dynamics remain unclear. In this study, we reconstituted interactions and compared binding kinetics of the E. coli TolC with AcrA, MacA, and EmrA, the periplasmic MFPs that function in multidrug efflux with transporters from the RND, ABC, and MF superfamilies, respectively. By using surface plasmon resonance, we demonstrate that TolC interactions with MFPs are highly dynamic and sensitive to pH. The affinity of TolC to MFPs decreases in the order MacA > EmrA > AcrA. We further show that MFPs are prone to oligomerization, but differ dramatically from each other in oligomerization kinetics and stability of oligomers. The propensity of MFPs to oligomerize correlates with the stability of MFP-TolC complexes and structural features of inner membrane transporters. We propose that recruitment of TolC by various MFPs is determined not only by kinetics of MFP-TolC interactions but also by oligomerization kinetics of MFPs and pH.
Multidrug-resistant strains of Salmonella enterica Typhimurium, United States, 1997-1998.

PubMed

Rabatsky-Ehr, Therese; Whichard, Jean; Rossiter, Shannon; Holland, Ben; Stamey, Karen; Headrick, Marcia L; Barrett, Timothy J; Angulo, Frederick J

2004-05-01

To evaluate multidrug-resistant strains of Salmonella enterica serotype Typhimurium, including definitive type 104 (DT104) in the United States, we reviewed data from the National Antimicrobial Resistance Monitoring System (NARMS). In 1997 to 1998, 703 (25%) of 2,767 serotyped Salmonella isolates received at NARMS were S. Typhimurium; antimicrobial susceptibility testing and phage typing were completed for 697. Fifty-eight percent (402) were resistant to > or = 1 antimicrobial agent. Three multidrug-resistant (> or = 5 drugs) strains accounted for (74%) 296 of all resistant isolates. Ceftriaxone resistance was present in 8 (3%), and nalidixic acid resistance in 4 (1%), of these multidrug-resistant strains. By phage typing, 259 (37%) of S. Typhimurium isolates were DT104, 209 (30%) were of undefined type and 103 (15%) were untypable. Fifty percent (202) of resistant (> or = 1 drug) isolates were DT104. Multidrug-resistant S. Typhimurium isolates, particularly DT104, account for a substantial proportion of S. Typhimurium isolates; ceftriaxone resistance is exhibited by some of these strains.
A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

PubMed Central

Liu, Binbin; Zhang, Jing; Koetzner, Cheri A.; Jones, Susan A.; Lin, Qishan

2017-01-01

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis
Optimization by Molecular Fine Tuning of Dihydro-β-agarofuran Sesquiterpenoids as Reversers of P-Glycoprotein-Mediated Multidrug Resistance.

PubMed

Callies, Oliver; Sánchez-Cañete, María P; Gamarro, Francisco; Jiménez, Ignacio A; Castanys, Santiago; Bazzocchi, Isabel L

2016-03-10

P-glycoprotein (P-gp) plays a crucial role in the development of multidrug resistance (MDR), a major obstacle for successful chemotherapy in cancer. Herein, we report on the development of a natural-product-based library of 81 dihydro-β-agarofuran sesquiterpenes (2-82) by optimization of the lead compound 1. The compound library was evaluated for its ability to inhibit P-gp-mediated daunomycin efflux in MDR cells. Selected analogues were further analyzed for their P-gp inhibition constant, intrinsic toxicity, and potency to reverse daunomycin and vinblastine resistances. Analogues 6, 24, 28, 59, and 66 were identified as having higher potency than compound 1 and verapamil, a first-generation P-gp modulator. SAR analysis revealed the size of the aliphatic chains and presence of nitrogen atoms are important structural characteristics to modulate reversal activity. The present study highlights the potential of these analogues as modulators of P-gp mediated MDR in cancer cells.
The influenza virus NS1 protein as a therapeutic target.

PubMed

Engel, Daniel A

2013-09-01

Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2'-5' oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN-β mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.
The influenza virus NS1 protein as a therapeutic target

PubMed Central

Engel, Daniel A.

2015-01-01

Nonstructural protein 1 (NS1) of influenza A virus plays a central role in virus replication and blockade of the host innate immune response, and is therefore being considered as a potential therapeutic target. The primary function of NS1 is to dampen the host interferon (IFN) response through several distinct molecular mechanisms that are triggered by interactions with dsRNA or specific cellular proteins. Sequestration of dsRNA by NS1 results in inhibition of the 2’-5’ oligoadenylate synthetase/RNase L antiviral pathway, and also inhibition of dsRNA-dependent signaling required for new IFN production. Binding of NS1 to the E3 ubiquitin ligase TRIM25 prevents activation of RIG-I signaling and subsequent IFN induction. Cellular RNA processing is also targeted by NS1, through recognition of cleavage and polyadenylation specificity factor 30 (CPSF30), leading to inhibition of IFN- mRNA processing as well as that of other cellular mRNAs. In addition NS1 binds to and inhibits cellular protein kinase R (PKR), thus blocking an important arm of the IFN system. Many additional proteins have been reported to interact with NS1, either directly or indirectly, which may serve its anti-IFN and additional functions, including the regulation of viral and host gene expression, signaling pathways and viral pathogenesis. Many of these interactions are potential targets for small-molecule intervention. Structural, biochemical and functional studies have resulted in hypotheses for drug discovery approaches that are beginning to bear experimental fruit, such as targeting the dsRNA-NS1 interaction, which could lead to restoration of innate immune function and inhibition of virus replication. This review describes biochemical, cell-based and nucleic acid-based approaches to identifying NS1 antagonists. PMID:23796981
[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

PubMed

Dong, Yun-qiao; Ma, Wen-li; Gu, Jin-bao; Zheng, Wen-ling

2009-12-01

To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.
The effect of classical swine fever virus NS5A and NS5A mutants on oxidative stress and inflammatory response in swine testicular cells.

PubMed

Dong, Wang; Lv, Huifang; Wang, Yifan; Li, Xiaomeng; Li, Cheng; Wang, Lu; Wang, Chengbao; Guo, Kangkang; Zhang, Yanming

2017-06-01

Infection with classical swine fever virus (CSFV) results in highly significant economic losses; this infection is characterized by being highly contagious and accompanied by hyperthermia and systemic bleeding. Oxidative stress (OS) plays a critical role in the pathological process of viral infection. The function of the nonstructural protein 5A (NS5A) in the pathogenesis of CSFV has not been completely understood. Here, OS and the inflammatory response were studied with NS5A and substitution mutants in swine testicular (ST) cells. ST cell lines stably expressing CSFV NS5A or substitution mutants were established. Reactive oxygen species (ROS) production, antioxidant protein expression and inflammatory response were analyzed by quantitative real-time PCR (qRT-PCR), ELISA and flow cytometry analysis. The results showed that CSFV NS5A did not increase ROS production or the antioxidant protein (Trx, HO-1 and PRDX-6) expression in ST cells. However, NS5A inhibited cyclooxygenase-2 (COX-2) expression, a pro-inflammatory protein related to OS. Further studies have shown that NS5A mutants S15A and S92A increased ROS production and inhibited antioxidant protein expression. S15A, S81A and T274A affected the inflammatory response. This study suggested that CSFV NS5A did not induce OS, and amino acids Ser15 and Ser92 of CSFV NS5A were essential for inhibiting OS. Additionally, Ser15, Ser81 and Thr274 played important roles in the inflammatory response in ST cells. These observations provided insight into the function of CSFV NS5A and the mechanism of CSFV persistent infection in ST cells. Copyright © 2017 Elsevier Ltd. All rights reserved.
A conserved predicted pseudoknot in the NS2A-encoding sequence of West Nile and Japanese encephalitis flaviviruses suggests NS1' may derive from ribosomal frameshifting

PubMed Central

Firth, Andrew E; Atkins, John F

2009-01-01

Japanese encephalitis, West Nile, Usutu and Murray Valley encephalitis viruses form a tight subgroup within the larger Flavivirus genus. These viruses utilize a single-polyprotein expression strategy, resulting in ~10 mature proteins. Plotting the conservation at synonymous sites along the polyprotein coding sequence reveals strong conservation peaks at the very 5' end of the coding sequence, and also at the 5' end of the sequence encoding the NS2A protein. Such peaks are generally indicative of functionally important non-coding sequence elements. The second peak corresponds to a predicted stable pseudoknot structure whose biological importance is supported by compensatory mutations that preserve the structure. The pseudoknot is preceded by a conserved slippery heptanucleotide (Y CCU UUU), thus forming a classical stimulatory motif for -1 ribosomal frameshifting. We hypothesize, therefore, that the functional importance of the pseudoknot is to stimulate a portion of ribosomes to shift -1 nt into a short (45 codon), conserved, overlapping open reading frame, termed foo. Since cleavage at the NS1-NS2A boundary is known to require synthesis of NS2A in cis, the resulting transframe fusion protein is predicted to be NS1-NS2AN-term-FOO. We hypothesize that this may explain the origin of the previously identified NS1 'extension' protein in JEV-group flaviviruses, known as NS1'. PMID:19196463
Nanodrug delivery in reversing multidrug resistance in cancer cells

PubMed Central

Kapse-Mistry, Sonali; Govender, Thirumala; Srivastava, Rohit; Yergeri, Mayur

2014-01-01

Different mechanisms in cancer cells become resistant to one or more chemotherapeutics is known as multidrug resistance (MDR) which hinders chemotherapy efficacy. Potential factors for MDR includes enhanced drug detoxification, decreased drug uptake, increased intracellular nucleophiles levels, enhanced repair of drug induced DNA damage, overexpression of drug transporter such as P-glycoprotein(P-gp), multidrug resistance-associated proteins (MRP1, MRP2), and breast cancer resistance protein (BCRP). Currently nanoassemblies such as polymeric/solid lipid/inorganic/metal nanoparticles, quantum dots, dendrimers, liposomes, micelles has emerged as an innovative, effective, and promising platforms for treatment of drug resistant cancer cells. Nanocarriers have potential to improve drug therapeutic index, ability for multifunctionality, divert ABC-transporter mediated drug efflux mechanism and selective targeting to tumor cells, cancer stem cells, tumor initiating cells, or cancer microenvironment. Selective nanocarrier targeting to tumor overcomes dose-limiting side effects, lack of selectivity, tissue toxicity, limited drug access to tumor tissues, high drug doses, and emergence of multiple drug resistance with conventional or combination chemotherapy. Current review highlights various nanodrug delivery systems to overcome mechanism of MDR by neutralizing, evading, or exploiting the drug efflux pumps and those independent of drug efflux pump mechanism by silencing Bcl-2 and HIF1α gene expressions by siRNA and miRNA, modulating ceramide levels and targeting NF-κB. “Theragnostics” combining a cytotoxic agent, targeting moiety, chemosensitizing agent, and diagnostic imaging aid are highlighted as effective and innovative systems for tumor localization and overcoming MDR. Physical approaches such as combination of drug with thermal/ultrasound/photodynamic therapies to overcome MDR are focused. The review focuses on newer drug delivery systems developed to overcome
Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I.

PubMed

Gack, Michaela Ulrike; Albrecht, Randy Allen; Urano, Tomohiko; Inn, Kyung-Soo; Huang, I-Chueh; Carnero, Elena; Farzan, Michael; Inoue, Satoshi; Jung, Jae Ung; García-Sastre, Adolfo

2009-05-08

The ubiquitin ligase TRIM25 mediates Lysine 63-linked ubiquitination of the N-terminal CARD domains of the viral RNA sensor RIG-I to facilitate type I interferon (IFN) production and antiviral immunity. Here, we report that the influenza A virus nonstructural protein 1 (NS1) specifically inhibits TRIM25-mediated RIG-I CARD ubiquitination, thereby suppressing RIG-I signal transduction. A novel domain in NS1 comprising E96/E97 residues mediates its interaction with the coiled-coil domain of TRIM25, thus blocking TRIM25 multimerization and RIG-I CARD domain ubiquitination. Furthermore, a recombinant influenza A virus expressing an E96A/E97A NS1 mutant is defective in blocking TRIM25-mediated antiviral IFN response and loses virulence in mice. Our findings reveal a mechanism by which influenza virus inhibits host IFN response and also emphasize the vital role of TRIM25 in modulating antiviral defenses.
Enhancing the pH sensitivity by laterally synergic modulation in dual-gate electric-double-layer transistors

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

Liu, Ning; Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201; Hui Liu, Yang

2015-02-16

The sensitivity of a standard ion-sensitive field-effect transistor is limited to be 59.2 mV/pH (Nernst limit) at room temperature. Here, a concept based on laterally synergic electric-double-layer (EDL) modulation is proposed in order to overcome the Nernst limit. Indium-zinc-oxide EDL transistors with two laterally coupled gates are fabricated, and the synergic modulation behaviors of the two asymmetric gates are investigated. A high sensitivity of ∼168 mV/pH is realized in the dual-gate operation mode. Laterally synergic modulation in oxide-based EDL transistors is interesting for high-performance bio-chemical sensors.
Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus

PubMed Central

Zhu, Shaomei; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre

2016-01-01

ABSTRACT A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. IMPORTANCE HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model
Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus.

PubMed

Zhu, Shaomei; Li, Tingting; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre; Li, Chengyao

2016-09-15

A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection
Acidic pH modulation of Na+ channels in trigeminal mesencephalic nucleus neurons.

PubMed

Kang, In-Sik; Cho, Jin-Hwa; Choi, In-Sun; Kim, Do-Yeon; Jang, Il-Sung

2016-12-07

Cell bodies of trigeminal mesencephalic nucleus (Vmes) neurons are located within the central nervous system, and therefore, peripheral as well as central acidosis can modulate the excitability of Vmes neurons. Here, we report the effect of acidic pH on voltage-gated Na channels in acutely isolated rat Vmes neurons using a conventional whole-cell patch clamp technique. Acidic pH (pH 6.0) slightly but significantly shifted both the activation and steady-state fast inactivation relationships toward depolarized potentials. However, acidic pH (pH 6.0) had a minor effect on the inactivation kinetics of voltage-gated Na channels. Less sensitivity of voltage-gated Na channels to acidic pH may allow Vmes neurons to transduce the precise proprioceptive information even under acidic pH conditions.
Temporal Fluctuation of Multidrug Resistant Salmonella Typhi Haplotypes in the Mekong River Delta Region of Vietnam

PubMed Central

Chau, Tran Thuy; Duy, Pham Thanh; La, Tran Thi Phi; Hoang, Nguyen Van Minh; Nga, Tran Vu Thieu; Campbell, James I.; Manh, Bui Huu; Vinh Chau, Nguyen Van; Hien, Tran Tinh; Farrar, Jeremy; Dougan, Gordon; Baker, Stephen

2011-01-01

Background Typhoid fever remains a public health problem in Vietnam, with a significant burden in the Mekong River delta region. Typhoid fever is caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi), which is frequently multidrug resistant with reduced susceptibility to fluoroquinolone-based drugs, the first choice for the treatment of typhoid fever. We used a GoldenGate (Illumina) assay to type 1,500 single nucleotide polymorphisms (SNPs) and analyse the genetic variation of S. Typhi isolated from 267 typhoid fever patients in the Mekong delta region participating in a randomized trial conducted between 2004 and 2005. Principal Findings The population of S. Typhi circulating during the study was highly clonal, with 91% of isolates belonging to a single clonal complex of the S. Typhi H58 haplogroup. The patterns of disease were consistent with the presence of an endemic haplotype H58-C and a localised outbreak of S. Typhi haplotype H58-E2 in 2004. H58-E2-associated typhoid fever cases exhibited evidence of significant geo-spatial clustering along the Sông H u branch of the Mekong River. Multidrug resistance was common in the established clone H58-C but not in the outbreak clone H58-E2, however all H58 S. Typhi were nalidixic acid resistant and carried a Ser83Phe amino acid substitution in the gyrA gene. Significance The H58 haplogroup dominates S. Typhi populations in other endemic areas, but the population described here was more homogeneous than previously examined populations, and the dominant clonal complex (H58-C, -E1, -E2) observed in this study has not been detected outside Vietnam. IncHI1 plasmid-bearing S. Typhi H58-C was endemic during the study period whilst H58-E2, which rarely carried the plasmid, was only transient, suggesting a selective advantage for the plasmid. These data add insight into the outbreak dynamics and local molecular epidemiology of S. Typhi in southern Vietnam. PMID:21245916

Multidrug-Resistant Gram-Negative Bacilli: Infection Control Implications.

PubMed

Adler, Amos; Friedman, N Deborah; Marchaim, Dror

2016-12-01

Antimicrobial resistance is a common iatrogenic complication of both modern life and medical care. Certain multidrug resistant and extensively drug resistant Gram-negative organisms pose the biggest challenges to health care today, predominantly owing to a lack of therapeutic options. Containing the spread of these organisms is challenging, and in reality, the application of multiple control measures during an evolving outbreak makes it difficult to measure the relative impact of each measure. This article reviews the usefulness of various infection control measures in containing the spread of multidrug-resistant Gram-negative bacilli. Copyright © 2016 Elsevier Inc. All rights reserved.
Synergistic combination of valproic acid and oncolytic parvovirus H-1PV as a potential therapy against cervical and pancreatic carcinomas.

PubMed

Li, Junwei; Bonifati, Serena; Hristov, Georgi; Marttila, Tiina; Valmary-Degano, Séverine; Stanzel, Sven; Schnölzer, Martina; Mougin, Christiane; Aprahamian, Marc; Grekova, Svitlana P; Raykov, Zahari; Rommelaere, Jean; Marchini, Antonio

2013-10-01

The rat parvovirus H-1PV has oncolytic and tumour-suppressive properties potentially exploitable in cancer therapy. This possibility is being explored and results are encouraging, but it is necessary to improve the oncotoxicity of the virus. Here we show that this can be achieved by co-treating cancer cells with H-1PV and histone deacetylase inhibitors (HDACIs) such as valproic acid (VPA). We demonstrate that these agents act synergistically to kill a range of human cervical carcinoma and pancreatic carcinoma cell lines by inducing oxidative stress, DNA damage and apoptosis. Strikingly, in rat and mouse xenograft models, H-1PV/VPA co-treatment strongly inhibits tumour growth promoting complete tumour remission in all co-treated animals. At the molecular level, we found acetylation of the parvovirus nonstructural protein NS1 at residues K85 and K257 to modulate NS1-mediated transcription and cytotoxicity, both of which are enhanced by VPA treatment. These results warrant clinical evaluation of H-1PV/VPA co-treatment against cervical and pancreatic ductal carcinomas. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.
Fast hepatitis C virus RNA elimination and NS5A redistribution by NS5A inhibitors studied by a multiplex assay approach.

PubMed

Liu, Dandan; Ji, Juan; Ndongwe, Tanya P; Michailidis, Eleftherios; Rice, Charles M; Ralston, Robert; Sarafianos, Stefan G

2015-01-01

While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID50]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
An 8-PSK TDMA uplink modulation and coding system

NASA Technical Reports Server (NTRS)

Ames, S. A.

1992-01-01

The combination of 8-phase shift keying (8PSK) modulation and greater than 2 bits/sec/Hz drove the design of the Nyquist filter to one specified to have a rolloff factor of 0.2. This filter when built and tested was found to produce too much intersymbol interference and was abandoned for a design with a rolloff factor of 0.4. The preamble is limited to 100 bit periods of the uncoded bit period of 5 ns for a maximum preamble length of 500 ns or 40 8PSK symbol times at 12.5 ns per symbol. For 8PSK modulation, the required maximum degradation of 1 dB in -20 dB cochannel interference (CCI) drove the requirement for forward error correction coding. In this contract, the funding was not sufficient to develop the proposed codec so the codec was limited to a paper design during the preliminary design phase. The mechanization of the demodulator is digital, starting from the output of the analog to digital converters which quantize the outputs of the quadrature phase detectors. This approach is amenable to an application specific integrated circuit (ASIC) replacement in the next phase of development.
H2O2 modulates the energetic metabolism of the cloud microbiome

NASA Astrophysics Data System (ADS)

Wirgot, Nolwenn; Vinatier, Virginie; Deguillaume, Laurent; Sancelme, Martine; Delort, Anne-Marie

2017-12-01

Chemical reactions in clouds lead to oxidation processes driven by radicals (mainly HO⚫, NO3⚫, or HO2⚫) or strong oxidants such as H2O2, O3, nitrate, and nitrite. Among those species, hydrogen peroxide plays a central role in the cloud chemistry by driving its oxidant capacity. In cloud droplets, H2O2 is transformed by microorganisms which are metabolically active. Biological activity can therefore impact the cloud oxidant capacity. The present article aims at highlighting the interactions between H2O2 and microorganisms within the cloud system. First, experiments were performed with selected strains studied as a reference isolated from clouds in microcosms designed to mimic the cloud chemical composition, including the presence of light and iron. Biotic and abiotic degradation rates of H2O2 were measured and results showed that biodegradation was the most efficient process together with the photo-Fenton process. H2O2 strongly impacted the microbial energetic state as shown by adenosine triphosphate (ATP) measurements in the presence and absence of H2O2. This ATP depletion was not due to the loss of cell viability. Secondly, correlation studies were performed based on real cloud measurements from 37 cloud samples collected at the PUY station (1465 m a.s.l., France). The results support a strong correlation between ATP and H2O2 concentrations and confirm that H2O2 modulates the energetic metabolism of the cloud microbiome. The modulation of microbial metabolism by H2O2 concentration could thus impact cloud chemistry, in particular the biotransformation rates of carbon compounds, and consequently can perturb the way the cloud system is modifying the global atmospheric chemistry.
Multidrug-Resistant Shigella Infections in Patients with Diarrhea, Cambodia, 2014-2015.

PubMed

Poramathikul, Kamonporn; Bodhidatta, Ladaporn; Chiek, Sivhour; Oransathid, Wilawan; Ruekit, Sirigade; Nobthai, Panida; Lurchachaiwong, Woradee; Serichantalergs, Oralak; Lon, Chanthap; Swierczewski, Brett

2016-09-01

We observed multidrug resistance in 10 (91%) of 11 Shigella isolates from a diarrheal surveillance study in Cambodia. One isolate was resistant to fluoroquinolones and cephalosporins and showed decreased susceptibility to azithromycin. We found mutations in gyrA, parC, β-lactamase, and mphA genes. Multidrug resistance increases concern about shigellosis treatment options.
XUV pulse effect on signal modulations of harmonic spectra from H2+ and T2+

NASA Astrophysics Data System (ADS)

Feng, Liqiang; Liu, Hang; Kapteyn, Henry J.; Feng, April Y.

2018-05-01

The effects of signal modulations on the molecular high-order harmonic generations in H2^{+ } and T2+ have been theoretically investigated. It is found that with the introduction of the XUV pulse, due to the absorption of the extra XUV photons in the recombination process, multiplateaus on the harmonic spectra, separated by the XUV photon energy can be found. Moreover, this multiplateau structure is insensitive to the wavelength of the XUV pulse. In shorter pulse duration, the intensities of the multiplateaus from H2+ are higher than those from T2+; while in longer pulse duration, the opposite results can be found. Finally, by changing the delay time of the XUV pulse, the signal modulations (including the amplitude and the frequency modulations) of the multiplateaus can be controlled.
High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

PubMed Central

Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Conseil, Gwenaëlle; Maitrejean, Mathias; Comte, Gilles; Barron, Denis; Di Pietro, Attilio; Castanys, Santiago; Gamarro, Francisco

2001-01-01

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. PMID:11158738
Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

PubMed Central

Sheaffer, Amy K.; Friborg, Jacques; Hernandez, Dennis; Falk, Paul; Zhai, Guangzhi; Levine, Steven; Chaniewski, Susan; Yu, Fei; Barry, Diana; Chen, Chaoqun; Lee, Min S.; Mosure, Kathy; Sun, Li-Qiang; Sinz, Michael; Meanwell, Nicholas A.; Colonno, Richard J.; Knipe, Jay; Scola, Paul

2012-01-01

Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with Ki values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC50s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC50, 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC50, >12 μM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC50s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients. PMID:22869577
Multidrug-resistant malaria and the impact of mass drug administration.

PubMed

Zuber, Janie Anne; Takala-Harrison, Shannon

2018-01-01

Based on the emergence and spread throughout the Greater Mekong Subregion (GMS) of multiple artemisinin-resistant lineages, the prevalence of multidrug resistance leading to high rates of artemisinin-based combination treatment failure in parts of the GMS, and the declining malaria burden in the region, the World Health Organization has recommended complete elimination of falciparum malaria from the GMS. Mass drug administration (MDA) is being piloted as one elimination intervention to be employed as part of this effort. However, concerns remain as to whether MDA might exacerbate the already prevalent problem of multidrug resistance in the region. In this review, we briefly discuss challenges of MDA, the use of MDA in the context of multidrug-resistant malaria, and the potential of different drug combinations and drug-based elimination strategies for mitigating the emergence and spread of resistance.
Cationic compounds with activity against multidrug-resistant bacteria: interest of a new compound compared with two older antiseptics, hexamidine and chlorhexidine.

PubMed

Grare, M; Dibama, H Massimba; Lafosse, S; Ribon, A; Mourer, M; Regnouf-de-Vains, J-B; Finance, C; Duval, R E

2010-05-01

Use of antiseptics and disinfectants is essential in infection control practices in hospital and other healthcare settings. In this study, the in vitro activity of a new promising compound, para-guanidinoethylcalix[4]arene (Cx1), has been evaluated in comparison with hexamidine (HX) and chlorhexidine (CHX), two older cationic antiseptics. The MICs for 69 clinical isolates comprising methicillin-resistant Staphylococcus aureus, methicillin-sensitive S. aureus, coagulase-negative staphylococci (CoNS) (with or without mecA), vancomycin-resistant enterococci, Enterobacteriaceae producing various beta-lactamases and non-fermenting bacilli (Pseudomonas aeruginosa, Acinetobacter baumanii, Stenotrophomonas maltophilia) were determined. Cx1 showed similar activity against S. aureus, CoNS and Enterococcus spp., irrespective of the presence of mecA or van genes, or associated resistance genes, with very good activity against CoNS (MIC <1 mg/L). Variable activities were observed against Enterobacteriaceae; the MICs determined seemed to be dependent both on the genus (MICs of 2, 8 and 64 mg/L for Escherichia coli, Klebsiella pneumoniae and Yersinia enterocolitica, respectively) and on the resistance phenotype production of [Extended Spectrum beta-Lactase (ESBLs) or other beta-lactamases; overproduction of AmpC]. Poor activity was found against non-fermenting bacilli, irrespective of the resistance phenotype. CHX appeared to be the most active compound against all strains, with broad-spectrum and conserved activity against multidrug-resistant strains. HX showed a lower activity, essentially against Gram-positive strains. Consequently, the differences observed with respect to Cx1 suggest that they are certainly not the consequence of antibiotic resistance phenotypes, but rather the result of membrane composition modifications (e.g. of lipopolysaccharide), or of the presence of (activated) efflux-pumps. These results raise the possibility that Cx1 may be a potent new antibacterial
Multidrug-resistant Strains of Salmonella enterica Typhimurium, United States, 1997–19981

PubMed Central

Whichard, Jean; Rossiter, Shannon; Holland, Ben; Stamey, Karen; Headrick, Marcia L.; Barrett, Timothy J.; Angulo, Frederick J.

2004-01-01

To evaluate multidrug-resistant strains of Salmonella enterica Typhimurium, including definitive type 104 (DT104) in the United States, we reviewed data from the National Antimicrobial Resistance Monitoring System (NARMS). In 1997–1998, 25% (703) of 2,767 serotyped Salmonella isolates received at NARMS were S. Typhimurium; antimicrobial susceptibility testing and phage typing were completed for 697. Fifty-eight percent (402) were resistant to >1 antimicrobial agent. Three multidrug-resistant (>5 drugs) strains accounted for 74% (296) of all resistant isolates. Ceftriaxone resistance was present in 3% (8), and nalidixic acid resistance in 1% (4), of these multidrug-resistant strains. By phage typing, 37% (259) of S. Typhimurium isolates were DT104, 30% (209) were of undefined type and 15% (103) were untypable. Fifty percent (202) of resistant (>1 drug) isolates were DT104. Multidrug-resistant S. Typhimurium isolates, particularly DT104, account for a substantial proportion of S. Typhimurium isolates; ceftriaxone resistance is exhibited by some of these strains. PMID:15200811
Roles of the phosphorylation of specific serines and threonines in the NS1 protein of human influenza A viruses.

PubMed

Hsiang, Tien-Ying; Zhou, Ligang; Krug, Robert M

2012-10-01

We demonstrate that phosphorylation of the NS1 protein of a human influenza A virus occurs not only at the threonine (T) at position 215 but also at serines (Ss), specifically at positions 42 and 48. By generating recombinant influenza A/Udorn/72 (Ud) viruses that encode mutant NS1 proteins, we determined the roles of these phosphorylations in virus replication. At position 215 only a T-to-A substitution attenuated replication, whereas other substitutions (T to E to mimic constitutive phosphorylation, T to N, and T to P, the amino acid in avian influenza A virus NS1 proteins) had no effect. We conclude that attenuation resulting from the T-to-A substitution at position 215 is attributable to a deleterious structural change in the NS1 protein that is not caused by other amino acid substitutions and that phosphorylation of T215 does not affect virus replication. At position 48 neither an S-to-A substitution nor an S-to-D substitution that mimics constitutive phosphorylation affected virus replication. In contrast, at position 42, an S-to-D, but not an S-to-A, substitution caused attenuation. The S-to-D substitution eliminates detectable double-stranded RNA binding by the NS1 protein, accounting for attenuation of virus replication. We show that protein kinase C α (PKCα) catalyzes S42 phosphorylation. Consequently, the only phosphorylation of the NS1 protein of this human influenza A virus that regulates its replication is S42 phosphorylation catalyzed by PKCα. In contrast, phosphorylation of Ts or Ss in the NS1 protein of the 2009 H1N1 pandemic virus was not detected, indicating that NS1 phosphorylation probably does not play any role in the replication of this virus.
Combination Approaches to Combat Multi-Drug Resistant Bacteria

PubMed Central

Worthington, Roberta J.; Melander, Christian

2013-01-01

The increasing prevalence of infections caused by multi-drug resistant bacteria is a global health problem that is exacerbated by the dearth of novel classes of antibiotics entering the clinic over the past 40 years. Herein we describe recent developments toward combination therapies for the treatment of multi-drug resistant bacterial infections. These efforts include antibiotic-antibiotic combinations, and the development of adjuvants that either directly target resistance mechanisms such as the inhibition of β-lactamase enzymes, or indirectly target resistance by interfering with bacterial signaling pathways such as two-component systems. We also discuss screening of libraries of previously approved drugs to identify non-obvious antimicrobial adjuvants. PMID:23333434
Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux*

PubMed Central

Radchenko, Martha; Nie, Rongxin; Lu, Min

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism. PMID:26975373
Novel quinolone chalcones targeting colchicine-binding pocket kill multidrug-resistant cancer cells by inhibiting tubulin activity and MRP1 function.

PubMed

Lindamulage, I Kalhari; Vu, Hai-Yen; Karthikeyan, Chandrabose; Knockleby, James; Lee, Yi-Fang; Trivedi, Piyush; Lee, Hoyun

2017-08-31

Agents targeting colchicine-binding pocket usually show a minimal drug-resistance issue, albeit often associated with high toxicity. Chalcone-based compounds, which may bind to colchicine-binding site, are found in many edible fruits, suggesting that they can be effective drugs with less toxicity. Therefore, we synthesized and examined 24 quinolone chalcone compounds, from which we identified ((E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-17) and ((E)-6-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-20) as promising leads. In particular, CTR-20 was effective against 65 different cancer cell lines originated from 12 different tissues, largely in a cancer cell-specific manner. We found that both CTR-17 and CTR-20 reversibly bind to the colchicine-binding pocket on β-tubulin. Interestingly however, both the CTRs were highly effective against multidrug-resistant cancer cells while colchicine, paclitaxel and vinblastine were not. Our study with CTR-20 showed that it overcomes multidrug-resistance through its ability to impede MRP1 function while maintaining strong inhibition against microtubule activity. Data from mice engrafted with the MDA-MB-231 triple-negative breast cancer cells showed that both CTR-17 and CTR-20 possess strong anticancer activity, alone or in combination with paclitaxel, without causing any notable side effects. Together, our data demonstrates that both the CTRs can be effective and safe drugs against many different cancers, especially against multidrug-resistant tumors.
Timing module for MTCA MCH

NASA Astrophysics Data System (ADS)

Gumiński, M.; Kasprowicz, G.

2016-09-01

White Rabbit is an extension of Precise Time Protocol for synchronous Ethernet networks. Network created with dedicated WR switches enable synchronisation of WR capable devices with 1 ns precision. MicroTCA on the other hand is open standard defining cost efficient shelves capable of housing AMC modules used for data processing. Presented article give further introduction to WR and MTCA standard. The most important aspects of MTCA system are described, with focus on shelf controller and its functionality. Following part describes timing difficulties in MTCA systems and possible solutions. Main section describes extension module for MCH, capable of implementing White Rabbit node and distributing acquired timing to all modules connected to MTCA. Conclusions are given at the end of the article.
Development and Evaluation of an On-Line Educational Module for Volunteer Leaders on Bio-Security in Washington State 4-H Livestock Projects

ERIC Educational Resources Information Center

Stevenson, Jill L.; Moore, Dale A.; Newman, Jerry; Schmidt, Janet L.; Smith, Sarah M.; Smith, Jean; Kerr, Susan; Wallace, Michael; BoyEs, Pat

2011-01-01

A module on disease prevention was created for 4-H volunteer leaders who work with livestock projects in Washington to better prepare them to teach youth about bio-security and its importance in 4-H livestock projects. Evaluation of the module and usage statistics since the module's debut were collected and evaluated. The module increases…
H19 promotes endometrial cancer progression by modulating epithelial-mesenchymal transition

PubMed Central

Zhao, Le; Li, Zhen; Chen, Wei; Zhai, Wen; Pan, Jingjing; Pang, Huan; Li, Xu

2017-01-01

Endometrial cancer is one of the most common types of gynecological malignancy worldwide. Novel biomarkers and therapeutic targets are imperative for improving patients' survival. Previous studies have suggested the long non-coding RNA H19 as a potential cancer biomarker. To investigate the role of H19 in endometrial cancer, the present study examined the expression pattern of H19 in endometrial cancer tissues by quantitative polymerase chain reaction, and characterized its function in the endometrial cancer cell line via knocking down its expression with small interfering RNAs. It was found that H19 level was significantly higher in tumor tissues than in paratumoral tissues. Knockdown of H19 did not affect the growth rate of HEC-1-B endometrial cancer cells, but significantly suppressed in vitro migration and invasion of HEC-1-B cells. Furthermore, H19 downregulation decreased Snail level and increased E-cadherin expression without affecting vimentin level, indicating partial reversion of epithelial-mesenchymal transition (EMT). The present findings suggested that H19 contributed to the aggressiveness of endometrial cancer by modulating EMT process. PMID:28123568
Development and Evaluation of an On-Line Educational Module for Volunteer Leaders on Bio-Security in Washington State 4-H Livestock Projects

ERIC Educational Resources Information Center

Stevenson, Jill L.; Moore, Dale A.; Newman, Jerry; Schmidt, Janet L.; Smith, Sarah M.; Smith, Jean; Kerr, Susan; Wallace, Michael; BoyEs, Pat

2011-01-01

An on-line module on disease prevention was created for 4-H volunteer leaders who work with livestock projects in Washington to better prepare them to teach youth about bio-security and its importance in 4-H livestock projects. Evaluation of the module and usage statistics since the module's debut were collected and evaluated. The module increases…

AGILE Observations of the Gravitational-wave Source GW170817: Constraining Gamma-Ray Emission from an NS-NS Coalescence

NASA Astrophysics Data System (ADS)

Verrecchia, F.; Tavani, M.; Donnarumma, I.; Bulgarelli, A.; Evangelista, Y.; Pacciani, L.; Ursi, A.; Piano, G.; Pilia, M.; Cardillo, M.; Parmiggiani, N.; Giuliani, A.; Pittori, C.; Longo, F.; Lucarelli, F.; Minervini, G.; Feroci, M.; Argan, A.; Fuschino, F.; Labanti, C.; Marisaldi, M.; Fioretti, V.; Trois, A.; Del Monte, E.; Antonelli, L. A.; Barbiellini, G.; Caraveo, P.; Cattaneo, P. W.; Colafrancesco, S.; Costa, E.; D'Amico, F.; Ferrari, A.; Giommi, P.; Morselli, A.; Paoletti, F.; Pellizzoni, A.; Picozza, P.; Rappoldi, A.; Soffitta, P.; Vercellone, S.; Baroncelli, L.; Zollino, G.

2017-12-01

The LIGO-Virgo Collaboration (LVC) detected, on 2017 August 17, an exceptional gravitational-wave (GW) event temporally consistent within ˜ 1.7 {{s}} with the GRB 1708117A observed by Fermi-GBM and INTEGRAL. The event turns out to be compatible with a neutron star-neutron star (NS-NS) coalescence that subsequently produced a radio/optical/X-ray transient detected at later times. We report the main results of the observations by the AGILE satellite of the GW170817 localization region (LR) and its electromagnetic (EM) counterpart. At the LVC detection time T 0, the GW170817 LR was occulted by the Earth. The AGILE instrument collected useful data before and after the GW/GRB event because in its spinning observation mode it can scan a given source many times per hour. The earliest exposure of the GW170817 LR by the gamma-ray imaging detector started about 935 s after T 0. No significant X-ray or gamma-ray emission was detected from the LR that was repeatedly exposed over timescales of minutes, hours, and days before and after GW170817, also considering Mini-calorimeter and Super-AGILE data. Our measurements are among the earliest ones obtained by space satellites on GW170817 and provide useful constraints on the precursor and delayed emission properties of the NS-NS coalescence event. We can exclude with high confidence the existence of an X-ray/gamma-ray emitting magnetar-like object with a large magnetic field of {10}15 {{G}}. Our data are particularly significant during the early stage of evolution of the EM remnant.
Daunorubicin and gambogic acid coloaded cysteamine-CdTe quantum dots minimizing the multidrug resistance of lymphoma in vitro and in vivo

PubMed Central

Zhou, Yi; Wang, Ruju; Chen, Bing; Sun, Dan; Hu, Yong; Xu, Peipei

2016-01-01

To minimize the side effects and the multidrug resistance (MDR) arising from daunorubicin (DNR) treatment of malignant lymphoma, a chemotherapy formulation of cysteamine-modified cadmium tellurium (Cys-CdTe) quantum dots coloaded with DNR and gambogic acid (GA) nanoparticles (DNR-GA-Cys-CdTe NPs) was developed. The physical property, drug-loading efficiency and drug release behavior of these DNR-GA-Cys-CdTe NPs were evaluated, and their cytotoxicity was explored by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide assay. These DNR-GA-Cys-CdTe NPs possessed a pH-responsive behavior, and displayed a dose-dependent antiproliferative activity on multidrug-resistant lymphoma Raji/DNR cells. The accumulation of DNR inside the cells, revealed by flow cytometry assay, and the down-regulated expression of P-glycoprotein inside the Raji/DNR cells measured by Western blotting assay indicated that these DNR-GA-Cys-CdTe NPs could minimize the MDR of Raji/DNR cells. This multidrug delivery system would be a promising strategy for minimizing MDR against the lymphoma. PMID:27799767
Marine Natural Products as Models to Circumvent Multidrug Resistance.

PubMed

Long, Solida; Sousa, Emília; Kijjoa, Anake; Pinto, Madalena M M

2016-07-08

Multidrug resistance (MDR) to anticancer drugs is a serious health problem that in many cases leads to cancer treatment failure. The ATP binding cassette (ABC) transporter P-glycoprotein (P-gp), which leads to premature efflux of drugs from cancer cells, is often responsible for MDR. On the other hand, a strategy to search for modulators from natural products to overcome MDR had been in place during the last decades. However, Nature limits the amount of some natural products, which has led to the development of synthetic strategies to increase their availability. This review summarizes the research findings on marine natural products and derivatives, mainly alkaloids, polyoxygenated sterols, polyketides, terpenoids, diketopiperazines, and peptides, with P-gp inhibitory activity highlighting the established structure-activity relationships. The synthetic pathways for the total synthesis of the most promising members and analogs are also presented. It is expected that the data gathered during the last decades concerning their synthesis and MDR-inhibiting activities will help medicinal chemists develop potential drug candidates using marine natural products as models which can deliver new ABC transporter inhibitor scaffolds.
Novel dihydro-beta-agarofuran sesquiterpenes as potent modulators of human P-glycoprotein dependent multidrug resistance.

PubMed

Torres-Romero, David; Muñoz-Martínez, Francisco; Jiménez, Ignacio A; Castanys, Santiago; Gamarro, Francisco; Bazzocchi, Isabel L

2009-12-21

P-Glycoprotein (Pgp) overexpression is one factor contributing to multidrug resistance (MDR) in cancer cells and represents one drawback in the treatment of cancer. In an attempt to find more specific and less toxic anticancer MDR-reversal agents, we report herein the isolation, structure elucidation and biological activity of nine new (, and ) and seven known (, and ) dihydro-beta-agarofuran sesquiterpenes from the leaves of Celastrus vulcanicola. Their stereostructures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques, CD studies and biogenetic means. All the compounds were assayed on human MDR1-transfected NIH-3T3 cells, in order to determine their ability to reverse the MDR phenotype due to Pgp overexpression. Six compounds from these series (, , , , and ) showed an effectiveness that was similar to (or higher than) the classical Pgp reversal agent verapamil for the reversal of resistance to daunomycin and vinblastine. The structure-activity relationships are discussed.
A module located at a chromosomal integration hot spot is responsible for the multidrug resistance of a reference strain from Escherichia coli clonal group A.

PubMed

Lescat, Mathilde; Calteau, Alexandra; Hoede, Claire; Barbe, Valérie; Touchon, Marie; Rocha, Eduardo; Tenaillon, Olivier; Médigue, Claudine; Johnson, James R; Denamur, Erick

2009-06-01

Escherichia coli clonal group A (CGA) commonly exhibits a distinctive multidrug antimicrobial resistance phenotype-i.e., resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim (ACSSuTTp)-and has accounted for up to 50% of trimethoprim-sulfamethoxazole-resistant E. coli urinary tract infections in some locales. Annotation of the whole-genome sequencing of UMN026, a reference CGA strain, clarified the genetic basis for this strain's ACSSuTTp antimicrobial resistance phenotype. Most of the responsible genes were clustered in a unique 23-kbp chromosomal region, designated the genomic resistance module (GRM), which occurred within a 105-kbp genomic island situated at the leuX tRNA. The GRM is characterized by numerous remnants of mobilization and rearrangement events suggesting multiple horizontal transfers. Additionally, comparative genomic analysis of the leuX tRNA genomic island in 14 sequenced E. coli genomes showed that this region is a hot spot of integration, with the presence/absence of specific subregions being uncorrelated with either the phylogenetic group or the pathotype. Our data illustrate the importance of whole-genome sequencing in the detection of genetic elements involved in antimicrobial resistance. Additionally, this is the first documentation of the bla(TEM) and dhfrVII genes in a chromosomal location in E. coli strains.
Nuclear Multidrug-Resistance Related Protein 1 Contributes to Multidrug-Resistance of Mucoepidermoid Carcinoma Mainly via Regulating Multidrug-Resistance Protein 1: A Human Mucoepidermoid Carcinoma Cells Model and Spearman's Rank Correlation Analysis

PubMed Central

Liu, Yuan; Xu, Xiaofang; Guan, Sumin; Wu, Junzheng; Liu, Yanpu

2013-01-01

Background Multidrug resistance-related protein 1 (MRP1/ABCC1) and multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1) are both membrane-bound drug transporters. In contrast to MDR1, MRP1 also transports glutathione (GSH) and drugs conjugated to GSH. Due to its extraordinary transport properties, MRP1/ABCC1 contributes to several physiological functions and pathophysiological incidents. We previously found that nuclear translocation of MRP1 contributes to multidrug-resistance (MDR) of mucoepidermoid carcinoma (MEC). The present study investigated how MRP1 contributes to MDR in the nuclei of MEC cells. Methods Western blot and RT-PCR was carried out to investigate the change of multidrug-resistance protein 1 (MDR1) in MC3/5FU cells after MRP1 was downregulated through RNA interference (RNAi). Immunohistochemistry (IHC) staining of 127 cases of MEC tissues was scored with the expression index (EI). The EI of MDR1 and MRP1 (or nuclear MRP1) was analyzed with Spearman's rank correlation analysis. Using multiple tumor tissue assays, the location of MRP1 in other tissues was checked by HIC. Luciferase reporter assays of MDR1 promoter was carried out to check the connection between MRP1 and MDR1 promoter. Results MRP1 downregulation led to a decreased MDR1 expression in MC3/5FU cells which was caused by decreased activity of MDR1 promoter. IHC study of 127 cases of MEC tissues demonstrated a strong positive correlation between nuclear MRP1 expression and MDR1 expression. Furthermore, IHC study of multiple tumor tissue array sections showed that although nuclear MRP1 widely existed in MEC tissues, it was not found in normal tissues or other tumor tissues. Conclusions Our findings indicate that nuclear MRP1 contributes to MDR mainly through regulating MDR1 expression in MEC. And the unique location of MRP1 made it an available target in identifying MEC from other tumors. PMID:24013781
Role of RNA Interference (RNAi) in Dengue Virus Replication and Identification of NS4B as an RNAi Suppressor

PubMed Central

Kakumani, Pavan Kumar; Ponia, Sanket Singh; S, Rajgokul K.; Sood, Vikas; Chinnappan, Mahendran; Banerjea, Akhil C.; Medigeshi, Guruprasad R.; Malhotra, Pawan

2013-01-01

RNA interference (RNAi) is an important antiviral defense response in plants and invertebrates; however, evidences for its contribution to mammalian antiviral defense are few. In the present study, we demonstrate the anti-dengue virus role of RNAi in mammalian cells. Dengue virus infection of Huh 7 cells decreased the mRNA levels of host RNAi factors, namely, Dicer, Drosha, Ago1, and Ago2, and in corollary, silencing of these genes in virus-infected cells enhanced dengue virus replication. In addition, we observed downregulation of many known human microRNAs (miRNAs) in response to viral infection. Using reversion-of-silencing assays, we further showed that NS4B of all four dengue virus serotypes is a potent RNAi suppressor. We generated a series of deletion mutants and demonstrated that NS4B mediates RNAi suppression via its middle and C-terminal domains, namely, transmembrane domain 3 (TMD3) and TMD5. Importantly, the NS4B N-terminal region, including the signal sequence 2K, which has been implicated in interferon (IFN)-antagonistic properties, was not involved in mediating RNAi suppressor activity. Site-directed mutagenesis of conserved residues revealed that a Phe-to-Ala (F112A) mutation in the TMD3 region resulted in a significant reduction of the RNAi suppression activity. The green fluorescent protein (GFP)-small interfering RNA (siRNA) biogenesis of the GFP-silenced line was considerably reduced by wild-type NS4B, while the F112A mutant abrogated this reduction. These results were further confirmed by in vitro dicer assays. Together, our results suggest the involvement of miRNA/RNAi pathways in dengue virus establishment and that dengue virus NS4B protein plays an important role in the modulation of the host RNAi/miRNA pathway to favor dengue virus replication. PMID:23741001
Last 20 aa of the West Nile virus NS1' protein are responsible for its retention in cells and the formation of unique heat-stable dimers.

PubMed

Young, Lucy B; Melian, Ezequiel Balmori; Setoh, Yin Xiang; Young, Paul R; Khromykh, Alexander A

2015-05-01

West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS1', a C-terminal extended product of NS1 generated as the result of a -1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS1', resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS1', or a combination of both, remains undetermined. Here, we showed that WNV NS1' formed a unique subpopulation of heat- and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS1'-expressing plasmid showed that stability of NS1' dimers was linked to the penultimate 10 aa. To examine the role of NS1' heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1' to create a WNV producing a truncated version of NS1' lacking the last 20 aa, but not affecting the PRF. NS1' protein produced by this mutant virus was secreted more efficiently than WT NS1', indicating that the sequence of the last 20 aa of NS1' was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNVKUN, suggesting that full-length NS1' was not essential for virus replication in vitro and for virulence in mice. © 2015 The Authors.
[Clinical significance of NS1-BP expression in esophageal squamous cell carcinoma].

PubMed

Ren, K; Qian, D; Wang, Y W; Pang, Q S; Zhang, W C; Yuan, Z Y; Wang, P

2018-01-23

Objective: To investigate the clinical significance of NS1-BP expression in patients with esophageal squamous cell carcinoma (ESCC), and to study the roles of NS1-BP in proliferation and apoptosis of ESCC cells. Methods: A total of 98 tumor tissues and 30 adjacent normal tissues from 98 ESCC patients were used as study group and control group, and these samples were collected in Sun Yat-Sen University Cancer Center between 2002 and 2008. In addition, 46 ESCC tissues which were collected in Cancer Institute and Hospital of Tianjin Medical University were used as validation group. Expression of mucosal NS1-BP was detected by immunohistochemistry. Kaplan-Meier curve and log-rank test were used to analyze the survival rate. Multivariate Cox proportional hazard model was used to analyze the prognostic factors. Furthermore, NS1-BP was over expressed or knocked down in ESCC cells by transient transfection. Protein levels of c-Myc were detected by western blot. Cell viability and apoptosis was analyzed by MTT assay and flow cytometry. Results: Among all of tested samples, NS1-BP were down-regulated in 9 out of 30 non-tumorous normal esophageal tissues (30.0%) and 85 out of 144 ESCC tissues (59.0%), respectively, showing a statistically significant difference ( P =0.012). In the study group, three-year disease-free survival rate of NS1-BP high expression group (53.2%) was significantly higher than that of NS1-BP low expression group (27.6%; P =0.009). In the validation group, the three-year disease-free survival rates were 57.8% and 25.5% in NS1-BP high and low levels groups, respectively, showing a similar results ( P =0.016). Importantly, multivariate analyses showed that low expression of NS1-BP was an independent predictor for chemoradiotherapy sensitivity and shorter disease-free survival time in ESCC patients( P <0.05 for all). Furthermore, overexpressed NS1-BP in TE-1 cells repressed c-Myc expression, inhibited cell proliferation and promoted apoptosis. In contrast
Multidrug resistant tuberculosis diagnosed by synovial fluid analysis.

PubMed

van Zeller, M; Monteiro, R; Ramalho, J; Almeida, I; Duarte, R

2012-01-01

Tuberculosis remains a major public health problem worldwide. HIV co-infection is contributing to an increased incidence of the disease, particularly that caused by multidrug resistant strains of Mycobacterium tuberculosis (MT). We describe an HIV-infected patient with pleural and lymph node tuberculosis diagnosed by pleural effusion characteristics and biopsy specimens, without MT identification, that further presented with knee-joint involvement. Arthrocentesis allowed MT isolation and drug susceptibility testing, resulting in a diagnosis of multidrug-resistant tuberculosis and an appropriate treatment regimen. MT identification and drug susceptibility tests are very important, especially for HIV co-infected patients. Copyright © 2011 Sociedade Portuguesa de Pneumologia. Published by Elsevier España. All rights reserved.
Antibacterial activity of epigallocatechin-3-gallate (EGCG) and its synergism with β-lactam antibiotics sensitizing carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii.

PubMed

Lee, Spencer; Razqan, Ghaida Saleh Al; Kwon, Dong H

2017-01-15

Infections caused by Acinetobacter baumannii were responsive to conventional antibiotic therapy. However, recently, carbapenem-associated multidrug resistant isolates have been reported worldwide and present a major therapeutic challenge. Epigallocatechin-3-Gallate (EGCG) extracted from green tea exhibits antibacterial activity. We evaluated the antibacterial activity of EGCG and possible synergism with antibiotics in carbapenem-associated multidrug resistant A. baumannii. A potential mechanism for synergism was also explored. Seventy clinical isolates of A. baumannii collected from geographically different areas were analyzed by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EGCG. Checkerboard and time-killing assays were performed to exam the synergism between EGCG and antibiotics. The effects of EGCG on a multidrug efflux pump inhibitor (1-[1-naphthylmethyl] piperazine; NMP) and β-lactamase production were also examined in A. baumannii. Sixty-three of 70 clinical isolates of A. baumannii carried carbapenemase-encoding genes with carbapenem-associated multidrug resistance. Levels of MIC and MBC of EGCG ranged from 64 to 512µg/ml and from 128 to ≥1024µg/ml, respectively among the clinical isolates. MIC 90 and MBC 86 levels were 256µg/ml and 512µg/ml of EGCG, respectively. Subinhibitory concentration of EGCG in combination with all antibiotics tested, including carbapenem, sensitized (MICs fall≤1.0µg/ml) all carbapenem-associated multidrug resistant isolates. Checkerboard and time-killing assays showed synergism between EGCG and meropenem (or carbenicillin) counted as fractional inhibitory concentration of < 0.5 and cell numbers' decrease per ml of >2log10 within 12h, respectively. EGCG significantly increased the effect of NMP but was unrelated to β-lactamase production in A. baumannii, suggesting EGCG may be associated with inhibition of efflux pumps. Overall we suggest that EGCG-antibiotic combinations might
Mechanistic Characterization of GS-9190 (Tegobuvir), a Novel Nonnucleoside Inhibitor of Hepatitis C Virus NS5B Polymerase▿

PubMed Central

Shih, I-hung; Vliegen, Inge; Peng, Betty; Yang, Huiling; Hebner, Christy; Paeshuyse, Jan; Pürstinger, Gerhard; Fenaux, Martijn; Tian, Yang; Mabery, Eric; Qi, Xiaoping; Bahador, Gina; Paulson, Matthew; Lehman, Laura S.; Bondy, Steven; Tse, Winston; Reiser, Hans; Lee, William A.; Schmitz, Uli; Neyts, Johan; Zhong, Weidong

2011-01-01

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain. PMID:21746939
Recruitment of DNA Replication and Damage Response Proteins to Viral Replication Centers during Infection with NS2 Mutants of Minute Virus of Mice (MVM)

PubMed Central

Ruiz, Zandra; Mihaylov, Ivailo S.; Cotmore, Susan F.; Tattersall, Peter

2010-01-01

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA32, which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins. PMID:21193212
Development and characterization of serotype-specific monoclonal antibodies against the dengue virus-4 (DENV-4) non-structural protein (NS1).

PubMed

Gelanew, Tesfaye; Hunsperger, Elizabeth

2018-02-06

Dengue, caused by one of the four serologically distinct dengue viruses (DENV-1 to - 4), is a mosquito-borne disease of serious global health significance. Reliable and cost-effective diagnostic tests, along with effective vaccines and vector-control strategies, are highly required to reduce dengue morbidity and mortality. Evaluation studies revealed that many commercially available NS1 antigen (Ag) tests have limited sensitivity to DENV-4 serotype compared to the other three serotypes. These studies indicated the need for development of new NS1 Ag detection test with improved sensitivity to DENV-4. An NS1 capture enzyme linked immunoassay (ELISA) specific to DENV-4 may improve the detection of DENV-4 cases worldwide. In addition, a serotype-specific NS1 Ag test identifies both DENV and the infecting serotype. In this study, we used a small-ubiquitin-like modifier (SUMO*) cloning vector to express a SUMO*-DENV-4 rNS1 fusion protein to develop NS1 DENV-4 specific monoclonal antibodies (MAbs). These newly developed MAbs were then optimized for use in an anti-NS1 DENV-4 capture ELISA. The serotype specificity and sensitivity of this ELISA was evaluated using (i) supernatants from DENV (1-4)-infected Vero cell cultures, (ii) rNS1s from all the four DENV (1-4) and, (iii) rNS1s of related flaviviruses (yellow fever virus; YFV and West Nile virus; WNV). From the evaluation studies of the newly developed MAbs, we identified three DENV-4 specific anti-NS1 MAbs: 3H7A9, 8A6F2 and 6D4B10. Two of these MAbs were optimal for use in a DENV-4 serotype-specific NS1 capture ELISA: MAb 8A6F2 as the capture antibody and 6D4B10 as a detection antibody. This ELISA was sensitive and specific to DENV-4 with no cross-reactivity to other three DENV (1-3) serotypes and other heterologous flaviviruses. Taken together these data indicated that our MAbs are useful reagents for the development of DENV-4 immunodiagnostic tests.
Pharmacophoric characteristics of dengue virus NS2B/NS3pro inhibitors: a systematic review of the most promising compounds.

PubMed

Leonel, Camyla Alves; Lima, William Gustavo; Dos Santos, Michelli; Ferraz, Ariane Coelho; Taranto, Alex Gutterres; de Magalhães, José Carlos; Dos Santos, Luciana Lara; Ferreira, Jaqueline Maria Siqueira

2018-03-01

Dengue virus (DENV) infection can lead to a wide range of clinical manifestations, including fatal hemorrhagic complications. There is a need to find effective pharmacotherapies to treat this disease due to the lack of specific immunotherapies and antiviral drugs. That said, the DENV NS2B/NS3pro protease complex is essential in both the viral multiplication cycle and in disease pathogenesis, and is considered a promising target for new antiviral therapies. Here, we performed a systematic review to evaluate the pharmacophoric characteristics of promising compounds against NS2B/NS3pro reported in the past 10 years. Online searches in the PUBMED/MEDLINE and SCOPUS databases resulted in 165 articles. Eight studies, which evaluated 3,384,268 molecules exhibiting protease inhibition activity, were included in this review. These studies evaluated anti-dengue activity in vitro and the IC 50 and EC 50 values were provided. Most compounds exhibited non-competitive inhibition. Cytotoxicity was evaluated in BHK-21, Vero, and LLC-MK2 cells, and the CC 50 values obtained ranged from < 1.0 to 780.5 µM. Several groups were associated with biological activity against dengue, including nitro, catechol, halogen and ammonium quaternaries. Thus, these groups seem to be potential pharmacophores that can be further investigated to treat dengue infections.
Natural products modulating the hERG channel: heartaches and hope.

PubMed

Kratz, Jadel M; Grienke, Ulrike; Scheel, Olaf; Mann, Stefan A; Rollinger, Judith M

2017-08-02

Covering: 1996-December 2016The human Ether-à-go-go Related Gene (hERG) channel is a voltage-gated potassium channel playing an essential role in the normal electrical activity in the heart. It is involved in the repolarization and termination of action potentials in excitable cardiac cells. Mutations in the hERG gene and hERG channel blockage by small molecules are associated with increased risk of fatal arrhythmias. Several drugs have been withdrawn from the market due to hERG channel-related cardiotoxicity. Moreover, as a result of its notorious ligand promiscuity, this ion channel has emerged as an important antitarget in early drug discovery and development. Surprisingly, the hERG channel blocking profile of natural compounds present in frequently consumed botanicals (i.e. dietary supplements, spices, and herbal medicinal products) is not routinely assessed. This comprehensive review will address these issues and provide a critical compilation of hERG channel data for isolated natural products and extracts over the past two decades (1996-2016). In addition, the review will provide (i) a solid basis for the molecular understanding of the physiological functions of the hERG channel, (ii) the translational potential of in vitro/in vivo results to cardiotoxicity in humans, (iii) approaches for the identification of hERG channel blockers from natural sources, (iv) future perspectives for cardiac safety guidelines and their applications within phytopharmaceuticals and dietary supplements, and (v) novel applications of hERG channel modulation (e.g. as a drug target).
Bioprospecting marine actinomycetes for multidrug-resistant pathogen control from Rameswaram coastal area, Tamil Nadu, India.

PubMed

Wahaab, Femina; Subramaniam, Kalidass

2018-01-01

A potent Streptomyces bacillaris strain RAM25C4 was isolated for controlling methicillin-resistant Staphylococcus aureus and multidrug-resistant bacteria such as Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa. A total of 131 actinomycetes were isolated from the Rameswaram coastal region, Tamil Nadu, India. Among 131 actinomycetes, maximum number of actinomycetes (55%) isolated at the distance of 3-6 m from seashore. Out of 131 actinomycetes, 85% of the actinomycetes exhibited different degree of antagonistic activity against test pathogens. The antagonistic activity evaluated using actinomycetes direct culture filtrate and culture filtrate extracts. Among these culture filtrate, extracts had supreme antagonistic activity against multidrug-resistant bacteria and the solvent ethyl acetate was the best for extracting secondary metabolites from actinomycetes. In HPTLC analysis, the presence of macrolides, terpenoids, and quinolones was identified in RAM25C4 extract. In GC-MS analysis, various potent compounds such as phenolic compound-2,6-di-tert-butylphenol, alkaloid compound-1H, 5H, pyrrolo (1' 2':3, 4) imidazo, and quinolone compound-1,4-benzenediol, 2,5-bis(1,1-dimethylethyl) were identified in the ethyl acetate extract of RAM25C4. The phylogenetic analysis of 16S rRNA gene sequence of RAM25C4 isolate was deposited in NCBI with name Streptomyces bacillaris strain RAM25C4 and accession number KM513543.
Structure-based discovery of clinically approved drugs as Zika virus NS2B-NS3 protease inhibitors that potently inhibit Zika virus infection in vitro and in vivo.

PubMed

Yuan, Shuofeng; Chan, Jasper Fuk-Woo; den-Haan, Helena; Chik, Kenn Ka-Heng; Zhang, Anna Jinxia; Chan, Chris Chung-Sing; Poon, Vincent Kwok-Man; Yip, Cyril Chik-Yan; Mak, Winger Wing-Nga; Zhu, Zheng; Zou, Zijiao; Tee, Kah-Meng; Cai, Jian-Piao; Chan, Kwok-Hung; de la Peña, Jorge; Pérez-Sánchez, Horacio; Cerón-Carrasco, José Pedro; Yuen, Kwok-Yung

2017-09-01

Zika virus (ZIKV) infection may be associated with severe complications in fetuses and adults, but treatment options are limited. We performed an in silico structure-based screening of a large chemical library to identify potential ZIKV NS2B-NS3 protease inhibitors. Clinically approved drugs belonging to different drug classes were selected among the 100 primary hit compounds with the highest predicted binding affinities to ZIKV NS2B-NS3-protease for validation studies. ZIKV NS2B-NS3 protease inhibitory activity was validated in most of the selected drugs and in vitro anti-ZIKV activity was identified in two of them (novobiocin and lopinavir-ritonavir). Molecular docking and molecular dynamics simulations predicted that novobiocin bound to ZIKV NS2B-NS3-protease with high stability. Dexamethasone-immunosuppressed mice with disseminated ZIKV infection and novobiocin treatment had significantly (P < 0.05) higher survival rate (100% vs 0%), lower mean blood and tissue viral loads, and less severe histopathological changes than untreated controls. This structure-based drug discovery platform should facilitate the identification of additional enzyme inhibitors of ZIKV. Copyright © 2017 Elsevier B.V. All rights reserved.
Masitinib antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance

PubMed Central

KATHAWALA, RISHIL J.; CHEN, JUN-JIANG; ZHANG, YUN-KAI; WANG, YI-JUN; PATEL, ATISH; WANG, DE-SHEN; TALELE, TANAJI T.; ASHBY, CHARLES R.; CHEN, ZHE-SHENG

2014-01-01

In this in vitro study, we determined whether masitinib could reverse multidrug resistance (MDR) in cells overexpressing the ATP binding cassette subfamily G member 2 (ABCG2) transporter. Masitinib (1.25 and 2.5 μM) significantly decreases the resistance to mitoxantrone (MX), SN38 and doxorubicin in HEK293 and H460 cells overexpressing the ABCG2 transporter. In addition, masitinib (2.5 μM) significantly increased the intracellular accumulation of [3H]-MX, a substrate for ABCG2, by inhibiting the function of ABCG2 and significantly decreased the efflux of [3H]-MX. However, masitinib (2.5 μM) did not significantly alter the expression of the ABCG2 protein. In addition, a docking model suggested that masitinib binds within the transmembrane region of a homology-modeled human ABCG2 transporter. Overall, our in vitro findings suggest that masitinib reverses MDR to various anti-neoplastic drugs in HEK293 and H460 cells overexpressing ABCG2 by inhibiting their transport activity as opposed to altering their levels of expression. PMID:24626598
Construction of plasmid, bacterial expression, purification, and assay of dengue virus type 2 NS5 methyltransferase.

PubMed

Boonyasuppayakorn, Siwaporn; Padmanabhan, Radhakrishnan

2014-01-01

Dengue virus (DENV), a member of mosquito-borne flavivirus, causes self-limiting dengue fever as well as life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its positive sense RNA genome has a cap at the 5'-end and no poly(A) tail at the 3'-end. The viral RNA encodes a single polyprotein, C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5. The polyprotein is processed into 3 structural proteins (C, prM, and E) and 7 nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). NS3 and NS5 are multifunctional enzymes performing various tasks in viral life cycle. The N-terminal domain of NS5 has distinct GTP and S-adenosylmethionine (SAM) binding sites. The role of GTP binding site is implicated in guanylyltransferase (GTase) activity of NS5. The SAM binding site is involved in both N-7 and 2'-O-methyltransferase (MTase) activities involved in formation of type I cap. The C-terminal domain of NS5 catalyzes RNA-dependent RNA polymerase (RdRp) activity involved in RNA synthesis. We describe the construction of the MTase domain of NS5 in an E. coli expression vector, purification of the enzyme, and conditions for enzymatic assays of N7- and 2'O-methyltransferase activities that yield the final type I 5'-capped RNA ((7Me)GpppA2'OMe-RNA).

CD44-engineered mesoporous silica nanoparticles for overcoming multidrug resistance in breast cancer

NASA Astrophysics Data System (ADS)

Wang, Xin; Liu, Ying; Wang, Shouju; Shi, Donghong; Zhou, Xianguang; Wang, Chunyan; Wu, Jiang; Zeng, Zhiyong; Li, Yanjun; Sun, Jing; Wang, Jiandong; Zhang, Longjiang; Teng, Zhaogang; Lu, Guangming

2015-03-01

Multidrug resistance is a major impediment for the successful chemotherapy in breast cancer. CD44 is over-expressed in multidrug resistant human breast cancer cells. CD44 monoclonal antibody exhibits anticancer potential by inhibiting proliferation and regulating P-glycoprotein-mediated drug efflux activity in multidrug resistant cells. Thereby, CD44 monoclonal antibody in combination with chemotherapeutic drug might be result in enhancing chemosensitivity and overcoming multidrug resistance. The purpose of this study is to investigate the effects of the CD44 monoclonal antibody functionalized mesoporous silica nanoparticles containing doxorubicin on human breast resistant cancer MCF-7 cells. The data showed that CD44-modified mesoporous silica nanoparticles increased cytotoxicity and enhanced the downregulation of P-glycoprotein in comparison to CD44 antibody. Moreover, CD44-engineered mesoporous silica nanoparticles provided active target, which promoted more cellular uptake of DOX in the resistant cells and more retention of DOX in tumor tissues than unengineered counterpart. Animal studies of the resistant breast cancer xenografts demonstrated that CD44-engineered drug delivery system remarkably induced apoptosis and inhibited the tumor growth. Our results indicated that the CD44-engineered mesoporous silica nanoparticle-based drug delivery system offers an effective approach to overcome multidrug resistance in human breast cancer.
In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model

PubMed Central

Szentpáli-Gavallér, Katalin; Lim, Stephanie M.; Dencső, László; Bányai, Krisztián; Koraka, Penelope; Osterhaus, Albert D.M.E.; Martina, Byron E.E.; Bakonyi, Tamás; Bálint, Ádám

2016-01-01

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA. PMID:26907325
In Vitro and in Vivo Evaluation of Mutations in the NS Region of Lineage 2 West Nile Virus Associated with Neuroinvasiveness in a Mammalian Model.

PubMed

Szentpáli-Gavallér, Katalin; Lim, Stephanie M; Dencső, László; Bányai, Krisztián; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E E; Bakonyi, Tamás; Bálint, Ádám

2016-02-19

West Nile virus (WNV) strains may differ significantly in neuroinvasiveness in vertebrate hosts. In contrast to genetic lineage 1 WNVs, molecular determinants of pathogenic lineage 2 strains have not been experimentally confirmed so far. A full-length infectious clone of a neurovirulent WNV lineage 2 strain (578/10; Central Europe) was generated and amino acid substitutions that have been shown to attenuate lineage 1 WNVs were introduced into the nonstructural proteins (NS1 (P250L), NS2A (A30P), NS3 (P249H) NS4B (P38G, C102S, E249G)). The mouse neuroinvasive phenotype of each mutant virus was examined following intraperitoneal inoculation of C57BL/6 mice. Only the NS1-P250L mutation was associated with a significant attenuation of virulence in mice compared to the wild-type. Multiplication kinetics in cell culture revealed significantly lower infectious virus titres for the NS1 mutant compared to the wild-type, as well as significantly lower amounts of positive and negative stranded RNA.
Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells.

PubMed

Gelhaus, Stacy L; Gilad, Oren; Hwang, Wei-Ting; Penning, Trevor M; Blair, Ian A

2012-02-25

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (-)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2'-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (-)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (-)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
Photoionization and ionic dissociation of the C3 H3 NS molecule induced by soft X-ray near the C1s edge.

PubMed

Lago, A F; Januário, R D; Cavasso Filho, R L; Simon, M; Dávalos, J Z

2017-10-01

Time of flight mass spectrometry, electron-ion coincidence, and ion yield spectroscopy were employed to investigate for the first time the thiazole (C 3 H 3 NS) molecule in the gas phase excited by synchrotron radiation in the soft X-ray domain. Total ion yield (TIY) and photoelectron-photoion coincidence (PEPICO) spectra were recorded as a function of the photon energy in the vicinity of the carbon K edge (C1s). The C1s resonant transitions as well as the core ionization thresholds have been determined from the profile of TIY spectrum, and the features were discussed. The corresponding partial ion yields were determined from the PEPICO spectra for the cation species produced upon the molecular photodissociation. Additional ab initio calculations have also been performed from where relevant structural and electronic configuration parameters were obtained for this molecule. Copyright © 2017 John Wiley & Sons, Ltd.
Crystal structure of full-length Zika virus NS5 protein reveals a conformation similar to Japanese encephalitis virus NS5

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

Upadhyay, Anup K.; Cyr, Matthew; Longenecker, Kenton

The rapid spread of the recentZika virus(ZIKV) epidemic across various countries in the American continent poses a major health hazard for the unborn fetuses of pregnant women. To date, there is no effective medical intervention. The nonstructural protein 5 ofZika virus(ZIKV-NS5) is critical for ZIKV replication through the 5'-RNA capping and RNA polymerase activities present in its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains, respectively. The crystal structure of the full-length ZIKV-NS5 protein has been determined at 3.05 Å resolution from a crystal belonging to space groupP2 12 12 and containing two protein molecules in the asymmetricmore » unit. The structure is similar to that reported for the NS5 protein fromJapanese encephalitis virusand suggests opportunities for structure-based drug design targeting either its MTase or RdRp domain.« less
Logistics support of the Japanese Experiment Module by the H-II rocket

NASA Astrophysics Data System (ADS)

Shibato, Yoji; Eto, Takao; Fukushima, Yukio; Takatsuka, Hitoshi

1988-10-01

This paper describes salient design features of the Japanese Experiment Module (JEM), which will be attached to the Space Station. Special attention is given to the logistic support of the JEM (which is planned to become operational in 1990s) by the HOPE orbiter, which will be used for the resupply and the retrieval of the JEM, and the H-II rocket, which will be used to launch the HOPE. The concepts of HOPE and the H-II rocket are discussed together with the estimated logistics requirements of this system. Configuration diagrams are included.
NS6180, a new KCa3.1 channel inhibitor prevents T-cell activation and inflammation in a rat model of inflammatory bowel disease

PubMed Central

Strøbæk, D; Brown, DT; Jenkins, DP; Chen, Y-J; Coleman, N; Ando, Y; Chiu, P; Jørgensen, S; Demnitz, J; Wulff, H; Christophersen, P

2013-01-01

Background and Purpose The KCa3.1 channel is a potential target for therapy of immune disease. We identified a compound from a new chemical class of KCa3.1 inhibitors and assessed in vitro and in vivo inhibition of immune responses. Experimental Approach We characterized the benzothiazinone NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) with respect to potency and molecular site of action on KCa3.1 channels, selectivity towards other targets, effects on T-cell activation as well as pharmacokinetics and inflammation control in colitis induced by 2,4-dinitrobenzene sulfonic acid, a rat model of inflammatory bowel disease (IBD). Key Results NS6180 inhibited cloned human KCa3.1 channels (IC50 = 9 nM) via T250 and V275, the same amino acid residues conferring sensitivity to triarylmethanes such as like TRAM-34. NS6180 inhibited endogenously expressed KCa3.1 channels in human, mouse and rat erythrocytes, with similar potencies (15–20 nM). NS6180 suppressed rat and mouse splenocyte proliferation at submicrolar concentrations and potently inhibited IL-2 and IFN-γ production, while exerting smaller effects on IL-4 and TNF-α and no effect on IL-17 production. Antibody staining showed KCa3.1 channels in healthy colon and strong up-regulation in association with infiltrating immune cells after induction of colitis. Despite poor plasma exposure, NS6180 (3 and 10 mg·kg−1 b.i.d.) dampened colon inflammation and improved body weight gain as effectively as the standard IBD drug sulfasalazine (300 mg·kg−1 q.d.). Conclusions and Implications NS6180 represents a novel class of KCa3.1 channel inhibitors which inhibited experimental colitis, suggesting KCa3.1 channels as targets for pharmacological control of intestinal inflammation. PMID:22891655
Secretome profile analysis of multidrug-resistant, monodrug-resistant and drug-susceptible Mycobacterium tuberculosis.

PubMed

Putim, Chanyanuch; Phaonakrop, Narumon; Jaresitthikunchai, Janthima; Gamngoen, Ratikorn; Tragoolpua, Khajornsak; Intorasoot, Sorasak; Anukool, Usanee; Tharincharoen, Chayada Sitthidet; Phunpae, Ponrut; Tayapiwatana, Chatchai; Kasinrerk, Watchara; Roytrakul, Sittiruk; Butr-Indr, Bordin

2018-03-01

The emergence of drug-resistant tuberculosis has generated great concern in the control of tuberculosis and HIV/TB patients have established severe complications that are difficult to treat. Although, the gold standard of drug-susceptibility testing is highly accurate and efficient, it is time-consuming. Diagnostic biomarkers are, therefore, necessary in discriminating between infection from drug-resistant and drug-susceptible strains. One strategy that aids to effectively control tuberculosis is understanding the function of secreting proteins that mycobacteria use to manipulate the host cellular defenses. In this study, culture filtrate proteins from Mycobacterium tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains were gathered and profiled by shotgun-proteomics technique. Mass spectrometric analysis of the secreted proteome identified several proteins, of which 837, 892, 838 and 850 were found in M. tuberculosis H37Rv, isoniazid-resistant, rifampicin-resistant and multidrug-resistant strains, respectively. These proteins have been implicated in various cellular processes, including biological adhesion, biological regulation, developmental process, immune system process localization, cellular process, cellular component organization or biogenesis, metabolic process, and response to stimulus. Analysis based on STITCH database predicted the interaction of DNA topoisomerase I, 3-oxoacyl-(acyl-carrier protein) reductase, ESAT-6-like protein, putative prophage phiRv2 integrase, and 3-phosphoshikimate 1-carboxyvinyltransferase with isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin, suggesting putative roles in controlling the anti-tuberculosis ability. However, several proteins with no interaction with all first-line anti-tuberculosis drugs might be used as markers for mycobacterial identification.
Variability of cutaneous and nasal population levels between patients colonized and infected by multidrug-resistant bacteria in two Brazilian intensive care units.

PubMed

Damaceno, Quésia; Nicoli, Jacques R; Oliveira, Adriana

2015-01-01

To compare cutaneous and nasal population levels between patients colonized and infected by multidrug-resistant organisms in two intensive care units. A prospective cohort study was performed in adult intensive care units of two hospitals in Belo Horizonte, Brazil (April 2012 to February 2013). Clinical and demographic data were first collected by reviewing patients' charts. Then, samples collected with nasal, groin, and perineum swabs were cultivated in selective media for 48 h at 37°C. After isolation, determination of antimicrobial susceptibility and biochemical identification were performed. A total of 53 cases of colonization were observed by the following bacteria in decreasing frequencies: imipenem-resistant Acinetobacter baumannii (50.9%), vancomycin-resistant Enterococcus faecalis (43.4%), extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (37.7%), imipenem-resistant Pseudomonas aeruginosa (32.1%), oxacillin-resistant Staphylococcus aureus (7.5%), and imipenem-resistant Klebsiella pneumoniae (5.7%). Among these colonization cases, 26 (49.0%) were followed by infection with bacteria phenotypically similar to those of the colonization. A relation between high population levels of colonization by most of the multidrug-resistant organisms at anatomical sites and a subsequent infection was observed. After colonization/infection, bacterial population levels decreased progressively and spontaneously until disappearance by day 45 in all the anatomical sites and for all the multidrug-resistant organisms. There was a correlation between high population levels of colonization by multidrug-resistant organisms at anatomical sites and a subsequent infection. Reduction in multidrug-resistant organism populations after colonization at anatomical sites could be a preventive measure to reduce evolution to infection as well as transmission of these bacteria between patients in intensive care unit.
Prevalence and behavior of multidrug-resistant shiga toxin-producing Escherichia coli, enteropathogenic E. coli and enterotoxigenic E. coli on coriander.

PubMed

Gómez-Aldapa, Carlos A; Segovia-Cruz, Jesús A; Cerna-Cortes, Jorge F; Rangel-Vargas, Esmeralda; Salas-Rangel, Laura P; Gutiérrez-Alcántara, Eduardo J; Castro-Rosas, Javier

2016-10-01

The prevalence and behavior of multidrug-resistant diarrheagenic Escherichia coli pathotypes on coriander was determined. One hundred coriander samples were collected from markets. Generic E. coli were determined using the most probable number procedure. Diarrheagenic E. coli pathotypes (DEPs) were identified using two multiplex polymerase chain reaction procedures. Susceptibility to sixteen antibiotics was tested for the isolated DEPs strains by standard test. The behavior of multidrug-resistant DEPs isolated from coriander was determined on coriander leaves and chopped coriander at 25°± 2 °C and 3°± 2 °C. Generic E. coli and DEPs were identified, respectively, in 43 and 7% of samples. Nine DEPs strains were isolated from positive coriander samples. The identified DEPs included Shiga toxin-producing E. coli (STEC, 4%) enterotoxigenic E. coli (ETEC, 2%) and enteropathogenic E. coli (EPEC, 1%). All isolated DEPs strains exhibited multi-resistance to antibiotics. On inoculated coriander leaves stored at 25°± 2 °C or 3°± 2 °C, no growth was observed for multidrug-resistant DEPs strains. However, multidrug-resistant DEPs strains grew in chopped coriander: after 24 h at 25° ± 2 °C, DEPs strains had grown to approximately 3 log CFU/g. However, at 3°± 2 °C the bacterial growth was inhibited. To the best of our knowledge, this is the first report of the presence and behavior of multidrug-resistant STEC, ETEC and EPEC on coriander and chopped coriander. Copyright © 2016 Elsevier Ltd. All rights reserved.
Extracellular pH modulates GABAergic neurotransmission in rat hypothalamus.

PubMed

Chen, Z L; Huang, R Q

2014-06-20

Changes in extracellular pH have a modulatory effect on GABAA receptor function. It has been reported that pH sensitivity of the GABA receptor is dependent on subunit composition and GABA concentration. Most of previous investigations focused on GABA-evoked currents, which only reflect the postsynaptic receptors. The physiological relevance of pH modulation of GABAergic neurotransmission is not fully elucidated. In the present studies, we examined the influence of extracellular pH on the GABAA receptor-mediated inhibitory neurotransmission in rat hypothalamic neurons. The inhibitory postsynaptic currents (IPSCs), tonic currents, and the GABA-evoked currents were recorded with whole-cell patch techniques on the hypothalamic slices from Sprague-Dawley rats at 15-26 postnatal days. The amplitude and frequency of spontaneous GABA IPSCs were significantly increased while the external pH was changed from 7.3 to 8.4. In the acidic pH (6.4), the spontaneous GABA IPSCs were reduced in amplitude and frequency. The pH induced changes in miniature GABA IPSCs (mIPSCs) similar to that in spontaneous IPSCs. The pH effect on the postsynaptic GABA receptors was assessed with exogenously applied varying concentrations of GABA. The tonic currents and the currents evoked by sub-saturating concentration of GABA ([GABA]) (10 μM) were inhibited by acidic pH and potentiated by alkaline pH. In contrast, the currents evoked by saturating [GABA] (1mM) were not affected by pH changes. We also investigated the influence of pH buffers and buffering capacity on pH sensitivity of GABAA receptors on human recombinant α1β2γ2 GABAA receptors stably expressed in HEK 293 cells. The pH influence on GABAA receptors was similar in HEPES- and MES-buffered media, and not dependent on protonated buffers, suggesting that the observed pH effect on GABA response is a specific consequence of changes in extracellular protons. Our data suggest that the hydrogen ions suppress the GABAergic neurotransmission
A Modular Coassembly Approach to All-In-One Multifunctional Nanoplatform for Synergistic Codelivery of Doxorubicin and Curcumin

PubMed Central

Yang, Muyang; Yu, Lixia; Guo, Ruiwei; Dong, Anjie; Lin, Cunguo

2018-01-01

Synergistic combination therapy by integrating chemotherapeutics and chemosensitizers into nanoparticles has demonstrated great potential to reduce side effects, overcome multidrug resistance (MDR), and thus improve therapeutic efficacy. However, with regard to the nanocarriers for multidrug codelivery, it remains a strong challenge to maintain design simplicity, while incorporating the desirable multifunctionalities, such as coloaded high payloads, targeted delivery, hemodynamic stability, and also to ensure low drug leakage before reaching the tumor site, but simultaneously the corelease of drugs in the same cancer cell. Herein, we developed a facile modular coassembly approach to construct an all-in-one multifunctional multidrug delivery system for the synergistic codelivery of doxorubicin (DOX, chemotherapeutic agent) and curcumin (CUR, MDR modulator). The acid-cleavable PEGylated polymeric prodrug (DOX-h-PCEC), tumor cell-specific targeting peptide (CRGDK-PEG-PCL), and natural chemosensitizer (CUR) were ratiometrically assembled into in one single nanocarrier (CUR/DOX-h-PCEC@CRGDK NPs). The resulting CUR/DOX-h-PCEC@CRGDK NPs exhibited several desirable characteristics, such as efficient and ratiometric drug loading, high hemodynamic stability and low drug leakage, tumor intracellular acid-triggered cleavage, and subsequent intracellular simultaneous drug corelease, which are expected to maximize a synergistic effect of chemotherapy and chemosensitization. Collectively, the multifunctional nanocarrier is feasible for the creation of a robust nanoplatform for targeted multidrug codelivery and efficient MDR modulation. PMID:29543780
Rational design of cancer-targeted selenium nanoparticles to antagonize multidrug resistance in cancer cells.

PubMed

Liu, Ting; Zeng, Lilan; Jiang, Wenting; Fu, Yuanting; Zheng, Wenjie; Chen, Tianfeng

2015-05-01

Multidrug resistance is one of the greatest challenges in cancer therapy. Herein we described the synthesis of folate (FA)-conjugated selenium nanoparticles (SeNPs) as cancer-targeted nano-drug delivery system for ruthenium polypyridyl (RuPOP) exhibits strong fluorescence, which allows the direct imaging of the cellular trafficking of the nanosystem. This nanosystem could effectively antagonize against multidrug resistance in liver cancer. FA surface conjugation significantly enhanced the cellular uptake of SeNPs by FA receptor-mediated endocytosis through nystain-dependent lipid raft-mediated and clathrin-mediated pathways. The nanomaterials overcame the multidrug resistance in R-HepG2 cells through inhibition of ABC family proteins expression. Internalized nanoparticles triggered ROS overproduction and induced apoptosis by activating p53 and MAPKs pathways. Moreover, FA-SeNPs exhibited low in vivo acute toxicity, which verified the safety and application potential of FA-SeNPs as nanodrugs. This study provides an effective strategy for the design of cancer-targeted nanodrugs against multidrug resistant cancers. In the combat against hepatocellular carcinoma, multidrug resistance remains one of the obstacles to be overcome. The authors designed and synthesized folate (FA)-conjugated selenium nanoparticles (SeNPs) with enhanced cancer-targeting capability. This system carried ruthenium polypyridyl (RuPOP), an efficient metal-based anti-cancer drug with strong fluorescence. It was shown that this combination was effective in antagonizing against multidrug resistance in vitro. Copyright © 2015 Elsevier Inc. All rights reserved.
Virtual screening of commercial cyclic peptides as NS2B-NS3 protease inhibitor of dengue virus serotype 2 through molecular docking simulation

NASA Astrophysics Data System (ADS)

Nasution, M. A. F.; Aini, R. N.; Tambunan, U. S. F.

2017-04-01

A disease caused by dengue virus infection has become one of the major health problems in the world, particularly in Asia, Africa, and South America. This disease has become endemic in more than 100 countries, and approximately 100 million cases occur each year with 2.5 billion people or 40% of the world population at risk of having this virus infection. Therefore, we need an antiviral drug that can inhibit the activity of the enzymes that involved in the virus replication in the body. Lately, the peptide-based drug design has been developed and proved to have interesting pharmacological properties. This study uses commercially cyclic peptides that have already marketed. The purpose of this study is to screen the commercial cyclic peptides that can be used as an inhibitor of the NS2B-NS3 protease of dengue virus serotype 2 (DENV-2) through molecular docking simulations. Inhibition of NS3 protease enzyme can lead to enzymatic inhibition activity so the formed polyprotein from the translation of RNA cannot be cut into pieces and remain in the long strand form. Consequently, proteins that are vital for the sustainability of dengue virus replication cannot be formed. This research resulted in [alpha]-ANF (1-28), rat, Brain Natriuretic Peptide, porcine, Atrial Natriuretic Factor (3-28) (human) and Atrial Natriuretic Peptide (126-150) (rat) as the best drug candidate for inhibiting the NS2B-NS3 protease of DENV-2.
MarA-Like Regulator of Multidrug Resistance in Yersinia pestis

PubMed Central

Udani, Rupa A.; Levy, Stuart B.

2006-01-01

MarA47Yp from Yersinia pestis, showing 47% identity to Escherichia coli MarA in its N terminus, caused resistance to antibiotics and to organic solvents when expressed in both E. coli and Y. pestis. Resistance was linked to increased expression of the AcrAB multidrug efflux pump. In four of five spontaneous multidrug-resistant mutants of Y. pestis independently selected by growth on tetracycline, the marA47Yp gene was overexpressed. The findings suggest that marA47Yp is a marA ortholog in Y. pestis. PMID:16940090
MarA-like regulator of multidrug resistance in Yersinia pestis.

PubMed

Udani, Rupa A; Levy, Stuart B

2006-09-01

MarA47(Yp) from Yersinia pestis, showing 47% identity to Escherichia coli MarA in its N terminus, caused resistance to antibiotics and to organic solvents when expressed in both E. coli and Y. pestis. Resistance was linked to increased expression of the AcrAB multidrug efflux pump. In four of five spontaneous multidrug-resistant mutants of Y. pestis independently selected by growth on tetracycline, the marA47(Yp) gene was overexpressed. The findings suggest that marA47(Yp) is a marA ortholog in Y. pestis.
Complete genome sequence of new bacteriophage phiE142, which causes simultaneously lysis of multidrug-resistant Escherichia coli O157:H7 and Salmonella enterica.

PubMed

Amarillas, Luis; Chaidez, Cristobal; González-Robles, Arturo; León-Félix, Josefina

2016-01-01

The emergence of antibiotic-resistant foodborne bacteria is a global health problem that requires immediate attention. Bacteriophages are a promising biotechnological alternative approach against bacterial pathogens. However, a detailed analysis of phage genomes is essential to assess the safety of the phages prior to their use as biocontrol agents. Therefore, here we report the complete genome sequence of bacteriophage phiE142, which is able to lyse Salmonella and multidrug-resistant Escherichia coli O157:H7 strains. Bacteriophage phiE142 belongs to the Myoviridae family due to the presence of long non-flexible tail and icosahedral head. The genome is composed of 121,442 bp and contains 194 ORFs, and 2 tRNAs. Furthermore, the phiE142 genome does not contain any genes coding for food-borne allergens, antibiotics resistance, virulence factors, or associated with lysogenic conversion. The bacteriophage phiE142 is characterized by broad host range and compelling genetic attributes making them potential candidates as a biocontrol agent.
The effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on protein-protein interactions.

PubMed

Yates, Christopher M; Sternberg, Michael J E

2013-11-01

Non-synonymous single nucleotide polymorphisms (nsSNPs) are single base changes leading to a change to the amino acid sequence of the encoded protein. Many of these variants are associated with disease, so nsSNPs have been well studied, with studies looking at the effects of nsSNPs on individual proteins, for example, on stability and enzyme active sites. In recent years, the impact of nsSNPs upon protein-protein interactions has also been investigated, giving a greater insight into the mechanisms by which nsSNPs can lead to disease. In this review, we summarize these studies, looking at the various mechanisms by which nsSNPs can affect protein-protein interactions. We focus on structural changes that can impair interaction, changes to disorder, gain of interaction, and post-translational modifications before looking at some examples of nsSNPs at human-pathogen protein-protein interfaces and the analysis of nsSNPs from a network perspective. © 2013.
Long implosion time (240 ns) Z-pinch experiments with a large diameter (12 cm) double-shell nozzle

NASA Astrophysics Data System (ADS)

Levine, J. S.; Banister, J. W.; Failor, B. H.; Qi, N.; Song, Y.; Sze, H. M.; Fisher, A.

2004-05-01

Recently, an 8 cm diameter double-shell nozzle has produced argon Z pinches with high K-shell yields with implosion time of 210 ns. To produce even longer implosion time Z pinches for facilities such as Decade Quad [D. Price, et al., "Electrical and Mechanical Design of the Decade Quad in PRS Mode," in Proceedings of the 12th IEEE Pulsed Power Conference, Monterey, CA, edited by C. Stallings and H. Kirbie (IEEE, New York, 1999), p. 489] (9 MA short circuit current at 300 ns), a larger nozzle (12 cm outer diameter) was designed and fabricated. During initial testing on Double-EAGLE [P. Sincerny et al., Proceedings of the 5th IEEE Pulsed Power Conference, Arlington, VA, edited by M. F. Rose and P. J. Turchi (IEEE, New York, 1985), p. 151], 9 kJ of argon K-shell radiation in a 6 ns full width at half maximum pulse was produced with a 240 ns implosion. The initial gas distributions produced by various nozzle configurations have been measured and their impact on the final radiative characteristics of the pinch are presented. The addition of a central jet to increase the initial gas density near the axis is observed to enhance the pinch quality, increasing K-shell yield by 17% and power by 40% in the best configuration tested.

Nodding syndrome (NS) and Onchocerca Volvulus (OV) in Northern Uganda.

PubMed

Lagoro, David Kitara; Arony, Denis Anywar

2017-01-01

Nodding Syndrome (NS) is a childhood neurological disorder characterized by atonic seizures, cognitive decline, school dropout, muscle weakness, thermal dysfunction, wasting and stunted growth. There are recent published information suggesting associations between Nodding Syndrome (NS) with cerebrospinal fluid (CSF) VGKC antibodies and serum leiomidin-1 antibody cross reacting with Onchocerca Volvulus ( OV ). These findings suggest a neuro-inflammatory cause of NS and they are important findings in the search for the cause of Nodding Syndrome. These observations perhaps provide further, the unique explanation for the association between Nodding Syndrome and Onchocerca Volvulus . Many clinical and epidemiological studies had shown a significant correlation between NS and infestation with a nematode, Onchocerca volvulus which causes a disease, Onchocerciasis , some of which when left untreated can develop visual defect ("River Blindness"). While these studies conducted in Northern Uganda and Southern Sudan indicate a statistically significant association with ( OV infection (using positive skin snips), we observe that ( OV is generally endemic in many parts of Sub Saharan Africa and Latin America and that to date, no NS cases have been recorded in those regions. This letter to the Editor is to provide additional information on the current view about the relationship between Nodding Syndrome and Onchocerca Volvulus as seen in Northern Uganda.
Nodding syndrome (NS) and Onchocerca Volvulus (OV) in Northern Uganda

PubMed Central

Lagoro, David Kitara; Arony, Denis Anywar

2017-01-01

Nodding Syndrome (NS) is a childhood neurological disorder characterized by atonic seizures, cognitive decline, school dropout, muscle weakness, thermal dysfunction, wasting and stunted growth. There are recent published information suggesting associations between Nodding Syndrome (NS) with cerebrospinal fluid (CSF) VGKC antibodies and serum leiomidin-1 antibody cross reacting with Onchocerca Volvulus (OV). These findings suggest a neuro-inflammatory cause of NS and they are important findings in the search for the cause of Nodding Syndrome. These observations perhaps provide further, the unique explanation for the association between Nodding Syndrome and Onchocerca Volvulus. Many clinical and epidemiological studies had shown a significant correlation between NS and infestation with a nematode, Onchocerca volvulus which causes a disease, Onchocerciasis, some of which when left untreated can develop visual defect ("River Blindness"). While these studies conducted in Northern Uganda and Southern Sudan indicate a statistically significant association with (OV infection (using positive skin snips), we observe that (OV is generally endemic in many parts of Sub Saharan Africa and Latin America and that to date, no NS cases have been recorded in those regions. This letter to the Editor is to provide additional information on the current view about the relationship between Nodding Syndrome and Onchocerca Volvulus as seen in Northern Uganda. PMID:29138647
Prevalence of NS5B resistance-associated variants in treatment-naïve Asian patients with chronic hepatitis C.

PubMed

Yang, Song; Xing, Huichun; Feng, Shenghu; Ju, Wei; Liu, Shunai; Wang, Xiaomei; Ou, Weini; Cheng, Jun; Pan, Calvin Q

2018-02-01

There is little information on the association between baseline non-structural protein (NS) 5b resistance-associated variants (RAVs) and treatment failure in hepatitis C patients. This study examined the frequencies of natural hepatitis C virus (HCV) NS5B resistance-associated variants (RAVs) in an Asian cohort. Samples from Asian HCV patients enrolled between October 2009 and September 2014 were analyzed for NS5B RAVs within the region from amino acid 230 to 371. Serum samples were tested by PCR genotyping, with sequence alignment performed using the neighbor-joining method. NS5B was detected by Sanger sequencing followed by Geno2pheno analysis. NS5B RAVs were detected in 80.52% (1199/1489) of patients; 68.4% (1019/1489) and 79.7% (1186/1489) were associated with resistance to sofosbuvir (SOF) and dasabuvir (DSV), respectively. These RAVs were present in 95% (1004/1058) of genotype 1b patients. When genotypes 1b and 2a were compared, SOF-associated RAVs were detected at a higher frequency in genotype 1b (94.8% [1004/1058] vs. 2.9% [9/309]; χ 2 = 1054.433, P < 0.001), C316H/N was more common in genotype 1b (94.7% [1002/1058] vs. 0% [0/309]; χ 2 = 1096.014, P < 0.001), M289F/L/I/W/V had a higher frequency in genotype 2a (0.7% [7/309] vs. 2.3% [7/1058]; χ 2 = 4.589, P = 0.032), DSV-associated RAVs were most often found in genotype 1b (95.0% [1005/1058] vs. 40.1% 124/309]; χ 2 = 500.577, P < 0.001), and frequency of C316Y/H/N/W was higher in genotype 1b (94.7% [1002/1058] vs. 0% [0/309]; χ 2 = 1096.014, P < 0.001). In conclusion, baseline SOF and DSV RAVs are common in Asian HCV patients and predominantly occur in genotype 1b.
Herschel Planetary Nebula Survey (HerPlaNS). hydrogen recombination laser lines in Mz 3

NASA Astrophysics Data System (ADS)

Aleman, Isabel; Exter, Katrina; Ueta, Toshiya; Walton, Samuel; Tielens, A. G. G. M.; Zijlstra, Albert; Montez, Rodolfo; Abraham, Zulema; Otsuka, Masaaki; Beaklini, Pedro P. B.; van Hoof, Peter A. M.; Villaver, Eva; Leal-Ferreira, Marcelo L.; Mendoza, Edgar; Lépine, Jacques D. R.

2018-07-01

The bipolar nebula Menzel 3 (Mz 3) was observed as part of the Herschel Planetary Nebula Survey (HerPlaNS), which used the PACS and SPIRE instruments aboard the Herschel Space Observatory to study a sample of planetary nebulae (PNe). In this paper, one of the series describing HerPlaNS results, we report the detection of H I recombination lines (HRLs) in the spectrum of Mz 3. Inspection of the spectrum reveals the presence of 12 HRLs in the 55-680 µm range covered by the PACS and SPIRE instruments (H11α to H21α and H14β). The presence of HRLs in this range is unusual for PNe and has not been reported in Mz 3 before. Our analysis indicates that the HRLs we observed are enhanced by laser effect occurring in the core of Mz 3. Our arguments for this are (i) the available Mz 3 optical to submillimetre HRL α line intensity ratios are not well reproduced by the spontaneous emission of optically thin ionized gas, as would be typical for nebular gas in PNe; (ii) the compact core of Mz 3 is responsible for a large fraction of the Herschel HRLs emission; (iii) the line intensity ratios for Mz 3 are very similar to those in the core emission of the well known star MWC 349A, where laser effect is responsible for the enhancement of HRLs in the Herschel wavelength range; (iv) the physical characteristics relevant to cause laser effect in the core of MWC 349A are very similar to those in the core of Mz 3.
Daclatasvir inhibits hepatitis C virus NS5A motility and hyper-accumulation of phosphoinositides

PubMed Central

Chukkapalli, Vineela; Berger, Kristi L.; Kelly, Sean M.; Thomas, Meryl; Deiters, Alexander; Randall, Glenn

2014-01-01

Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies are HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation. PMID:25546252
Production of recombinant dengue non-structural 1 (NS1) proteins from clinical virus isolates.

PubMed

Yohan, Benediktus; Wardhani, Puspa; Aryati; Trimarsanto, Hidayat; Sasmono, R Tedjo

2017-01-01

Dengue is a febrile disease caused by infection of dengue virus (DENV). Early diagnosis of dengue infection is important for better management of the disease. The DENV Non-Structural Protein 1 (NS1) antigen has been routinely used for the early dengue detection. In dengue epidemic countries such as Indonesia, clinicians are increasingly relying on the NS1 detection for confirmation of dengue infection. Various NS1 diagnostic tests are commercially available, however different sensitivities and specificities were observed in various settings. This study was aimed to generate dengue NS1 recombinant protein for the development of dengue diagnostic tests. Four Indonesian DENV isolates were used as the source of the NS1 gene cloning, expression, and purification in bacterial expression system. Recombinant NS1 proteins were successfully purified and their antigenicities were assessed. Immunization of mice with recombinant proteins observed the immunogenicity of the NS1 protein. The generated recombinant proteins can be potentially used in the development of NS1 diagnostic test. With minimal modifications, this method can be used for producing NS1 recombinant proteins from isolates obtained from other geographical regions. Copyright © 2016 Elsevier Inc. All rights reserved.
Multidrug-Resistant Candida: Epidemiology, Molecular Mechanisms, and Treatment.

PubMed

Arendrup, Maiken Cavling; Patterson, Thomas F

2017-08-15

Invasive Candida infections remain an important cause of morbidity and mortality, especially in hospitalized and immunocompromised or critically ill patients. A limited number of antifungal agents from only a few drug classes are available to treat patients with these serious infections. Resistance can be either intrinsic or acquired. Resistance mechanisms are not exchanged between Candida; thus, acquired resistance either emerges in response to an antifungal selection pressure in the individual patient or, more rarely, occur due to horizontal transmission of resistant strains between patients. Although multidrug resistance is uncommon, increasing reports of multidrug resistance to the azoles, echinocandins, and polyenes have occurred in several Candida species, most notably Candida glabrata and more recently Candida auris. Drivers are overall antifungal use, subtherapeutic drug levels at sites of infection/colonization, drug sequestration in the biofilm matrix, and, in the setting of outbreaks, suboptimal infection control. Moreover, recent research suggests that DNA mismatch repair gene mutations may facilitate acquisition of resistance mutations in C. glabrata specifically. Diagnosis of antifungal-resistant Candida infections is critical to the successful management of patients with these infections. Reduction of unnecessary use of antifungals via antifungal stewardship is critical to limit multidrug resistance emergence. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
Multidrug resistant Acinetobacter baumannii in veterinary medicine--emergence of an underestimated pathogen?

PubMed

Müller, Stefanie; Janssen, Traute; Wieler, Lothar H

2014-01-01

The proportion of multidrug resistant bacteria causing infections in animals has continuously been increasing. While the relevance of ESBL (extended spectrum beta-lactamase)-producing Enterobacteriaceae spp. and MRSA (methicillin resistant Staphylococcus aureus) is unquestionable, knowledge about multidrug resistant Acinetobacter baumannii in veterinary medicine is scarce. This is a worrisome situation, as A. baumannii are isolated from veterinary clinical specimens with rising frequency. The remarkable ability of A. baumannii to develop multidrug resistance and the high risk of transmission are known in human medicine for years. Despite this, data regarding A. baumannii isolates of animal origin are missing. Due to the changing role of companion animals with closer contact between animal and owner, veterinary intensive care medicine is steadily developing. It can be assumed that the number of "high risk" patients with an enhanced risk for hospital acquired infections will be rising simultaneously. Thus, development and spread of multidrug resistant pathogens is envisioned to rise. It is possible, that A. baumannii will evolve into a veterinary nosocomial pathogen similar to ESBL-producing Enterobacteriaceae and MRSA. The lack of attention paid to A. baumannii in veterinary medicine is even more worrying, as first reports indicate a transmission between humans and animals. Essential questions regarding the role of livestock, especially as a potential source of multidrug resistant isolates, remain unanswered. This review summarizes the current knowledge on A. baumannii in veterinary medicine for the first time. It underlines the utmost significance of further investigations of A. baumannii animal isolates, particularly concerning epidemiology and resistance mechanisms.
Effective Targeted Photothermal Ablation of Multidrug Resistant Bacteria and Their Biofilms with NIR-Absorbing Gold Nanocrosses.

PubMed

Teng, Choon Peng; Zhou, Tielin; Ye, Enyi; Liu, Shuhua; Koh, Leng Duei; Low, Michelle; Loh, Xian Jun; Win, Khin Yin; Zhang, Lianhui; Han, Ming-Yong

2016-08-01

With the rapid evolution of antibiotic resistance in bacteria, antibiotic-resistant bacteria (in particular, multidrug-resistant bacteria) and their biofilms have been becoming more and more difficult to be effectively treated with conventional antibiotics. As such, there is a great demand to develop a nonantibiotic approach in efficiently eliminating such bacteria. Here, multibranched gold nanocrosses with strong near-infrared absorption falling in the biological window, which heat up quickly under near-infrared-light irradiation are presented. The gold nanocrosses are conjugated to secondary and primary antibodies for targeting PcrV, a type III secretion protein, which is uniquely expressed on the bacteria superbug, Pseudomonas aeruginosa. The conjugated gold nanocrosses are capable of completely destroying P. aeruginosa and its biofilms upon near-infrared-light irradiation for 5 min with an 800 nm laser at a low power density of ≈3.0 W cm(-2) . No bacterial activity is detected after 48 h postirradiation, which indicates that the heat generated from the irradiated plasmonic gold nanocrosses attached to bacteria is effective in eliminating and preventing the re-growth of the bacteria. Overall, the conjugated gold nanocrosses allow targeted and effective photothermal ablation of multidrug-resistant bacteria and their biofilms in the localized region with reduced nonspecific damage to normal tissue. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Multidrug-resistant tuberculosis, Somalia, 2010-2011.

PubMed

Sindani, Ireneaus; Fitzpatrick, Christopher; Falzon, Dennis; Suleiman, Bashir; Arube, Peter; Adam, Ismail; Baghdadi, Samiha; Bassili, Amal; Zignol, Matteo

2013-03-01

In a nationwide survey in 2011, multidrug-resistant tuberculosis (MDR TB) was found in 5.2% and 40.8% of patients with new and previously treated TB, respectively. These levels of drug resistance are among the highest ever documented in Africa and the Middle East. This finding presents a serious challenge for TB control in Somalia.
Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM).

PubMed

Ruiz, Zandra; Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter

2011-02-20

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins. Copyright © 2010 Elsevier Inc. All rights reserved.
Histone deacetylase inhibitor (HDACI) PCI-24781 enhances chemotherapy induced apoptosis in multidrug resistant sarcoma cell lines

PubMed Central

Yang, Cao; Choy, Edwin; Hornicek, Francis J.; Wood, Kirkham B; Schwab, Joseph H; Liu, Xianzhe; Mankin, Henry; Duan, Zhenfeng

2013-01-01

The anti-tumor activity of histone deacetylase inhibitors (HDACI) on multi-drug resistant sarcoma cell lines has never been previously described. Four multidrug resistant sarcoma cell lines treated with HDACI PCI-24781 resulted in dose-dependent accumulation of acetylated histones, p21 and PARP cleavage products. Growth of these cell lines was inhibited by PCI-24781 at IC50 of 0.43 to 2.7. When we looked for synergy of PCI-24781 with chemotherapeutic agents, we found that PCI-24781 reverses drug resistance in all four multidrug resistant sarcoma cell lines and synergizes with chemotherapeutic agents to enhance caspase-3/7 activity. Expression of RAD51 (a marker for DNA double-strand break repair) was inhibited and the expression of GADD45α (a marker for growth arrest and DNA-damage) was induced by PCI-24781 in multidrug resistant sarcoma cell lines. In conclusion, HDACI PCI-24781 synergizes with chemotherapeutic drugs to induce apoptosis and reverses drug resistance in multidrug resistant sarcoma cell lines. PMID:21508354
A comparative study for the inactivation of multidrug resistance bacteria using dielectric barrier discharge and nano-second pulsed plasma

PubMed Central

Hoon Park, Ji; Kumar, Naresh; Hoon Park, Dae; Yusupov, Maksudbek; Neyts, Erik C.; Verlackt, Christof C. W.; Bogaerts, Annemie; Ho Kang, Min; Sup Uhm, Han; Ha Choi, Eun; Attri, Pankaj

2015-01-01

Bacteria can be inactivated through various physical and chemical means, and these have always been the focus of extensive research. To further improve the methodology for these ends, two types of plasma systems were investigated: nano-second pulsed plasma (NPP) as liquid discharge plasma and an Argon gas-feeding dielectric barrier discharge (Ar-DBD) as a form of surface plasma. To understand the sterilizing action of these two different plasma sources, we performed experiments with Staphylococcus aureus (S. aureus) bacteria (wild type) and multidrug resistant bacteria (Penicillum-resistant, Methicillin-resistant and Gentamicin-resistant). We observed that both plasma sources can inactivate both the wild type and multidrug-resistant bacteria to a good extent. Moreover, we observed a change in the surface morphology, gene expression and β-lactamase activity. Furthermore, we used X-ray photoelectron spectroscopy to investigate the variation in functional groups (C-H/C-C, C-OH and C=O) of the peptidoglycan (PG) resulting from exposure to plasma species. To obtain atomic scale insight in the plasma-cell interactions and support our experimental observations, we have performed molecular dynamics simulations to study the effects of plasma species, such as OH, H2O2, O, O3, as well as O2 and H2O, on the dissociation/formation of above mentioned functional groups in PG. PMID:26351132
Management of multidrug-resistant tuberculosis in human immunodeficiency virus patients

NASA Astrophysics Data System (ADS)

Jamil, K. F.

2018-03-01

Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis(MTB). 10.4 million new TB cases will appear in 2015 worldwide. There were an estimated 1.4 million TB deaths in 2015, and an additional 0.4 million deaths resulting from TB disease among people living with human immunodeficiency virus (HIV). Multidrug- resistant and extensively drug-resistant tuberculosis (MDR and XDR-TB) are major public health concerns worldwide. 480.000 new cases of MDR-TB will appear in 2015 and an additional 100,000 people with rifampicin-resistant TB (RR-TB) who were also newly eligible for MDR-TB treatment. Their association with HIV infection has contributed to the slowing down of TB incidence decline over the last two decades, therefore representing one important barrier to reach TB elimination. Patients infected with MDR-TB require more expensive treatment regimens than drug-susceptible TB, with poor treatment.Patients with multidrug- resistant tuberculosis do not receive rifampin; drug interactions risk is markedly reduced. However, overlapping toxicities may limit options for co-treatment of HIV and multidrug- resistant tuberculosis.
Natural History of Multi-Drug Resistant Organisms in a New Military Medical Facility

DTIC Science & Technology

2013-12-01

environment plays in the transmission of multidrug- resistant Gram-negative bacteria and methicillin - resistant Staphylococcus aureus (MDRO) is increasingly...Pseudomonas aeruginosa, methicillin - resistant Staphylococcus aureus (MRSA); Klebsiella pneumoniea; and Clostridium difficile. Multidrug- resistance (MDR...target organism infection was Staphylococcus aureus (n=77), followed by E coli (56), Klebsiella pneumoniae (28), and Pseudomonas aeruginosa (11
Worldwide Endemicity of a Multidrug-Resistant Staphylococcus capitis Clone Involved in Neonatal Sepsis.

PubMed

Butin, Marine; Martins-Simões, Patricia; Rasigade, Jean-Philippe; Picaud, Jean-Charles; Laurent, Frédéric

2017-03-01

A multidrug-resistant Staphylococcus capitis clone, NRCS-A, has been isolated from neonatal intensive care units in 17 countries throughout the world. S. capitis NRCS-A prevalence is high in some neonatal intensive care units in France. These data highlight the worldwide endemicity and epidemiologic relevance of this multidrug-resistant, coagulase-negative staphylococci clone.
Potential strategies for the eradication of multidrug-resistant Gram-negative bacterial infections.

PubMed

Huwaitat, Rawan; McCloskey, Alice P; Gilmore, Brendan F; Laverty, Garry

2016-07-01

Antimicrobial resistance is one of the leading threats to society. The increasing burden of multidrug-resistant Gram-negative infection is particularly concerning as such bacteria are demonstrating resistance to nearly all currently licensed therapies. Various strategies have been hypothesized to treat multidrug-resistant Gram-negative infections including: targeting the Gram-negative outer membrane; neutralization of lipopolysaccharide; inhibition of bacterial efflux pumps and prevention of protein folding. Silver and silver nanoparticles, fusogenic liposomes and nanotubes are potential strategies for extending the activity of licensed, Gram-positive selective, antibiotics to Gram-negatives. This may serve as a strategy to fill the current void in pharmaceutical development in the short term. This review outlines the most promising strategies that could be implemented to solve the threat of multidrug-resistant Gram-negative infections.
Multidrug Efflux Pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus Bacterial Food Pathogens

PubMed Central

Andersen, Jody L.; He, Gui-Xin; Kakarla, Prathusha; KC, Ranjana; Kumar, Sanath; Lakra, Wazir Singh; Mukherjee, Mun Mun; Ranaweera, Indrika; Shrestha, Ugina; Tran, Thuy; Varela, Manuel F.

2015-01-01

Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations. PMID:25635914
Multidrug efflux pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus bacterial food pathogens.

PubMed

Andersen, Jody L; He, Gui-Xin; Kakarla, Prathusha; K C, Ranjana; Kumar, Sanath; Lakra, Wazir Singh; Mukherjee, Mun Mun; Ranaweera, Indrika; Shrestha, Ugina; Tran, Thuy; Varela, Manuel F

2015-01-28

Foodborne illnesses caused by bacterial microorganisms are common worldwide and constitute a serious public health concern. In particular, microorganisms belonging to the Enterobacteriaceae and Vibrionaceae families of Gram-negative bacteria, and to the Staphylococcus genus of Gram-positive bacteria are important causative agents of food poisoning and infection in the gastrointestinal tract of humans. Recently, variants of these bacteria have developed resistance to medically important chemotherapeutic agents. Multidrug resistant Escherichia coli, Salmonella enterica, Vibrio cholerae, Enterobacter spp., and Staphylococcus aureus are becoming increasingly recalcitrant to clinical treatment in human patients. Of the various bacterial resistance mechanisms against antimicrobial agents, multidrug efflux pumps comprise a major cause of multiple drug resistance. These multidrug efflux pump systems reside in the biological membrane of the bacteria and actively extrude antimicrobial agents from bacterial cells. This review article summarizes the evolution of these bacterial drug efflux pump systems from a molecular biological standpoint and provides a framework for future work aimed at reducing the conditions that foster dissemination of these multidrug resistant causative agents through human populations.
In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir

PubMed Central

Tian, Yang; Doehle, Brian; Peng, Betty; Corsa, Amoreena; Lee, Yu-Jen; Gong, Ruoyu; Yu, Mei; Han, Bin; Xu, Simin; Dvory-Sobol, Hadas; Perron, Michel; Xu, Yili; Mo, Hongmei; Pagratis, Nikos; Link, John O.; Delaney, William

2016-01-01

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type. PMID:26824950

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir.

PubMed

Cheng, Guofeng; Tian, Yang; Doehle, Brian; Peng, Betty; Corsa, Amoreena; Lee, Yu-Jen; Gong, Ruoyu; Yu, Mei; Han, Bin; Xu, Simin; Dvory-Sobol, Hadas; Perron, Michel; Xu, Yili; Mo, Hongmei; Pagratis, Nikos; Link, John O; Delaney, William

2016-01-11

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Multidrug-resistant Salmonella Typhimurium in Four Animal Facilities

PubMed Central

Wright, Jennifer G.; Tengelsen, Leslie A.; Smith, Kirk E.; Bender, Jeff B.; Frank, Rodney K.; Grendon, John H.; Rice, Daniel H.; Thiessen, Ann Marie B.; Gilbertson, Catherine Jo; Sivapalasingam, Sumathi; Barrett, Timothy J.; Besser, Thomas E.; Hancock, Dale D.

2005-01-01

In 1999 and 2000, 3 state health departments reported 4 outbreaks of gastrointestinal illness due to Salmonella enterica serotype Typhimurium in employees, clients, and client animals from 3 companion animal veterinary clinics and 1 animal shelter. More than 45 persons and companion animals became ill. Four independent investigations resulted in the testing of 19 human samples and >200 animal samples; 18 persons and 36 animals were culture-positive for S. Typhimurium. One outbreak was due to multidrug-resistant S. Typhimurium R-type ACKSSuT, while the other 3 were due to multidrug-resistant S. Typhimurium R-type ACSSuT DT104. This report documents nosocomial transmission of S. Typhimurium and demonstrates that companion animal facilities may serve as foci of transmission for salmonellae between animals and humans if adequate precautions are not followed. PMID:16102313
In-silico identification and evaluation of plant flavonoids as dengue NS2B/NS3 protease inhibitors using molecular docking and simulation approach.

PubMed

Qamar, Muhammad Tahirul; Ashfaq, Usman Ali; Tusleem, Kishver; Mumtaz, Arooj; Tariq, Quratulain; Goheer, Alina; Ahmed, Bilal

2017-11-01

Dengue infection is prevailing among the people not only from the developing countries but also from the developed countries due to its high morbidity rate around the globe. Hence, due to the unavailability of any suitable vaccine for rigorous dengue virus (DENV), the only mode of its treatment is prevention. The circumstances require an urgent development of efficient and practical treatment to deal with these serotypes. The severe effects and cost of synthetic vaccines simulated researchers to find anti-viral agents from medicinal plants. Flavonoids present in medicinal plants, holds anti-viral activity and can be used as vaccine against viruses. Therefore, present study was planned to find anti-viral potential of 2500 flavonoids inhibitors against the DENVNS2B/NS3 protease through computational screening which can hinder the viral replication within the host cell. By using molecular docking, it was revealed that flavonoids showed strong and stable bonding in the binding pocket of DENV NS2B/NS3 protease and had strong interactions with catalytic triad. Drug capability and anti-dengue potential of the flavonoids was also evaluated by using different bioinformatics tools. Some flavonoids effectively blocked the catalytic triad of DENV NS2B/NS3 protease and also passed through drug ability evaluation. It can be concluded from this study that these flavonoids could act as potential inhibitors to stop the replication of DENV and there is a need to study the action of these molecules in-vitro to confirm their action and other properties.
Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells.

PubMed

Stegajev, Vasili; Kouri, Vesa-Petteri; Salem, Abdelhakim; Rozov, Stanislav; Stark, Holger; Nordström, Dan C E; Konttinen, Yrjö T

2014-12-01

Apoptosis is involved in the pathogenesis of Sjögren's syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H₄R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H₄R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H₄R agonist HST-10. Expression and internalization of H₄R were studied by immunofluorescence staining ± clathrin inhibitor methyl-β-cyclodextrin (MβCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H₄R internalization was inhibited by MβCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H₄R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H4R-stimulated samples. H₄R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H₄R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H₄R activation on apoptosis of human salivary gland cells. Diminished H₄R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.
Tackling Threats and Future Problems of Multidrug-Resistant Bacteria.

PubMed

Medina, Eva; Pieper, Dietmar Helmut

With the advent of the antibiotic era, the overuse and inappropriate consumption and application of antibiotics have driven the rapid emergence of multidrug-resistant pathogens. Antimicrobial resistance increases the morbidity, mortality, length of hospitalization and healthcare costs. Among Gram-positive bacteria, Staphylococcus aureus (MRSA) and multidrug-resistant (MDR) Mycobacterium tuberculosis, and among the Gram-negative bacteria, extended-spectrum beta-lactamase (ESBLs)-producing bacteria have become a major global healthcare problem in the 21st century. The pressure to use antibiotics guarantees that the spread and prevalence of these as well as of future emerging multidrug-resistant pathogens will be a persistent phenomenon. The unfeasibility of reversing antimicrobial resistance back towards susceptibility and the critical need to treat bacterial infection in modern medicine have burdened researchers and pharmaceutical companies to develop new antimicrobials effective against these difficult-to-treat multidrug-resistant pathogens. However, it can be anticipated that antibiotic resistance will continue to develop more rapidly than new agents to treat these infections become available and a better understanding of the molecular, evolutionary and ecological mechanisms governing the spread of antibiotic resistance is needed. The only way to curb the current crisis of antimicrobial resistance will be to develop entirely novel strategies to fight these pathogens such as combining antimicrobial drugs with other agents that counteract and obstruct the antibiotic resistant mechanisms expressed by the pathogen. Furthermore, as many antibiotics are often inappropriately prescribed, a more personalized approach based on precise diagnosis tools will ensure that proper treatments can be promptly applied leading to more targeted and effective therapies. However, in more general terms, also the overall use and release of antibiotics in the environment needs to be
Fluorescence behaviour of 5,10-methenyltetrahydrofolate, 10-formyltetrahydrofolate, 10-formyldihydrofolate, and 10-formylfolate in aqueous solution at pH 8

NASA Astrophysics Data System (ADS)

Tyagi, A.; Penzkofer, A.; Batschauer, A.; Wolf, E.

2009-06-01

The fluorescence spectroscopic behaviour of (6R,S)-5,10-methenyltetrahydrofolate (MTHF), (6R,S)-10-formyltetrahydrofolate (10-HCO-H4folate), 10-formyldihydrofolate (10-HCO-H2folate), and 10-formylfolate (10-HCO-folate) in aqueous Tris-HCl buffer at pH 8 is studied. MTHF and 10-HCO-folate were commercially available. 10-HCO-H4folate was prepared from MTHF by hydrolysis at room temperature under anaerobic conditions. 10-HCO-H2folate was prepared by oxidation of 10-HCO-H4folate under aerobic conditions. Fluorescence quantum distributions at room temperature and fluorescence signal decays at room temperature and liquid nitrogen temperature were measured. The fluorescence lifetimes determined at room temperature (liquid nitrogen temperature) are 10 ps (2.9 ns) for MTHF, 38 ps (3.7 ns) for 10-HCO-H4folate, 80 ps (10.5 ns) for 10-HCO-H2folate, and 7.1 ns (20 ns) for 10-HCO-folate. The results are discussed in terms of dyadic (pterin-benzoyl-glutamate) photo-induced electron transfer and dyadic fluorescent dynamics.
Bacillus subtilis from Soybean Food Shows Antimicrobial Activity for Multidrug-Resistant Acinetobacter baumannii by Affecting the adeS Gene.

PubMed

Wang, Tieshan; Su, Jianrong

2016-12-28

Exploring novel antibiotics is necessary for multidrug-resistant pathogenic bacteria. Because the probiotics in soybean food have antimicrobial activities, we investigated their effects on multidrug-resistant Acinetobacter baumannii . Nineteen multidrug-resistant A. baumannii strains were clinifcally isolated as an experimental group and 11 multidrug-sensitive strains as controls. The growth rates of all bacteria were determined by using the analysis for xCELLigence Real-Time Cell. The combination of antibiotics showed synergistic effects on the strains in the control group but no effect on the strains in the experimental group. Efflux pump gene adeS was absent in all the strains from the control group, whereas it exists in all the strains from the experimental group. Furthermore, all the strains lost multidrug resistance when an adeS inhibitor was used. One strain of probiotics isolated from soybean food showed high antimicrobial activity for multidrug-resistant A. baumannii . The isolated strain belongs to Bacillus subtilis according to 16S RNA analysis. Furthermore, E. coli showed multidrug resistance when it was transformed with the adeS gene from A. baumannii whereas the resistant bacteria could be inhibited completely by isolated Bacillus subtilis . Thus, probiotics from soybean food provide potential antibiotics against multidrug-resistant pathogenic bacteria.
Gelatin nanoparticles enhance delivery of hepatitis C virus recombinant NS2 gene

PubMed Central

George, Marina A.; El-Shorbagy, Haidan M.; Bassiony, Heba; Farroh, Khaled Y.; Youssef, Tareq; Salaheldin, Taher A.

2017-01-01

Background Development of an effective non-viral vaccine against hepatitis C virus infection is of a great importance. Gelatin nanoparticles (Gel.NPs) have an attention and promising approach as a viable carrier for delivery of vaccine, gene, drug and other biomolecules in the body. Aim of work The present study aimed to develop stable Gel.NPs conjugated with nonstructural protein 2 (NS2) gene of Hepatitis C Virus genotype 4a (HCV4a) as a safe and an efficient vaccine delivery system. Methods and results Gel.NPs were synthesized and characterized (size: 150±2 nm and zeta potential +17.6 mv). NS2 gene was successfully cloned and expressed into E. coli M15 using pQE-30 vector. Antigenicity of the recombinant NS2 protein was confirmed by Western blotting to verify the efficiency of NS2 as a possible vaccine. Then NS2 gene was conjugated to gelatin nanoparticles and a successful conjugation was confirmed by labeling and imaging using Confocal Laser Scanning Microscope (CLSM). Interestingly, the transformation of the conjugated NS2/Gel.NPs complex into E. coli DH5-α was 50% more efficient than transformation with the gene alone. In addition, conjugated NS2/Gel.NPs with ratio 1:100 (w/w) showed higher transformation efficiency into E. coli DH5-α than the other ratios (1:50 and 2:50). Conclusion Gel.NPs effectively enhanced the gene delivery in bacterial cells without affecting the structure of NS2 gene and could be used as a safe, easy, rapid, cost-effective and non-viral vaccine delivery system for HCV. PMID:28746382
Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components

PubMed Central

Beatty, P. Robert

2017-01-01

Dengue virus (DENV) is the most prevalent, medically important mosquito-borne virus. Disease ranges from uncomplicated dengue to life-threatening disease, characterized by endothelial dysfunction and vascular leakage. Previously, we demonstrated that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability in a systemic mouse model and human pulmonary endothelial cells, where NS1 disrupts the endothelial glycocalyx-like layer. NS1 also triggers release of inflammatory cytokines from PBMCs via TLR4. Here, we examined the relative contributions of inflammatory mediators and endothelial cell-intrinsic pathways. In vivo, we demonstrated that DENV NS1 but not the closely-related West Nile virus NS1 triggers localized vascular leak in the dorsal dermis of wild-type C57BL/6 mice. In vitro, we showed that human dermal endothelial cells exposed to DENV NS1 do not produce inflammatory cytokines (TNF-α, IL-6, IL-8) and that blocking these cytokines does not affect DENV NS1-induced endothelial hyperpermeability. Further, we demonstrated that DENV NS1 induces vascular leak in TLR4- or TNF-α receptor-deficient mice at similar levels to wild-type animals. Finally, we blocked DENV NS1-induced vascular leak in vivo using inhibitors targeting molecules involved in glycocalyx disruption. Taken together, these data indicate that DENV NS1-induced endothelial cell-intrinsic vascular leak is independent of inflammatory cytokines but dependent on endothelial glycocalyx components. PMID:29121099
Synergistic and Additive Effect of Oregano Essential Oil and Biological Silver Nanoparticles against Multidrug-Resistant Bacterial Strains

PubMed Central

Scandorieiro, Sara; de Camargo, Larissa C.; Lancheros, Cesar A. C.; Yamada-Ogatta, Sueli F.; Nakamura, Celso V.; de Oliveira, Admilton G.; Andrade, Célia G. T. J.; Duran, Nelson; Nakazato, Gerson; Kobayashi, Renata K. T.

2016-01-01

Bacterial resistance to conventional antibiotics has become a clinical and public health problem, making therapeutic decisions more challenging. Plant compounds and nanodrugs have been proposed as potential antimicrobial alternatives. Studies have shown that oregano (Origanum vulgare) essential oil (OEO) and silver nanoparticles have potent antibacterial activity, also against multidrug-resistant strains; however, the strong organoleptic characteristics of OEO and the development of resistance to these metal nanoparticles can limit their use. This study evaluated the antibacterial effect of a two-drug combination of biologically synthesized silver nanoparticles (bio-AgNP), produced by Fusarium oxysporum, and OEO against Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. OEO and bio-AgNP showed bactericidal effects against all 17 strains tested, with minimal inhibitory concentrations (MIC) ranging from 0.298 to 1.193 mg/mL and 62.5 to 250 μM, respectively. Time-kill curves indicated that OEO acted rapidly (within 10 min), while the metallic nanoparticles took 4 h to kill Gram-negative bacteria and 24 h to kill Gram-positive bacteria. The combination of the two compounds resulted in a synergistic or additive effect, reducing their MIC values and reducing the time of action compared to bio-AgNP used alone, i.e., 20 min for Gram-negative bacteria and 7 h for Gram-positive bacteria. Scanning electron microscopy (SEM) revealed similar morphological alterations in Staphylococcus aureus (non-methicillin-resistant S. aureus, non-MRSA) cells exposed to three different treatments (OEO, bio-AgNP and combination of the two), which appeared cell surface blebbing. Individual and combined treatments showed reduction in cell density and decrease in exopolysaccharide matrix compared to untreated bacterial cells. It indicated that this composition have an antimicrobial activity against S. aureus by disrupting cells. Both compounds showed very low
Synergistic and Additive Effect of Oregano Essential Oil and Biological Silver Nanoparticles against Multidrug-Resistant Bacterial Strains.

PubMed

Scandorieiro, Sara; de Camargo, Larissa C; Lancheros, Cesar A C; Yamada-Ogatta, Sueli F; Nakamura, Celso V; de Oliveira, Admilton G; Andrade, Célia G T J; Duran, Nelson; Nakazato, Gerson; Kobayashi, Renata K T

2016-01-01

Bacterial resistance to conventional antibiotics has become a clinical and public health problem, making therapeutic decisions more challenging. Plant compounds and nanodrugs have been proposed as potential antimicrobial alternatives. Studies have shown that oregano (Origanum vulgare) essential oil (OEO) and silver nanoparticles have potent antibacterial activity, also against multidrug-resistant strains; however, the strong organoleptic characteristics of OEO and the development of resistance to these metal nanoparticles can limit their use. This study evaluated the antibacterial effect of a two-drug combination of biologically synthesized silver nanoparticles (bio-AgNP), produced by Fusarium oxysporum, and OEO against Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. OEO and bio-AgNP showed bactericidal effects against all 17 strains tested, with minimal inhibitory concentrations (MIC) ranging from 0.298 to 1.193 mg/mL and 62.5 to 250 μM, respectively. Time-kill curves indicated that OEO acted rapidly (within 10 min), while the metallic nanoparticles took 4 h to kill Gram-negative bacteria and 24 h to kill Gram-positive bacteria. The combination of the two compounds resulted in a synergistic or additive effect, reducing their MIC values and reducing the time of action compared to bio-AgNP used alone, i.e., 20 min for Gram-negative bacteria and 7 h for Gram-positive bacteria. Scanning electron microscopy (SEM) revealed similar morphological alterations in Staphylococcus aureus (non-methicillin-resistant S. aureus, non-MRSA) cells exposed to three different treatments (OEO, bio-AgNP and combination of the two), which appeared cell surface blebbing. Individual and combined treatments showed reduction in cell density and decrease in exopolysaccharide matrix compared to untreated bacterial cells. It indicated that this composition have an antimicrobial activity against S. aureus by disrupting cells. Both compounds showed very low
Biomimetic RNA-silencing nanocomplexes: overcoming multidrug resistance in cancer cells.

PubMed

Wang, Zhongliang; Wang, Zhe; Liu, Dingbin; Yan, Xuefeng; Wang, Fu; Niu, Gang; Yang, Min; Chen, Xiaoyuan

2014-02-10

RNA interference (RNAi) is an RNA-dependent gene silencing approach controlled by an RNA-induced silencing complex (RISC). Herein, we present a synthetic RISC-mimic nanocomplex, which can actively cleave its target RNA in a sequence-specific manner. With high enzymatic stability and efficient self-delivery to target cells, the designed nanocomplex can selectively and potently induce gene silencing without cytokine activation. These nanocomplexes, which target multidrug resistance, are not only able to bypass the P-glycoprotein (Pgp) transporter, due to their nano-size effect, but also effectively suppress Pgp expression, thus resulting in successful restoration of drug sensitivity of OVCAR8/ADR cells to Pgp-transportable cytotoxic agents. This nanocomplex approach has the potential for both functional genomics and cancer therapy. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Conclusions and future directions for the REiNS International Collaboration

PubMed Central

Blakeley, Jaishri O.; Dombi, Eva; Fisher, Michael J.; Hanemann, Clemens O.; Walsh, Karin S.; Wolters, Pamela L.; Plotkin, Scott R.

2013-01-01

The Response Evaluation in Neurofibromatosis and Schwannomatosis (REiNS) International Collaboration was established with the goal to develop consensus recommendations for the use of endpoints in neurofibromatosis (NF) clinical trials. This supplement includes the first series of REiNS recommendations for the use of patient-reported, functional, and visual outcomes, and for the evaluation of imaging response in NF clinical trials. Recommendations for neurocognitive outcome measures, the use of whole-body MRI in NF, the evaluation of potential biomarkers of disease, and the comprehensive evaluation of functional and patient-reported outcomes in NF are in development. The REiNS recommendations are made based on current knowledge. Experience with the use of the recommended endpoints in clinical trials, development of new tools and technologies, new knowledge of the natural history of NF, and advances in the methods used to analyze endpoints will likely lead to modifications of the currently proposed guidelines, which will be shared with the NF research community through the REiNS Web site www.reinscollaboration.org. Due to the clinical complexity of NF, there is a need to seek expertise from multiple medical disciplines, regulatory agencies, and industry to develop trial endpoints and designs, which will lead to the identification and approval of effective treatments for NF tumor and nontumor manifestations. The REiNS Collaboration welcomes anyone interested in providing his or her expertise toward this effort. PMID:24249805
A 64ch readout module for PPD/MPPC/SiPM using EASIROC ASIC

NASA Astrophysics Data System (ADS)

Nakamura, Isamu; Ishijima, N.; Hanagaki, K.; Yoshimura, K.; Nakai, Y.; Ueno, K.

2015-07-01

A readout module for PPD/MPPC/GAPD/SiPM is developed using EASIROC ASIC. The module can handle 64 PPDs and has on-board bias power supply, ADC for energy measurement, 1 ns TDC on FPGA as well as 64ch Logic output for external trigger. Controls and data transfer are through SiTCP technology implemented in FPGA. The module has NIM format for convenience, but can be operated without crate with 5 V AC/DC converter. Basic performance of production module was tested and the results are presented in the poster.
Wound infections with multi-drug resistant bacteria.

PubMed

Pîrvănescu, H; Bălăşoiu, M; Ciurea, M E; Bălăşoiu, A T; Mănescu, R

2014-01-01

Wound infections remain a public health problem, despite the progress made on improving surgical techniques and antibiotic prophylaxis application. Misuse of antibiotics to prevent bacterial infections leads to increased bacterial resistance and their dissemination. The study refers to 470 samples taken from wound infections of which only multi-drug resistant strains were selected for study, using two special culture mediums (Metistaph-2 for methicillin-resistant staphylococci and ESBLs-Agar for extended-spectrum betalactamases secreting bacteria). Sensitivity of these strains was tested using the diffusion method. Of all studied samples, a rate of 27.6 bacterial strains showed multi-drug resistance. Among them stood primarily Staphylococcus aureus; both MRSA strains and ESBL Gram negative bacteria studied showed high resistance to aminoglycosides, quinolones, third generation cephalosporins and low to fourth generation cephalosporins. No vancomycin resitant nor vancomycin-intermediate Staphylococcus aureus strains were isolated. Knowing the antibiotic resistance is very useful in antibiotic "cycling"application, avoiding this way the emergence of increased resistant strains. Celsius.
Lack of Influence of Substrate on Ligand Interaction with the Human Multidrug and Toxin Extruder, MATE1

PubMed Central

Martínez-Guerrero, Lucy J.; Morales, Mark; Ekins, Sean

2016-01-01

Multidrug and toxin extruder (MATE) 1 plays a central role in mediating renal secretion of organic cations, a structurally diverse collection of compounds that includes ∼40% of prescribed drugs. Because inhibition of transport activity of other multidrug transporters, including the organic cation transporter (OCT) 2, is influenced by the structure of the transported substrate, the present study screened over 400 drugs as inhibitors of the MATE1-mediated transport of four structurally distinct organic cation substrates: the commonly used drugs: 1) metformin and 2) cimetidine; and two prototypic cationic substrates, 3) 1-methyl-4-phenylpyridinium (MPP), and 4) the novel fluorescent probe, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]ethanaminium iodide. Transport was measured in Chinese hamster ovary cells that stably expressed the human ortholog of MATE1. Comparison of the resulting inhibition profiles revealed no systematic influence of substrate structure on inhibitory efficacy. Similarly, IC50 values for 26 structurally diverse compounds revealed no significant influence of substrate structure on the kinetic interaction of inhibitor with MATE1. The IC50 data were used to generate three-dimensional quantitative pharmacophores that identified hydrophobic regions, H-bond acceptor sites, and an ionizable (cationic) feature as key determinants for ligand binding to MATE1. In summary, in contrast to the behavior observed with some other multidrug transporters, including OCT2, the results suggest that substrate identity exerts comparatively little influence on ligand interaction with MATE1. PMID:27418674
The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids.

PubMed

Eckford, Paul D W; Sharom, Frances J

2005-07-15

The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a 'flippase' for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD-GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD-GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD-GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the Kd for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD-lactosylceramide drastically reduced the rate of flipping compared with NBD-PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids.
Effect of curcumin on human colon cancer multidrug resistance in vitro and in vivo.

PubMed

Lu, Wei-Dong; Qin, Yong; Yang, Chuang; Li, Lei; Fu, Zhong-Xue

2013-05-01

To determine whether curcumin reverses the multidrug resistance of human colon cancer cells in vitro and in vivo. In a vincristine-resistant cell line of human colon cancer, the cell viability of curcumin-treated cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Rhodamine123 efflux was evaluated to detect P-glycoprotein transporter activity, and expression of the multidrug resistance protein 1 and survivin genes was analyzed by reverse transcription polymerase chain reaction and western blotting. In addition, xenograft mouse tumors were grown and treated with curcumin. The morphology of the xenografts was investigated by hematoxylin-eosin staining. The in vivo expression of the multidrug resistance gene and P-glycoprotein and survivin genes and proteins was observed using reverse transcription-polymerase chain reaction and western blotting, respectively. Curcumin was not obviously toxic to the vincristine-resistant human colon cancer cells at concentrations less than 25 μM, but the growth of cells was significantly inhibited. At concentrations greater than 25 μM, curcumin was toxic in a concentration-dependent manner. The sensitivity of cells to vincristine, cisplatin, fluorouracil, and hydroxycamptothecin was enhanced, intracellular Rhodamine123 accumulation was increased (p<0.05), and the expression of the multidrug resistance gene and P-glycoprotein were significantly suppressed (p<0.05). The combination of curcumin and vincristine significantly inhibited xenograft growth. The expression of the multidrug resistance protein 1 and survivin genes was significantly reduced in xenografts of curcumin-treated mice and mice treated with both curcumin and vincristine relative to control mice. Curcumin has strong reversal effects on the multidrug resistance of human colon carcinoma in vitro and in vivo.
The emergence and outbreak of multidrug-resistant typhoid fever in China.

PubMed

Yan, Meiying; Li, Xinlan; Liao, Qiaohong; Li, Fang; Zhang, Jing; Kan, Biao

2016-06-22

Typhoid fever remains a severe public health problem in developing countries. The emergence of resistant typhoid, particularly multidrug-resistant typhoid infections, highlights the necessity of monitoring the resistance characteristics of this invasive pathogen. In this study, we report a typhoid fever outbreak caused by multidrug-resistant Salmonella enterica serovar Typhi strains with an ACSSxtT pattern. Resistance genes conferring these phenotypes were harbored by a large conjugative plasmid, which increases the threat of Salmonella Typhi and thus requires close surveillance for dissemination of strains containing such genes.
Engineered Cationic Antimicrobial Peptides To Overcome Multidrug Resistance by ESKAPE Pathogens

PubMed Central

Deslouches, Berthony; Steckbeck, Jonathan D.; Craigo, Jodi K.; Doi, Yohei; Burns, Jane L.

2014-01-01

Multidrug resistance constitutes a threat to the medical achievements of the last 50 years. In this study, we demonstrated the abilities of two de novo engineered cationic antibiotic peptides (eCAPs), WLBU2 and WR12, to overcome resistance from 142 clinical isolates representing the most common multidrug-resistant (MDR) pathogens and to display a lower propensity to select for resistant bacteria in vitro compared to that with colistin and LL37. The results warrant an exploration of eCAPs for use in clinical settings. PMID:25421473

A compact 7-cell Si-drift detector module for high-count rate X-ray spectroscopy.

PubMed

Hansen, K; Reckleben, C; Diehl, I; Klär, H

2008-05-01

A new Si-drift detector module for fast X-ray spectroscopy experiments was developed and realized. The Peltier-cooled module comprises a sensor with 7 × 7-mm 2 active area, an integrated circuit for amplification, shaping and detection, storage, and derandomized readout of signal pulses in parallel, and amplifiers for line driving. The compactness and hexagonal shape of the module with a wrench size of 16mm allow very short distances to the specimen and multi-module arrangements. The power dissipation is 186mW. At a shaper peaking time of 190 ns and an integration time of 450 ns an electronic rms noise of ~11 electrons was achieved. When operated at 7 °C, FWHM line widths around 260 and 460 eV (Cu-K α ) were obtained at low rates and at sum-count rates of 1.7 MHz, respectively. The peak shift is below 1% for a broad range of count rates. At 1.7-MHz sum-count rate the throughput loss amounts to 30%.
The structure of Zika virus NS5 reveals a conserved domain conformation

DOE PAGES

Wang, Boxiao; Tan, Xiao -Feng; Thurmond, Stephanie; ...

2017-03-27

The recent outbreak of Zika virus (ZIKV) has imposed a serious threat to public health. Here we report the crystal structure of the ZIKV NS5 protein in complex with S-adenosyl-L-homocysteine, in which the tandem methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains stack into one of the two alternative conformations of flavivirus NS5 proteins. In conclusion, the activity of this NS5 protein is verified through a de novo RdRp assay on a subgenomic ZIKV RNA template. Importantly, our structural analysis leads to the identification of a potential drug-binding site of ZIKV NS5, which might facilitate the development of novel antiviralsmore » for ZIKV.« less
Polymeric micelles for multi-drug delivery in cancer.

PubMed

Cho, Hyunah; Lai, Tsz Chung; Tomoda, Keishiro; Kwon, Glen S

2015-02-01

Drug combinations are common in cancer treatment and are rapidly evolving, moving beyond chemotherapy combinations to combinations of signal transduction inhibitors. For the delivery of drug combinations, i.e., multi-drug delivery, major considerations are synergy, dose regimen (concurrent versus sequential), pharmacokinetics, toxicity, and safety. In this contribution, we review recent research on polymeric micelles for multi-drug delivery in cancer. In concurrent drug delivery, polymeric micelles deliver multi-poorly water-soluble anticancer agents, satisfying strict requirements in solubility, stability, and safety. In sequential drug delivery, polymeric micelles participate in pretreatment strategies that "prime" solid tumors and enhance the penetration of secondarily administered anticancer agent or nanocarrier. The improved delivery of multiple poorly water-soluble anticancer agents by polymeric micelles via concurrent or sequential regimens offers novel and interesting strategies for drug combinations in cancer treatment.
Identification of potential hit compounds for Dengue virus NS2B/NS3 protease inhibitors by combining virtual screening and binding free energy calculations.

PubMed

Wichapong, K; Nueangaudom, A; Pianwanit, S; Sippl, W; Kokpol, S

2013-09-01

Dengue virus (DV) infections are a serious public health problem and there is currently no vaccine or drug treatment. NS2B/NS3 protease, an essential enzyme for viral replication, is one of the promising targets in the search for drugs against DV. In this research work, virtual screening (VS) was carried out on four multi-conformational databases using several criteria. Firstly, molecular dynamics simulations of the NS2B/NS3 protease and four known inhibitors, which reveal an importance of both electrostatic and van der Waals interactions in stabilizing the ligand-enzyme interaction, were used to generate three different pharmacophore models (a structure-based, a static and a dynamic). Subsequently, these three models were employed for pharmacophore search in the VS. Secondly, compounds passing the first criterion were further reduced using the Lipinski's rule of five to keep only compounds with drug-like properties. Thirdly, molecular docking calculations were performed to remove compounds with unsuitable ligand-enzyme interactions. Finally, binding free energy of each compound was calculated. Compounds having better energy than the known inhibitors were selected and thus 20 potential hits were obtained.
The Spectral Signatures Of BH Versus NS Sources

NASA Astrophysics Data System (ADS)

Seifina, E.; Titarchuk, L.

2011-09-01

We present a comparative analysis of spectral properties of Black Hole (BH) and Neutron Star (NS) X-ray binaries during transition events observed with BeppoSAX and RXTE satellites. In particular, we investigated the behavior of Comptonized component of X-ray spectra when object evolves from the low to high spectral states. The basic models to fit X-ray spectra of these objects are upscattering models (so called BMC and COMPTB models) which are the first principal models. These models taking into account both dynamical and thermal Comptonization and allow to study separate contributions of thermal component and Comptonization component (bulk and thermal effect of Comptonization processes). Specifically, we tested quite a few observations of BHs (GRS 1915+105 and SS 433) and NSs (4U 1728-34 and GX 3+1) applying BMC and COMPTB models. In this way it was found a crucial difference in behavior of photon index vs mass accretion rate (mdot) for BHs and NSs. Namely, we revealed the stability of the photon index around typical value of Gamma=2 versus mdot (or electron temperature) during spectral evolution of NS sources. This stability effect was previously suggested for a number of other neutron binaries (see Farinelli and Titarchuk, 2011). This intrinsic property of NS is fundamentally different from that in BH binary sources for which the index demonstrates monotonic growth with mass accretion rate followed by its saturation at high values of mdot. These index-mass accretion rate behavior during X-ray spectral transition events can be considered as signatures, which allow to differ NS from BH.
Immunoliposomal delivery of doxorubicin can overcome multidrug resistance mechanisms in EGFR-overexpressing tumor cells.

PubMed

Mamot, Christoph; Ritschard, Reto; Wicki, Andreas; Küng, Willy; Schuller, Jan; Herrmann, Richard; Rochlitz, Christoph

2012-06-01

Immunoliposomes (ILs) can be constructed to target the epidermal growth factor receptor (EGFR) to provide efficient intracellular drug delivery in tumor cells. We hypothesized that this approach might be able to overcome drug resistance mechanisms, which remain an important obstacle to better outcomes in cancer therapy. ILs were evaluated in vitro and in vivo against EGFR-overexpressing pairs of human cancer cells (HT-29 and MDA-MB-231) that either lack or feature the multidrug resistance (mdr) phenotype. In multidrug-resistant cell lines, ILs loaded with doxorubicin (DOX) produced 19-216-fold greater cytotoxicity than free DOX, whereas in nonresistant cells, immunoliposomal cytotoxicity of DOX was comparable with that of the free drug. In intracellular distribution studies, free DOX was efficiently pumped out of the multidrug-resistant tumor cells, whereas immunoliposomal DOX leads to 3.5-8 times higher accumulation of DOX in the cytoplasm and 3.5-4.9 times in the nuclei compared with the free drug. Finally, in vivo studies in the MDA-MB-231 Vb100 xenograft model confirmed the ability of anti-EGFR ILs-DOX to efficiently target multidrug-resistant cells and showed impressive antitumor effects, clearly superior to all other treatments. In conclusion, ILs provide efficient and targeted drug delivery to EGFR-overexpressing tumor cells and are capable of completely reversing the multidrug-resistant phenotype of human cancer cells.
Imidazoles and benzimidazoles as tubulin-modulators for anti-cancer therapy.

PubMed

Torres, Fernando C; García-Rubiño, M Eugenia; Lozano-López, César; Kawano, Daniel F; Eifler-Lima, Vera L; von Poser, Gilsane L; Campos, Joaquín M

2015-01-01

Imidazoles and benzimidazoles are privileged heterocyclic bioactive compounds used with success in the clinical practice of innumerous diseases. Although there are many advancements in cancer therapy, microtubules remain as one of the few macromolecular targets validated for planning active anti-cancer compounds, and the design of drugs that modulate microtubule dynamics in unknown sites of tubulin is one of the goals of the medicinal chemistry. The discussion of the role of new and commercially available imidazole and benzimidazole derivatives as tubulin modulators is scattered throughout scientific literature, and indicates that these compounds have a tubulin modulation mechanism different from that of tubulin modulators clinically available, such as paclitaxel, docetaxel, vincristine and vinblastine. In fact, recent literature indicates that these derivatives inhibit microtubule formation binding to the colchicine site, present good pharmacokinetic properties and are capable of overcoming multidrug resistance in many cell lines. The understanding of the mechanisms involved in the imidazoles/benzimidazoles modulation of microtubule dynamics is very important to develop new strategies to overcome the resistance to anti-cancer drugs and to discover new biomarkers and targets for cancer chemotherapy.
Isolation and Structure Elucidation, Molecular Docking Studies of Screlotiumol from Soil Borne Fungi Screlotium rolfsii and their Reversal of Multidrug Resistance in Mouse Lymphoma Cells.

PubMed

Ahmad, Bashir; Rizwan, Muhammad; Rauf, Abdur; Raza, Muslim; Azam, Sadiq; Bashir, Shumaila; Molnar, Joseph; Csonka, Akos; Szabo, Diana

2016-01-01

A new compound namely (13-(3,3-dihydroxypropyl)-1,6-dihydroxy-3,4-dihydro-1H-isochromen-8(5H)-one (1) was isolated from an ethyl acetate extract of the borne fungi Screlotium rolfsii. Its chemical structure was elucidated by spectroscopic analysis. Screlotiumol 1 were evaluated for their effects on the reversion of multidrug resistant (MDR) mediated by P-glycoprotein (P-gp) of the soil borne fungi. The multidrug resistant P-glycoprotein is a target for chemotherapeutic drugs in cancer cells. In the present study rhodamine-123 exclusion screening test on human mdr1 gene transfected mouse gene transfected L5178 and L5178Y mouse T-cell lymphoma which showed excellent MDR reversing effect in a dose dependent manner against mouse T-lymphoma cell line. Moreover, molecular docking studies of compound-1 also showed better results as compared with the standard. Therefore the preliminary results obtained from this study suggest that screlotiumol 1 could be used as a potential agent for the treatment of cancer.
Utility of dengue NS1 antigen rapid diagnostic test for use in difficult to reach areas and its comparison with dengue NS1 ELISA and qRT-PCR.

PubMed

Shukla, Mohan K; Singh, Neeru; Sharma, Ravendra K; Barde, Pradip V

2017-07-01

The objective of this study was to demonstrate the utility of dengue virus (DENV) non structural protein 1 (NS1) based rapid diagnostic test (RDT) for use in tribal and difficult to reach areas for early dengue (DEN) diagnosis in acute phase patients and evaluate its sensitivity and specificity against DENV NS1 enzyme linked immune sorbent assay (ELISA) and real time reverse transcriptase polymerase chain reaction (qRT-PCR). The DENV NS1 RDT was used for preliminary diagnosis during outbreaks in difficult to reach rural and tribal areas. The diagnosis was confirmed by DENV NS1 ELISA in the laboratory. The samples were also tested and serotyped by qRT-PCR. The results were evaluated using statistical tests. The DENV NS1 RDT showed 99.2% sensitivity and 96.0% specificity when analyzed using DENV NS1 ELISA as standard. The specificity and sensitivity of the RDT when compared with qRT-PCR was 93.6% and 91.1%, respectively. The serotype specific evaluation showed more than 90% sensitivity and specificity for DENV-1, 2, and 3. The RDT proved a good diagnostic tool in difficult to reach rural and tribal areas. Further evaluation studies with different commercially available RDTs in different field conditions are essential, that will help clinicians and patients for treatment and programme managers for timely intervention. © 2017 Wiley Periodicals, Inc.
Structure of the transcriptional regulator LmrR and its mechanism of multidrug recognition.

PubMed

Madoori, Pramod Kumar; Agustiandari, Herfita; Driessen, Arnold J M; Thunnissen, Andy-Mark W H

2009-01-21

LmrR is a PadR-related transcriptional repressor that regulates the production of LmrCD, a major multidrug ABC transporter in Lactococcus lactis. Transcriptional regulation is presumed to follow a drug-sensitive induction mechanism involving the direct binding of transporter ligands to LmrR. Here, we present crystal structures of LmrR in an apo state and in two drug-bound states complexed with Hoechst 33342 and daunomycin. LmrR shows a common topology containing a typical beta-winged helix-turn-helix domain with an additional C-terminal helix involved in dimerization. Its dimeric organization is highly unusual with a flat-shaped hydrophobic pore at the dimer centre serving as a multidrug-binding site. The drugs bind in a similar manner with their aromatic rings sandwiched in between the indole groups of two dimer-related tryptophan residues. Multidrug recognition is facilitated by conformational plasticity and the absence of drug-specific hydrogen bonds. Combined analyses using site-directed mutagenesis, fluorescence-based drug binding and protein-DNA gel shift assays reveal an allosteric coupling between the multidrug- and DNA-binding sites of LmrR that most likely has a function in the induction mechanism.
The Intimin-Like Protein FdeC Is Regulated by H-NS and Temperature in Enterohemorrhagic Escherichia coli

PubMed Central

Easton, Donna M.; Allsopp, Luke P.; Phan, Minh-Duy; Moriel, Danilo Gomes; Goh, Guan Kai; Beatson, Scott A.; Mahony, Timothy J.; Cobbold, Rowland N.

2014-01-01

Enterohemorrhagic Escherichia coli (EHEC) is a Shiga-toxigenic pathogen capable of inducing severe forms of enteritis (e.g., hemorrhagic colitis) and extraintestinal sequelae (e.g., hemolytic-uremic syndrome). The molecular basis of colonization of human and animal hosts by EHEC is not yet completely understood, and an improved understanding of EHEC mucosal adherence may lead to the development of interventions that could disrupt host colonization. FdeC, also referred to by its IHE3034 locus tag ECOK1_0290, is an intimin-like protein that was recently shown to contribute to kidney colonization in a mouse urinary tract infection model. The expression of FdeC is tightly regulated in vitro, and FdeC shows promise as a vaccine candidate against extraintestinal E. coli strains. In this study, we characterized the prevalence, regulation, and function of fdeC in EHEC. We showed that the fdeC gene is conserved in both O157 and non-O157 EHEC and encodes a protein that is expressed at the cell surface and promotes biofilm formation under continuous-flow conditions in a recombinant E. coli strain background. We also identified culture conditions under which FdeC is expressed and showed that minor alterations of these conditions, such as changes in temperature, can significantly alter the level of FdeC expression. Additionally, we demonstrated that the transcription of the fdeC gene is repressed by the global regulator H-NS. Taken together, our data suggest a role for FdeC in EHEC when it grows at temperatures above 37°C, a condition relevant to its specialized niche at the rectoanal junctions of cattle. PMID:25239893
The emergence and outbreak of multidrug-resistant typhoid fever in China

PubMed Central

Yan, Meiying; Li, Xinlan; Liao, Qiaohong; Li, Fang; Zhang, Jing; Kan, Biao

2016-01-01

Typhoid fever remains a severe public health problem in developing countries. The emergence of resistant typhoid, particularly multidrug-resistant typhoid infections, highlights the necessity of monitoring the resistance characteristics of this invasive pathogen. In this study, we report a typhoid fever outbreak caused by multidrug-resistant Salmonella enterica serovar Typhi strains with an ACSSxtT pattern. Resistance genes conferring these phenotypes were harbored by a large conjugative plasmid, which increases the threat of Salmonella Typhi and thus requires close surveillance for dissemination of strains containing such genes. PMID:27329848
Engineered cationic antimicrobial peptides to overcome multidrug resistance by ESKAPE pathogens.

PubMed

Deslouches, Berthony; Steckbeck, Jonathan D; Craigo, Jodi K; Doi, Yohei; Burns, Jane L; Montelaro, Ronald C

2015-02-01

Multidrug resistance constitutes a threat to the medical achievements of the last 50 years. In this study, we demonstrated the abilities of two de novo engineered cationic antibiotic peptides (eCAPs), WLBU2 and WR12, to overcome resistance from 142 clinical isolates representing the most common multidrug-resistant (MDR) pathogens and to display a lower propensity to select for resistant bacteria in vitro compared to that with colistin and LL37. The results warrant an exploration of eCAPs for use in clinical settings. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Understanding institutional stakeholders’ perspectives on multidrug-resistant bacterial organism at the end of life: a qualitative study

PubMed Central

Heckel, Maria; Herbst, Franziska A; Adelhardt, Thomas; Tiedtke, Johanna M; Sturm, Alexander; Stiel, Stephanie; Ostgathe, Christoph

2017-01-01

Background Information lacks about institutional stakeholders’ perspectives on management approaches of multidrug-resistant bacterial organism in end-of-life situations. The term “institutional stakeholder” includes persons in leading positions with responsibility in hospitals’ multidrug-resistant bacterial organism management. They have great influence on how strategies on multidrug-resistant bacterial organism management approaches in institutions of the public health system are designed. This study targeted institutional stakeholders’ individual perspectives on multidrug-resistant bacterial organism colonization or infection and isolation measures at the end of life. Methods Between March and December 2014, institutional stakeholders of two study centers, a German palliative care unit and a geriatric ward, were queried in semistructured interviews. Interviews were audiotaped, transcribed verbatim, and analyzed qualitatively with the aid of the software MAXQDA for qualitative data analysis using principles of Grounded Theory. In addition, two external stakeholders were interviewed to enrich data. Results Key issues addressed by institutional stakeholders (N=18) were the relevance of multidrug-resistant bacterial organism in palliative and geriatric care, contradictions between hygiene principles and patients’ and family caregivers’ needs and divergence from standards, frame conditions, and reflections on standardization of multidrug-resistant bacterial organism end-of-life care procedures. Results show that institutional stakeholders face a dilemma between their responsibility in protecting third persons and ensuring patients’ quality of life. Until further empirical evidence establishes a clear multidrug-resistant bacterial organism management approach in end-of-life care, stakeholders suggest a case-based approach. Conclusion The institutional stakeholders’ perspectives and their suggestion of a case-based approach advance the development
Understanding institutional stakeholders' perspectives on multidrug-resistant bacterial organism at the end of life: a qualitative study.

PubMed

Heckel, Maria; Herbst, Franziska A; Adelhardt, Thomas; Tiedtke, Johanna M; Sturm, Alexander; Stiel, Stephanie; Ostgathe, Christoph

2017-01-01

Information lacks about institutional stakeholders' perspectives on management approaches of multidrug-resistant bacterial organism in end-of-life situations. The term "institutional stakeholder" includes persons in leading positions with responsibility in hospitals' multidrug-resistant bacterial organism management. They have great influence on how strategies on multidrug-resistant bacterial organism management approaches in institutions of the public health system are designed. This study targeted institutional stakeholders' individual perspectives on multidrug-resistant bacterial organism colonization or infection and isolation measures at the end of life. Between March and December 2014, institutional stakeholders of two study centers, a German palliative care unit and a geriatric ward, were queried in semistructured interviews. Interviews were audiotaped, transcribed verbatim, and analyzed qualitatively with the aid of the software MAXQDA for qualitative data analysis using principles of Grounded Theory. In addition, two external stakeholders were interviewed to enrich data. Key issues addressed by institutional stakeholders (N=18) were the relevance of multidrug-resistant bacterial organism in palliative and geriatric care, contradictions between hygiene principles and patients' and family caregivers' needs and divergence from standards, frame conditions, and reflections on standardization of multidrug-resistant bacterial organism end-of-life care procedures. Results show that institutional stakeholders face a dilemma between their responsibility in protecting third persons and ensuring patients' quality of life. Until further empirical evidence establishes a clear multidrug-resistant bacterial organism management approach in end-of-life care, stakeholders suggest a case-based approach. The institutional stakeholders' perspectives and their suggestion of a case-based approach advance the development process of a patient-, family-, staff-, and institutional
[Determination of drug resistance mutations of NS3 inhibitors in chronic hepatitis C patients infected with genotype 1].

PubMed

Şanlıdağ, Tamer; Sayan, Murat; Akçalı, Sinem; Kasap, Elmas; Buran, Tahir; Arıkan, Ayşe

2017-04-01

Direct-acting antiviral agents (DAA) such as NS3 protease inhibitors is the first class of drugs used for chronic hepatitis C (CHC) treatment. NS3 inhibitors (PI) with low genetic barrier have been approved to be used in the CHC genotype 1 infections, and in the treatment of compensated liver disease including cirrhosis together with pegile interferon and ribavirin. Consequently, the development of drug resistance during DAA treatment of CHC is a major problem. NS3 resistant variants can be detected before treatment as they can occurnaturally. The aim of this study was to investigate new and old generation NS3 inhibitors resistance mutations before DAA treatment in hepatitis C virus (HCV) that were isolated from CHC. The present study was conducted in 2015 and included 97 naive DAA patients infected with HCV genotype 1, who were diagnosed in Manisa and Kocaeli cities of Turkey. Magnetic particle based HCV RNA extraction and than RNA detection and quantification were performed using commercial real-time PCR assay QIASypmhony + Rotorgene Q/ArtusHCV QS-RGQ and COBAS Ampliprep/COBAS TaqMan HCV Tests. HCV NS3 viral protease genome region was amplified with PCR and mutation analysis was performed by Sanger dideoxy sequencing technique of NS3 protease codons (codon 32-185). HCV NS3 protease inhibitors; asunaprevir, boceprevir, faldaprevir, grazoprevir, pariteprevir, simeprevir and telaprevir were analysed for resistant mutations by Geno2pheno-HCV resistance tool. HCV was genotyped in all patients and 88 patients (n= 88/97, 91%) had genotype 1. Eight (n= 8/97, 8.2%) and 80 (n= 80/97, 82.4%) HCC patients were subgenotyped as 1a and 1b, respectively. Many aminoacid substitutions and resistance mutations were determined in 39/88 (44%) patients in the study group. Q80L, S122C/N, S138W were defined as potential substitutions (6/88 patients; 7%); R109K, R117C, S122G, I132V, I170V, N174S were described as potential resistance (34/88 patients; 39%); V36L, T54S, V55A, Q80H were
Delamanid for multidrug-resistant pulmonary tuberculosis.

PubMed

Gler, Maria Tarcela; Skripconoka, Vija; Sanchez-Garavito, Epifanio; Xiao, Heping; Cabrera-Rivero, Jose L; Vargas-Vasquez, Dante E; Gao, Mengqiu; Awad, Mohamed; Park, Seung-Kyu; Shim, Tae Sun; Suh, Gee Young; Danilovits, Manfred; Ogata, Hideo; Kurve, Anu; Chang, Joon; Suzuki, Katsuhiro; Tupasi, Thelma; Koh, Won-Jung; Seaworth, Barbara; Geiter, Lawrence J; Wells, Charles D

2012-06-07

Delamanid (OPC-67683), a nitro-dihydro-imidazooxazole derivative, is a new antituberculosis medication that inhibits mycolic acid synthesis and has shown potent in vitro and in vivo activity against drug-resistant strains of Mycobacterium tuberculosis. In this randomized, placebo-controlled, multinational clinical trial, we assigned 481 patients (nearly all of whom were negative for the human immunodeficiency virus) with pulmonary multidrug-resistant tuberculosis to receive delamanid, at a dose of 100 mg twice daily (161 patients) or 200 mg twice daily (160 patients), or placebo (160 patients) for 2 months in combination with a background drug regimen developed according to World Health Organization guidelines. Sputum cultures were assessed weekly with the use of both liquid broth and solid medium; sputum-culture conversion was defined as a series of five or more consecutive cultures that were negative for growth of M. tuberculosis. The primary efficacy end point was the proportion of patients with sputum-culture conversion in liquid broth medium at 2 months. Among patients who received a background drug regimen plus 100 mg of delamanid twice daily, 45.4% had sputum-culture conversion in liquid broth at 2 months, as compared with 29.6% of patients who received a background drug regimen plus placebo (P=0.008). Likewise, as compared with the placebo group, the group that received the background drug regimen plus 200 mg of delamanid twice daily had a higher proportion of patients with sputum-culture conversion (41.9%, P=0.04). The findings were similar with assessment of sputum-culture conversion in solid medium. Most adverse events were mild to moderate in severity and were evenly distributed across groups. Although no clinical events due to QT prolongation on electrocardiography were observed, QT prolongation was reported significantly more frequently in the groups that received delamanid. Delamanid was associated with an increase in sputum-culture conversion at 2
Crystal Structure of a Plant Multidrug and Toxic Compound Extrusion Family Protein.

PubMed

Tanaka, Yoshiki; Iwaki, Shigehiro; Tsukazaki, Tomoya

2017-09-05

The multidrug and toxic compound extrusion (MATE) family of proteins consists of transporters responsible for multidrug resistance in prokaryotes. In plants, a number of MATE proteins were identified by recent genomic and functional studies, which imply that the proteins have substrate-specific transport functions instead of multidrug extrusion. The three-dimensional structure of eukaryotic MATE proteins, including those of plants, has not been reported, preventing a better understanding of the molecular mechanism of these proteins. Here, we describe the crystal structure of a MATE protein from the plant Camelina sativa at 2.9 Å resolution. Two sets of six transmembrane α helices, assembled pseudo-symmetrically, possess a negatively charged internal pocket with an outward-facing shape. The crystal structure provides insight into the diversity of plant MATE proteins and their substrate recognition and transport through the membrane. Copyright © 2017 Elsevier Ltd. All rights reserved.
Forthcoming therapeutic perspectives for infections due to multidrug-resistant Gram-positive pathogens.

PubMed

Cornaglia, G; Rossolini, G M

2009-03-01

Multidrug resistance in Gram-positive pathogens emerged as a major therapeutic challenge over two decades ago. The worldwide spread of methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-resistant enterococci and other resistant Gram-positive pathogens had a major impact on antibiotic policies, and prompted the discovery and development of new antibiotics to combat difficult-to-treat infections caused by such pathogens. Several new antibiotics active against multidrug-resistant Gram-positive pathogens have recently been introduced into clinical practice, and the antibiotic pipeline contains additional anti-Gram-positive drugs at an advanced stage of development, including new glycopeptides (dalbavancin, oritavancin, and telavancin), new anti-MRSA beta-lactams (ceftobiprole), and new diaminopyrimidines (iclaprim). This article provides a brief overview of these upcoming agents, partially based on the material presented at the ESCMID Conference entitled 'Fighting infections due to multidrug-resistant Gram-positives' (Venice, Italy, 29-31 May 2008) and on the most recent literature.
Effect of curcumin on human colon cancer multidrug resistance in vitro and in vivo

PubMed Central

Lu, Wei-Dong; Qin, Yong; Yang, Chuang; Li, Lei

2013-01-01

OBJECTIVE: To determine whether curcumin reverses the multidrug resistance of human colon cancer cells in vitro and in vivo. METHODS: In a vincristine-resistant cell line of human colon cancer, the cell viability of curcumin-treated cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Rhodamine123 efflux was evaluated to detect P-glycoprotein transporter activity, and expression of the multidrug resistance protein 1 and survivin genes was analyzed by reverse transcription polymerase chain reaction and western blotting. In addition, xenograft mouse tumors were grown and treated with curcumin. The morphology of the xenografts was investigated by hematoxylin-eosin staining. The in vivo expression of the multidrug resistance gene and P-glycoprotein and survivin genes and proteins was observed using reverse transcription-polymerase chain reaction and western blotting, respectively. RESULTS: Curcumin was not obviously toxic to the vincristine-resistant human colon cancer cells at concentrations less than 25 μM, but the growth of cells was significantly inhibited. At concentrations greater than 25 μM, curcumin was toxic in a concentration-dependent manner. The sensitivity of cells to vincristine, cisplatin, fluorouracil, and hydroxycamptothecin was enhanced, intracellular Rhodamine123 accumulation was increased (p<0.05), and the expression of the multidrug resistance gene and P-glycoprotein were significantly suppressed (p<0.05). The combination of curcumin and vincristine significantly inhibited xenograft growth. The expression of the multidrug resistance protein 1 and survivin genes was significantly reduced in xenografts of curcumin-treated mice and mice treated with both curcumin and vincristine relative to control mice. CONCLUSION: Curcumin has strong reversal effects on the multidrug resistance of human colon carcinoma in vitro and in vivo. PMID:23778405

The trypanocidal benznidazole promotes adaptive response to oxidative injury: Involvement of the nuclear factor-erythroid 2-related factor-2 (Nrf2) and multidrug resistance associated protein 2 (MRP2)

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

Rigalli, Juan Pablo

Oxidative stress is a frequent cause underlying drug-induced hepatotoxicity. Benznidazole (BZL) is the only trypanocidal agent available for treatment of Chagas disease in endemic areas. Its use is associated with side effects, including increases in biomarkers of hepatotoxicity. However, BZL potential to cause oxidative stress has been poorly investigated. Here, we evaluated the effect of a pharmacologically relevant BZL concentration (200 μM) at different time points on redox status and the counteracting mechanisms in the human hepatic cell line HepG2. BZL increased reactive oxygen species (ROS) after 1 and 3 h of exposure, returning to normality at 24 h. Additionally,more » BZL increased glutathione peroxidase activity at 12 h and the oxidized glutathione/total glutathione (GSSG/GSSG + GSH) ratio that reached a peak at 24 h. Thus, an enhanced detoxification of peroxide and GSSG formation could account for ROS normalization. GSSG/GSSG + GSH returned to control values at 48 h. Expression of the multidrug resistance-associated protein 2 (MRP2) and GSSG efflux via MRP2 were induced by BZL at 24 and 48 h, explaining normalization of GSSG/GSSG + GSH. BZL activated the nuclear erythroid 2-related factor 2 (Nrf2), already shown to modulate MRP2 expression in response to oxidative stress. Nrf2 participation was confirmed using Nrf2-knockout mice in which MRP2 mRNA expression was not affected by BZL. In summary, we demonstrated a ROS increase by BZL in HepG2 cells and a glutathione peroxidase- and MRP2 driven counteracting mechanism, being Nrf2 a key modulator of this response. Our results could explain hepatic alterations associated with BZL therapy. - Highlights: • BZL triggers a redox imbalance in the human hepatic cell line HepG2. • Concomitantly BZL triggers compensatory mechanisms to alleviate the redox injury. • Response mechanisms comprise an enhanced glutathione peroxidase and MRP2 activity. • Transcription factor Nrf2 plays a key role orchestrating
BOREAS Level-2 NS001 TMS Imagery: Reflectance and Temperature in BSQ Format

NASA Technical Reports Server (NTRS)

Lobitz, Brad; Spanner, Michael; Hall, Forrest G. (Editor); Newcomer, Jeffrey A. (Editor); Strub, Richard

2000-01-01

For BOREAS, the NS001 TMS images, along with the other remotely sensed data, were collected to provide spatially extensive information over the primary study areas. This information includes detailed land cover and biophysical parameter maps such as fPAR and LAI. Collection of the NS001 images occurred over the study areas during the 1994 field campaigns. The level-2 NS001 data are atmospherically corrected versions of some of the best original NS001 imagery and cover the dates of 19-Apr-1994, 07-Jun-1994, 21-Jul-1994, 08-Aug-1994, and 16-Sep-1994. The data are not geographically/geometrically corrected; however, files of relative X and Y coordinates for each image pixel were derived by using the C130 INS data in an NS001 scan model. The data are provided in binary image format files.
Induction of apoptosis and reversal of permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM by ginsenoside Rh2.

PubMed

Zhang, Hui; Gong, Jian; Zhang, Huilai; Kong, Di

2015-01-01

Multidrug resistance is a phenomenon that cancer cells develop a cross-resistant phenotype against several unrelated drugs, and permeability glycoprotein derived from the overexpression of multidrug resistance gene 1 has been taken as the most significant cause of multidrug resistance. In the present study, ginsenoside Rh2 was used to reverse permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM cell line. Effects of ginsenoside Rh2 on the apoptotic process and caspase-3 activity of MCF-7 and MCF-7/ADM cell lines were determined using flow cytometry and microplate reader. Methyl thiazolyl tetrazolium test was conducted to assess the IC50 values of ginsenoside Rh2 and adriamycin on MCF-7 and MCF-7/ADM cultures; Rhodamin 123 assay was used to assess the retention of permeability glycoprotein after ginsenoside Rh2 treatment; flow cytometry and real time polymerase chain reaction were used to determine the expression levels of permeability glycoprotein and multidrug resistance gene 1 in drug-resistant cells and their parental cells after exposure to ginsenoside Rh2. The results showed that ginsenoside Rh2, except for inducing apoptosis, had the ability to reverse multidrug resistance in MCF-7/ADM cell line without changing the expression levels of permeability glycoprotein and multidrug resistance gene 1. Our findings provided some valuable information for the application of ginsenoside Rh2 in cancer therapy, especially for multidrug resistance reversal in clinic.
Comparative Analysis of Disruption Tolerant Network Routing Simulations in the One and NS-3

DTIC Science & Technology

2017-12-01

real systems with less work compared to ns-2. In order to meet the design goals of ns-3, the entire code structure changed to a modular design . As a...NAVAL POSTGRADUATE SCHOOL MONTEREY, CALIFORNIA THESIS COMPARATIVE ANALYSIS OF DISRUPTION TOLERANT NETWORK ROUTING SIMULATIONS IN THE ONE AND NS-3...Thesis 03-23-2016 to 12-15-2017 4. TITLE AND SUBTITLE COMPARATIVE ANALYSIS OF DISRUPTION TOLERANT NETWORK ROUTING SIMULATIONS IN THE ONE AND NS-3 5
HIST1H1C Regulates Interferon-β and Inhibits Influenza Virus Replication by Interacting with IRF3

PubMed Central

Liu, Xiaokun; Yang, Cha; Hu, Yong; Lei, Erming; Lin, Xian; Zhao, Lianzhong; Zou, Zhong; Zhang, Anding; Zhou, Hongbo; Chen, Huanchun; Qian, Ping; Jin, Meilin

2017-01-01

Influenza virus NS2 is well known for its role in viral ribonucleoprotein nuclear export; however, its function has not been fully understood. A recent study showed that NS2 might interact with HIST1H1C (H1C, H1.2). Histones have been found to affect influenza virus replication, such as the H2A, H2B, H3, and H4, but H1 has not been detected. Here, we found that H1C interacts with NS2 via its C-terminal in the nucleus and that H1C affects influenza virus replication. The H1N1 influenza virus replicates better in H1C knockout A549 cells compared to wild-type A549 cells, primarily because of the regulation of H1C on interferon-β (IFN-β). Further studies showed that the H1C phosphorylation mutant (T146A) decreases IFN-β, while H1C methylation mutants (K34A, K187A) increases IFN-β by releasing the nucleosome and promoting IRF3 binding to the IFN-β promoter. Interestingly, NS2 interacts with H1C, which reduces H1C–IRF3 interaction and results in the inhibition of IFN-β enhanced by H1C. In summary, our study reveals a novel function of H1C to regulate IFN-β and uncovers an underlying mechanism, which suggests H1C plays a role in epigenetic regulation. Moreover, our results suggest a novel mechanism for the influenza virus to antagonize the innate immune response by NS2. PMID:28392790
Purification and crystallization of dengue and West Nile virus NS2B-NS3 complexes.

PubMed

D'Arcy, Allan; Chaillet, Maxime; Schiering, Nikolaus; Villard, Frederic; Lim, Siew Pheng; Lefeuvre, Peggy; Erbel, Paul

2006-02-01

Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B-NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.
Management of multi-drug resistant Helicobacter pylori infection by supplementary, complementary and alternative medicine; a review

PubMed Central

Rezaeimanesh, Nasim; Farzi, Nastaran; Pirmanesh, Samira; Emami, Saeed; Yadegar, Abbas

2017-01-01

Helicobacter pylori is recognized as the most common bacterial pathogens colonizing the gastric epithelium of nearly half of the world’s population. This bacterium is the main etiological cause of gastroduodenal ulcers, and more importantly as the substantial risk factor for development of gastric cancer. The emergence and rapid increase in the prevalence of multi-drug resistant phenotypes have posed major pitfalls in effectiveness of various treatment regimens and eradication strategies against H. pylori infections. Several natural products and supplementary food components have been reported to have established anti-H. pylori activity. Herein, we review the application and efficacy of some specific natural products and foodstuffs such as milk, bee products (honey and propolis), fish oil, vitamins C and E, and also a nickel free-diet used as anti-H. pylori alternative treatment regimens. PMID:29511465
Characterization of Pressure Transients Generated by Nanosecond Electrical Pulse (nsEP) Exposure

PubMed Central

Roth, Caleb C.; Barnes Jr., Ronald A.; Ibey, Bennett L.; Beier, Hope T.; Christopher Mimun, L.; Maswadi, Saher M.; Shadaram, Mehdi; Glickman, Randolph D.

2015-01-01

The mechanism(s) responsible for the breakdown (nanoporation) of cell plasma membranes after nanosecond pulse (nsEP) exposure remains poorly understood. Current theories focus exclusively on the electrical field, citing electrostriction, water dipole alignment and/or electrodeformation as the primary mechanisms for pore formation. However, the delivery of a high-voltage nsEP to cells by tungsten electrodes creates a multitude of biophysical phenomena, including electrohydraulic cavitation, electrochemical interactions, thermoelastic expansion, and others. To date, very limited research has investigated non-electric phenomena occurring during nsEP exposures and their potential effect on cell nanoporation. Of primary interest is the production of acoustic shock waves during nsEP exposure, as it is known that acoustic shock waves can cause membrane poration (sonoporation). Based on these observations, our group characterized the acoustic pressure transients generated by nsEP and determined if such transients played any role in nanoporation. In this paper, we show that nsEP exposures, equivalent to those used in cellular studies, are capable of generating high-frequency (2.5 MHz), high-intensity (>13 kPa) pressure transients. Using confocal microscopy to measure cell uptake of YO-PRO®-1 (indicator of nanoporation of the plasma membrane) and changing the electrode geometry, we determined that acoustic waves alone are not responsible for poration of the membrane. PMID:26450165
Characterization of Pressure Transients Generated by Nanosecond Electrical Pulse (nsEP) Exposure.

PubMed

Roth, Caleb C; Barnes, Ronald A; Ibey, Bennett L; Beier, Hope T; Christopher Mimun, L; Maswadi, Saher M; Shadaram, Mehdi; Glickman, Randolph D

2015-10-09

The mechanism(s) responsible for the breakdown (nanoporation) of cell plasma membranes after nanosecond pulse (nsEP) exposure remains poorly understood. Current theories focus exclusively on the electrical field, citing electrostriction, water dipole alignment and/or electrodeformation as the primary mechanisms for pore formation. However, the delivery of a high-voltage nsEP to cells by tungsten electrodes creates a multitude of biophysical phenomena, including electrohydraulic cavitation, electrochemical interactions, thermoelastic expansion, and others. To date, very limited research has investigated non-electric phenomena occurring during nsEP exposures and their potential effect on cell nanoporation. Of primary interest is the production of acoustic shock waves during nsEP exposure, as it is known that acoustic shock waves can cause membrane poration (sonoporation). Based on these observations, our group characterized the acoustic pressure transients generated by nsEP and determined if such transients played any role in nanoporation. In this paper, we show that nsEP exposures, equivalent to those used in cellular studies, are capable of generating high-frequency (2.5 MHz), high-intensity (>13 kPa) pressure transients. Using confocal microscopy to measure cell uptake of YO-PRO®-1 (indicator of nanoporation of the plasma membrane) and changing the electrode geometry, we determined that acoustic waves alone are not responsible for poration of the membrane.
Multidrug-resistant opportunistic pathogens challenging veterinary infection control.

PubMed

Walther, Birgit; Tedin, Karsten; Lübke-Becker, Antina

2017-02-01

Although the problems associated with healthcare-associated infections (HAI) and the emergence of zoonotic and multidrug-resistant pathogens in companion animal (dogs, cats and horses) medicine have been well-known for decades, current progress with respect to practical implementation of infection control programs in veterinary clinics has been limited. Clinical outbreak events reported for methicillin-resistant Staphylooccus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP), extended spectrum beta-lactamase (ESBL)-producing Escherichia coli and multidrug-resistant (MDR) Salmonella Serovars indicate the necessity of infection control strategies for protecting animal patients at risk as well as veterinary personnel. The close bond between humans and their companion animals provides opportunities for exchange of microorganisms, including MDR pathogens. This particular aspect of the "One Health" idea requires more representative surveillance efforts and infection control strategies with respect to animal-species specific characters. Copyright © 2016 Elsevier B.V. All rights reserved.
Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation.

PubMed

Ni, Duan; Song, Kun; Zhang, Jian; Lu, Shaoyong

2017-10-26

Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras-NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras.
Molecular Dynamics Simulations and Dynamic Network Analysis Reveal the Allosteric Unbinding of Monobody to H-Ras Triggered by R135K Mutation

PubMed Central

Song, Kun; Zhang, Jian; Lu, Shaoyong

2017-01-01

Ras proteins, as small GTPases, mediate cell proliferation, survival and differentiation. Ras mutations have been associated with a broad spectrum of human cancers and thus targeting Ras represents a potential way forward for cancer therapy. A recently reported monobody NS1 allosterically disrupts the Ras-mediated signaling pathway, but its efficacy is reduced by R135K mutation in H-Ras. However, the detailed mechanism is unresolved. Here, using molecular dynamics (MD) simulations and dynamic network analysis, we explored the molecular mechanism for the unbinding of NS1 to H-Ras and shed light on the underlying allosteric network in H-Ras. MD simulations revealed that the overall structures of the two complexes did not change significantly, but the H-Ras–NS1 interface underwent significant conformational alteration in the mutant Binding free energy analysis showed that NS1 binding was unfavored after R135K mutation, which resulted in the unfavorable binding of NS1. Furthermore, the critical residues on H-Ras responsible for the loss of binding of NS1 were identified. Importantly, the allosteric networks for these important residues were revealed, which yielded a novel insight into the allosteric regulatory mechanism of H-Ras. PMID:29072601
Discovery of Dengue Virus NS4B Inhibitors

PubMed Central

Wang, Qing-Yin; Dong, Hongping; Zou, Bin; Karuna, Ratna; Wan, Kah Fei; Zou, Jing; Susila, Agatha; Yip, Andy; Shan, Chao; Yeo, Kim Long; Xu, Haoying; Ding, Mei; Chan, Wai Ling; Gu, Feng; Seah, Peck Gee; Liu, Wei; Lakshminarayana, Suresh B.; Kang, CongBao; Lescar, Julien; Blasco, Francesca; Smith, Paul W.

2015-01-01

ABSTRACT The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50, >20 μM). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients. IMPORTANCE Dengue virus (DENV) threatens up to 2.5 billion people and is now spreading in many regions in the world where it was not previously endemic. While there are several promising vaccine candidates in clinical trials, approved vaccines or antivirals are not yet available. Here we describe the identification and characterization of a spiropyrazolopyridone as a novel inhibitor of DENV by targeting the viral NS4B protein. The compound potently inhibits two of the four serotypes of DENV (DENV-2 and -3) both in vitro and in vivo. Our
Multidrug resistance in pediatric urinary tract infections.

PubMed

Gaspari, Romolo J; Dickson, Eric; Karlowsky, James; Doern, Gary

2006-01-01

Urinary tract infections (UTIs) represent a common infection in the pediatric population. Escherichia coli is the most common uropathogen in children, and antimicrobial resistance in this species complicates the treatment of pediatric UTIs. Despite the impact of resistance on empiric antibiotic choice, there is little data on multidrug resistance in pediatric patients. In this paper, we describe characteristics of multidrug-resistant E. coli in pediatric patients using a large national database of uropathogens antimicrobial sensitivities. Antimicrobial susceptibility patterns to commonly prescribed antibiotics were performed on uropathogens isolated from children presenting to participating hospitals between 1999 and 2001. Data were analyzed separately for four pediatric age groups. Single and multidrug resistance to ampicillin, amoxicillin-clavulanate, cefazolin, ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole (TMP-SMX) were performed on all specimens. There were a total of 11,341 E. coli urine cultures from 343 infants (0-4 weeks), 1,801 toddlers (5 weeks-24 months), 6,742 preteens (2-12 years), and 2,455 teens (13-17 years). E. coli resistance to ampicillin peaked in toddlers (52.8%) but was high in preteens (52.1%), infants (50.4%), and teens (40.6%). Resistance to two or more antibiotics varied across age groups, with toddlers (27%) leading preteens (23.1%), infants (21%), and teens (15.9%). Resistance to three or more antibiotics was low in all age groups (range 3.1-5.2%). The most common co-resistance in all age groups was ampicillin/TMP-SMZ. In conclusion, less than half of all pediatric UTIs are susceptible to all commonly used antibiotics. In some age groups, there is a significant percentage of co-resistance between the two most commonly used antibiotics (ampicillin and TMP-SMZ).
Clonal Multidrug-Resistant Corynebacterium striatum Strains, Italy

PubMed Central

Campanile, Floriana; Carretto, Edoardo; Barbarini, Daniela; Grigis, Annalisa; Falcone, Marco; Goglio, Antonio; Venditti, Mario

2009-01-01

We assessed the clinical relevance and performed molecular characterization of 36 multidrug-resistant strains of Corynebacterium striatum. Pulsed-field gel electrophoresis confirmed a single clone, possessing erm(X), tetA/B, cmxA/B, and aphA1 genes, but few related subclones. This strain is emerging as a pathogen in Italy. PMID:19116057
Multidrug-Resistant Pathogens in Hospitalized Syrian Children.

PubMed

Kassem, Diana Faour; Hoffmann, Yoav; Shahar, Naama; Ocampo, Smadar; Salomon, Liora; Zonis, Zeev; Glikman, Daniel

2017-01-01

Since 2013, wounded and ill children from Syria have received treatment in Israel. Screening cultures indicated that multidrug-resistant (MDR) pathogens colonized 89 (83%) of 107 children. For 58% of MDR infections, the pathogen was similar to that identified during screening. MDR screening of these children is valuable for purposes of isolation and treatment.
[The effect of modulators of SK channels on simple spike firing frequency in the discharge of the cerebellar Purkinje cells in laboratory mice].

PubMed

Egorova, P A; Karelina, T V; Vlasova, O L; Antonov, S M; Besprozvanny, I B

2014-01-01

The effect of CyPPA, a positive modulator of small conductance calcium-activated potassium channels of type 3 and 2 (SK3/SK2), and of NS309, an activator of intermediate and small conductance calcium-activated potassium channels (IK/SK), on the activity of cerebellar Purkinje cells was studied in 2-month-old male mice. The use of 1 mM of CyPPA has led to a decrease of simple spike firing frequency in the discharge of Purkinje cells by 25%, on average, during 1 h after application. At the same time, application of 100 μM of NS309 has promoted a decrease in simple spike firing frequency by 47 %, on average, during 1 h after the beginning of the action. The obtained results confirm the hypothesis that SK channels participate in regulation of simple spike firing frequency in the discharge of Purkinje cells and are responsible for restriction of signal frequency. The effect of NS309 on simple spike firing frequency was more pronounced; therefore, the IK/SK channels may be suggested to play the cardinal role in regulation of spike activity of Purkinje cells. Since increasing simple spike frequency in the discharge of Purkinje cells is observed at many disturbances of motor activity, in particular, at spinocerebellar ataxia, it can be suggested that the studied compounds or substances of similar action are of interest as potential medicinal agents.
Computational screening and molecular dynamics simulation of disease associated nsSNPs in CENP-E.

PubMed

Kumar, Ambuj; Purohit, Rituraj

2012-01-01

Aneuploidy and chromosomal instability (CIN) are hallmarks of most solid tumors. Mutations in centroemere proteins have been observed in promoting aneuploidy and tumorigenesis. Recent studies reported that Centromere-associated protein-E (CENP-E) is involved in inducing cancers. In this study we investigated the pathogenic effect of 132 nsSNPs reported in CENP-E using computational platform. Y63H point mutation found to be associated with cancer using SIFT, Polyphen, PhD-SNP, MutPred, CanPredict and Dr. Cancer tools. Further we investigated the binding affinity of ATP molecule to the CENP-E motor domain. Complementarity scores obtained from docking studies showed significant loss in ATP binding affinity of mutant structure. Molecular dynamics simulation was carried to examine the structural consequences of Y63H mutation. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (R(g)), solvent accessibility surface area (SASA), energy value, hydrogen bond (NH Bond), eigenvector projection, trace of covariance matrix and atom density analysis results showed notable loss in stability for mutant structure. Y63H mutation was also shown to disrupt the native conformation of ATP binding region in CENP-E motor domain. Docking studies for remaining 18 mutations at 63rd residue position as well as other two computationally predicted disease associated mutations S22L and P69S were also carried to investigate their affect on ATP binding affinity of CENP-E motor domain. Our study provided a promising computational methodology to study the tumorigenic consequences of nsSNPs that have not been characterized and clear clue to the wet lab scientist. Copyright © 2012 Elsevier B.V. All rights reserved.
Molecular Mechanism by Which a Potent Hepatitis C Virus NS3-NS4A Protease Inhibitor Overcomes Emergence of Resistance

PubMed Central

O'Meara, Jeff A.; Lemke, Christopher T.; Godbout, Cédrickx; Kukolj, George; Lagacé, Lisette; Moreau, Benoît; Thibeault, Diane; White, Peter W.; Llinàs-Brunet, Montse

2013-01-01

Although optimizing the resistance profile of an inhibitor can be challenging, it is potentially important for improving the long term effectiveness of antiviral therapy. This work describes our rational approach toward the identification of a macrocyclic acylsulfonamide that is a potent inhibitor of the NS3-NS4A proteases of all hepatitis C virus genotypes and of a panel of genotype 1-resistant variants. The enhanced potency of this compound versus variants D168V and R155K facilitated x-ray determination of the inhibitor-variant complexes. In turn, these structural studies revealed a complex molecular basis of resistance and rationalized how such compounds are able to circumvent these mechanisms. PMID:23271737
Antibacterial and antibiotic resistance modifying activity of the extracts from Allanblackia gabonensis, Combretum molle and Gladiolus quartinianus against Gram-negative bacteria including multi-drug resistant phenotypes.

PubMed

Fankam, Aimé G; Kuiate, Jules R; Kuete, Victor

2015-06-30

Bacterial resistance to antibiotics is becoming a serious problem worldwide. The discovery of new and effective antimicrobials and/or resistance modulators is necessary to tackle the spread of resistance or to reverse the multi-drug resistance. We investigated the antibacterial and antibiotic-resistance modifying activities of the methanol extracts from Allanblackia gabonensis, Gladiolus quartinianus and Combretum molle against 29 Gram-negative bacteria including multi-drug resistant (MDR) phenotypes. The broth microdilution method was used to determine the minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of the samples meanwhile the standard phytochemical methods were used for the preliminary phytochemical screening of the plant extracts. Phytochemical analysis showed the presence of alkaloids, flavonoids, phenols and tannins in all studied extracts. Other chemical classes of secondary metabolites were selectively presents. Extracts from A. gabonensis and C. molle displayed a broad spectrum of activity with MICs varying from 16 to 1024 μg/mL against about 72.41% of the tested bacteria. The extract from the fruits of A. gabonensis had the best activity, with MIC values below 100 μg/mL on 37.9% of tested bacteria. Percentages of antibiotic-modulating effects ranging from 67 to 100% were observed against tested MDR bacteria when combining the leaves extract from C. molle (at MIC/2 and MIC/4) with chloramphenicol, kanamycin, streptomycin and tetracycline. The overall results of the present study provide information for the possible use of the studied plant, especially Allanblackia gabonensis and Combretum molle in the control of Gram-negative bacterial infections including MDR species as antibacterials as well as resistance modulators.

Modulational instabilities in acetanilide taking into account both the N H and the C=O vibrational self-trappings

NASA Astrophysics Data System (ADS)

Simo, Elie

2007-02-01

A model of crystalline acetanilide, ACN accounting for the C=O and N-H vibrational self-trappings is presented. We develop a fully discrete version of ACN. We show that ACN can be described by a set of two coupled discrete nonlinear Schrödinger (DNLS) equations. Modulational instabilities (MI) are studied both theoretically and numerically. Dispersion laws for the wavenumbers and frequencies of the linear modulation waves are determined. We also derived the criterion for the existence of MI. Numerical simulations are carried out for a variety of selected wave amplitudes in the unstable zone. It is shown that instabilities grow as the wavenumbers and amplitudes of the modulated waves increase. MI grow faster in the N-H mode than in the C=O mode. Temporal evolution of the density probabilities of the vibrational excitons are obtained by the numerical integration of the coupled DNLS equations governing the ACN molecule. These investigations confirm the generation of localized modes by the phenomenon of MI and the predominance of the N-H vibrational mode in the MI process of the ACN.
Molecular characterization of multidrug-resistant Shigella spp. of food origin.

PubMed

Ahmed, Ashraf M; Shimamoto, Tadashi

2015-02-02

Shigella spp. are the causative agents of food-borne shigellosis, an acute enteric infection. The emergence of multidrug-resistant clinical isolates of Shigella presents an increasing challenge for clinicians in the treatment of shigellosis. Several studies worldwide have characterized the molecular basis of antibiotic resistance in clinical Shigella isolates of human origin, however, to date, no such characterization has been reported for Shigella spp. of food origin. In this study, we characterized the genetic basis of multidrug resistance in Shigella spp. isolated from 1600 food samples (800 meat products and 800 dairy products) collected from different street venders, butchers, retail markets, and slaughterhouses in Egypt. Twenty-four out of 27 Shigella isolates (88.9%) showed multidrug resistance phenotypes to at least three classes of antimicrobials. The multidrug-resistant Shigella spp. were as follows: Shigella flexneri (66.7%), Shigella sonnei (18.5%), and Shigella dysenteriae (3.7%). The highest resistance was to streptomycin (100.0%), then to kanamycin (95.8%), nalidixic acid (95.8%), tetracycline (95.8%), spectinomycin (93.6%), ampicillin (87.5%), and sulfamethoxazole/trimethoprim (87.5%). PCR and DNA sequencing were used to screen and characterize integrons and antibiotic resistance genes. Our results indicated that 11.1% and 74.1% of isolates were positive for class 1 and class 2 integrons, respectively. Beta-lactamase-encoding genes were identified in 77.8% of isolates, and plasmid-mediated quinolone resistance genes were identified in 44.4% of isolates. These data provide useful information to better understand the molecular basis of antimicrobial resistance in Shigella spp. To the best of our knowledge, this is the first report of the molecular characterization of antibiotic resistance in Shigella spp. isolated from food. Copyright © 2014 Elsevier B.V. All rights reserved.
Rough Interface Effects on N-S Proximity-Contact Systems

NASA Astrophysics Data System (ADS)

Nagato, Yasushi; Nagai, Katsuhiko

2003-03-01

We discuss the influence of atomic scale roughness of the interface on the properties of the N-S contact systems. To treat the interface roughness effects we extend our previous quasi-classical theory of the rough surface effect and construct a formal solution for the quasi-classical Green's function. We apply the formulation to N-S systems with two-dimensional anisotropic dx2-y2 superconductor and calculate the self-consistent pair potential and the density of states at the interface.
Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

PubMed Central

Heng, Jie; Zhao, Yan; Liu, Ming; Liu, Yue; Fan, Junping; Wang, Xianping; Zhao, Yongfang; Zhang, Xuejun C

2015-01-01

Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters. PMID:26238402
Induction of apoptosis of liver cancer cells by nanosecond pulsed electric fields (nsPEFs).

PubMed

He, Ling; Xiao, Deyou; Feng, Jianguo; Yao, Chenguo; Tang, Liling

2017-02-01

The application of nanosecond pulsed electric fields (nsPEFs) is a novel method to induce the death of cancer cells. NsPEFs could directly function on the cell membrane and activate the apoptosis pathways, then induce apoptosis in various cell lines. However, the nsPEFs-inducing-apoptosis action sites and the exact pathways are not clear now. In this study, nsPEFs were applied to the human liver cancer cells HepG2 with different parameters. By apoptosis assay, morphological observation, detecting the mitochondrial membrane potential (ΔΨ m ), intracellular calcium ion concentration ([Ca 2+ ]i) and the expressions of key apoptosis factors, we demonstrated that nsPEFs could induce the morphology of cell apoptosis, the change in ΔΨ m , [Ca 2+ ]i and the upregulation of some key apoptosis factors, which revealed the responses of liver cancer cells and indicated that cells may undergo apoptosis through the mitochondria-dependent pathway after nsPEFs were applied.
pH-modulated self-assembly of colloidal nanoparticles in a dual-droplet inkjet printing process.

PubMed

Al-Milaji, Karam Nashwan; Radhakrishnan, Vinod; Kamerkar, Prajakta; Zhao, Hong

2018-06-05

Interfacial self-assembly has been demonstrated as a powerful driving mechanism for creating various nanostructured assemblies. In this work, we employed a dual-droplet printing process and interfacial self-assembly mechanism to produce deposits with controlled assembly structures of colloidal nanoparticles. We hypothesize that pH modulation of the droplet will influence the interfacial self-assembly through the multibody interactions, e.g. particle-particle, particle-interface, and particle-substrate interactions, correspondingly affecting the deposition morphology of the colloidal nanoparticles. During the dual-droplet printing, a wetting droplet, containing colloidal nanoparticles, was jetted over a supporting droplet that contains water only. pH modulation was carried out to the supporting droplet. The self-assembly of two kinds of functionalized polystyrene (PS) nanoparticles (carboxyl-PS and sulfate-PS) was systematically investigated under various pH conditions. Depending on the pH level of the supporting droplet, deposits of carboxyl-PS particles ranging from clear ring-like patterns to nearly uniform monolayer depositions have been obtained. On the other hand, the sulfate-PS particles, even at extreme basic and acidic environments, successfully assemble into nearly monolayer depositions. The multibody interactions are discussed. Such findings can be harnessed in manufacturing high-performance optical and electronic devices. Copyright © 2018 Elsevier Inc. All rights reserved.
Multidrug-Resistant Pathogens in Hospitalized Syrian Children

PubMed Central

Kassem, Diana Faour; Hoffmann, Yoav; Shahar, Naama; Ocampo, Smadar; Salomon, Liora; Zonis, Zeev

2017-01-01

Since 2013, wounded and ill children from Syria have received treatment in Israel. Screening cultures indicated that multidrug-resistant (MDR) pathogens colonized 89 (83%) of 107 children. For 58% of MDR infections, the pathogen was similar to that identified during screening. MDR screening of these children is valuable for purposes of isolation and treatment. PMID:27618479
Multidrug-Resistant Tuberculosis, Somalia, 2010–2011

PubMed Central

Sindani, Ireneaus; Fitzpatrick, Christopher; Falzon, Dennis; Suleiman, Bashir; Arube, Peter; Adam, Ismail; Baghdadi, Samiha; Bassili, Amal

2013-01-01

In a nationwide survey in 2011, multidrug-resistant tuberculosis (MDR TB) was found in 5.2% and 40.8% of patients with new and previously treated TB, respectively. These levels of drug resistance are among the highest ever documented in Africa and the Middle East. This finding presents a serious challenge for TB control in Somalia. PMID:23621911
Structure and sequence based functional annotation of Zika virus NS2b protein: Computational insights.

PubMed

Aguilera-Pesantes, Daniel; Méndez, Miguel A

2017-10-28

While Zika virus (ZIKV) outbreaks are a growing concern for global health, a deep understanding about the virus is lacking. Here we report a contribution to the basic science on the virus- a detailed computational analysis of the non structural protein NS2b. This protein acts as a cofactor for the NS3 protease (NS3Pro) domain that is important on the viral life cycle, and is an interesting target for drug development. We found that ZIKV NS2b cofactor is highly similar to other virus within the Flavivirus genus, especially to West Nile Virus, suggesting that it is completely necessary for the protease complex activity. Furthermore, the ZIKV NS2b has an important role to the function and stability of the ZIKV NS3 protease domain even when presents a low conservation score. In addition, ZIKV NS2b is mostly rigid, which could imply a non dynamic nature in substrate recognition. Finally, by performing a computational alanine scanning mutagenesis, we found that residues Gly 52 and Asp 83 in the NS2b could be important in substrate recognition. Copyright © 2017 Elsevier Inc. All rights reserved.
Reversion of the P-glycoprotein-mediated multidrug resistance of cancer cells by FK-506 derivatives.

PubMed

Jachez, B; Boesch, D; Grassberger, M A; Loor, F

1993-04-01

FK-506 is a resistance-modulating agent (RMA) for tumor cells whose multidrug resistance (MDR) involves a P-glycoprotein (Pgp)-mediated anti-cancer drug efflux. The family of FK-506 relatives and derivatives includes analogs which display a whole range of chemosensitizing strengths, from no detectable RMA activity to a complete reversion of the MDR phenotype. Similarly, FK-506 analogs display a whole range of immunosuppressive activities, including inactive ones. FK-506 was compared for RMA activity with 11 FK-506 analogs which were at least 20-fold less active than FK-506 for the inhibition of the bi-directional mixed lymphocyte reaction displayed the whole range of RMA activity. One such strong RMA derivative of FK-506 (SDZ 280-629) was further shown able to restore completely daunomycin retention by highly resistant MDR P388 tumor cells.
Controlling biofilm formation, prophage excision and cell death by rewiring global regulator H‐NS of Escherichia coli

PubMed Central

Hong, Seok Hoon; Wang, Xiaoxue; Wood, Thomas K.

2010-01-01

Summary The global regulator H‐NS of Escherichia coli controls genes related to stress response, biofilm formation and virulence by recognizing curved DNA and by silencing acquired genes. Here, we rewired H‐NS to control biofilm formation using protein engineering; H‐NS variant K57N was obtained that reduces biofilm formation 10‐fold compared with wild‐type H‐NS (wild‐type H‐NS increases biofilm formation whereas H‐NS K57N reduces it). Whole‐transcriptome analysis revealed that H‐NS K57N represses biofilm formation through its interaction with the nucleoid‐associated proteins Cnu and StpA and in the absence of these proteins, H‐NS K57N was unable to reduce biofilm formation. Significantly, H‐NS K57N enhanced the excision of defective prophage Rac while wild‐type H‐NS represses excision, and H‐NS controlled only Rac excision among the nine resident E. coli K‐12 prophages. Rac prophage excision not only led to the change in biofilm formation but also resulted in cell lysis through the expression of toxin HokD. Hence, the H‐NS regulatory system may be evolved through a single‐amino‐acid change in its N‐terminal oligomerization domain to control biofilm formation, prophage excision and apoptosis. PMID:21255333
Multidrug-resistant organisms in military wounds from Iraq and Afghanistan.

PubMed

Calhoun, Jason H; Murray, Clinton K; Manring, M M

2008-06-01

Mortality from battlefield wounds has historically declined, thanks to better surgical management, faster transport of casualties, and improved antibiotics. Today, one of the major challenges facing U.S. military caregivers is the presence of multidrug-resistant organisms in orthopaedic extremity wounds. The most frequently identified resistant strains of bacteria are Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus-baumannii complex. Overuse of broad-spectrum antibiotics may be an important factor in building resistant strains. Acinetobacter infections appear to hospital-acquired and not from an initial colonization of the injury. More research is required to give military physicians the tools they require to reduce the infection rate and defeat multidrug-resistant organisms.
Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing.

PubMed

Bergfors, Assar; Leenheer, Daniël; Bergqvist, Anders; Ameur, Adam; Lennerstrand, Johan

2016-02-01

Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naïve-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naïve patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual. Copyright © 2015 Elsevier B.V. All rights reserved.
Effects of NS lactobacillus strains on lipid metabolism of rats fed a high-cholesterol diet

PubMed Central

2013-01-01

Background Elevated serum cholesterol level is generally considered to be a risk factor for the development of cardiovascular diseases which seriously threaten human health. The cholesterol-lowering effects of lactic acid bacteria have recently become an area of great interest and controversy for many researchers. In this study, we investigated the effects of two NS lactobacillus strains, Lactobacillus plantarum NS5 and Lactobacillus delbrueckii subsp. bulgaricus NS12, on lipid metabolism of rats fed a high cholesterol diet. Methods Thirty-two SD rats were assigned to four groups and fed either a normal or a high-cholesterol diet. The NS lactobacillus treated groups received the high-cholesterol diet supplemented with Lactobacillus plantarum NS5 or Lactobacillus delbrueckii subsp. bulgaricus NS12 in drinking water. The rats were sacrificed after a 6-week feeding period. Body weights, visceral organ and fat weights, serum and liver cholesterol and lipid levels, intestinal microbiota and liver mRNA expression levels related to cholesterol metabolism were analyzed. Liver lipid deposition and adipocyte size were evaluated histologically. Results Compared with rats fed a high cholesterol diet, serum total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B and free fatty acids levels were decreased and apolipoprotein A-I level was increased in NS5 or NS12 strain treated rats, and with no significant change in high-density lipoprotein cholesterol level. Liver cholesterol and triglyceride levels were also significantly decreased in NS lactobacillus strains treated groups. Meanwhile, the NS lactobacillus strains obviously alleviated hepatic injuries, decreased liver lipid deposition and reduced adipocyte size of high cholesterol diet fed rats. NS lactobacillus strains restored the changes in intestinal microbiota compositions, such as the increase in Bacteroides and the decrease in Clostridium. NS lactobacillus strains also regulated the mRNA expression
The 2NS Translocation from Aegilops ventricosa Confers Resistance to the Triticum Pathotype of Magnaporthe oryzae

PubMed Central

Cruz, C.D.; Peterson, G.L.; Bockus, W.W.; Kankanala, P.; Dubcovsky, J.; Jordan, K.W.; Akhunov, E.; Chumley, F.; Baldelomar, F.D.; Valent, B.

2016-01-01

Wheat blast is a serious disease caused by the fungus Magnaporthe oryzae (Triticum pathotype) (MoT). The objective of this study was to determine the effect of the 2NS translocation from Aegilops ventricosa (Zhuk.) Chennav on wheat head and leaf blast resistance. Disease phenotyping experiments were conducted in growth chamber, greenhouse, and field environments. Among 418 cultivars of wheat (Triticum aestivum L.), those with 2NS had 50.4 to 72.3% less head blast than those without 2NS when inoculated with an older MoT isolate under growth chamber conditions. When inoculated with recently collected isolates, cultivars with 2NS had 64.0 to 80.5% less head blast. Under greenhouse conditions when lines were inoculated with an older MoT isolate, those with 2NS had a significant head blast reduction. With newer isolates, not all lines with 2NS showed a significant reduction in head blast, suggesting that the genetic background and/or environment may influence the expression of any resistance conferred by 2NS. However, when near-isogenic lines (NILs) with and without 2NS were planted in the field, there was strong evidence that 2NS conferred resistance to head blast. Results from foliar inoculations suggest that the resistance to head infection that is imparted by the 2NS translocation does not confer resistance to foliar disease. In conclusion, the 2NS translocation was associated with significant reductions in head blast in both spring and winter wheat. PMID:27814405
Discovery of the Ubiquitous Cation NS+ in Space Confirmed by Laboratory Spectroscopy

NASA Astrophysics Data System (ADS)

Cernicharo, J.; Lefloch, B.; Agúndez, M.; Bailleux, S.; Margulès, L.; Roueff, E.; Bachiller, R.; Marcelino, N.; Tercero, B.; Vastel, C.; Caux, E.

2018-02-01

We report the detection in space of a new molecular species that has been characterized spectroscopically and fully identified from astrophysical data. The observations were carried out with the IRAM 30 m telescope. The molecule is ubiquitous as its J=2\\to 1 transition has been found in cold molecular clouds, prestellar cores, and shocks. However, it is not found in the hot cores of Orion-KL and in the carbon-rich evolved star IRC+10216. Three rotational transitions in perfect harmonic relation J\\prime =2/3/5 have been identified in the prestellar core B1b. The molecule has a 1Σ electronic ground state and its J=2\\to 1 transition presents the hyperfine structure characteristic of a molecule containing a nucleus with spin 1. A careful analysis of possible carriers shows that the best candidate is NS+. The derived rotational constant agrees within 0.3%–0.7% with ab initio calculations. NS+ was also produced in the laboratory to unambiguously validate the astrophysical assignment. The observed rotational frequencies and determined molecular constants confirm the discovery of the nitrogen sulfide cation in space. The chemistry of NS+ and related nitrogen-bearing species has been analyzed by means of a time-dependent gas-phase model. The model reproduces well the observed NS/NS+ abundance ratio, in the range 30–50, and indicates that NS+ is formed by reactions of the neutral atoms N and S with the cations SH+ and NH+, respectively.
Photoexcited quantum dots for killing multidrug-resistant bacteria

NASA Astrophysics Data System (ADS)

Courtney, Colleen M.; Goodman, Samuel M.; McDaniel, Jessica A.; Madinger, Nancy E.; Chatterjee, Anushree; Nagpal, Prashant

2016-05-01

Multidrug-resistant bacterial infections are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics. Metal nanoparticles can induce cell death, yet the toxicity effect is typically nonspecific. Here, we show that photoexcited quantum dots (QDs) can kill a wide range of multidrug-resistant bacterial clinical isolates, including methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Salmonella typhimurium. The killing effect is independent of material and controlled by the redox potentials of the photogenerated charge carriers, which selectively alter the cellular redox state. We also show that the QDs can be tailored to kill 92% of bacterial cells in a monoculture, and in a co-culture of E. coli and HEK 293T cells, while leaving the mammalian cells intact, or to increase bacterial proliferation. Photoexcited QDs could be used in the study of the effect of redox states on living systems, and lead to clinical phototherapy for the treatment of infections.
Prevalence of polymorphisms with significant resistance to NS5A inhibitors in treatment-naive patients with hepatitis C virus genotypes 1a and 3a in Sweden.

PubMed

Lindström, Ida; Kjellin, Midori; Palanisamy, Navaneethan; Bondeson, Kåre; Wesslén, Lars; Lannergard, Anders; Lennerstrand, Johan

2015-08-01

The future treatment of hepatitis C virus (HCV) infection will be combinations of direct-acting antivirals (DAAs) that not only target multiple viral targets, but are also effective against different HCV genotypes. Of the many drug targets in HCV, one promising target is the non-structural 5A protein (NS5A), against which inhibitors, namely daclatasvir, ledipasvir and ombitasvir, have shown potent efficacy. However, since HCV is known to have very high sequence diversity, development of resistance is a problem against but not limited to NS5A inhibitors (i.e. resistance also found against NS3-protease and NS5B non-nucleoside inhibitors), when used in suboptimal combinations. Furthermore, it has been shown that natural resistance against DAAs is present in treatment-naïve patients and such baseline resistance will potentially complicate future treatment strategies. A pan-genotypic population-sequencing method with degenerated primers targeting the NS5A region was developed. We have investigated the prevalence of baseline resistant variants in 127 treatment-naïve patients of HCV genotypes 1a, 1b, 2b and 3a. The method could successfully sequence more than 95% of genotype 1a, 1b and 3a samples. Interpretation of fold resistance data against the NS5A inhibitors was done with the help of earlier published phenotypic data. Baseline resistance variants associated with high resistance (1000-50,000-fold) was found in three patients: Q30H or Y93N in genotype 1a patients and further Y93H in a genotype 3a patient. Using this method, baseline resistance can be examined and the data could have a potential role in selecting the optimal and cost-efficient treatment for the patient.
Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro.

PubMed

Sikora, Bartek; Chen, Yingfeng; Lichti, Cheryl F; Harrison, Melody K; Jennings, Thomas A; Tang, Yong; Tackett, Alan J; Jordan, John B; Sakon, Joshua; Cameron, Craig E; Raney, Kevin D

2008-04-25

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.
Interaction of dengue virus nonstructural protein 5 with Daxx modulates RANTES production

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

Khunchai, Sasiprapa; Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok; Junking, Mutita

Highlights: Black-Right-Pointing-Pointer For the first time how DENV NS5 increases RANTES production. Black-Right-Pointing-Pointer DENV NS5 physically interacts with human Daxx. Black-Right-Pointing-Pointer Nuclear localization of NS5 is required for Daxx interaction and RANTES production. -- Abstract: Dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), caused by dengue virus (DENV) infection, are important public health problems in the tropical and subtropical regions. Abnormal hemostasis and plasma leakage are the main patho-physiological changes in DHF/DSS. A remarkably increased production of cytokines, the so called 'cytokine storm', is observed in the patients with DHF/DSS. A complex interaction between DENV proteinsmore » and the host immune response contributes to cytokine production. However, the molecular mechanism(s) by which DENV nonstructural protein 5 (NS5) mediates these responses has not been fully elucidated. In the present study, yeast two-hybrid assay was performed to identify host proteins interacting with DENV NS5 and a death-domain-associate protein (Daxx) was identified. The in vivo relevance of this interaction was suggested by co-immunoprecipitation and nuclear co-localization of these two proteins in HEK293 cells expressing DENV NS5. HEK293 cells expressing DENV NS5-K/A, which were mutated at the nuclear localization sequences (NLS), were created to assess its functional roles in nuclear translocation, Daxx interaction, and cytokine production. In the absence of NLS, DENV NS5 could neither translocate into the nucleus nor interact with Daxx to increase the DHF-associated cytokine, RANTES (CCL5) production. This work demonstrates the interaction between DENV NS5 and Daxx and the role of the interaction on the modulation of RANTES production.« less

The Enigmatic Alphavirus Non-Structural Protein 3 (nsP3) Revealing Its Secrets at Last

PubMed Central

Götte, Benjamin; Liu, Lifeng

2018-01-01

Alphaviruses encode 4 non-structural proteins (nsPs), most of which have well-understood functions in capping and membrane association (nsP1), polyprotein processing and RNA helicase activity (nsP2) and as RNA-dependent RNA polymerase (nsP4). The function of nsP3 has been more difficult to pin down and it has long been referred to as the more enigmatic of the nsPs. The protein comprises three domains, an N-terminal macro domain, a central zinc-binding domain and a C-terminal hypervariable domain (HVD). In this article, we review old and new literature about the functions of the three domains. Much progress in recent years has contributed to a picture of nsP3, particularly through its HVD as a hub for interactions with host cell molecules, with multiple effects on the biology of the host cell at early points in infection. These and many future discoveries will provide targets for anti-viral therapies as well as strategies for modification of vectors for vaccine and oncolytic interventions. PMID:29495654
Structural basis for the inhibition of bacterial multidrug exporters.

PubMed

Nakashima, Ryosuke; Sakurai, Keisuke; Yamasaki, Seiji; Hayashi, Katsuhiko; Nagata, Chikahiro; Hoshino, Kazuki; Onodera, Yoshikuni; Nishino, Kunihiko; Yamaguchi, Akihito

2013-08-01

The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens. However, despite efforts to develop efflux inhibitors, clinically useful inhibitors are not available at present. Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa. We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics. Drugs were shown to be exported by a functionally rotating mechanism through tandem proximal and distal multisite drug-binding pockets. Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π-π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa.
Protein dynamics observed by tunable mid-IR quantum cascade lasers across the time range from 10ns to 1s.

PubMed

Schultz, Bernd-Joachim; Mohrmann, Hendrik; Lorenz-Fonfria, Victor A; Heberle, Joachim

2018-01-05

We have developed a spectrometer based on tunable quantum cascade lasers (QCLs) for recording time-resolved absorption spectra of proteins in the mid-infrared range. We illustrate its performance by recording time-resolved difference spectra of bacteriorhodopsin in the carboxylic range (1800-1700cm -1 ) and on the CO rebinding reaction of myoglobin (1960-1840cm -1 ), at a spectral resolution of 1cm -1 . The spectrometric setup covers the time range from 4ns to nearly a second with a response time of 10-15ns. Absorption changes as low as 1×10 -4 are detected in single-shot experiments at t>1μs, and of 5×10 -6 in kinetics obtained after averaging 100 shots. While previous time-resolved IR experiments have mostly been conducted on hydrated films of proteins, we demonstrate here that the brilliance of tunable quantum cascade lasers is superior to perform ns time-resolved experiments even in aqueous solution (H 2 O). Copyright © 2017 Elsevier B.V. All rights reserved.
Genome-based insights into the resistome and mobilome of multidrug-resistant Aeromonas sp. ARM81 isolated from wastewater.

PubMed

Adamczuk, Marcin; Dziewit, Lukasz

2017-01-01

The draft genome of multidrug-resistant Aeromonas sp. ARM81 isolated from a wastewater treatment plant in Warsaw (Poland) was obtained. Sequence analysis revealed multiple genes conferring resistance to aminoglycosides, β-lactams or tetracycline. Three different β-lactamase genes were identified, including an extended-spectrum β-lactamase gene bla PER-1 . The antibiotic susceptibility was experimentally tested. Genome sequencing also allowed us to investigate the plasmidome and transposable mobilome of ARM81. Four plasmids, of which two carry phenotypic modules (i.e., genes encoding a zinc transporter ZitB and a putative glucosyltransferase), and 28 putative transposase genes were identified. The mobility of three insertion sequences (isoforms of previously identified elements ISAs12, ISKpn9 and ISAs26) was confirmed using trap plasmids.
Modeling the association of space, time, and host species with variation of the HA, NA, and NS genes of H5N1 highly pathogenic avian influenza viruses isolated from birds in Romania in 2005-2007.

PubMed

Alkhamis, Mohammad; Perez, Andres; Batey, Nicole; Howard, Wendy; Baillie, Greg; Watson, Simon; Franz, Stephanie; Focosi-Snyman, Raffaella; Onita, Iuliana; Cioranu, Raluca; Turcitu, Mihai; Kellam, Paul; Brown, Ian H; Breed, Andrew C

2013-09-01

Molecular characterization studies of a diverse collection of avian influenza viruses (AIVs) have demonstrated that AIVs' greatest genetic variability lies in the HA, NA, and NS genes. The objective here was to quantify the association between geographical locations, periods of time, and host species and pairwise nucleotide variation in the HA, NA, and NS genes of 70 isolates of H5N1 highly pathogenic avian influenza virus (HPAIV) collected from October 2005 to December 2007 from birds in Romania. A mixed-binomial Bayesian regression model was used to quantify the probability of nucleotide variation between isolates and its association with space, time, and host species. As expected for the three target genes, a higher probability of nucleotide differences (odds ratios [ORs] > 1) was found between viruses sampled from places at greater geographical distances from each other, viruses sampled over greater periods of time, and viruses derived from different species. The modeling approach in the present study maybe useful in further understanding the molecular epidemiology of H5N1 HPAI virus in bird populations. The methodology presented here will be useful in predicting the most likely genetic distance for any of the three gene segments of viruses that have not yet been isolated or sequenced based on space, time, and host species during the course of an epidemic.
Detection of Multi-drug Resistant Acinetobacter Lwoffii Isolated from Soil of Mink Farm.

PubMed

Sun, Na; Wen, Yong Jun; Zhang, Shu Qin; Zhu, Hong Wei; Guo, Li; Wang, Feng Xue; Chen, Qiang; Ma, Hong Xia; Cheng, Shi Peng

2016-07-01

There were 4 Acinetobacter lwoffii obtained from soil samples. The antimicrobial susceptibility of the strains to 16 antimicrobial agents was investigated using K-B method. Three isolates showed the multi-drug resistance. The presence of resistance genes and integrons was determined using PCR. The aadA1, aac(3')-IIc, aph(3')-VII, aac(6')-Ib, sul2, cat2, floR, and tet(K) genes were detected, respectively. Three class 1 integrons were obtained. The arr-3-aacA4 and blaPSE-1 gene cassette, which cause resistance to aminoglycoside and beta-lactamase antibiotics. Our results reported the detection of multi-drug resistant and carried resistant genes Acinetobacter lwoffii from soil. The findings suggested that we should pay close attention to the prevalence of multi-drug resistant bacterial species of environment. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.
Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod.

PubMed

Lüders, Ann-Katrin; Saborowski, Reinhard; Bickmeyer, Ulf

2009-09-01

Multidrug/xenobiotic resistance transporters are present in living organisms as a first line defence system against small, potentially harmful molecules from the environment or from internal metabolic reactions. Multidrug resistance associated proteins (MRP) are one type of ATP-Binding-Cassette (ABC) transporters, which also transport dyes such as Fura 2, a calcium chelating fluorescence indicator. The specific MRP inhibitor MK571 was used to investigate the fluorescence intensity of cells in tissues of the brain and the midgut gland of the crustaceans Homarus gammarus (lobster), Crangon crangon (brown shrimp) and Idotea emarginata (isopod) during incubation with Fura 2AM (1 microM). In the presence of the inhibitor MK571 (50 microM), the fluorescence of brain tissue significantly increased in all of the three species. The midgut gland of H. gammarus showed a significant increase of fluorescence, whereas there was no effect in the midgut glands of C. crangon and I. baltica. The half maximal concentration of MK571 was 50 microM as measured in the midgut gland of H. gammarus. In conclusion, MRP transporters are present in the three investigated crustacean nervous systems. Using the midgut glands of the three species, only in H. gammarus MK571 inhibited dye extrusion, indicating species-specific differences of transporter systems, their specificity, or tissue specific expression.
The reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in a membrane environment

PubMed Central

Condic-Jurkic, Karmen; Subramanian, Nandhitha; Mark, Alan E.

2018-01-01

Despite decades of research, the mechanism of action of the ABC multidrug transporter P-glycoprotein (P-gp) remains elusive. Due to experimental limitations, many researchers have turned to molecular dynamics simulation studies in order to investigate different aspects of P-gp function. However, such studies are challenging and caution is required when interpreting the results. P-gp is highly flexible and the time scale on which it can be simulated is limited. There is also uncertainty regarding the accuracy of the various crystal structures available, let alone the structure of the protein in a physiologically relevant environment. In this study, three alternative structural models of mouse P-gp (3G5U, 4KSB, 4M1M), all resolved to 3.8 Å, were used to initiate sets of simulations of P-gp in a membrane environment in order to determine: a) the sensitivity of the results to differences in the starting configuration; and b) the extent to which converged results could be expected on the times scales commonly simulated for this system. The simulations suggest that the arrangement of the nucleotide binding domains (NBDs) observed in the crystal structures is not stable in a membrane environment. In all simulations, the NBDs rapidly associated (within 10 ns) and changes within the transmembrane helices were observed. The secondary structure within the transmembrane domain was best preserved in the 4M1M model under the simulation conditions used. However, the extent to which replicate simulations diverged on a 100 to 200 ns timescale meant that it was not possible to draw definitive conclusions as to which structure overall was most stable, or to obtain converged and reliable results for any of the properties examined. The work brings into question the reliability of conclusions made in regard to the nature of specific interactions inferred from previous simulation studies on this system involving similar sampling times. It also highlights the need to demonstrate the
The reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in a membrane environment.

PubMed

Condic-Jurkic, Karmen; Subramanian, Nandhitha; Mark, Alan E; O'Mara, Megan L

2018-01-01

Despite decades of research, the mechanism of action of the ABC multidrug transporter P-glycoprotein (P-gp) remains elusive. Due to experimental limitations, many researchers have turned to molecular dynamics simulation studies in order to investigate different aspects of P-gp function. However, such studies are challenging and caution is required when interpreting the results. P-gp is highly flexible and the time scale on which it can be simulated is limited. There is also uncertainty regarding the accuracy of the various crystal structures available, let alone the structure of the protein in a physiologically relevant environment. In this study, three alternative structural models of mouse P-gp (3G5U, 4KSB, 4M1M), all resolved to 3.8 Å, were used to initiate sets of simulations of P-gp in a membrane environment in order to determine: a) the sensitivity of the results to differences in the starting configuration; and b) the extent to which converged results could be expected on the times scales commonly simulated for this system. The simulations suggest that the arrangement of the nucleotide binding domains (NBDs) observed in the crystal structures is not stable in a membrane environment. In all simulations, the NBDs rapidly associated (within 10 ns) and changes within the transmembrane helices were observed. The secondary structure within the transmembrane domain was best preserved in the 4M1M model under the simulation conditions used. However, the extent to which replicate simulations diverged on a 100 to 200 ns timescale meant that it was not possible to draw definitive conclusions as to which structure overall was most stable, or to obtain converged and reliable results for any of the properties examined. The work brings into question the reliability of conclusions made in regard to the nature of specific interactions inferred from previous simulation studies on this system involving similar sampling times. It also highlights the need to demonstrate the
pH Modulates the Binding of EGR1 Transcription Factor to DNA

PubMed Central

Mikles, David C.; Bhat, Vikas; Schuchardt, Brett J.; Deegan, Brian J.; Seldeen, Kenneth L.; McDonald, Caleb B.; Farooq, Amjad

2013-01-01

EGR1 transcription factor orchestrates a plethora of signaling cascades involved in cellular homeostasis and its down-regulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with increasing pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as H382 by virtue of the fact that its substitution to non-ionizable residues abolishes pH-dependence of the binding of EGR1 to DNA. Notably, H382 inserts into the major groove of DNA and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, H382 is predominantly conserved across other members of EGR1 family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating protein-DNA interactions central to this family of transcription factors. Collectively, our findings uncover an unexpected but a key step in the molecular recognition of EGR1 family of transcription factors and suggest that they may act as sensors of pH within the intracellular environment. PMID:23718776
Operation of low-noise single-gap RPC modules exposed to ionisation rates up to 1 kHz /cm2

NASA Astrophysics Data System (ADS)

Ćwiok, M.; Dominik, W.; Górski, M.; Królikowski, J.

2004-11-01

Two single gap medium-size RPC modules, made of bakelite plates of very good mechanical quality of the surface and having initial volume resistivity of 1 ×1010 Ω cm, were tested in the Gamma Irradiation Facility at CERN at ionisation rates up to 1 kHz /cm2. The internal surfaces facing the gas volume of one RPC module were cladded with a thin layer of linseed oil varnish for comparison of oiled and non-oiled RPC operation. The results refer to the gas mixture of C2H2F4/isobutane (97:3) with SF6 addition below 1%. The single gap modules exhibited full detection efficiency plateau for the high voltage range of about 1 kV at full intensity of gamma rays. Good timing characteristics allowed to reach 95% efficiency at fully opened irradiation source with time window of 20 ns. The intrinsic noise rate for a non-oiled and an oiled RPC gap was, respectively, below 5 and 1 Hz /cm2 at full efficiency over 1 kV voltage range.
Prevalence and risk factors for carriage of multi-drug resistant Staphylococci in healthy cats and dogs

PubMed Central

Regula, Gertraud; Petrini, Orlando; Zinsstag, Jakob; Schelling, Esther

2013-01-01

We investigated the distribution of commensal staphylococcal species and determined the prevalence of multi-drug resistance in healthy cats and dogs. Risk factors associated with the carriage of multi-drug resistant strains were explored. Isolates from 256 dogs and 277 cats were identified at the species level using matrix-assisted laser desorption ionisation-time of flight mass spectrometry. The diversity of coagulase-negative Staphylococci (CNS) was high, with 22 species in dogs and 24 in cats. Multi-drug resistance was frequent (17%) and not always associated with the presence of the mecA gene. A stay in a veterinary clinic in the last year was associated with an increased risk of colonisation by multi-drug resistant Staphylococci (OR = 2.4, 95% CI: 1.1~5.2, p value LRT = 0.04). When identifying efficient control strategies against antibiotic resistance, the presence of mechanisms other than methicillin resistance and the possible role of CNS in the spread of resistance determinants should be considered. PMID:23820161
Small-angle X-Ray analysis of macromolecular structure: the structure of protein NS2 (NEP) in solution

NASA Astrophysics Data System (ADS)

Shtykova, E. V.; Bogacheva, E. N.; Dadinova, L. A.; Jeffries, C. M.; Fedorova, N. V.; Golovko, A. O.; Baratova, L. A.; Batishchev, O. V.

2017-11-01

A complex structural analysis of nuclear export protein NS2 (NEP) of influenza virus A has been performed using bioinformatics predictive methods and small-angle X-ray scattering data. The behavior of NEP molecules in a solution (their aggregation, oligomerization, and dissociation, depending on the buffer composition) has been investigated. It was shown that stable associates are formed even in a conventional aqueous salt solution at physiological pH value. For the first time we have managed to get NEP dimers in solution, to analyze their structure, and to compare the models obtained using the method of the molecular tectonics with the spatial protein structure predicted by us using the bioinformatics methods. The results of the study provide a new insight into the structural features of nuclear export protein NS2 (NEP) of the influenza virus A, which is very important for viral infection development.
High-bandwidth scanned-wavelength-modulation spectroscopy sensors for temperature and H2O in a rotating detonation engine

NASA Astrophysics Data System (ADS)

Goldenstein, Christopher S.; Almodóvar, Christopher A.; Jeffries, Jay B.; Hanson, Ronald K.; Brophy, Christopher M.

2014-10-01

The design and use of two-color tunable diode laser (TDL) absorption sensors for measurements of temperature and H2O in a rotating detonation engine (RDE) are presented. Both sensors used first-harmonic-normalized scanned-wavelength-modulation spectroscopy with second-harmonic detection (scanned-WMS-2f/1f) to account for non-absorbing transmission losses and emission encountered in the harsh combustion environment. One sensor used two near-infrared (NIR) TDLs near 1391.7 nm and 1469.3 nm that were modulated at 225 kHz and 285 kHz, respectively, and sinusoidally scanned across the peak of their respective H2O absorption transitions to provide a measurement rate of 50 kHz and a detection limit in the RDE of 0.2% H2O by mole. The other sensor used two mid-infrared (MIR) TDLs near 2551 nm and 2482 nm that were modulated at 90 kHz and 112 kHz, respectively, and sinusoidally scanned across the peak of their respective H2O transitions to provide a measurement rate of 10 kHz and a detection limit in the RDE of 0.02% H2O by mole. Four H2O absorption transitions with different lower-state energies were used to assess the homogeneity of temperature in the measurement plane. Experimentally derived spectroscopic parameters that enable temperature and H2O sensing to within 1.5-3.5% of known values are reported. The sensor design enabling the high-bandwidth scanned-WMS-2f/1f measurements is presented. The two sensors were deployed across two orthogonal and coplanar lines-of-sight (LOS) located in the throat of a converging-diverging nozzle at the RDE combustor exit. Measurements in the non-premixed H2-fueled RDE indicate that the temperature and H2O oscillate at the detonation frequency (≈3.25 kHz) and that production of H2O is a weak function of global equivalence ratio.
Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri*

PubMed Central

Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H+ or Na+ electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri. Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4′,6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402
Influence of multidrug resistance and drug transport proteins on chemotherapy drug metabolism.

PubMed

Joyce, Helena; McCann, Andrew; Clynes, Martin; Larkin, Annemarie

2015-05-01

Chemotherapy involving the use of anticancer drugs remains an important strategy in the overall management of patients with metastatic cancer. Acquisition of multidrug resistance remains a major impediment to successful chemotherapy. Drug transporters in cell membranes and intracellular drug metabolizing enzymes contribute to the resistance phenotype and determine the pharmacokinetics of anticancer drugs in the body. ATP-binding cassette (ABC) transporters mediate the transport of endogenous metabolites and xenobiotics including cytotoxic drugs out of cells. Solute carrier (SLC) transporters mediate the influx of cytotoxic drugs into cells. This review focuses on the substrate interaction of these transporters, on their biology and what role they play together with drug metabolizing enzymes in eliminating therapeutic drugs from cells. The majority of anticancer drugs are substrates for the ABC transporter and SLC transporter families. Together, these proteins have the ability to control the influx and the efflux of structurally unrelated chemotherapeutic drugs, thereby modulating the intracellular drug concentration. These interactions have important clinical implications for chemotherapy because ultimately they determine therapeutic efficacy, disease progression/relapse and the success or failure of patient treatment.
Reovirus Nonstructural Protein σNS Acts as an RNA-Stability Factor Promoting Viral Genome Replication.

PubMed

Zamora, Paula F; Hu, Liya; Knowlton, Jonathan J; Lahr, Roni M; Moreno, Rodolfo A; Berman, Andrea J; Prasad, B V Venkataram; Dermody, Terence S

2018-05-16

Viral nonstructural proteins, which are not packaged into virions, are essential for replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. Reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered that σNS increases RNA half-life using in vitro and cell-based RNA degradation experiments. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication. IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the Reoviridae family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different Reoviridae family viruses are diverged in primary sequence, these proteins are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell-culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new
ANTIGENIC MODULATION

PubMed Central

Old, Lloyd J.; Stockert, Elisabeth; Boyse, Edward A.; Kim, Jae Ho

1968-01-01

Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro. An ascites leukemia, phenotype TL.1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon. Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr. The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test. The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C. Thus modulation apparently is an active cellular process. Antigens TL. 1,2, and 3 are all modulated by anti-TL.1,3 serum and by anti-TL.3 serum. This modulation affects all three TL components together, even when antibody to one or two of them is lacking. aAnti-TL.2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies. The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro. Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region. Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions. PMID:5636556
A single-chip 32-channel analog beamformer with 4-ns delay resolution and 768-ns maximum delay range for ultrasound medical imaging with a linear array transducer.

PubMed

Um, Ji-Yong; Kim, Yoon-Jee; Cho, Seong-Eun; Chae, Min-Kyun; Kim, Byungsub; Sim, Jae-Yoon; Park, Hong-June

2015-02-01

A single-chip 32-channel analog beamformer is proposed. It achieves a delay resolution of 4 ns and a maximum delay range of 768 ns. It has a focal-point based architecture, which consists of 7 sub-analog beamformers (sub-ABF). Each sub-ABF performs a RX focusing operation for a single focal point. Seven sub-ABFs perform a time-interleaving operation to achieve the maximum delay range of 768 ns. Phase interpolators are used in sub-ABFs to generate sampling clocks with the delay resolution of 4 ns from a low frequency system clock of 5 MHz. Each sub-ABF samples 32 echo signals at different times into sampling capacitors, which work as analog memory cells. The sampled 32 echo signals of each sub-ABF are originated from one target focal point at one instance. They are summed at one instance in a sub-ABF to perform the RX focusing for the target focal point. The proposed ABF chip has been fabricated in a 0.13- μ m CMOS process with an active area of 16 mm (2). The total power consumption is 287 mW. In measurement, the digital echo signals from a commercial ultrasound medical imaging machine were applied to the fabricated chip through commercial DAC chips. Due to the speed limitation of the DAC chips, the delay resolution was relaxed to 10 ns for the real-time measurement. A linear array transducer with no steering operation is used in this work.
Virulence, Speciation and Antibiotic Susceptibility of Ocular Coagualase Negative Staphylococci (CoNS)

PubMed Central

Priya, Ravindran; Mythili, Arumugam; Singh, Yendremban Randhir Babu; Sreekumar, Haridas; Manikandan, Palanisamy; Panneerselvam, Kanesan

2014-01-01

Background: Coagulase negative Staphylococci (CoNS) are common inhabitants of human skin and mucous membranes. With the emergence of these organisms as prominent pathogens in patients with ocular infections, investigation has intensified in an effort to identify important virulence factors and to inform new approaches to treatment and prevention. Aim: To isolate CoNS from ocular specimens; to study the possible virulence factors; speciation of coagulase negative staphylococci (CoNS) which were isolated from ocular complications; antibiotic susceptibility testing of ocular CoNS. Materials and Methods: The specimens were collected from the target patients who attended the Microbiology Laboratory of a tertiary care eye hospital in Coimbatore, Tamilnadu state, India. The isolates were subjected to tube and slide coagulase tests for the identification of CoNS. All the isolates were subjected to screening for lipase and protease activities. Screening for other virulence factors viz., slime production on Congo red agar medium and haemagglutination assay with use of 96-well microtitre plates. These isolates were identified upto species level by performing biochemical tests such as phosphatase test, arginine test, maltose and trehalose fermentation tests and novobiocin sensitivity test. The isolates were subjected to antibiotic susceptibility studies, based on the revised standards of Clinical and Laboratory Standards Institutes (CLSI). Results: During the one year of study, among the total 260 individuals who were screened, 100 isolates of CoNS were obtained. Lipolytic activity was seen in all the isolates, whereas 38 isolates showed a positive result for protease. A total of 63 isolates showed slime production. Of 100 isolates, 30 isolates were analyzed for haemagglutination, where 4 isolates showed the capacity to agglutinate the erythrocytes. The results of the biochemical analysis revealed that of the 100 isolates of CoNS, 43% were Staphylococcus epidermidis. The other

pH-Dependent doxorubicin release from terpolymer of starch, polymethacrylic acid and polysorbate 80 nanoparticles for overcoming multi-drug resistance in human breast cancer cells.

PubMed

Shalviri, Alireza; Raval, Gaurav; Prasad, Preethy; Chan, Carol; Liu, Qiang; Heerklotz, Heiko; Rauth, Andrew Michael; Wu, Xiao Yu

2012-11-01

This work investigated the capability of a new nanoparticulate system, based on terpolymer of starch, polymethacrylic acid and polysorbate 80, to load and release doxorubicin (Dox) as a function of pH and to evaluate the anticancer activity of Dox-loaded nanoparticles (Dox-NPs) to overcome multidrug resistance (MDR) in human breast cancer cells in vitro. The Dox-NPs were characterized by Fourier transform infrared spectroscopy (FTIR), isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The cellular uptake and cytotoxicity of the Dox-loaded nanoparticles were investigated using fluorescence microscopy, flow cytometry, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The nanoparticles were able to load up to 49.7±0.3% of Dox with a high loading efficiency of 99.9±0.1%, while maintaining good colloidal stability. The nanoparticles released Dox at a higher rate at acidic pH attributable to weaker Dox-polymer molecular interactions evidenced by ITC. The Dox-NPs were taken up by the cancer cells in vitro and significantly enhanced the cytotoxicity of Dox against human MDR1 cells with up to a 20-fold decrease in the IC50 values. The results suggest that the new terpolymeric nanoparticles are a promising vehicle for the controlled delivery of Dox for treatment of drug resistant breast cancer. Copyright © 2012 Elsevier B.V. All rights reserved.
A first-principles based study of ns2 containing ternary iodides and their possibility of scintillation

NASA Astrophysics Data System (ADS)

Kang, Byungkyun; Fang, C. M.; Biswas, Koushik

2016-10-01

A recently investigated scintillator material CsBa2I5 showed promising properties when activated with ns2 ions In+, Tl+ or the lanthanide Eu2+. This sparked our interest in an analogous group of materials, e.g. InBa2I5 or TlBa2I5 where the ns2 ion is part of the crystal framework, replacing the alkali ion. Many of these compounds of the type AB2X5 (X = halogen) have been previously synthesized and have interesting stereochemical activity. Using density functional calculations we have studied the stable monoclinic phase of the aforementioned ns2 containing iodides. One objective is to explore them as scintillators where the ns2 ions, now appearing as part of the crystal, play a central role. Compared to CsBa2I5, their reduced fundamental band gap and possibility of higher light yield may be attributed to an induced degree of covalency in the ns2-I bonds. The valence and conduction band edges have discernible contributions from the ns2 ions’ s and p orbitals which is crucial in carrier localization. The antibonding Ga or In s sates near valence edge may be a favored site for a hole trap, as against a {{V}k} center. Additional differences among the ns2 compounds lead to qualitatively different self-trapped excitons that may fundamentally affect luminescence. The possibility of fast electron capture at the ns2 sites and the prospect of self-activated scintillation via ns2-p → {{V}k} or ns2-p → ns2-s transitions may draw interest in related applications.
A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

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

Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain

2016-09-15

The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc.more » The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.« less
Detection and characterisation of multi-drug resistance protein 1 (MRP-1) in human mitochondria.

PubMed

Roundhill, E A; Burchill, S A

2012-03-13

Overexpression of plasma membrane multi-drug resistance protein 1 (MRP-1) can lead to multidrug resistance. In this study, we describe for the first time the expression of mitochondrial MRP-1 in untreated human normal and cancer cells and tissues. MRP-1 expression and subcellular localisation in normal and cancer cells and tissues was examined by differential centrifugation and western blotting, and immunofluorescence microscopy. Viable mitochondria were isolated and MRP-1 efflux activity measured using the calcein-AM functional assay. MRP-1 expression was increased using retroviral infection and specific overexpression confirmed by RNA array. Cell viability was determined by trypan blue exclusion and annexin V-propidium iodide labelling of cells. MRP-1 was detected in the mitochondria of cancer and normal cells and tissues. The efflux activity of mitochondrial MRP-1 was more efficient (55-64%) than that of plasma membrane MRP-1 (11-22%; P<0.001). Induced MRP-1 expression resulted in a preferential increase in mitochondrial MRP-1, suggesting selective targeting to this organelle. Treatment with a non-lethal concentration of doxorubicin (0.85 nM, 8 h) increased mitochondrial and plasma membrane MRP-1, increasing resistance to MRP-1 substrates. For the first time, we have identified MRP-1 with efflux activity in human mitochondria. Mitochondrial MRP-1 may be an exciting new therapeutic target where historically MRP-1 inhibitor strategies have limited clinical success.
Changes of ns-soot mixing states and shapes in an urban area during CalNex

NASA Astrophysics Data System (ADS)

Adachi, Kouji; Buseck, Peter R.

2013-05-01

Aerosol particles from megacities influence the regional and global climate as well as the health of their occupants. We used transmission electron microscopes (TEMs) to study aerosol particles collected from the Los Angeles area during the 2010 CalNex campaign. We detected major amounts of ns-soot, defined as consisting of carbon nanospheres, sulfate, sea salt, and organic aerosol (OA) and lesser amounts of brochosome particles from leaf hoppers. Ns-soot-particle shapes, mixing states, and abundances varied significantly with sampling times and days. Within plumes having high CO2 concentrations, much ns-soot was compacted and contained a relatively large number of carbon nanospheres. Ns-soot particles from both CalNex samples and Mexico City, the latter collected in 2006, had a wide range of shapes when mixed with other aerosol particles, but neither sets showed spherical ns-soot nor the core-shell configuration that is commonly used in optical calculations. Our TEM observations and light-absorption calculations of modeled particles indicate that, in contrast to ns-soot particles that are embedded within other materials or have the hypothesized core-shell configurations, those attached to other aerosol particles hardly enhance their light absorption. We conclude that the ways in which ns-soot mixes with other particles explain the observations of smaller light amplification by ns-soot coatings than model calculations during the CalNex campaign and presumably in other areas.
Broadband Fan Noise Prediction System for Turbofan Engines. Volume 2; BFaNS User's Manual and Developer's Guide

NASA Technical Reports Server (NTRS)

Morin, Bruce L.

2010-01-01

Pratt & Whitney has developed a Broadband Fan Noise Prediction System (BFaNS) for turbofan engines. This system computes the noise generated by turbulence impinging on the leading edges of the fan and fan exit guide vane, and noise generated by boundary-layer turbulence passing over the fan trailing edge. BFaNS has been validated on three fan rigs that were tested during the NASA Advanced Subsonic Technology Program (AST). The predicted noise spectra agreed well with measured data. The predicted effects of fan speed, vane count, and vane sweep also agreed well with measurements. The noise prediction system consists of two computer programs: Setup_BFaNS and BFaNS. Setup_BFaNS converts user-specified geometry and flow-field information into a BFaNS input file. From this input file, BFaNS computes the inlet and aft broadband sound power spectra generated by the fan and FEGV. The output file from BFaNS contains the inlet, aft and total sound power spectra from each noise source. This report is the second volume of a three-volume set documenting the Broadband Fan Noise Prediction System: Volume 1: Setup_BFaNS User s Manual and Developer s Guide; Volume 2: BFaNS User s Manual and Developer s Guide; and Volume 3: Validation and Test Cases. The present volume begins with an overview of the Broadband Fan Noise Prediction System, followed by step-by-step instructions for installing and running BFaNS. It concludes with technical documentation of the BFaNS computer program.
The crystal structure of Zika virus NS5 reveals conserved drug targets.

PubMed

Duan, Wenqian; Song, Hao; Wang, Haiyuan; Chai, Yan; Su, Chao; Qi, Jianxun; Shi, Yi; Gao, George F

2017-04-03

Zika virus (ZIKV) has emerged as major health concern, as ZIKV infection has been shown to be associated with microcephaly, severe neurological disease and possibly male sterility. As the largest protein component within the ZIKV replication complex, NS5 plays key roles in the life cycle and survival of the virus through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent RNA polymerase (RdRp) domains. Here, we present the crystal structures of ZIKV NS5 MTase in complex with an RNA cap analogue ( m7 GpppA) and the free NS5 RdRp. We have identified the conserved features of ZIKV NS5 MTase and RdRp structures that could lead to development of current antiviral inhibitors being used against flaviviruses, including dengue virus and West Nile virus, to treat ZIKV infection. These results should inform and accelerate the structure-based design of antiviral compounds against ZIKV. © 2017 The Authors.
Structure and function of the Zika virus full-length NS5 protein

DOE PAGES

Zhao, Baoyu; Yi, Guanghui; Du, Fenglei; ...

2017-03-27

The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions tomore » those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Altogether, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.« less
The CENNS-10 liquid argon detector to measure CEvNS at the Spallation Neutron Source

NASA Astrophysics Data System (ADS)

Tayloe, R.

2018-04-01

The COHERENT collaboration is deploying a suite of low-energy detectors in a low-background corridor of the ORNL Spallation Neutron Source (SNS) to measure coherent elastic neutrino-nucleus scattering (CEvNS) on an array of nuclear targets employing different detector technologies. A measurement of CEvNS on different nuclei will test the N2-dependence of the CEvNS cross section and further the physics reach of the COHERENT effort. The first step of this program has been realized recently with the observation of CEvNS in a 14.6 kg CsI detector. Operation and deployment of Ge and NaI detectors are also underway. A 22 kg, single-phase, liquid argon detector (CENNS-10) started data-taking in Dec. 2016 and will provide results on CEvNS from a lighter nucleus. Initial results indicate that light output, pulse-shape discrimination, and background suppression are sufficient for a measurement of CEvNS on argon.
R-Flurbiprofen Traps Prostaglandins within Cells by Inhibition of Multidrug Resistance-Associated Protein-4.

PubMed

Wobst, Ivonne; Ebert, Lisa; Birod, Kerstin; Wegner, Marthe-Susanna; Hoffmann, Marika; Thomas, Dominique; Angioni, Carlo; Parnham, Michael J; Steinhilber, Dieter; Tegeder, Irmgard; Geisslinger, Gerd; Grösch, Sabine

2016-12-30

R -flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S -flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E₂ (PGE₂) in cancer or immune cell cultures and human extracellular fluid. Here, we show that R -flurbiprofen acts through a dual mechanism: (i) it inhibits the translocation of cPLA 2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii) R -flurbiprofen traps PGE₂ inside of the cells by inhibiting multidrug resistance-associated protein 4 (MRP4, ABCC4), which acts as an outward transporter for prostaglandins. Consequently, the effects of R -flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R -flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.
Diverse and abundant multi-drug resistant E. coli in Matang mangrove estuaries, Malaysia

PubMed Central

Ghaderpour, Aziz; Ho, Wing Sze; Chew, Li-Lee; Bong, Chui Wei; Chong, Ving Ching; Thong, Kwai-Lin; Chai, Lay Ching

2015-01-01

E.coli, an important vector distributing antimicrobial resistance in the environment, was found to be multi-drug resistant, abundant, and genetically diverse in the Matang mangrove estuaries, Malaysia. One-third (34%) of the estuarine E. coli was multi-drug resistant. The highest antibiotic resistance prevalence was observed for aminoglycosides (83%) and beta-lactams (37%). Phylogenetic groups A and B1, being the most predominant E. coli, demonstrated the highest antibiotic resistant level and prevalence of integrons (integron I, 21%; integron II, 3%). Detection of phylogenetic group B23 downstream of fishing villages indicates human fecal contamination as a source of E. coli pollution. Enteroaggregative E. coli (1%) were also detected immediately downstream of the fishing village. The results indicated multi-drug resistance among E. coli circulating in Matang estuaries, which could be reflective of anthropogenic activities and aggravated by bacterial and antibiotic discharges from village lack of a sewerage system, aquaculture farms and upstream animal husbandry. PMID:26483759
In vitro antibacterial activity of rifampicin in combination with imipenem, meropenem and doripenem against multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

PubMed

Hu, Yi-Fan; Liu, Chang-Pan; Wang, Nai-Yu; Shih, Shou-Chuan

2016-08-24

Multidrug-resistant Pseudomonas aeruginosa has emerged as one of the most important healthcare-associated pathogens. Colistin is regarded as the last-resort antibiotic for multidrug-resistant Gram-negative bacteria, but is associated with high rates of acute kidney injury. The aim of this in vitro study is to search for an alternative treatment to colistin for multidrug-resistant P. aeruginosa infections. Multidrug and carbapenem-resistant P. aeruginosa isolates were collected between January 2009 and December 2012 at MacKay Memorial Hospital. Minimal inhibitory concentrations (MICs) were determined for various antibiotic combinations. Carbapenemase-producing genes including bla VIM, other β-lactamase genes and porin mutations were screened by PCR and sequencing. The efficacy of carbapenems (imipenem, meropenem, doripenem) with or without rifampicin was correlated with the type of porin mutation (frameshift mutation, premature stop codon mutation) in multidrug-resistant P. aeruginosa isolates without carbapenemase-producing genes. Of the 71 multidrug-resistant clinical P. aeruginosa isolates, only six harboured the bla VIM gene. Imipenem, meropenem and doripenem were significantly more effective (reduced fold-change of MICs) when combined with rifampicin in bla VIM-negative isolates, especially in isolates with porin frameshift mutation. Imipenem + rifampicin combination has a low MIC against multidrug-resistant P. aeruginosa, especially in isolates with porin frameshift mutation. The imipenem + rifampicin combination may provide an alternative treatment to colistin for multidrug -resistant P. aeruginosa infections, especially for patients with renal insufficiency.
Pomegranate ( Punica granatum L.) expresses several nsLTP isoforms characterized by different immunoglobulin E-binding properties.

PubMed

Bolla, Michela; Zenoni, Sara; Scheurer, Stephan; Vieths, Stefan; San Miguel Moncin, Maria Del Mar; Olivieri, Mario; Antico, Andrea; Ferrer, Marta; Berroa, Felicia; Enrique, Ernesto; Avesani, Linda; Marsano, Francesco; Zoccatelli, Gianni

2014-01-01

Pomegranate allergy is associated with sensitization to non-specific lipid transfer proteins (nsLTPs). Our aim was to identify and characterize the non-specific nsLTPs expressed in pomegranate at the molecular level and to study their allergenic properties in terms of immunoglobulin E (IgE)-binding and cross-reactivity with peach nsLTP (Pru p 3). A non-equilibrium two-dimensional (2-D) electrophoretic approach based on acid-urea PAGE and sodium dodecyl sulfate PAGE was set up to separate pomegranate nsLTPs. Their immunoreactivity was tested by immunoblotting carried out with anti-Pru p 3 polyclonal antibodies and sera from pomegranate-allergic patients. For final identification, pomegranate nsLTPs were purified by chromatography and subjected to trypsin digestion and mass spectrometry (MS) analysis. For this purpose, the sequences obtained by cDNA cloning of three pomegranate nsLTPs were integrated in the database that was subsequently searched for MS data interpretation. Four nsLTPs were identified by 2-D immunoblotting. The detected proteins showed different IgE-binding capacity and partial cross-reactivity with Pru p 3. cDNA cloning and MS analyses led to the identification of three nsLTP isoforms with 66-68% amino acid sequence identity named Pun g 1.0101, Pun g 1.0201 and Pun g 1.0301. By 2-D electrophoresis, we could separate different nsLTP isoforms possessing different IgE-binding properties, which might reflect peculiar allergenic potencies. The contribution of Pru p 3 to prime sensitization is not central as in other plant nsLTPs. © 2014 S. Karger AG, Basel.
Reversal of multidrug resistance by surfactants.

PubMed Central

Woodcock, D. M.; Linsenmeyer, M. E.; Chojnowski, G.; Kriegler, A. B.; Nink, V.; Webster, L. K.; Sawyer, W. H.

1992-01-01

Cremophor EL, a pharmacologically inactive solubilising agent, has been shown to reverse multidrug resistance (MDR). Using flow cytometric evaluation of equilibrium intracellular levels of daunorubicin (DNR), we found that eight other surface active agents will also reverse MDR. All the active detergents contain polyethoxylated moieties but have no similarities in their hydrophobic components. The properties of three polyethoxylated surfactants that showed the lowest toxicities, Cremophor, Tween 80 and Solutol HS15, were examined in more detail. The concentrations of Tween 80 and Solutol required to reverse DNR exclusion were 10-fold lower than for Cremophor. However while concentrations greater than or equal to 1:10(2) of the former two surfactants resulted in breakdown of cells, even 1:10 of Cremophor did not lyse cells. Studies of the effects of Cremophor on the uptake and efflux of DNR in normal and MDR cell types showed that Cremophor increases intracellular DNR primarily by locking the rapid efflux from the cells. This blockage of drug efflux may be mediated by a substantial alteration in the fluidity of cell membranes induced by Cremophor, as shown by decreased fluorescence anisotropy of a membrane probe. Consistent with these data, coinjection of adriamycin plus Cremophor into mice carrying a multidrug resistant P388 transplantable tumour significantly increased the survival time of the mice compared with adriamycin treatment alone. PMID:1637678
Lethal neonatal meningoencephalitis caused by multi-drug resistant, highly virulent Escherichia coli.

PubMed

Iqbal, Junaid; Dufendach, Kevin R; Wellons, John C; Kuba, Maria G; Nickols, Hilary H; Gómez-Duarte, Oscar G; Wynn, James L

2016-01-01

Neonatal meningitis is a rare but devastating condition. Multi-drug resistant (MDR) bacteria represent a substantial global health risk. This study reports on an aggressive case of lethal neonatal meningitis due to a MDR Escherichia coli (serotype O75:H5:K1). Serotyping, MDR pattern and phylogenetic typing revealed that this strain is an emergent and highly virulent neonatal meningitis E. coli isolate. The isolate was resistant to both ampicillin and gentamicin; antibiotics currently used for empiric neonatal sepsis treatment. The strain was also positive for multiple virulence genes including K1 capsule, fimbrial adhesion fimH, siderophore receptors iroN, fyuA and iutA, secreted autotransporter toxin sat, membrane associated proteases ompA and ompT, type II polysaccharide synthesis genes (kpsMTII) and pathogenicity-associated island (PAI)-associated malX gene. The presence of highly-virulent MDR organisms isolated in neonates underscores the need to implement rapid drug resistance diagnostic methods and should prompt consideration of alternate empiric therapy in neonates with Gram negative meningitis.
A basic cluster in the N terminus of yellow fever virus NS2A contributes to infectious particle production.

PubMed

Voßmann, Stephanie; Wieseler, Janett; Kerber, Romy; Kümmerer, Beate Mareike

2015-05-01

The flavivirus NS2A protein is involved in the assembly of infectious particles. To further understand its role in this process, a charged-to-alanine scanning analysis was performed on NS2A encoded by an infectious cDNA clone of yellow fever virus (YFV). Fifteen mutants containing single, double, or triple charged-to-alanine changes were tested. Five of them did not produce infectious particles, whereas efficient RNA replication was detectable for two of the five NS2A mutants (R22A-K23A-R24A and R99A-E100A-R101A mutants). Prolonged cultivation of transfected cells resulted in the recovery of pseudorevertants. Besides suppressor mutants in NS2A, a compensating second-site mutation in NS3 (D343G) arose for the NS2A R22A-K23A-R24A mutant. We found this NS3 mutation previously to be suppressive for the NS2Aα cleavage site Q189S mutant, also deficient in virion assembly. In this study, the subsequently suggested interaction between NS2A and NS3 was proven by coimmunoprecipitation analyses. Using selectively permeabilized cells, we could demonstrate that the regions encompassing R22A-K23A-R24A and Q189S in NS2A are localized to the cytoplasm, where NS3 is also known to reside. However, the defect in particle production observed for the NS2A R22A-K23A-R24A and Q189S mutants was not due to a defect in physical interaction between NS2A and NS3, as the NS2A mutations did not interrupt NS3 interaction. In fact, a region just upstream of R22-K23-R24 was mapped to be critical for NS2A-NS3 interaction. Taken together, these data support a complex interplay between YFV NS2A and NS3 in virion assembly and identify a basic cluster in the NS2A N terminus to be critical in this process. Despite an available vaccine, yellow fever remains endemic in tropical areas of South America and Africa. To control the disease, antiviral drugs are required, and an understanding of the determinants of virion assembly is central to their development. In this study, we identified a basic cluster of
A Basic Cluster in the N Terminus of Yellow Fever Virus NS2A Contributes to Infectious Particle Production

PubMed Central

Voßmann, Stephanie; Wieseler, Janett; Kerber, Romy

2015-01-01

ABSTRACT The flavivirus NS2A protein is involved in the assembly of infectious particles. To further understand its role in this process, a charged-to-alanine scanning analysis was performed on NS2A encoded by an infectious cDNA clone of yellow fever virus (YFV). Fifteen mutants containing single, double, or triple charged-to-alanine changes were tested. Five of them did not produce infectious particles, whereas efficient RNA replication was detectable for two of the five NS2A mutants (R22A-K23A-R24A and R99A-E100A-R101A mutants). Prolonged cultivation of transfected cells resulted in the recovery of pseudorevertants. Besides suppressor mutants in NS2A, a compensating second-site mutation in NS3 (D343G) arose for the NS2A R22A-K23A-R24A mutant. We found this NS3 mutation previously to be suppressive for the NS2Aα cleavage site Q189S mutant, also deficient in virion assembly. In this study, the subsequently suggested interaction between NS2A and NS3 was proven by coimmunoprecipitation analyses. Using selectively permeabilized cells, we could demonstrate that the regions encompassing R22A-K23A-R24A and Q189S in NS2A are localized to the cytoplasm, where NS3 is also known to reside. However, the defect in particle production observed for the NS2A R22A-K23A-R24A and Q189S mutants was not due to a defect in physical interaction between NS2A and NS3, as the NS2A mutations did not interrupt NS3 interaction. In fact, a region just upstream of R22-K23-R24 was mapped to be critical for NS2A-NS3 interaction. Taken together, these data support a complex interplay between YFV NS2A and NS3 in virion assembly and identify a basic cluster in the NS2A N terminus to be critical in this process. IMPORTANCE Despite an available vaccine, yellow fever remains endemic in tropical areas of South America and Africa. To control the disease, antiviral drugs are required, and an understanding of the determinants of virion assembly is central to their development. In this study, we identified
Detergent-resistant membrane association of NS2 and E2 during hepatitis C virus replication.

PubMed

Shanmugam, Saravanabalaji; Saravanabalaji, Dhanaranjani; Yi, MinKyung

2015-04-01

Previously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-β-cyclodextrin (MβCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MβCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production. The biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these
Extra-pulmonary primary multidrug-resistant tubercular lymphadenitis in an HIV negative patient

PubMed Central

Kant, Surya; Saheer, S; Hassan, Ghulam; Parengal, Jabeed

2012-01-01

A 28-year-old woman without any history of prior antituberculosis treatment presented with cervical lymphadenopathy and a cold abscess near medial end of clavicle of 5 months duration. Pus culture and sensitivity revealed Mycobacterium tuberculosis resistant to rifampicin and isoniazid. Thus she was diagnosed as a case of primary multidrug-resistant tuberculosis and treated with second line drugs according to culture susceptibility pattern. On completion of therapy, patent showed good clinical response. This case highlights the observation that even extra-pulmonary primary multidrug-resistant tuberculosis can be successfully treated with currently available second line drugs. PMID:22605844
Influence of modulation periods on the tribological behavior of Si/a-C: H multilayer film

NASA Astrophysics Data System (ADS)

Zhu, Linan; Wu, Yanxia; Zhang, Shujiao; Yu, Shengwang; Tang, Bin; Liu, Ying; Zhou, Bing; Shen, Yanyan

2018-01-01

A series of Si/a-C: H multilayer films with different modulation periods were fabricated on stainless steel and silicon substrates by radio-frequency magnetron sputtering. The influence of the modulation period on the structure, morphology, mechanical properties and tribological behaviors in different environments (air, simulated acid rain, and NaCl solution) was investigated. The results show that the content of the sp2 hybrid carbon, surface roughness and hardness of the multilayer film increased firstly and then decreased with the decreased modulation period. Furthermore, the combination of the sublayer agrees well with the formation of the SiC crystal at the interface. Interestingly, the films show quite substantially different tribological properties in various test environments. The lowest friction coefficient is 0.2 for the S1 film in air. However, the lowest friction coefficient can reach 0.13 in solution. Importantly, the tribological behavior of the multilayer film is mainly determined by its hardness, as well as surface roughness in air while it is closely related with modulation period and interface structure in solution.

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