Alternative Nucleophilic Substrates for the Endonuclease Activities of Human Immunodeficiency Virus Type 1 Integrase

PubMed Central

Ealy, Julie B.; Sudol, Malgorzata; Krzeminski, Jacek; Amin, Shantu; Katzman, Michael

2012-01-01

Retroviral integrase can use water or some small alcohols as the attacking nucleophile to nick DNA. To characterize the range of compounds that human immunodeficiency virus type 1 integrase can accommodate for its endonuclease activities, we tested 45 potential electron donors (having varied size and number or spacing of nucleophilic groups) as substrates during site-specific nicking at viral DNA ends and during nonspecific nicking reactions. We found that integrase used 22 of the 45 compounds to nick DNA, but not all active compounds were used for both activities. In particular, 13 compounds were used for site-specific and nonspecific nicking, 5 only for site-specific nicking, and 4 only for nonspecific nicking; 23 other compounds were not used for either activity. Thus, integrase can accommodate a large number of nucleophilic substrates but has selective requirements for its different activities, underscoring its dynamic properties and providing new information for modeling and understanding integrase. PMID:22910593

A computational model for predicting integrase catalytic domain of retrovirus.

PubMed

Wu, Sijia; Han, Jiuqiang; Zhang, Xinman; Zhong, Dexing; Liu, Ruiling

2017-06-21

Integrase catalytic domain (ICD) is an essential part in the retrovirus for integration reaction, which enables its newly synthesized DNA to be incorporated into the DNA of infected cells. Owing to the crucial role of ICD for the retroviral replication and the absence of an equivalent of integrase in host cells, it is comprehensible that ICD is a promising drug target for therapeutic intervention. However, annotated ICDs in UniProtKB database have still been insufficient for a good understanding of their statistical characteristics so far. Accordingly, it is of great importance to put forward a computational ICD model in this work to annotate these domains in the retroviruses. The proposed model then discovered 11,660 new putative ICDs after scanning sequences without ICD annotations. Subsequently in order to provide much confidence in ICD prediction, it was tested under different cross-validation methods, compared with other database search tools, and verified on independent datasets. Furthermore, an evolutionary analysis performed on the annotated ICDs of retroviruses revealed a tight connection between ICD and retroviral classification. All the datasets involved in this paper and the application software tool of this model can be available for free download at https://sourceforge.net/projects/icdtool/files/?source=navbar. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors | Center for Cancer Research

Cancer.gov

Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistancecausing mutations that diminish the efficacy of DTG have appeared. Our current findings

Diketo acids derivatives as integrase inhibitors: the war against the acquired immunodeficiency syndrome.

PubMed

Henao-Mejia, Jorge; Góez, Yenny; Patiño, Pablo; Rugeles, Maria T

2006-06-01

Since the human immunodeficiency virus was identified as etiological agent of the acquired immunodeficiency syndrome, great advances have been accomplished in the therapeutic field leading to reduced morbidity and mortality among infected patients. However, the high mutation rate of the viral genome generates strains resistant to multiple drugs, pointing to the importance of finding new therapeutic targets. Among the HIV structural genes, the POL gene codes for three essential enzymes: reverse transcriptase, protease, and integrase; nineteen of the twenty drugs currently approved by the Food and Drug Administration to treat this viral infection, inhibit the reverse transcriptase and the protease. Although intense research has been carried out in this area during the last 10 years, HIV integrase inhibitors are not yet approved for clinical use; however the fact that presence of this enzyme is a sine qua non for a productive HIV life cycle joined to its unique properties makes it a promissory target for anti-HIV therapy. Many compounds have been claimed to inhibit integrase in vitro; however, few of them have proven to have antiviral activity and low cytotoxicity in cell systems. Diketoacid derivatives are the most promising integrase inhibitors so far reported. Initially discovered independently by Shionogi & Co. and the Merck Research Laboratories, these compounds are highly specific for the integrase with potent antiviral activity in vitro and in vivo, and low cytotoxicity in cell cultures. Some of these compounds have recently entered clinical trials. Due to the high relevance of integrase inhibitors, and specifically of diketoacid derivatives, we review the latest findings and patents in this important field of research.

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

PubMed Central

Rubtsova, Myroslava; Kumlehn, Jochen; Gils, Mario

2012-01-01

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity. PMID:23024817

Discovery of 2-Pyridinone Aminals: A Prodrug Strategy to Advance a Second Generation of HIV-1 Integrase Strand Transfer Inhibitors

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

Raheem, Izzat T.; Walji, Abbas M.; Klein, Daniel

The search for new molecular constructs that resemble the critical two-metal binding pharmacophore required for HIV integrase strand transfer inhibition represents a vibrant area of research within drug discovery. Here we present the discovery of a new class of HIV integrase strand transfer inhibitors based on the 2-pyridinone core of MK-0536. These efforts led to the identification of two lead compounds with excellent antiviral activity and preclinical pharmacokinetic profiles to support a once-daily human dose prediction. Dose escalating PK studies in dog revealed significant issues with limited oral absorption and required an innovative prodrug strategy to enhance the high-dose plasmamore » exposures of the parent molecules.« less

von Hippel–Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

PubMed Central

Mousnier, Aurélie; Kubat, Nicole; Massias-Simon, Aurélie; Ségéral, Emmanuel; Rain, Jean-Christophe; Benarous, Richard; Emiliani, Stéphane; Dargemont, Catherine

2007-01-01

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin–proteasome pathway. Here, we identify von Hippel–Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel–Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin–VHL–proteasome pathway in the integration–transcription transition of the viral replication cycle. PMID:17698809

Towards β-globin gene-targeting with integrase-defective lentiviral vectors.

PubMed

Inanlou, Davoud Nouri; Yakhchali, Bagher; Khanahmad, Hossein; Gardaneh, Mossa; Movassagh, Hesam; Cohan, Reza Ahangari; Ardestani, Mehdi Shafiee; Mahdian, Reza; Zeinali, Sirous

2010-11-01

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of β-thalassemia. The β-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the β-globin gene, a β-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the β-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for β-thalassemia.

Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery.

PubMed

Luo, Yan; Wang, Yongsheng; Liu, Jun; Lan, Hui; Shao, Minghao; Yu, Yuan; Quan, Fusheng; Zhang, Yong

2015-10-01

Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

HIV‑1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases | Center for Cancer Research

Cancer.gov

On the cover: Mutant forms of HIV-1 IN reduce the therapeutic effectiveness of integrase strand transfer inhibitors (INSTIs). The cover figure shows the IN of prototype foamy virus complexed to a novel INSTI (gold) that retains potency against resistant mutants of HIV-1 IN. Overlain are the host and viral DNA substrates (blue and green, respectively), showing substrate mimicry

Lack of integrase inhibitors associated resistance mutations among HIV-1C isolates.

PubMed

Mulu, Andargachew; Maier, Melanie; Liebert, Uwe Gerd

2015-12-01

Although biochemical analysis of HIV-1 integrase enzyme suggested the use of integrase inhibitors (INIs) against HIV-1C, different viral subtypes may favor different mutational pathways potentially leading to varying levels of drug resistance. Thus, the aim of this study was to search for the occurrence and natural evolution of integrase polymorphisms and/or resistance mutations in HIV-1C Ethiopian clinical isolates prior to the introduction of INIs. Plasma samples from chronically infected drug naïve patients (N = 45), of whom the PR and RT sequence was determined previously, were used to generate population based sequences of HIV-1 integrase. HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. Resistance mutations were interpreted according to the Stanford HIV drug resistance database ( http://hivdb.stanford.edu ) and the updated International Antiviral Society (IAS)-USA mutation lists. Moreover, rates of polymorphisms in the current isolates were compared with South African and global HIV-1C isolates. All subjects were infected with HIV-1C concordant to the protease (PR) and reverse transcriptase (RT) regions. Neither major resistance-associated IN mutations (T66I/A/K, E92Q/G, T97A, Y143HCR, S147G, Q148H/R/K, and N155H) nor silent mutations known to change the genetic barrier were observed. Moreover, the DDE-catalytic motif (D64G/D116G/E152 K) and signature HHCC zinc-binding motifs at codon 12, 16, 40 and 43 were found to be highly conserved. However, compared to other South African subtype C isolates, the rate of polymorphism was variable at various positions. Although the sample size is small, the findings suggest that this drug class could be effective in Ethiopia and other southern African countries where HIV-1C is predominantly circulating. The data will contribute to define the importance of integrase polymorphism and to improve resistance interpretation algorithms in HIV-1C isolates.

QSAR study of curcumine derivatives as HIV-1 integrase inhibitors.

PubMed

Gupta, Pawan; Sharma, Anju; Garg, Prabha; Roy, Nilanjan

2013-03-01

A QSAR study was performed on curcumine derivatives as HIV-1 integrase inhibitors using multiple linear regression. The statistically significant model was developed with squared correlation coefficients (r(2)) 0.891 and cross validated r(2) (r(2) cv) 0.825. The developed model revealed that electronic, shape, size, geometry, substitution's information and hydrophilicity were important atomic properties for determining the inhibitory activity of these molecules. The model was also tested successfully for external validation (r(2) pred = 0.849) as well as Tropsha's test for model predictability. Furthermore, the domain analysis was carried out to evaluate the prediction reliability of external set molecules. The model was statistically robust and had good predictive power which can be successfully utilized for screening of new molecules.

A Mos1 transposase in vivo assay to screen new HIV-1 integrase inhibitors.

PubMed

Cancian, Mariana; Loreto, Elgion L S

2018-04-01

The integrase and transposase enzymes of retrovirus and transposons, respectively, share the catalytic DDE domain. In vitro assays showed that inhibitors of HIV-1 integrase generally inhibit the mariner Mos1 transposase. Using a Drosophila strain in which the mobilisation of the mariner element can be quantified by mosaic eyes, we showed that flies maintained in medium containing 210 µM to 4 mM of raltegravir, or 1 or 2 mM of dolutegravir, which are HIV-1 integrase inhibitor used in AIDS treatment, have 23-33% less somatic mobilisation in mosaic eyes when treated with raltegravir and 28-32% when treated with dolutegravir. The gene expression of the mariner transposase gene, estimated by qPCR, is similar among treated and control flies. The results suggest that in vivo assays using Drosophila can be used as a primary screening of inhibitory drugs for transposase and retroviral integrase. The advantages of this assay are that it is easy, quick, cheap and is an in vivo test, meaning that the tested substance has to have been taken in by cells and has arrived at the target site, which is not the case when in vitro assays are applied.

Molecular dynamics simulation studies of the wild type and E92Q/N155H mutant of Elvitegravir-resistance HIV-1 integrase.

PubMed

Chen, Qi; Cheng, Xiaolin; Wei, Dongqing; Xu, Qin

2015-03-01

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we herein use the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was used to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.

Development of Tricyclic Hydroxy-1H-pyrrolopyridine-trione Containing HIV-1 Integrase Inhibitors

PubMed Central

Zhao, Xue Zhi; Maddali, Kasthuraiah; Metifiot, Mathieu; Smith, Steven J.; Vu, B. Christie; Marchand, Christophe; Hughes, Stephen H.; Pommier, Yves; Burke, Terrence R.

2011-01-01

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3′-processing. PMID:21493066

Developing a Dynamic Pharmacophore Model for HIV-1 Integrase

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

Carlson, Heather A.; Masukawa, Keven M.; Rubins, Kathleen

2000-05-11

We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of ''dynamic'' pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is amore » multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a ''static'' pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors.« less

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

PubMed Central

2013-01-01

Background Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. Results We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. Conclusions We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase. PMID:24139482

In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1.

PubMed

Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae; Chang, Hyo-Ihl; Ha, Chang Hoon

2016-04-22

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. Copyright © 2016 Elsevier Inc. All rights reserved.

High avidity anti-integrase antibodies discriminate recent and non-recent HIV infection: Implications for HIV incidence assay.

PubMed

Rikhtegaran Tehrani, Zahra; Azadmanesh, Kayhan; Mostafavi, Ehsan; Gharibzadeh, Safoora; Soori, Shahrzad; Azizi, Mohammad; Khabiri, Alireza

2018-03-01

Estimation of HIV incidence provides real-time information of HIV transmission trends for decision makers. Anti-integrase antibodies are the last ones produced during seroconversion and presence of high-avidity anti-integrase antibodies indicates the chronicity of HIV infection. This study aimed to evaluate the performance of these antibodies in discriminating of recent from non-recent HIV infection. For this purpose, different ELISA formats were developed to detect high-avidity anti-integrase antibodies in a commercially available performance panel, and the best assay was selected for further evaluation. The false recent rate of the selected assay was evaluated in a panel of Iranian patients and compared to two commercial assays, BED-EIA and LAg-Avidity. While the false recent rate of the developed assay was 3.8%, it was 14.1% and 1.3% for BED-EIA and LAg-Avidity, respectively. To our knowledge, this is the first report to study the performance of high-avidity anti-integrase antibodies for classification of HIV infection. The preliminary results showed that the specificity of the newly developed assay is markedly higher than BED-EIA and is comparable with LAg-Avidity. The promising results point to the potential use of anti-integrase antibodies as a biomarker in HIV incidence laboratory tests or algorithms. The developed assay needs further evaluation in future. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimization of Streptomyces bacteriophage phi C31 integrase system to prevent post integrative gene silencing in pulmonary type II cells.

PubMed

Aneja, Manish Kumar; Geiger, Johannes; Imker, Rabea; Uzgun, Senta; Kormann, Michael; Hasenpusch, Guenther; Maucksch, Christof; Rudolph, Carsten

2009-12-31

phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

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

Iri-Sofla, Farnoush Jafari; Rahbarizadeh, Fatemeh, E-mail: rahbarif@modares.ac.ir; Ahmadvand, Davoud

2011-11-01

The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3{zeta}/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of Fc{gamma}RII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for highmore » and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.« less

Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by φC31 integrase.

PubMed

Iri-Sofla, Farnoush Jafari; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud; Rasaee, Mohammad J

2011-11-01

The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy. Copyright © 2011 Elsevier Inc. All rights reserved.

Recombination directionality factor gp3 binds ϕC31 integrase via the zinc domain, potentially affecting the trajectory of the coiled-coil motif

PubMed Central

Younger, Ellen; Fernando, Booshini D; Khaleel, Thanafez; Stark, W Marshall; Smith, Margaret C M

2018-01-01

Abstract To establish a prophage state, the genomic DNA of temperate bacteriophages normally becomes integrated into the genome of their host bacterium by integrase-mediated, site-specific DNA recombination. Serine integrases catalyse a single crossover between an attachment site in the host (attB) and a phage attachment site (attP) on the circularized phage genome to generate the integrated prophage DNA flanked by recombinant attachment sites, attL and attR. Exiting the prophage state and entry into the lytic growth cycle requires an additional phage-encoded protein, the recombination directionality factor or RDF, to mediate recombination between attL and attR and excision of the phage genome. The RDF is known to bind integrase and switch its activity from integration (attP x attB) to excision (attL x attR) but its precise mechanism is unclear. Here, we identify amino acid residues in the RDF, gp3, encoded by the Streptomyces phage ϕC31 and within the ϕC31 integrase itself that affect the gp3:Int interaction. We show that residue substitutions in integrase that reduce gp3 binding adversely affect both excision and integration reactions. The mutant integrase phenotypes are consistent with a model in which the RDF binds to a hinge region at the base of the coiled-coil motif in ϕC31 integrase. PMID:29228292

[Isolation, idetification and anti-HIV-1 integrase activity of culturable endophytic fungi from Tibetan medicinal plant Phlomis younghusbandii Mukerjee].

PubMed

Zhang, Da-Wei; Zhao, Ming-Ming; Chen, Juan; Li, Chao; Guo, Shun-Xing

2013-05-01

A total of 52 endophytic fungi were isolated from roots and stems of Tibetan medicinal plant Phlomis younghusbandii Mukerjee. These fungal isolates were molecularly identified based on ITS sequnces and 28S sequences distributed to 12 genera, including Phoma, Chaetosphaeronema, Fusarium and Leptosphaeria, etc. Among them, the dominant genus was Phoma. Extracts of all strains were evaluated for anti-HIV-1 integrase activity by using soluable integrase expressed in E. coli BL21 (DE3). The results showed that seven samples from five fungal endophytes PHY-24, PHY-38, PHY-40, PHY-51, PHY-53, which belonged to genus Chaetosphaeronema, inhibited strand transfer reaction catalyzed by HIV-1 integrase with IC50 values, of 6.60, 5.20, 2.86, 7.86, 4.47, 4.56 and 3.23 microg x mL(-1) respectively. In conclusion, the endophytic fungi of Phlomis younghusbandii Mukerjee are valuable for further screening anti-HIV-1 integrase agents.

Novel Bifunctional Quinolonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis, Biological Activities and Mechanism of Action

PubMed Central

Di Santo, Roberto; Costi, Roberta; Roux, Alessandra; Artico, Marino; Lavecchia, Antonio; Marinelli, Luciana; Novellino, Ettore; Palmisano, Lucia; Andreotti, Mauro; Amici, Roberta; Galluzzo, Clementina Maria; Nencioni, Lucia; Palamara, Anna Teresa; Pommier, Yves; Marchand, Christophe

2008-01-01

The virally encoded integrase protein is an essential enzyme in the life cycle of the HIV-1 virus and represents an attractive and validated target in the development of therapeutics against HIV infection. Drugs that selectively inhibit this enzyme, when used in combination with inhibitors of reverse transcriptase and protease, are believed to be highly effective in suppressing the viral replication. Among the HIV-1 integrase inhibitors, the β-diketo acids (DKAs) represent a major lead for anti-HIV-1drug development. In this study, novel bifunctional quinolonyl diketo acid derivatives were designed, synthesized and tested for their inhibitory ability against HIV-1 integrase. The compounds are potent inhibitors of integrase activity. Particularly, derivative 8 is a potent IN inhibitor for both steps of the reaction (3′-processing and strand transfer) and exhibits both high antiviral activity against HIV-1 infected cells and low cytotoxicity. Molecular modeling studies provide a plausible mechanism of action, which is consistent with ligand SARs and enzyme photo-crosslinking experiments. PMID:16539381

Long-term increase in mVEGF164 in mouse hindlimb muscle mediated by phage phiC31 integrase after nonviral DNA delivery.

PubMed

Portlock, Joylette L; Keravala, Annahita; Bertoni, Carmen; Lee, Solomon; Rando, Thomas A; Calos, Michele P

2006-08-01

Peripheral vascular disease (PVD), characterized by insufficient blood supply to extremities, can be a devastating illness. Although many gene therapy strategies for PVD using vascular endothelial growth factor (VEGF) have resulted in increased blood vessel formation, the vessels are often impermanent and regress after therapy, probably because of the short-lived VEGF expression mediated by gene therapy vectors (14 days or less). phiC31 integrase is a recombinase originally isolated from a bacteriophage of Streptomyces. This integrase performs efficient chromosomal integration of plasmid DNA into mammalian genomes that results in long-term transgene expression. In this study, gene transfer was achieved by intramuscular injection of VEGF and integrase plasmid DNAs into the tibialis anterior muscle in the mouse hindlimb, followed by electroporation of the muscle with needle electrodes. We observed VEGF levels significantly above background 40 days after injection in animals that received phiC31 integrase and the VEGF plasmid. Site-specific integration of plasmid DNA in the chromosomes of muscle tissue was verified by polymerase chain reaction at a common integration site. These results suggest the possible utility of the phiC31 integrase system to treat ischemic disease.

The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency.

PubMed

Feng, Lei; Dharmarajan, Venkatasubramanian; Serrao, Erik; Hoyte, Ashley; Larue, Ross C; Slaughter, Alison; Sharma, Amit; Plumb, Matthew R; Kessl, Jacques J; Fuchs, James R; Bushman, Frederic D; Engelman, Alan N; Griffin, Patrick R; Kvaratskhelia, Mamuka

2016-05-20

Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein-protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.

Postexposure protection of macaques from vaginal SHIV infection by topical integrase inhibitors.

PubMed

Dobard, Charles; Sharma, Sunita; Parikh, Urvi M; West, Rolieria; Taylor, Andrew; Martin, Amy; Pau, Chou-Pong; Hanson, Debra L; Lipscomb, Jonathan; Smith, James; Novembre, Francis; Hazuda, Daria; Garcia-Lerma, J Gerardo; Heneine, Walid

2014-03-12

Coitally delivered microbicide gels containing antiretroviral drugs are important for HIV prevention. However, to date, microbicides have contained entry or reverse transcriptase inhibitors that block early steps in virus infection and thus need to be given as a preexposure dose that interferes with sexual practices and may limit compliance. Integrase inhibitors block late steps after virus infection and therefore are more suitable for post-coital dosing. We first determined the kinetics of strand transfer in vitro and confirmed that integration begins about 6 hours after infection. We then used a repeat-challenge macaque model to assess efficacy of vaginal gels containing integrase strand transfer inhibitors when applied before or after simian/human immunodeficiency virus (SHIV) challenge. We showed that gel containing the strand transfer inhibitor L-870812 protected two of three macaques when applied 30 min before SHIV challenge. We next evaluated the efficacy of 1% raltegravir gel and demonstrated its ability to protect macaques when applied 3 hours after SHIV exposure (five of six protected; P < 0.05, Fisher's exact test). Breakthrough infections showed no evidence of drug resistance in plasma or vaginal secretions despite continued gel dosing after infection. We documented rapid vaginal absorption reflecting a short pharmacological lag time and noted that vaginal, but not plasma, virus load was substantially reduced in the breakthrough infection after raltegravir gel treatment. We provide a proof of concept that topically applied integrase inhibitors protect against vaginal SHIV infection when administered shortly before or 3 hours after virus exposure.

Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA).

PubMed

Merrick, C A; Wardrope, C; Paget, J E; Colloms, S D; Rosser, S J

2016-01-01

Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways. © 2016 Elsevier Inc. All rights reserved.

The integrase of the long terminal repeat-retrotransposon tf1 has a chromodomain that modulates integrase activities.

PubMed

Hizi, Amnon; Levin, Henry L

2005-11-25

Chromodomains in a variety of proteins mediate the formation of heterochromatin by interacting directly with histone H3, DNA, or RNA. A diverse family of long terminal repeat (LTR)-retrotransposons possesses chromodomains in their integrases (IN), suggesting that the chromodomains may control integration. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is highly active and possesses a chromodomain in the COOH terminus of its IN. To test this chromodomain for a role in integration, recombinant INs with and without the chromodomain were assayed for activity in in vitro reactions. The full-length IN had integration activity with oligonucleotide substrates that modeled both the insertion reaction and a reverse reaction known as disintegration. The INs of retroviruses possess an additional activity termed 3' processing that must remove 2-3 nucleotides from the 3' ends of the viral cDNA before insertion can occur. These additional nucleotides are added during reverse transcription because of the position of the minus strand primer downstream of the LTR. The position of the primer for Tf1 suggests no nucleotides are added 3' of the LTR. It was therefore surprising that Tf1 IN was capable of 3' cleavage. The most unexpected result reported here was that the IN lacking the chromodomain had significantly higher activity and substantially reduced substrate specificity. These results reveal that both the activity and specificity of enzymes can be modulated by their chromodomains.

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase.

PubMed

Mandali, Sridhar; Gupta, Kushol; Dawson, Anthony R; Van Duyne, Gregory D; Johnson, Reid C

2017-06-01

The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes , but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine. IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that

Substrate mimicry—overcoming HIV-1 integrase resistance mutations | Center for Cancer Research

Cancer.gov

HIV integrase (IN) strand transfer inhibitors (INSTIs) are among the newest anti-AIDS drugs; however, mutant forms of IN can confer resistance. We developed noncytotoxic naphthyridine-containing INSTIs that retain low nanomolar IC50 values against HIV-1 variants harboring all of the major INSTI-resistant mutations. We found by analyzing crystal structures of inhibitors bound

Performance of the Abbott RealTime HIV-1 Viral Load Assay Is Not Impacted by Integrase Inhibitor Resistance-Associated Mutations▿

PubMed Central

Young, Thomas P.; Cloherty, Gavin; Fransen, Signe; Napolitano, Laura; Swanson, Priscilla; Herman, Christine; Parkin, Neil T.; Hackett, John

2011-01-01

The Abbott RealTime HIV-1 viral load assay uses primers and probes targeted to integrase, which is also the target of integrase inhibitors such as raltegravir. Viral loads of 42 raltegravir-susceptible and 40 raltegravir-resistant specimens were determined using RealTime HIV-1 and Roche Monitor (v1.5). The differences in viral load measurements between assays were comparable in the two groups, demonstrating that the RealTime HIV-1 assay can tolerate raltegravir-selected mutations. PMID:21289145

Integrase inhibitor versus protease inhibitor based regimen for HIV-1 infected women (WAVES): a randomised, controlled, double-blind, phase 3 study

PubMed Central

Squires, Kathleen; Kityo, Cissy; Hodder, Sally; Johnson, Margaret; Voronin, Evgeny; Hagins, Debbie; Avihingsanon, Anchalee; Koenig, Ellen; Jiang, Shuping; White, Kirsten; Cheng, Andrew; Szwarcberg, Javier; Cao, Huyen

2018-01-01

Summary Background Women are under-represented in HIV antiretroviral therapy (ART) studies. Guidelines for selection of ART as initial therapy in patients with HIV-1 infection do not contain sex-specific treatment. We aimed to assess the safety and efficacy of the single tablet integrase inhibitor regimen containing elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate compared with a boosted protease inhibitor regimen of ritonavir-boosted atazanavir with emtricitabine and tenofovir disoproxil fumarate. Methods In this international, randomised, controlled, double-blind, phase 3 study (Women AntiretroViral Efficacy and Safety study [WAVES]), we recruited treatment-naive HIV-infected women with an estimated creatinine clearance of 70 mL/min or higher from 80 centres in 11 countries. Women were randomly assigned (1:1) to receive elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate (integrase inhibitor regimen) or ritonavir-boosted atazanavir with emtricitabine and tenofovir disoproxil fumarate (protease inhibitor based regimen); regimens were masked with matching placebos. Randomisation was done by a computer-generated allocation sequence (block size four) and was stratified by HIV-1 RNA viral load and race. Investigators, patients, study staff, and those assessing outcomes were masked to treatment group. All participants who received one dose of study drug were included in the primary efficacy and safety analyses. The main outcome was the proportion of patients with plasma HIV-1 RNA less than 50 copies per mL at week 48 as defined by US Food and Drug Administration snapshot algorithm (prespecified non-inferiority margin of 12%). This study is registered with ClinicalTrials.gov, number NCT01705574. Findings Between Nov 28, 2012, and March 12, 2014, 575 women were enrolled. 289 were randomly assigned to receive the integrase inhibitor regimen and 286 to receive the protease inhibitor based regimen. 252 (87%) women in the

Real-time monitoring of disintegration activity of catalytic core domain of HIV-1 integrase using molecular beacon.

PubMed

Zhang, Da-wei; Zhao, Ming-ming; He, Hong-qiu; Guo, Shun-xing

2013-09-15

HIV-1 integrase, an essential enzyme for retroviral replication, is a validated target for anti-HIV therapy development. The catalytic core domain of integrase (IN-CCD) is capable of catalyzing disintegration reaction. In this work, a hairpin-shaped disintegration substrate was designed and validated by enzyme-linked immunosorbent assay; a molecular beacon-based assay was developed for disintegration reaction of IN-CCD. Results showed that the disintegration substrate could be recognized and catalyzed by IN-CCD, and the disintegration reaction can be monitored according to the increase of fluorescent signal. The assay can be applied to real-time detection of disintegration with advantages of simplicity, high sensitivity, and excellent specificity. Copyright © 2013 Elsevier Inc. All rights reserved.

Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of integrase in HIV-1 Gag-Pol polyprotein proteolytic processing.

PubMed

Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka

2017-12-01

The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of the relationship between integrase, excisionase and antirepressor activities associated with a superinfecting Shiga toxin encoding bacteriophage

PubMed Central

Fogg, P. C. M.; Rigden, D. J.; Saunders, J. R.; McCarthy, A. J.; Allison, H. E.

2011-01-01

Shigatoxigenic Escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of Shiga toxin-encoding lambdoid bacteriophages. At least some of these Stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host's pathogenic profile. Here, increasing levels of Stx phage Φ24B integrase expression in multiple lysogen cultures are demonstrated along with apparently negligible repression of integrase expression by the cognate CI repressor. The Φ24B int transcription start site and promoter region were identified and found to differ from in silico predictions. The unidirectional activity of this integrase was determined in an in situ, inducible tri-partite reaction. This indicated that Φ24B must encode a novel directionality factor that is controlling excision events during prophage induction. This excisionase was subsequently identified and characterized through complementation experiments. In addition, the previous proposal that a putative antirepressor was responsible for the lack of immunity to superinfection through inactivation of CI has been revisited and a new hypothesis involving the role of this protein in promoting efficient induction of the Φ24B prophage is proposed. PMID:21062824

HIV-2 Integrase Polymorphisms and Longitudinal Genotypic Analysis of HIV-2 Infected Patients Failing a Raltegravir-Containing Regimen

PubMed Central

Cavaco-Silva, Joana; Abecasis, Ana; Miranda, Ana Cláudia; Poças, José; Narciso, Jorge; Águas, Maria João; Maltez, Fernando; Almeida, Isabel; Germano, Isabel; Diniz, António; Gonçalves, Maria de Fátima; Gomes, Perpétua; Cunha, Celso; Camacho, Ricardo Jorge

2014-01-01

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients. PMID:24681625

HIV-2 integrase polymorphisms and longitudinal genotypic analysis of HIV-2 infected patients failing a raltegravir-containing regimen.

PubMed

Cavaco-Silva, Joana; Abecasis, Ana; Miranda, Ana Cláudia; Poças, José; Narciso, Jorge; Águas, Maria João; Maltez, Fernando; Almeida, Isabel; Germano, Isabel; Diniz, António; Gonçalves, Maria de Fátima; Gomes, Perpétua; Cunha, Celso; Camacho, Ricardo Jorge

2014-01-01

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients.

Metagenomic exploration reveals a marked change in the river resistome and mobilome after treated wastewater discharges.

PubMed

Lekunberri, Itziar; Balcázar, José Luis; Borrego, Carles M

2018-03-01

Mobile genetic elements (MGEs) are key agents in the spread of antibiotic resistance genes (ARGs) across environments. Here we used metagenomics to compare the river resistome (collection of all ARGs) and mobilome (e.g., integrases, transposases, integron integrases and insertion sequence common region "ISCR" elements) between samples collected upstream (n = 6) and downstream (n = 6) of an urban wastewater treatment plant (UWWTP). In comparison to upstream metagenomes, downstream metagenomes showed a drastic increase in the abundance of ARGs, as well as markers of MGEs, particularly integron integrases and ISCR elements. These changes were accompanied by a concomitant prevalence of 16S rRNA gene signatures of bacteria affiliated to families encompassing well-known human and animal pathogens. Our results confirm that chronic discharges of treated wastewater severely impact the river resistome affecting not only the abundance and diversity of ARGs but also their potential spread by enriching the river mobilome in a wide variety of MGEs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Differential Effects of the G118R, H51Y, and E138K Resistance Substitutions in Different Subtypes of HIV Integrase

PubMed Central

Quashie, Peter K.; Oliviera, Maureen; Veres, Tamar; Osman, Nathan; Han, Ying-Shan; Hassounah, Said; Lie, Yolanda; Huang, Wei; Mesplède, Thibault

2014-01-01

ABSTRACT Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. IMPORTANCE Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how “minor” polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also

Differential effects of the G118R, H51Y, and E138K resistance substitutions in different subtypes of HIV integrase.

PubMed

Quashie, Peter K; Oliviera, Maureen; Veres, Tamar; Osman, Nathan; Han, Ying-Shan; Hassounah, Said; Lie, Yolanda; Huang, Wei; Mesplède, Thibault; Wainberg, Mark A

2015-03-01

Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how "minor" polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also highlights the importance

Role of the His-Cys finger of Moloney murine leukemia virus integrase protein in integration and disintegration.

PubMed Central

Jonsson, C B; Roth, M J

1993-01-01

Retroviral integrases mediate site-specific endonuclease and transesterification reactions in the absence of exogenous energy. The basis for the sequence specificity in these integrase-viral DNA recognition processes is unknown. Structural analogs of the disintegration substrate were made to analyze the disintegration reaction mechanism for the Moloney murine leukemia virus (M-MuLV) integrase (IN). Modifications in the target DNA portion of the disintegration substrate decreased enzymatic activity, while substitution of the highly conserved CA in the viral long terminal repeat portion had no effect on activity. The role of the His-Cys finger region in catalysis was addressed by N-ethylmaleimide (NEM) modification of the cysteine residues of M-MuLV IN as well as by mutations. Both integration activities, 3' processing, and strand transfer, were completely inhibited by NEM modification of M-MuLV IN, while disintegration activity was only partially sensitive. However, structural analogs of the disintegration substrates that were modified in the target DNA and had the conserved CA removed were not active with NEM-treated M-MuLV IN. In addition, mutants made in the His-Cys region of M-MuLV IN were examined and found to also be completely blocked in integration but not disintegration activity. These data suggest that the domains of M-MuLV IN that are required for the forward integration reaction substrate differ from those required for the reverse disintegration reaction substrate. Images PMID:8350412

Treatment with integrase inhibitor suggests a new interpretation of HIV RNA decay curves that reveals a subset of cells with slow integration

DOE PAGES

Cardozo, Erwing Fabian; Andrade, Adriana; Mellors, John W.; ...

2017-07-05

The kinetics of HIV-1 decay under treatment depends on the class of antiretrovirals used. Mathematical models are useful to interpret the different profiles, providing quantitative information about the kinetics of virus replication and the cell populations contributing to viral decay. We modeled proviral integration in short- and long-lived infected cells to compare viral kinetics under treatment with and without the integrase inhibitor raltegravir (RAL). We fitted the model to data obtained from participants treated with RAL-containing regimes or with a four-drug regimen of protease and reverse transcriptase inhibitors. Our model explains the existence and quantifies the three phases of HIV-1more » RNA decay in RAL-based regimens vs. the two phases observed in therapies without RAL. Our findings indicate that HIV-1 infection is mostly sustained by short-lived infected cells with fast integration and a short viral production period, and by long-lived infected cells with slow integration but an equally short viral production period. We propose that these cells represent activated and resting infected CD4+ T-cells, respectively, and estimate that infection of resting cells represent ~4% of productive reverse transcription events in chronic infection. RAL reveals the kinetics of proviral integration, showing that in short-lived cells the pre-integration population has a half-life of ~7 hours, whereas in long-lived cells this half-life is ~6 weeks. We also show that the efficacy of RAL can be estimated by the difference in viral load at the start of the second phase in protocols with and without RAL. Altogether, we provide a mechanistic model of viral infection that parsimoniously explains the kinetics of viral load decline under multiple classes of antiretrovirals.« less

Treatment with integrase inhibitor suggests a new interpretation of HIV RNA decay curves that reveals a subset of cells with slow integration

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

Cardozo, Erwing Fabian; Andrade, Adriana; Mellors, John W.

The kinetics of HIV-1 decay under treatment depends on the class of antiretrovirals used. Mathematical models are useful to interpret the different profiles, providing quantitative information about the kinetics of virus replication and the cell populations contributing to viral decay. We modeled proviral integration in short- and long-lived infected cells to compare viral kinetics under treatment with and without the integrase inhibitor raltegravir (RAL). We fitted the model to data obtained from participants treated with RAL-containing regimes or with a four-drug regimen of protease and reverse transcriptase inhibitors. Our model explains the existence and quantifies the three phases of HIV-1more » RNA decay in RAL-based regimens vs. the two phases observed in therapies without RAL. Our findings indicate that HIV-1 infection is mostly sustained by short-lived infected cells with fast integration and a short viral production period, and by long-lived infected cells with slow integration but an equally short viral production period. We propose that these cells represent activated and resting infected CD4+ T-cells, respectively, and estimate that infection of resting cells represent ~4% of productive reverse transcription events in chronic infection. RAL reveals the kinetics of proviral integration, showing that in short-lived cells the pre-integration population has a half-life of ~7 hours, whereas in long-lived cells this half-life is ~6 weeks. We also show that the efficacy of RAL can be estimated by the difference in viral load at the start of the second phase in protocols with and without RAL. Altogether, we provide a mechanistic model of viral infection that parsimoniously explains the kinetics of viral load decline under multiple classes of antiretrovirals.« less

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site | Center for Cancer Research

Cancer.gov

The lowest energy-binding conformation of an inhibitor bound to the dimeric interface of HIV-1 integrase core domain. The yellow region represents a unique allosteric binding site identified by affinity labeling and mass spectrometry and validated through mutagenesis. This site can provide a potential platform for the rational design of inhibitors selective for disruption of

Discovery of Novel HIV-1 Integrase Inhibitors Using QSAR-Based Virtual Screening of the NCI Open Database.

PubMed

Ko, Gene M; Garg, Rajni; Bailey, Barbara A; Kumar, Sunil

2016-01-01

Quantitative structure-activity relationship (QSAR) models can be used as a predictive tool for virtual screening of chemical libraries to identify novel drug candidates. The aims of this paper were to report the results of a study performed for descriptor selection, QSAR model development, and virtual screening for identifying novel HIV-1 integrase inhibitor drug candidates. First, three evolutionary algorithms were compared for descriptor selection: differential evolution-binary particle swarm optimization (DE-BPSO), binary particle swarm optimization, and genetic algorithms. Next, three QSAR models were developed from an ensemble of multiple linear regression, partial least squares, and extremely randomized trees models. A comparison of the performances of three evolutionary algorithms showed that DE-BPSO has a significant improvement over the other two algorithms. QSAR models developed in this study were used in consensus as a predictive tool for virtual screening of the NCI Open Database containing 265,242 compounds to identify potential novel HIV-1 integrase inhibitors. Six compounds were predicted to be highly active (plC50 > 6) by each of the three models. The use of a hybrid evolutionary algorithm (DE-BPSO) for descriptor selection and QSAR model development in drug design is a novel approach. Consensus modeling may provide better predictivity by taking into account a broader range of chemical properties within the data set conducive for inhibition that may be missed by an individual model. The six compounds identified provide novel drug candidate leads in the design of next generation HIV- 1 integrase inhibitors targeting drug resistant mutant viruses.

Polarization-basis tracking scheme for quantum key distribution using revealed sifted key bits.

PubMed

Ding, Yu-Yang; Chen, Wei; Chen, Hua; Wang, Chao; Li, Ya-Ping; Wang, Shuang; Yin, Zhen-Qiang; Guo, Guang-Can; Han, Zheng-Fu

2017-03-15

The calibration of the polarization basis between the transmitter and receiver is an important task in quantum key distribution. A continuously working polarization-basis tracking scheme (PBTS) will effectively promote the efficiency of the system and reduce the potential security risk when switching between the transmission and calibration modes. Here, we proposed a single-photon level continuously working PBTS using only sifted key bits revealed during an error correction procedure, without introducing additional reference light or interrupting the transmission of quantum signals. We applied the scheme to a polarization-encoding BB84 QKD system in a 50 km fiber channel, and obtained an average quantum bit error rate (QBER) of 2.32% and a standard derivation of 0.87% during 24 h of continuous operation. The stable and relatively low QBER validates the effectiveness of the scheme.

Docking studies on a new human immunodeficiency virus integrase-Mg-DNA complex: phenyl ring exploration and synthesis of 1H-benzylindole derivatives through fluorine substitutions.

PubMed

Ferro, Stefania; De Luca, Laura; Barreca, Maria Letizia; Iraci, Nunzio; De Grazia, Sara; Christ, Frauke; Witvrouw, Myriam; Debyser, Zeger; Chimirri, Alba

2009-01-22

A new model of HIV-1 integrase-Mg-DNA complex that is useful for docking experiments has been built. It was used to study the binding mode of integrase strand transfer inhibitor 1 (CHI-1043) and other fluorine analogues. Molecular modeling results prompted us to synthesize the designed derivatives which showed potent enzymatic inhibition at nanomolar concentration, high antiviral activity, and low toxicity. Microwave assisted organic synthesis (MAOS) was employed in several steps of the synthetic pathway, thus reducing reaction times and improving yields.

3-Hydroxypyrimidine-2,4-dione-5-N-benzylcarboxamides potently inhibit HIV-1 integrase and RNase H

PubMed Central

Wu, Bulan; Tang, Jing; Wilson, Daniel J.; Huber, Andrew D.; Casey, Mary C.; Ji, Juan; Kankanala, Jayakanth; Xie, Jiashu; Sarafianos, Stefan G.; Wang, Zhengqiang

2016-01-01

Resistance selection by human immunodeficiency virus (HIV) towards known drug regimens necessitates the discovery of structurally novel antivirals with a distinct resistance profile. Based on our previously reported 3-hydroxypyrimidine-2,4-dione (HPD) core we have designed and synthesized a new integrase strand transfer (INST) inhibitor type featuring a 5-N-benzylcarboxamide moiety. Significantly, the 6-alkylamino variant of this new chemotype consistently conferred low nanomolar inhibitory activity against HIV-1. Extended antiviral testing against a few raltegravir-resistant HIV-1 clones revealed a resistance profile similar to that of the second generation INST inhibitor (INSTIs) dolutegravir. Although biochemical testing and molecular modeling also strongly corroborate the inhibition of INST as the antiviral mechanism of action, selected antiviral analogues also potently inhibited reverse transcriptase (RT) associated RNase H, implying potential dual target inhibition. In vitro ADME assays demonstrated that this novel chemotype possesses largely favorable physicochemical properties suitable for further development. PMID:27283261

The chromodomain of Tf1 integrase promotes binding to cDNA and mediates target site selection.

PubMed

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D; Levin, Henry L

2009-03-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDDelta, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDDelta caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.

Small Molecule Inhibitors of Drug Resistant Forms of HIV-1 Integrase | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute discovered small-molecule compounds containing 1-hydroxy-2-oxo-1,8-naphthyridine moieties whose activity against HIV-1 integrase mutants confer resistance to currently approved INSTIs. Preliminary rodent efficacy, metabolic, and pharmacokinetic studies have been completed by the NCI researchers. The National Cancer Institute seeks partners to commercialize this class of compounds through licensing or co-development.

ILG1 : a new integrase-like gene that is a marker of bacterial contamination by the laboratory Escherichia coli strain TOP10F'.

PubMed Central

Tian, Wenzhi; Chua, Kevin; Strober, Warren; Chu, Charles C.

2002-01-01

BACKGROUND: Identification of differentially expressed genes between normal and diseased states is an area of intense current medical research that can lead to the discovery of new therapeutic targets. However, isolation of differentially expressed genes by subtraction often suffers from unreported contamination of the resulting subtraction library with clones containing DNA sequences not from the original RNA samples. MATERIALS AND METHODS: Subtraction using cDNA representational difference analysis (RDA) was performed on human B cells from normal or common variable immunodeficiency patients. The material remaining after the subtraction was cloned and individual clones were sequenced. The sequence of one clone with similarity to integrases (ILG1, integrase-like gene-1) was used to obtain the full length cDNA sequence and as a probe for the presence of this sequence in RNA or genomic DNA samples. RESULTS: After five rounds of cDNA RDA, 23.3% of the clones from the resulting subtraction library contained Escherichia coli DNA. In addition, three clones contained the sequence of a new integrase, ILG1. The full length cDNA sequence of ILG1 exhibits prokaryotic, but not eukaryotic, features. At the DNA level, ILG1 is not similar to any known gene. At the protein level, ILG1 has 58% similarity to integrases from the cryptic P4 bacteriophage family (S clade). The catalytic domain of ILG1 contains the conserved features found in site-specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R-H-R-Y hallmark of these recombinases. Interestingly, ILG1 was not present in the original B cell populations. By probing genomic DNA, ILG1 could only be detected in the E. coli TOP10F' strain used in our laboratory for molecular cloning, but not in any of its precursor strains, including TOP10. Furthermore, bacteria cultured from the mouth of the laboratory worker who performed cDNA RDA were also positive for

Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry.

PubMed

Borrenberghs, Doortje; Dirix, Lieve; De Wit, Flore; Rocha, Susana; Blokken, Jolien; De Houwer, Stéphanie; Gijsbers, Rik; Christ, Frauke; Hofkens, Johan; Hendrix, Jelle; Debyser, Zeger

2016-11-10

Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.

The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection▿ †

PubMed Central

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D.; Levin, Henry L.

2009-01-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDΔ, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDΔ caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting. PMID:19109383

LEDGINs, non-catalytic site inhibitors of HIV-1 integrase: a patent review (2006 - 2014).

PubMed

Demeulemeester, Jonas; Chaltin, Patrick; Marchand, Arnaud; De Maeyer, Marc; Debyser, Zeger; Christ, Frauke

2014-06-01

Integration of the viral genome into the host cell chromatin is a central step in the replication cycle of the HIV. Blocking the viral integrase (IN) enzyme therefore provides an attractive therapeutic strategy, as evidenced by the recent clinical approval of three IN strand transfer inhibitors. Viral resistance and cross-resistance among these inhibitors, however, warrant the search for compounds targeting HIV integration through alternative mechanisms of action. The most potent class of allosteric IN inhibitors was independently identified at the University of Leuven, Belgium, and at Boehringer Ingelheim, Canada. These compounds, coined LEDGINs (after the lens epithelium-derived growth factor/p75 cofactor binding pocket on IN) or non-catalytic site IN inhibitors (NCINIs) by the respective groups, have shown remarkable antiviral activity. This review provides a brief introduction to the compound class and discusses the recent patent literature (2006 to the present). LEDGINs are still early in development. Trials with clinical candidate BI-224436 were put on hold despite promising results. Literature, however, reveals that almost all major pharmaceutical companies active in the treatment of HIV/AIDS have taken a significant interest in this class. As a result, several of these inhibitors may soon enter clinical trials.

Efficacies of Cabotegravir and Bictegravir against drug-resistant HIV-1 integrase mutants.

PubMed

Smith, Steven J; Zhao, Xue Zhi; Burke, Terrence R; Hughes, Stephen H

2018-05-16

Integrase strand transfer inhibitors (INSTIs) are the class of antiretroviral (ARV) drugs most recently approved by the FDA for the treatment of HIV-1 infections. INSTIs block the strand transfer reaction catalyzed by HIV-1 integrase (IN) and have been shown to potently inhibit infection by wild-type HIV-1. Of the three current FDA-approved INSTIs, Dolutegravir (DTG), has been the most effective, in part because treatment does not readily select for resistant mutants. However, recent studies showed that when INSTI-experienced patients are put on a DTG-salvage therapy, they have reduced response rates. Two new INSTIs, Cabotegravir (CAB) and Bictegravir (BIC), are currently in late-stage clinical trials. Both CAB and BIC had much broader antiviral profiles than RAL and EVG against the INSTI-resistant single, double, and triple HIV-1 mutants used in this study. BIC was more effective than DTG against several INSTI-resistant mutants. Overall, in terms of their ability to inhibit a broad range of INSTI-resistant IN mutants, BIC was superior to DTG, and DTG was superior to CAB. Modeling the binding of CAB, BIC, and DTG within the active site of IN suggested that the "left side" of the INSTI pharmacophore (the side away from the viral DNA) was important in determining the ability of the compound to inhibit the IN mutants we tested. Of the two INSTIs in late stage clinical trials, BIC appears to be better able to inhibit the replication of a broad range of IN mutants. BIC retained potency against several of the INSTI-resistant mutants that caused a decrease in susceptibility to DTG.

Therapy-Emergent Drug Resistance to Integrase Strand Transfer Inhibitors in HIV-1 Patients: A Subgroup Meta-Analysis of Clinical Trials

PubMed Central

Wang, Hongren; Huang, Xiaojun; Qin, Zhen; Deng, Zhaomin; Luo, Jun; Wang, Baoning; Li, Mingyuan

2016-01-01

Background Integrase strand transfer inhibitors (INSTIs) are a novel class of anti-HIV agents that show high activity in inhibiting HIV-1 replication. Currently, licensed INSTIs include raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG); these drugs have played a critical role in AIDS therapy, serving as additional weapons in the arsenal for treating patients infected with HIV-1. To date, long-term data regarding clinical experience with INSTI use and the emergence of resistance remain scarce. However, the literature is likely now sufficiently comprehensive to warrant a meta-analysis of resistance to INSTIs. Methods Our team implemented a manuscript retrieval protocol using Medical Subject Headings (MeSH) via the Web of Science, MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials databases. We screened the literature based on inclusion and exclusion criteria and then performed a quality analysis and evaluation using RevMan software, Stata software, and the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). We also performed a subgroup analysis. Finally, we calculated resistance rates and risk ratios (RRs) for the three types of drugs. Results We identified 26 references via the database search. A meta-analysis of the RAL data revealed that the resistance rate was 3.9% (95% CI = 2.9%-4.9%) for the selected randomized controlled trials (RCTs). However, the RAL resistance rate reached 40.9% (95% CI = 8.8%-72.9%) for the selected observational studies (OBSs). The rates of resistance to RAL that were associated with HIV subtypes A, B, and C as well as with more complex subtypes were 0.1% (95% CI = -0.7%-0.9%), 2.5% (95% CI = 0.5%-4.5%), 4.6% (95% CI = 2.7%-6.6%) and 2.2% (95% CI = 0.7%-3.7%), respectively. The rates of resistance to EVG and DTG were 1.2% (95% CI = 0.2%-2.2%) and 0.1% (95% CI = -0.2%-0.5%), respectively. Furthermore, we found that the RRs for antiviral resistance were 0.414 (95% CI = 0.210–0

Interrogating HIV integrase for compounds that bind- a SAMPL challenge

NASA Astrophysics Data System (ADS)

Peat, Thomas S.; Dolezal, Olan; Newman, Janet; Mobley, David; Deadman, John J.

2014-04-01

Tremendous gains and novel methods are often developed when people are challenged to do something new or difficult. This process is enhanced when people compete against each other-this can be seen in sport as well as in science and technology (e.g. the space race). The SAMPL challenges, like the CASP challenges, aim to challenge modellers and software developers to develop new ways of looking at molecular interactions so the community as a whole can progress in the accurate prediction of these interactions. In order for this challenge to occur, data must be supplied so the prospective test can be done. We have supplied unpublished data related to a drug discovery program run several years ago on HIV integrase for the SAMPL4 challenge. This paper describes the methods used to obtain these data and the chemistry involved.

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

PubMed

Latham, Catherine F; La, Jennifer; Tinetti, Ricky N; Chalmers, David K; Tachedjian, Gilda

2016-01-01

Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay.

PubMed

Diamond, Tracy L; Bushman, Frederic D

2006-01-01

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5' hydroxyl groups as well as 3' hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold.

Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay

PubMed Central

Diamond, Tracy L.; Bushman, Frederic D.

2006-01-01

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5′ hydroxyl groups as well as 3′ hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold. PMID:17085478

A direct comparison of two nonviral gene therapy vectors for somatic integration: in vivo evaluation of the bacteriophage integrase phiC31 and the Sleeping Beauty transposase.

PubMed

Ehrhardt, Anja; Xu, Hui; Huang, Zan; Engler, Jeffrey A; Kay, Mark A

2005-05-01

In this study we performed a head-to-head comparison of the integrase phiC31 derived from a Streptomyces phage and the Sleeping Beauty (SB) transposase, a member of the TC1/mariner superfamily of transposable elements. Mouse liver was cotransfused with a vector containing our most robust human coagulation factor IX expression cassette and the appropriate recombinase recognition site and either a phiC31- or a SB transposase-expressing vector. To analyze transgene persistence and to prove somatic integration in vivo we induced cell cycling of mouse hepatocytes and found that the transgene expression levels dropped by only 16 to 21% and 56 to 66% in mice that received phiC31 and SB, respectively. Notably, no difference in the toxicity profile was detected in mice treated with either recombinase. Moreover we observed that with the integrase-mediated gene transfer, transgene expression levels were dependent on the remaining noncoding vector sequences, which also integrate into the host genome. Further analyses of a hot spot of integration after phiC31-mediated integration revealed small chromosomal deletions at the target site and that the recombination process was not dependent on the orientation in which the phiC31 recognition site attached to the pseudo-recognition sites in the host genome. Coupled together with ongoing improvements in both systems this study suggests that both nonviral vector systems will have important roles in achieving stable gene transfer in vivo.

Impact of resistance mutations on inhibitor binding to HIV-1 integrase

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

Chen, Qi; Buolamwini, John K.; Smith, Jeremy C.

2013-11-08

Here, HIV-1 integrase (IN) is essential for HIV-1 replication, catalyzing two key reaction steps termed 3' processing and strand transfer. Therefore, IN has become an important target for antiviral drug discovery. However, mutants have emerged, such as E92Q/N155H and G140S/Q148H, which confer resistance to raltegravir (RAL), the first IN strand transfer inhibitor (INSTI) approved by the FDA, and to the recently approved elvitegravir (EVG). To gain insights into the molecular mechanisms of ligand binding and drug resistance, we performed molecular dynamics (MD) simulations of homology models of the HIV-1 IN and four relevant mutants complexed with viral DNA and RAL.more » The results show that the structure and dynamics of the 140s loop, comprising residues 140 to 149, are strongly influenced by the IN mutations. In the simulation of the G140S/Q148H double mutant, we observe spontaneous dissociation of RAL from the active site, followed by an intrahelical swing-back of the 3' -OH group of nucleotide A17, consistent with the experimental observation that the G140S/Q148H mutant exhibits the highest resistance to RAL compared to other IN mutants. An important hydrogen bond between residues 145 and 148 is present in the wild-type IN but not in the G140S/Q148H mutant, accounting for the structural and dynamical differences of the 140s' loop and ultimately impairing RAL binding in the double mutant. End-point free energy calculations that broadly capture the experimentally known RAL binding profiles elucidate the contributions of the 140s' loop to RAL binding free energies and suggest possible approaches to overcoming drug resistance.« less

6-(1-Benzyl-1H-pyrrol-2-yl)-2,4-dioxo-5-hexenoic acids as dual inhibitors of recombinant HIV-1 integrase and ribonuclease H, synthesized by a parallel synthesis approach.

PubMed

Costi, Roberta; Métifiot, Mathieu; Esposito, Francesca; Cuzzucoli Crucitti, Giuliana; Pescatori, Luca; Messore, Antonella; Scipione, Luigi; Tortorella, Silvano; Zinzula, Luca; Novellino, Ettore; Pommier, Yves; Tramontano, Enzo; Marchand, Christophe; Di Santo, Roberto

2013-11-14

The increasing efficiency of HAART has helped to transform HIV/AIDS into a chronic disease. Still, resistance and drug-drug interactions warrant the development of new anti-HIV agents. We previously discovered hit 6, active against HIV-1 replication and targeting RNase H in vitro. Because of its diketo-acid moiety, we speculated that this chemotype could serve to develop dual inhibitors of both RNase H and integrase. Here, we describe a new series of 1-benzyl-pyrrolyl diketohexenoic derivatives, 7a-y and 8a-y, synthesized following a parallel solution-phase approach. Those 50 analogues have been tested on recombinant enzymes (RNase H and integrase) and in cell-based assays. Approximately half (22) exibited inhibition of HIV replication. Compounds 7b, 7u, and 8g were the most active against the RNase H activity of reverse-transcriptase, with IC50 values of 3, 3, and 2.5 μM, respectively. Compound 8g was also the most potent integrase inhibitor with an IC50 value of 26 nM.

The Hepatitis B Virus Ribonuclease H Is Sensitive to Inhibitors of the Human Immunodeficiency Virus Ribonuclease H and Integrase Enzymes

PubMed Central

Tavis, John E.; Totten, Michael; Cao, Feng; Michailidis, Eleftherios; Aurora, Rajeev; Meyers, Marvin J.; Jacobsen, E. Jon; Parniak, Michael A.; Sarafianos, Stefan G.

2013-01-01

Nucleos(t)ide analog therapy blocks DNA synthesis by the hepatitis B virus (HBV) reverse transcriptase and can control the infection, but treatment is life-long and has high costs and unpredictable long-term side effects. The profound suppression of HBV by the nucleos(t)ide analogs and their ability to cure some patients indicates that they can push HBV to the brink of extinction. Consequently, more patients could be cured by suppressing HBV replication further using a new drug in combination with the nucleos(t)ide analogs. The HBV ribonuclease H (RNAseH) is a logical drug target because it is the second of only two viral enzymes that are essential for viral replication, but it has not been exploited, primarily because it is very difficult to produce active enzyme. To address this difficulty, we expressed HBV genotype D and H RNAseHs in E. coli and enriched the enzymes by nickel-affinity chromatography. HBV RNAseH activity in the enriched lysates was characterized in preparation for drug screening. Twenty-one candidate HBV RNAseH inhibitors were identified using chemical structure-activity analyses based on inhibitors of the HIV RNAseH and integrase. Twelve anti-RNAseH and anti-integrase compounds inhibited the HBV RNAseH at 10 µM, the best compounds had low micromolar IC50 values against the RNAseH, and one compound inhibited HBV replication in tissue culture at 10 µM. Recombinant HBV genotype D RNAseH was more sensitive to inhibition than genotype H. This study demonstrates that recombinant HBV RNAseH suitable for low-throughput antiviral drug screening has been produced. The high percentage of compounds developed against the HIV RNAseH and integrase that were active against the HBV RNAseH indicates that the extensive drug design efforts against these HIV enzymes can guide anti-HBV RNAseH drug discovery. Finally, differential inhibition of HBV genotype D and H RNAseHs indicates that viral genetic variability will be a factor during drug development. PMID

Ferrocenyl chalcone difluoridoborates inhibit HIV-1 integrase and display low activity towards cancer and endothelial cells.

PubMed

Monserrat, Jean-Philippe; Al-Safi, Rasha I; Tiwari, Keshri Nath; Quentin, Lionel; Chabot, Guy G; Vessières, Anne; Jaouen, Gérard; Neamati, Nouri; Hillard, Elizabeth A

2011-10-15

We report here the discovery of a potent series of HIV-1 integrase (IN) inhibitors based on the ferrocenyl chalcone difluoridoborate structure. Ten new compounds have been synthesized and were generally found to have similar inhibitory activities against the IN 3' processing and strand transfer (ST) processes. IC(50) values were found to be in the low micromolar range, and significantly lower than those found for the non-coordinated ferrocenyl chalcones and other ferrocene molecules. The ferrocenyl chalcone difluoridoborates furthermore exhibited low cytotoxicity against cancer cells and low morphological activity against epithelial cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Successful Prevention of Transmission of Integrase Resistance in the Swiss HIV Cohort Study.

PubMed

Scherrer, Alexandra U; Yang, Wan-Lin; Kouyos, Roger D; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Cavassini, Matthias; Battegay, Manuel; Hauser, Christoph; Calmy, Alexandra; Schmid, Patrick; Bernasconi, Enos; Günthard, Huldrych F

2016-08-01

The prevalence of integrase strand transfer inhibitor (INSTI)-transmitted drug resistance (TDR) may increase with the increasing use of INSTIs. We analyzed the prevalence of INSTI TDR in the Swiss HIV Cohort Study (2008-2014). In 1 of 1316 drug-naive samples (0.1%), a major INSTI TDR mutation was detected. Prevalence was stable, although INSTIs were increasingly used. We showed that this is in contrast to the introduction of previous drug classes, in which more treatment failures with resistant strains occurred and TDR was observed more rapidly. We demonstrated on a population-level that it is possible to avoid TDR to a new drug class for years. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Reduction of antibiotic resistome and integron-integrase genes in laboratory-scale photobioreactors treating municipal wastewater.

PubMed

Nõlvak, Hiie; Truu, Marika; Oopkaup, Kristjan; Kanger, Kärt; Krustok, Ivo; Nehrenheim, Emma; Truu, Jaak

2018-06-08

Wastewater treatment systems receiving municipal wastewater are major dissemination nodes of antibiotic resistance genes (ARGs) between anthropogenic and natural environments. This study examined the fate of antibiotic resistome and class 1-3 integron-integrase genes in photobioreactors that were treating municipal wastewater diluted (70/30) with lake or tap water for the algal biomass production. A combined approach of metagenomic and quantitative (qPCR) analysis was undertaken. Municipal wastewater treatment in the photobioreactors led to reduced antibiotic resistome proportion, number of ARG subtypes, and abundances of individual ARGs in the bacterial community. The ARGs and intI1 gene abundances and relative abundances in the discharges of the photobioreactors were either comparable or lower than the respective values in the effluents of conventional wastewater treatment plants. The reduction of the resistome proved to be strongly related to the changes in the bacterial community composition during the wastewater treatment process as it was responding to rising pH levels caused by intense algal growth. Several bacterial genera (e.g., Azoarcus, Dechloromonas, and Sulfuritalea) were recognized as potential hosts of multiple antibiotic resistance types. Although the lake water contributed a diverse and abundant resistome and intI genes profile to the treatment system, it proved to be considerably more beneficial for wastewater dilution than the tap water. The diversity (number of detected resistance types and subtypes) and proportion of the antibiotic resistome, the amount of plasmid borne integron-integrase gene reads, and the abundances and relative abundances of the majority of quantified ARGs (aadA, sul1, tetQ, tetW, qnrS, ermB, blaOXA2-type) and intI1 gene as well as the amount of multi-resistance determinants were significantly lower in the discharges of photobioreactors where lake water was used to dilute wastewater. Copyright © 2018. Published by Elsevier

Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4.

PubMed Central

Auvray, F; Coddeville, M; Ritzenthaler, P; Dupont, L

1997-01-01

Bacteriophage mv4 is a temperate phage infecting Lactobacillus delbrueckii subsp. bulgaricus. During lysogenization, the phage integrates its genome into the host chromosome at the 3' end of a tRNA(Ser) gene through a site-specific recombination process (L. Dupont et al., J. Bacteriol., 177:586-595, 1995). A nonreplicative vector (pMC1) based on the mv4 integrative elements (attP site and integrase-coding int gene) is able to integrate into the chromosome of a wide range of bacterial hosts, including Lactobacillus plantarum, Lactobacillus casei (two strains), Lactococcus lactis subsp. cremoris, Enterococcus faecalis, and Streptococcus pneumoniae. Integrative recombination of pMC1 into the chromosomes of all of these species is dependent on the int gene product and occurs specifically at the pMC1 attP site. The isolation and sequencing of pMC1 integration sites from these bacteria showed that in lactobacilli, pMC1 integrated into the conserved tRNA(Ser) gene. In the other bacterial species where this tRNA gene is less or not conserved; secondary integration sites either in potential protein-coding regions or in intergenic DNA were used. A consensus sequence was deduced from the analysis of the different integration sites. The comparison of these sequences demonstrated the flexibility of the integrase for the bacterial integration site and suggested the importance of the trinucleotide CCT at the 5' end of the core in the strand exchange reaction. PMID:9068626

Metal-dependent inhibition of HIV-1 integrase by 5CITEP inhibitor: A theoretical QM/MM approach

NASA Astrophysics Data System (ADS)

do Nascimento, Josenaide P.; Araújo Silva, José Rogério; Lameira, Jerônimo; Alves, Cláudio N.

2013-09-01

HIV-1 integrase (IN) is a potential target for developing drugs against AIDS. In this letter, QM/MM approach was used to study the inhibition of IN by 5CITEP inhibitor in presence of divalent cations (Mg2+ or Mn2+). In addition, the main interactions occurring in 5CITEP-IN complex and the influence of divalent cations (Mg2+ or Mn2+) in enzymatic inhibition were investigated using B3LYP/6-31+G(d,p)/MM. The results suggest that the Asp64, Asp116 and four crystal water molecules plays a crucial role in cation (Mg2+ or Mn2+) coordination sphere.

Recent Advances in the Development of Small-Molecular Inhibitors Target HIV Integrase-LEDGF/p75 Interaction.

PubMed

Zhao, Yu; Luo, Zaigang

2015-01-01

Lens epithelium-derived growth factor (LEDGF/p75) plays an essential role in the HIV-1 replication. It acts by tethering integrase (IN) into the host cellular chromatin. Due to its significance of the IN-LEDGF/p75 interaction affords a novel therapeutic approach for the design of new classes of antiretroviral agents. To date, many small molecules have been found to be the inhibitors of INLEDGF/ p75 interaction. This review summarizes recent advances in the development of potential structure-based IN-LEDGF/p75 interaction inhibitors. The work will be helpful to shed light on the antiretroviral drug development pipeline in the next future.

Inorganic and organic fertilizers impact the abundance and proportion of antibiotic resistance and integron-integrase genes in agricultural grassland soil.

PubMed

Nõlvak, Hiie; Truu, Marika; Kanger, Kärt; Tampere, Mailiis; Espenberg, Mikk; Loit, Evelin; Raave, Henn; Truu, Jaak

2016-08-15

Soil fertilization with animal manure or its digestate may facilitate an important antibiotic resistance dissemination route from anthropogenic sources to the environment. This study examines the effect of mineral fertilizer (NH4NO3), cattle slurry and cattle slurry digestate amendment on the abundance and proportion dynamics of five antibiotic resistance genes (ARGs) and two classes of integron-integrase genes (intI1 and intI2) in agricultural grassland soil. Fertilization was performed thrice throughout one vegetation period. The targeted ARGs (sul1, tetA, blaCTX-M, blaOXA2 and qnrS) encode resistance to several major antibiotic classes used in veterinary medicine such as sulfonamides, tetracycline, cephalosporins, penicillin and fluoroquinolones, respectively. The non-fertilized grassland soil contained a stable background of tetA, blaCTX-M and sul1 genes. The type of applied fertilizer significantly affected ARGs and integron-integrase genes abundances and proportions in the bacterial community (p<0.001 in both cases), explaining 67.04% of the abundance and 42.95% of the proportion variations in the grassland soil. Both cattle slurry and cattle slurry digestate proved to be considerable sources of ARGs, especially sul1, as well as integron-integrases. Sul1, intI1 and intI2 levels in grassland soil were elevated in response to each organic fertilizer's application event, but this increase was followed by a stage of decrease, suggesting that microbes possessing these genes were predominantly entrained into soil via cattle slurry or its digestate application and had somewhat limited survival potential in a soil environment. However, the abundance of these three target genes did not decrease to a background level by the end of the study period. TetA was most abundant in mineral fertilizer treated soil and blaCTX-M in cattle slurry digestate amended soil. Despite significantly different abundances, the abundance dynamics of bacteria possessing these genes were

Comparative docking and CoMFA analysis of curcumine derivatives as HIV-1 integrase inhibitors.

PubMed

Gupta, Pawan; Garg, Prabha; Roy, Nilanjan

2011-08-01

The docking studies and comparative molecular field analysis (CoMFA) were performed on highly active molecules of curcumine derivatives against 3' processing activity of HIV-1 integrase (IN) enzyme. The optimum CoMFA model was selected with statistically significant cross-validated r(2) value of 0.815 and non-cross validated r (2) value of 0.99. The common pharmacophore of highly active molecules was used for screening of HIV-1 IN inhibitors. The high contribution of polar interactions in pharmacophore mapping is well supported by docking and CoMFA results. The results of docking, CoMFA, and pharmacophore mapping give structural insights as well as important binding features of curcumine derivatives as HIV-1 IN inhibitors which can provide guidance for the rational design of novel HIV-1 IN inhibitors.

Structural dynamics of native and V260E mutant C-terminal domain of HIV-1 integrase

NASA Astrophysics Data System (ADS)

Sangeetha, Balasubramanian; Muthukumaran, Rajagopalan; Amutha, Ramaswamy

2015-04-01

The C-terminal domain (CTD) of HIV-1 integrase is a five stranded β-barrel resembling an SH3 fold. Mutational studies on isolated CTD and full-length IN have reported V260E mutant as either homo-dimerization defective or affecting the stability and folding of CTD. In this study, molecular dynamics simulation techniques were used to unveil the effect of V260E mutation on isolated CTD monomer and dimer. Both monomeric and dimeric forms of wild type and V260E mutant are highly stable during the simulated period. However, the stabilizing π-stacking interaction between Trp243 and Trp243' at the dimer interface is highly disturbed in CTD-V260E (>6 Å apart). The loss in entropy for dimerization is -30 and -25 kcal/mol for CTD-wt and CTD-V260E respectively signifying a weak hydrophobic interaction and its perturbation in CTD-V260E. The mutant Glu260 exhibits strong attraction/repulsion with all the basic/acidic residues of CTD. In addition to this, the dynamics of CTD-wild type and V260E monomers at 498 K was analyzed to elucidate the effect of V260E mutation on CTD folding. Increase in SASA and reduction in the number of contacts in CTD-V260E during simulation highlights the instability caused by the mutation. In general, V260E mutation affects both multimerization and protein folding with a pronounced effect on protein folding rather than multimerization. This study emphasizes the importance of the hydrophobic nature and SH3 fold of CTD in proper functioning of HIV integrase and perturbing this nature would be a rational approach toward designing more selective and potent allosteric anti-HIV inhibitors.

Functional Coupling between HIV-1 Integrase and the SWI/SNF Chromatin Remodeling Complex for Efficient in vitro Integration into Stable Nucleosomes

PubMed Central

Lesbats, Paul; Botbol, Yair; Chevereau, Guillaume; Vaillant, Cédric; Calmels, Christina; Arneodo, Alain; Andreola, Marie-Line; Lavigne, Marc; Parissi, Vincent

2011-01-01

Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier. PMID:21347347

Impairment of Human Immunodeficiency Virus Type-1 Integrase SUMOylation Correlates with an Early Replication Defect*

PubMed Central

Zamborlini, Alessia; Coiffic, Audrey; Beauclair, Guillaume; Delelis, Olivier; Paris, Joris; Koh, Yashuiro; Magne, Fabian; Giron, Marie-Lou; Tobaly-Tapiero, Joelle; Deprez, Eric; Emiliani, Stephane; Engelman, Alan; de Thé, Hugues; Saïb, Ali

2011-01-01

HIV-1 integrase (IN) orchestrates the integration of the reverse transcribed viral cDNA into the host cell genome and participates also in other steps of HIV-1 replication. Cellular and viral factors assist IN in performing its multiple functions, and post-translational modifications contribute to modulate its activities. Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO acceptor sites. Viruses harboring SUMOylation site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Because SUMOylation-defective IN mutants retained WT catalytic activity, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication. PMID:21454548

DNA Physical Properties and Nucleosome Positions Are Major Determinants of HIV-1 Integrase Selectivity

PubMed Central

Naughtin, Monica; Haftek-Terreau, Zofia; Xavier, Johan; Meyer, Sam; Silvain, Maud; Jaszczyszyn, Yan; Levy, Nicolas; Miele, Vincent; Benleulmi, Mohamed Salah; Ruff, Marc; Parissi, Vincent; Vaillant, Cédric; Lavigne, Marc

2015-01-01

Retroviral integrases (INs) catalyse the integration of the reverse transcribed viral DNA into the host cell genome. This process is selective, and chromatin has been proposed to be a major factor regulating this step in the viral life cycle. However, the precise underlying mechanisms are still under investigation. We have developed a new in vitro integration assay using physiologically-relevant, reconstituted genomic acceptor chromatin and high-throughput determination of nucleosome positions and integration sites, in parallel. A quantitative analysis of the resulting data reveals a chromatin-dependent redistribution of the integration sites and establishes a link between integration sites and nucleosome positions. The co-activator LEDGF/p75 enhanced integration but did not modify the integration sites under these conditions. We also conducted an in cellulo genome-wide comparative study of nucleosome positions and human immunodeficiency virus type-1 (HIV-1) integration sites identified experimentally in vivo. These studies confirm a preferential integration in nucleosome-covered regions. Using a DNA mechanical energy model, we show that the physical properties of DNA probed by IN binding are important in determining IN selectivity. These novel in vitro and in vivo approaches confirm that IN has a preference for integration into a nucleosome, and suggest the existence of two levels of IN selectivity. The first depends on the physical properties of the target DNA and notably, the energy required to fit DNA into the IN catalytic pocket. The second depends on the DNA deformation associated with DNA wrapping around a nucleosome. Taken together, these results indicate that HIV-1 IN is a shape-readout DNA binding protein. PMID:26075397

Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31.

PubMed

Yin, Yajuan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2014-10-01

The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.

Quantification of the HIV-integrase inhibitor raltegravir (MK-0518) in human plasma by high-performance liquid chromatography with fluorescence detection.

PubMed

Poirier, Jean-Marie; Robidou, Pascal; Jaillon, Patrice

2008-05-15

A simple and sensitive HLPC method with fluorescence detection was developed for the accurate determination of the first licensed HIV integrase inhibitor raltegravir in human plasma. A 500-microL plasma sample was spiked with delavirdine as internal standard and subjected to liquid-liquid extraction based on a previously described assay i.e. using hexane/methylene chloride (1:1, v/v%) at pH 4.0. HPLC was performed using a Symmetry Shield RP18 column (150 mm x 4.6 mm), a gradient elution of acetonitrile -0.01% (v/v) triethylamine in water adjusted to pH 3.0 at a flow rate of 1 mL/min and a fluorimetric detector set at 299 and 396 nm as excitation and emission wavelengths, respectively. The retention time was 5.0 min for internal standard and 6.4 min for raltegravir. Calibration curves were linear in the range 5-1000 ng/mL and the accuracy of quality control samples in the range 10-750 ng/mL varied from 98.3 to 99.1% and 98.3 to 101.0% of the nominal concentrations for intra-day and day-to-day analysis, respectively with a precision of 6.3% or less. Among the other antiretroviral drugs which can be given in association to HIV-infected patients, none was found to interfere with internal standard or raltegravir. The described assay was developed for the purpose of therapeutic drug of this HIV integrase inhibitor.

Blocking the interaction between HIV-1 integrase and human LEDGF/p75: mutational studies, virtual screening and molecular dynamics simulations.

PubMed

Reddy, Karnati Konda; Singh, Poonam; Singh, Sanjeev Kumar

2014-03-04

HIV-1 integrase (IN) mediates integration of viral cDNA into the host cell genome, an essential step in the retroviral life cycle. The human lens epithelium-derived growth factor (LEDGF/p75) is a co-factor of HIV-1 IN that plays a crucial role in viral integration. Because of its crucial role in early steps of HIV replication, the IN-LEDGF/p75 interaction represents an attractive target for anti-HIV drug discovery. In this study, the IN-LEDGF/p75 interaction was studied by in silico mutational studies and molecular dynamics simulations. The results showed that all of the key residues in the LEDGF/p75 binding pocket of IN protein are important for stabilization of the complex. Structure-based virtual screening against HIV-1 IN using the ChemBridge database was performed through three different protocols of docking simulations with varying precisions and computational intensities. Six compounds based on the docking score, binding affinity and pharmacokinetic parameters were selected and an analysis of the interactions with key amino acid residues of IN was carried out. Subsequently, molecular dynamics simulations of these compounds in the LEDGF/p75 binding site of IN were carried out in order to study the stability of complexes and their hydrogen bonding interactions. IN residues Glu170, His171, and Thr174 in chain A as well as Gln95 and Thr125 in chain B were discovered to play important roles in the binding of compounds. These findings could be helpful for blocking IN-LEDGF/p75 interaction, and provide a method for avoiding viral resistance and cross-resistance.

ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome

PubMed Central

Bi, Yanzhen; Hua, Zaidong; Ren, Hongyan; Zhang, Liping; Xiao, Hongwei; Liu, Ximei; Hua, Wenjun; Mei, Shuqi; Molenaar, Adrian; Laible, Götz; Zheng, Xinmin

2018-01-01

Programmable nucleases have allowed the rapid development of gene editing and transgenics, but the technology still suffers from the lack of predefined genetic loci for reliable transgene expression and maintenance. To address this issue, we used ФC31 integrase to navigate the porcine genome and identify the pseudo attP sites suitable as safe harbors for sustained transgene expression. The combined ФC31 integrase mRNA and an enhanced green fluorescence protein (EGFP) reporter donor were microinjected into one-cell zygotes for transgene integration. Among the resulting seven EGFP-positive piglets, two had transgene integrations at pseudo attP sites, located in an intergenic region of chromosome 1 (chr1-attP) and the 6th intron of the TRABD2A gene on chromosome 3 (chr3-attP), respectively. The integration structure was determined by TAIL-PCR and Southern blotting. Primary fibroblast cells were isolated from the two piglets and examined using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA), which demonstrated that the chr1-attP site was more potent than chr3-attP site in supporting the EGFP expression. Both piglets had green feet under the emission of UV light, and pelleted primary fibroblast cells were green-colored under natural light, corroborating that the two pseudo attP sites are beneficial to transgene expression. The discovery of these two novel safe harbors for robust and durable transgene expression will greatly facilitate the use of transgenic pigs for basic, biomedical and agricultural studies and applications. PMID:29300364

Ultrasensitive liquid chromatography-tandem mass spectrometric methodologies for quantification of five HIV-1 integrase inhibitors in plasma for a microdose clinical trial.

PubMed

Sun, Li; Li, Hankun; Willson, Kenneth; Breidinger, Sheila; Rizk, Matthew L; Wenning, Larissa; Woolf, Eric J

2012-10-16

HIV-1 integrase strand transfer inhibitors are an important class of compounds targeted for the treatment of HIV-1 infection. Microdosing has emerged as an attractive tool to assist in drug candidate screening for clinical development, but necessitates extremely sensitive bioanalytical assays, typically in the pg/mL concentration range. Currently, accelerator mass spectrometry is the predominant tool for microdosing support, which requires a specialized facility and synthesis of radiolabeled compounds. There have been few studies attempted to comprehensively assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach in the context of microdosing applications. Herein, we describe the development of automated LC-MS/MS methods to quantify five integrase inhibitors in plasma with the limits of quantification at 1 pg/mL for raltegravir and 2 pg/mL for four proprietary compounds. The assays involved double extractions followed by UPLC coupled with negative ion electrospray MS/MS analysis. All methods were fully validated to the rigor of regulated bioanalysis requirements, with intraday precision between 1.20 and 14.1% and accuracy between 93.8 and 107% at the standard curve concentration range. These methods were successfully applied to a human microdose study and demonstrated to be accurate, reproducible, and cost-effective. Results of the study indicate that raltegravir displayed linear pharmacokinetics between a microdose and a pharmacologically active dose.

Coordinated disintegration reactions mediated by Moloney murine leukemia virus integrase.

PubMed Central

Donzella, G A; Jonsson, C B; Roth, M J

1996-01-01

The protein-DNA and protein-protein interactions important for function of the integrase (IN) protein of Moloney murine leukemia virus (M-MuLV) were investigated by using a coordinated-disintegration assay. A panel of M-MuLV IN mutants and substrate alterations highlighted distinctions between the intermolecular and intramolecular reactions of coordinated disintegration. Mispairing of the crossbone single-strand region and altered long terminal repeat (LTR) positioning affected the intermolecular, but not the intramolecular, reactions of coordinated disintegration. Partial components of the crossbone substrate were coordinated by M-MuLV IN, indicating a reliance on both LTR and target DNA determinants for substrate assembly. The intramolecular reaction was dependent on the presence of either the HHCC domain or a crossbone LTR 5' single-stranded tail. An M-MuLV IN mutant without the HHCC domain (Ndelta105) catalyzed reduced levels of double disintegration but not single disintegration. A separately purified HHCC domain protein (Cdelta232) stimulated double disintegration mediated by Ndelta105, suggesting a role of the N-terminal HHCC domain in stable IN-IN and IN-DNA interactions. Significantly, crossbone substrates lacking the LTR 5' tails were not recognized by the fingerless Ndelta105 protein. Collectively, these data suggest similar roles of the HHCC domain and 5' LTR tail in substrate recognition and modulation of IN activity. PMID:8648728

Site-specific integration of Streptomyces PhiC31 integrase-based vectors in the chromosome of Rhodococcus equi.

PubMed

Hong, Yang; Hondalus, Mary K

2008-10-01

Streptomyces PhiC31-based site-specific integration was used to transform the facultative intracellular pathogen Rhodococcus equi. The transformation efficiency of vectors incorporating the PhiC31 integrase and attP sites was comparable to that of replication plasmids using the same electroporation procedure. A single attB integration site was identified within an ORF encoding a pirin-like protein, which deviates slightly from the consensus sequence of Streptomyces attB sites. Vector integration was stably maintained in the R. equi chromosome for as many as 100 generations during unselected passage in vitro. In addition, integration does not appear to affect the replication of bacteria inside macrophages. Finally, this integration system was also used to successfully complement an R. equi mutant.

The phage integrase vector pIPI03 allows RecA-independent, site-specific labelling of Staphylococcus lugdunensis strains.

PubMed

Heilbronner, Simon; Monk, Ian R; Foster, Timothy J

2013-11-01

Staphylococcus lugdunensis is a coagulase negative staphylococcus that is a commensal of man and an opportunistic pathogen. A site-specific integrative plasmid for the use in S. lugdunensis was constructed and validated. The integrase gene ccrB of bacteriophage ϕSL01 together with its attachment site was cloned into the thermosensitive plasmid pIMAY. The resulting plasmid pIPI03 integrated RecA-independently, site-specifically and irreversibly into the S. lugdunensis chromosome. Two IPTG-inducible antibiotic resistance determinants were cloned into pIPI03 and the derivatives were used to construct strains suitable for competitive growth experiments in both in vitro and in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

Human immunodeficiency virus integrase inhibitors efficiently suppress feline immunodeficiency virus replication in vitro and provide a rationale to redesign antiretroviral treatment for feline AIDS

PubMed Central

Savarino, Andrea; Pistello, Mauro; D'Ostilio, Daniela; Zabogli, Elisa; Taglia, Fabiana; Mancini, Fabiola; Ferro, Stefania; Matteucci, Donatella; De Luca, Laura; Barreca, Maria Letizia; Ciervo, Alessandra; Chimirri, Alba; Ciccozzi, Massimo; Bendinelli, Mauro

2007-01-01

Background Treatment of feline immunodeficiency virus (FIV) infection has been hampered by the absence of a specific combination antiretroviral treatment (ART). Integrase strand transfer inhibitors (INSTIs) are emerging as a promising new drug class for HIV-1 treatment, and we evaluated the possibility of inhibiting FIV replication using INSTIs. Methods Phylogenetic analysis of lentiviral integrase (IN) sequences was carried out using the PAUP* software. A theoretical three-dimensional structure of the FIV IN catalytic core domain (CCD) was obtained by homology modeling based on a crystal structure of HIV-1 IN CCD. The interaction of the transferred strand of viral DNA with the catalytic cavity of FIV IN was deduced from a crystal structure of a structurally similar transposase complexed with transposable DNA. Molecular docking simulations were conducted using a genetic algorithm (GOLD). Antiviral activity was tested in feline lymphoblastoid MBM cells acutely infected with the FIV Petaluma strain. Circular and total proviral DNA was quantified by real-time PCR. Results The calculated INSTI-binding sites were found to be nearly identical in FIV and HIV-1 IN CCDs. The close similarity of primate and feline lentivirus IN CCDs was also supported by phylogenetic analysis. In line with these bioinformatic analyses, FIV replication was efficiently inhibited in acutely infected cell cultures by three investigational INSTIs, designed for HIV-1 and belonging to different classes. Of note, the naphthyridine carboxamide INSTI, L-870,810 displayed an EC50 in the low nanomolar range. Inhibition of FIV integration in situ was shown by real-time PCR experiments that revealed accumulation of circular forms of FIV DNA within cells treated with L-870,810. Conclusion We report a drug class (other than nucleosidic reverse transcriptase inhibitors) that is capable of inhibiting FIV replication in vitro. The present study helped establish L-870,810, a compound successfully tested in

1,2,3,4-Tetrahydroisoquinolines as inhibitors of HIV-1 integrase and human LEDGF/p75 interaction.

PubMed

George, Anu; Gopi Krishna Reddy, Alavala; Satyanarayana, Gedu; Raghavendra, Nidhanapati K

2018-06-01

Alkaloids are a class of organic compounds with a wide range of biological properties, including anti-HIV activity. The 1,2,3,4-tetrahydroisoquinoline is a ubiquitous structural motif of many alkaloids. Using a short and an efficient route for synthesis, a series of 1,2,3,4-tetrahydroisoquinolines/isoquinolines was developed. These compounds have been analysed for their ability to inhibit an important interaction between HIV-1 integrase enzyme (IN) and human LEDGF/p75 protein (p75) which assists in the viral integration into the active genes. A lead compound 6d is found to inhibit the LEDGF/p75-IN interaction in vitro with an IC 50 of ~10 μm. Molecular docking analysis of the isoquinoline 6d reveals its interactions with the LEDGF/p75-binding residues of IN. Based on an order of addition experiment, the binding of 6d or LEDGF/p75 to IN is shown to be mutually exclusive. Also, the activity of 6d in vitro is found to be unaffected by the presence of a non-specific DNA. As reported earlier for the inhibitors of LEDGF/p75-IN interaction, 6d exhibits a potent inhibition of both the early and late stages of HIV-1 replication. Compound 6d differing from the known inhibitors in the chemical moieties and interactions with CCD could potentially be explored further for developing small molecule inhibitors of LEDGF/p75-IN interaction having a higher potency. © 2018 John Wiley & Sons A/S.

The Microbiota and Abundance of the Class 1 Integron-Integrase Gene in Tropical Sewage Treatment Plant Influent and Activated Sludge

PubMed Central

Paiva, Magna C.; Ávila, Marcelo P.; Reis, Mariana P.; Costa, Patrícia S.; Nardi, Regina M. D.; Nascimento, Andréa M. A.

2015-01-01

Bacteria are assumed to efficiently remove organic pollutants from sewage in sewage treatment plants, where antibiotic-resistance genes can move between species via mobile genetic elements known as integrons. Nevertheless, few studies have addressed bacterial diversity and class 1 integron abundance in tropical sewage. Here, we describe the extant microbiota, using V6 tag sequencing, and quantify the class 1 integron-integrase gene (intI1) in raw sewage (RS) and activated sludge (AS). The analysis of 1,174,486 quality-filtered reads obtained from RS and AS samples revealed complex and distinct bacterial diversity in these samples. The RS sample, with 3,074 operational taxonomic units, exhibited the highest alpha-diversity indices. Among the 25 phyla, Proteobacteria, Bacteroidetes and Firmicutes represented 85% (AS) and 92% (RS) of all reads. Increased relative abundance of Micrococcales, Myxococcales, and Sphingobacteriales and reduced pathogen abundance were noted in AS. At the genus level, differences were observed for the dominant genera Simplicispira and Diaphorobacter (AS) as well as for Enhydrobacter (RS). The activated sludge process decreased (55%) the amount of bacteria harboring the intI1 gene in the RS sample. Altogether, our results emphasize the importance of biological treatment for diminishing pathogenic bacteria and those bearing the intI1 gene that arrive at a sewage treatment plant. PMID:26115093

B′-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase

PubMed Central

Maertens, Goedele N.

2016-01-01

To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B′ family of the regulatory subunits. B′-PP2A and HTLV-1 IN display nuclear co-localization, and the B′ subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein–protein interaction interface maps to a patch of highly conserved residues on B′, which when mutated render B′ incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity. PMID:26657642

Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration.

PubMed

Thierry, Sylvain; Munir, Soundasse; Thierry, Eloïse; Subra, Frédéric; Leh, Hervé; Zamborlini, Alessia; Saenz, Dyana; Levy, David N; Lesbats, Paul; Saïb, Ali; Parissi, Vincent; Poeschla, Eric; Deprez, Eric; Delelis, Olivier

2015-03-12

Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation. Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication. Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.

Using the class 1 integron-integrase gene as a proxy for anthropogenic pollution.

PubMed

Gillings, Michael R; Gaze, William H; Pruden, Amy; Smalla, Kornelia; Tiedje, James M; Zhu, Yong-Guan

2015-06-01

Around all human activity, there are zones of pollution with pesticides, heavy metals, pharmaceuticals, personal care products and the microorganisms associated with human waste streams and agriculture. This diversity of pollutants, whose concentration varies spatially and temporally, is a major challenge for monitoring. Here, we suggest that the relative abundance of the clinical class 1 integron-integrase gene, intI1, is a good proxy for pollution because: (1) intI1 is linked to genes conferring resistance to antibiotics, disinfectants and heavy metals; (2) it is found in a wide variety of pathogenic and nonpathogenic bacteria; (3) its abundance can change rapidly because its host cells can have rapid generation times and it can move between bacteria by horizontal gene transfer; and (4) a single DNA sequence variant of intI1 is now found on a wide diversity of xenogenetic elements, these being complex mosaic DNA elements fixed through the agency of human selection. Here we review the literature examining the relationship between anthropogenic impacts and the abundance of intI1, and outline an approach by which intI1 could serve as a proxy for anthropogenic pollution.

The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

PubMed

van Bel, Nikki; van der Velden, Yme; Bonnard, Damien; Le Rouzic, Erwann; Das, Atze T; Benarous, Richard; Berkhout, Ben

2014-01-01

The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

The discovery and preclinical evaluation of BMS-707035, a potent HIV-1 integrase strand transfer inhibitor.

PubMed

Naidu, B Narasimhulu; Walker, Michael A; Sorenson, Margaret E; Ueda, Yasutsugu; Matiskella, John D; Connolly, Timothy P; Dicker, Ira B; Lin, Zeyu; Bollini, Sagarika; Terry, Brian J; Higley, Helen; Zheng, Ming; Parker, Dawn D; Wu, Dedong; Adams, Stephen; Krystal, Mark R; Meanwell, Nicholas A

2018-07-01

BMS-707035 is an HIV-1 integrase strand transfer inhibitor (INSTI) discovered by systematic optimization of N-methylpyrimidinone carboxamides guided by structure-activity relationships (SARs) and the single crystal X-ray structure of compound 10. It was rationalized that the unexpectedly advantageous profiles of N-methylpyrimidinone carboxamides with a saturated C2-substitutent may be due, in part, to the geometric relationship between the C2-substituent and the pyrimidinone core. The single crystal X-ray structure of 10 provided support for this reasoning and guided the design of a spirocyclic series 12 which led to discovery of the morpholino-fused pyrimidinone series 13. Several carboxamides derived from this bicyclic scaffold displayed improved antiviral activity and pharmacokinetic profiles when compared with corresponding spirocyclic analogs. Based on the excellent antiviral activity, preclinical profiles and acceptable in vitro and in vivo toxicity profiles, 13a (BMS-707035) was selected for advancement into phase I clinical trials. Copyright © 2018 Elsevier Ltd. All rights reserved.

Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.

PubMed

Takahata, Tatsuro; Takeda, Eri; Tobiume, Minoru; Tokunaga, Kenzo; Yokoyama, Masaru; Huang, Yu-Lun; Hasegawa, Atsuhiko; Shioda, Tatsuo; Sato, Hironori; Kannagi, Mari; Masuda, Takao

2017-01-01

Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic

Kuwanon-L as a New Allosteric HIV-1 Integrase Inhibitor: Molecular Modeling and Biological Evaluation.

PubMed

Esposito, Francesca; Tintori, Cristina; Martini, Riccardo; Christ, Frauke; Debyser, Zeger; Ferrarese, Roberto; Cabiddu, Gianluigi; Corona, Angela; Ceresola, Elisa Rita; Calcaterra, Andrea; Iovine, Valentina; Botta, Bruno; Clementi, Massimo; Canducci, Filippo; Botta, Maurizio; Tramontano, Enzo

2015-11-01

HIV-1 integrase (IN) active site inhibitors are the latest class of drugs approved for HIV treatment. The selection of IN strand-transfer drug-resistant HIV strains in patients supports the development of new agents that are active as allosteric IN inhibitors. Here, a docking-based virtual screening has been applied to a small library of natural ligands to identify new allosteric IN inhibitors that target the sucrose binding pocket. From theoretical studies, kuwanon-L emerged as the most promising binder and was thus selected for biological studies. Biochemical studies showed that kuwanon-L is able to inhibit the HIV-1 IN catalytic activity in the absence and in the presence of LEDGF/p75 protein, the IN dimerization, and the IN/LEDGF binding. Kuwanon-L also inhibited HIV-1 replication in cell cultures. Overall, docking and biochemical results suggest that kuwanon-L binds to an allosteric binding pocket and can be considered an attractive lead for the development of new allosteric IN antiviral agents. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates*

PubMed Central

Bojja, Ravi Shankar; Andrake, Mark D.; Merkel, George; Weigand, Steven; Dunbrack, Roland L.; Skalka, Anna Marie

2013-01-01

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the “reaching dimer” previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition. PMID:23322775

Interrogating Key Positions of Size-Reduced TALE Repeats Reveals a Programmable Sensor of 5-Carboxylcytosine.

PubMed

Maurer, Sara; Giess, Mario; Koch, Oliver; Summerer, Daniel

2016-12-16

Transcription-activator-like effector (TALE) proteins consist of concatenated repeats that recognize consecutive canonical nucleobases of DNA via the major groove in a programmable fashion. Since this groove displays unique chemical information for the four human epigenetic cytosine nucleobases, TALE repeats with epigenetic selectivity can be engineered, with potential to establish receptors for the programmable decoding of all human nucleobases. TALE repeats recognize nucleobases via key amino acids in a structurally conserved loop whose backbone is positioned very close to the cytosine 5-carbon. This complicates the engineering of selectivities for large 5-substituents. To interrogate a more promising structural space, we engineered size-reduced repeat loops, performed saturation mutagenesis of key positions, and screened a total of 200 repeat-nucleobase interactions for new selectivities. This provided insight into the structural requirements of TALE repeats for affinity and selectivity, revealed repeats with improved or relaxed selectivity, and resulted in the first selective sensor of 5-carboxylcytosine.

Lens Epithelium-derived Growth Factor/p75 Interacts with the Transposase-derived DDE Domain of PogZ*S⃞

PubMed Central

Bartholomeeusen, Koen; Christ, Frauke; Hendrix, Jelle; Rain, Jean-Christophe; Emiliani, Stéphane; Benarous, Richard; Debyser, Zeger; Gijsbers, Rik; De Rijck, Jan

2009-01-01

Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular interaction partner of human immunodeficiency virus-1 (HIV-1) integrase, tethering the preintegration complex to the host chromosome. In light of the development of LEDGF/p75-integrase interaction inhibitors, it is essential to understand the cell biology of LEDGF/p75. We identified pogZ as new cellular interaction partner of LEDGF/p75. Analogous to lentiviral integrase, pogZ, a domesticated transposase, carries a DDE domain, the major determinant for LEDGF/p75 interaction. Using different in vitro and in vivo approaches, we corroborated the interaction between the C terminus of LEDGF/p75 and the DDE domain of pogZ, revealing an overlap in the binding of pogZ and HIV-1 integrase. Competition experiments showed that integrase is efficient in displacing pogZ from LEDGF/p75. Moreover, pogZ does not seem to play a role as a restriction factor of HIV. The finding that LEDGF/p75 is capable of interacting with a DDE domain protein that is not a lentiviral integrase points to a profound role of LEDGF/p75 in DDE domain protein function. PMID:19244240

Structural and sequencing analysis of local target DNA recognition by MLV integrase.

PubMed

Aiyer, Sriram; Rossi, Paolo; Malani, Nirav; Schneider, William M; Chandar, Ashwin; Bushman, Frederic D; Montelione, Gaetano T; Roth, Monica J

2015-06-23

Target-site selection by retroviral integrase (IN) proteins profoundly affects viral pathogenesis. We describe the solution nuclear magnetic resonance structure of the Moloney murine leukemia virus IN (M-MLV) C-terminal domain (CTD) and a structural homology model of the catalytic core domain (CCD). In solution, the isolated MLV IN CTD adopts an SH3 domain fold flanked by a C-terminal unstructured tail. We generated a concordant MLV IN CCD structural model using SWISS-MODEL, MMM-tree and I-TASSER. Using the X-ray crystal structure of the prototype foamy virus IN target capture complex together with our MLV domain structures, residues within the CCD α2 helical region and the CTD β1-β2 loop were predicted to bind target DNA. The role of these residues was analyzed in vivo through point mutants and motif interchanges. Viable viruses with substitutions at the IN CCD α2 helical region and the CTD β1-β2 loop were tested for effects on integration target site selection. Next-generation sequencing and analysis of integration target sequences indicate that the CCD α2 helical region, in particular P187, interacts with the sequences distal to the scissile bonds whereas the CTD β1-β2 loop binds to residues proximal to it. These findings validate our structural model and disclose IN-DNA interactions relevant to target site selection. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Resistance Analyses of Integrase Strand Transfer Inhibitors within Phase 3 Clinical Trials of Treatment-Naive Patients

PubMed Central

White, Kirsten L.; Raffi, Francois; Miller, Michael D.

2014-01-01

The integrase (IN) strand transfer inhibitors (INSTIs), raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG), comprise the newest drug class approved for the treatment of HIV-1 infection, which joins the existing classes of reverse transcriptase, protease and binding/entry inhibitors. The efficacy of first-line regimens has attained remarkably high levels, reaching undetectable viral loads in 90% of patients by Week 48; however, there remain patients who require a change in regimen due to adverse events, virologic failure with emergent resistance or other issues of patient management. Large, randomized clinical trials conducted in antiretroviral treatment-naive individuals are required for drug approval in this population in the US, EU and other countries, with the primary endpoint for virologic success at Week 48. However, there are differences in the definition of virologic failure and the evaluation of drug resistance among the trials. This review focuses on the methodology and tabulation of resistance to INSTIs in phase 3 clinical trials of first-line regimens and discusses case studies of resistance. PMID:25054884

Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration.

PubMed

Bonnard, Damien; Le Rouzic, Erwann; Eiler, Sylvia; Amadori, Céline; Orlov, Igor; Bruneau, Jean-Michel; Brias, Julie; Barbion, Julien; Chevreuil, Francis; Spehner, Danièle; Chasset, Sophie; Ledoussal, Benoit; Moreau, François; Saïb, Ali; Klaholz, Bruno P; Emiliani, Stéphane; Ruff, Marc; Zamborlini, Alessia; Benarous, Richard

2018-04-20

Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, whereas inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Because these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125-IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, which are responsible for inhibition of virus maturation, were lost, whereas inhibition of the IN-LEDGF/p75 interaction and consequently integration was fully retained. Hence, the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the Ala-125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125-IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Dual HIV-1 reverse transcriptase and integrase inhibitors from Limonium morisianum Arrigoni, an endemic species of Sardinia (Italy).

PubMed

Sanna, Cinzia; Rigano, Daniela; Corona, Angela; Piano, Dario; Formisano, Carmen; Farci, Domenica; Franzini, Genni; Ballero, Mauro; Chianese, Giuseppina; Tramontano, Enzo; Taglialatela-Scafati, Orazio; Esposito, Francesca

2018-02-04

During our search for potential templates of HIV-1 reverse transcriptase (RT) and integrase (IN) dual inhibitors, the methanolic extract obtained from aerial parts of Limonium morisianum was investigated. Repeated bioassay-guided chromatographic purifications led to the isolation of the following secondary metabolites: myricetin, myricetin 3-O-rutinoside, myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside, (-)-epigallocatechin 3-O-gallate, tryptamine, ferulic and phloretic acids. The isolated compounds were tested on both HIV-1 RT-associated RNase H and IN activities. Interestingly, (-)-epigallocatechin-3-O-gallate and myricetin-3-O-(6″-O-galloyl)-β-d-galactopyranoside potently inhibited both enzyme activities with IC 50 values ranging from 0.21 to 10.9 μM. Differently, tryptamine and ferulic acid exhibited a significant inhibition only on the IN strand transfer reaction, showing a selectivity for this viral enzyme. Taken together these results strongly support the potential of this plant as a valuable anti HIV-1 drugs source worthy of further investigations.

Pedigree-based analysis of derivation of genome segments of an elite rice reveals key regions during its breeding.

PubMed

Zhou, Degui; Chen, Wei; Lin, Zechuan; Chen, Haodong; Wang, Chongrong; Li, Hong; Yu, Renbo; Zhang, Fengyun; Zhen, Gang; Yi, Junliang; Li, Kanghuo; Liu, Yaoguang; Terzaghi, William; Tang, Xiaoyan; He, Hang; Zhou, Shaochuan; Deng, Xing Wang

2016-02-01

Analyses of genome variations with high-throughput assays have improved our understanding of genetic basis of crop domestication and identified the selected genome regions, but little is known about that of modern breeding, which has limited the usefulness of massive elite cultivars in further breeding. Here we deploy pedigree-based analysis of an elite rice, Huanghuazhan, to exploit key genome regions during its breeding. The cultivars in the pedigree were resequenced with 7.6× depth on average, and 2.1 million high-quality single nucleotide polymorphisms (SNPs) were obtained. Tracing the derivation of genome blocks with pedigree and information on SNPs revealed the chromosomal recombination during breeding, which showed that 26.22% of Huanghuazhan genome are strictly conserved key regions. These major effect regions were further supported by a QTL mapping of 260 recombinant inbred lines derived from the cross of Huanghuazhan and a very dissimilar cultivar, Shuanggui 36, and by the genome profile of eight cultivars and 36 elite lines derived from Huanghuazhan. Hitting these regions with the cloned genes revealed they include numbers of key genes, which were then applied to demonstrate how Huanghuazhan were bred after 30 years of effort and to dissect the deficiency of artificial selection. We concluded the regions are helpful to the further breeding based on this pedigree and performing breeding by design. Our study provides genetic dissection of modern rice breeding and sheds new light on how to perform genomewide breeding by design. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Solution structure of the His12 --> Cys mutant of the N-terminal zinc binding domain of HIV-1 integrase complexed to cadmium.

PubMed Central

Cai, M.; Huang, Y.; Caffrey, M.; Zheng, R.; Craigie, R.; Clore, G. M.; Gronenborn, A. M.

1998-01-01

The solution structure of His12 --> Cys mutant of the N-terminal zinc binding domain (residues 1-55; IN(1-55)) of HIV-1 integrase complexed to cadmium has been solved by multidimensional heteronuclear NMR spectroscopy. The overall structure is very similar to that of the wild-type N-terminal domain complexed to zinc. In contrast to the wild-type domain, however, which exists in two interconverting conformational states arising from different modes of coordination of the two histidine side chains to the metal, the cadmium complex of the His12 --> Cys mutant exists in only a single form at low pH. The conformation of the polypeptide chain encompassing residues 10-18 is intermediate between the two forms of the wild-type complex. PMID:9865962

Identification and analysis of integrons and cassette arrays in bacterial genomes

PubMed Central

Touchon, Marie; Néron, Bertrand; Rocha, Eduardo PC

2016-01-01

Abstract Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program – IntegronFinder – to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome. PMID:27130947

Anti-HIV-1 Integrase Activity and Molecular Docking Study of Compounds from Caesalpinia  sappan L.

PubMed

Tewtrakul, Supinya; Chaniad, Prapaporn; Pianwanit, Somsak; Karalai, Chatchanok; Ponglimanont, Chanita; Yodsaoue, Orapun

2015-05-01

Caesalpinia sappan L. (Caesalpiniaceae) has been traditionally used as blood tonic, expectorant, and astringent by boiling with water. Searching for HIV-1 integrase (IN) inhibitors from this plant is a promising approach. The EtOH extract of C. sappan and its isolated compounds were tested for their anti-HIV-1 IN effect using the multiplate integration assay, and the active compounds were determined for their mechanisms by molecular docking technique. Extraction from the heartwoods and roots of C. sappan led to the isolation of nine compounds. Among the compounds tested, sappanchalcone (2) displayed the strongest effect against HIV-1 IN with an IC50 value of 2.3 μM followed by protosappanin A (9, IC50  = 12.6 μM). Structure-activity relationships of compounds from C. sappan were found, in which the vicinal hydroxyl moiety were essential for anti-HIV-1 IN effect of compounds 2 and 9 by binding with the amino acid residues Gln148 and Thr66 in the core domain of the HIV- 1 IN enzyme, respectively. Copyright © 2015 John Wiley & Sons, Ltd.

Isolation and anti-HIV-1 integrase activity of lentzeosides A-F from extremotolerant lentzea sp. H45, a strain isolated from a high-altitude Atacama Desert soil.

PubMed

Wichner, Dominik; Idris, Hamidah; Houssen, Wael E; McEwan, Andrew R; Bull, Alan T; Asenjo, Juan A; Goodfellow, Michael; Jaspars, Marcel; Ebel, Rainer; Rateb, Mostafa E

2017-04-01

The extremotolerant isolate H45 was one of several actinomycetes isolated from a high-altitude Atacama Desert soil collected in northwest Chile. The isolate was identified as a new Lentzea sp. using a combination of chemotaxonomic, morphological and phylogenetic properties. Large scale fermentation of the strain in two different media followed by chromatographic purification led to the isolation of six new diene and monoene glycosides named lentzeosides A-F, together with the known compound (Z)-3-hexenyl glucoside. The structures of the new compounds were confirmed by HRESIMS and NMR analyses. Compounds 1-6 displayed moderate inhibitory activity against HIV integrase.

Insight into the fundamental interactions between LEDGF binding site inhibitors and integrase combining docking and molecular dynamics simulations.

PubMed

De Luca, Laura; Morreale, Francesca; Chimirri, Alba

2012-12-21

In recent years, HIV-1 integrase (IN) has emerged as an attractive target for novel anti-AIDS agents. In particular, nonactive-site-binding IN inhibitors would display synergy with current strand-transfer-specific IN inhibitors and other antiretroviral drugs in clinical use. An effective allosteric inhibitory approach would be the disruption of protein-protein interaction (PPI) between IN and cellular cofactors, such as LEDGF/p75. To date, several small molecules have been reported to be inhibitors of the PPI between IN and LEDGF/p75. In this study, we investigated the most relevant interactions between five selected PPI inhibitors and IN comparing them to the naturally occurring IN-LEDGF/p75 complex. We calculated the binding free energies by using the method of molecular mechanics-generalized Born surface area (MM-GBSA). Total energy was decomposed on per residue contribution, and hydrogen bond occupancies were monitored throughout the simulations. Considering all these results we obtained a good correlation with experimental activity and useful insights for the development of new inhibitors.

HIV Virions as Nanoscopic Test Tubes for Probing Oligomerization of the Integrase Enzyme

PubMed Central

2015-01-01

Employing viruses as nanoscopic lipid-enveloped test tubes allows the miniaturization of protein–protein interaction (PPI) assays while preserving the physiological environment necessary for particular biological processes. Applied to the study of the human immunodeficiency virus type 1 (HIV-1), viral biology and pathology can also be investigated in novel ways, both in vitro as well as in infected cells. In this work we report on an experimental strategy that makes use of engineered HIV-1 viral particles, to allow for probing PPIs of the HIV-1 integrase (IN) inside viruses with single-molecule Förster resonance energy transfer (FRET) using fluorescent proteins (FP). We show that infectious fluorescently labeled viruses can be obtained and that the quantity of labels can be accurately measured and controlled inside individual viral particles. We demonstrate, with proper control experiments, the formation of IN oligomers in single viral particles and inside viral complexes in infected cells. Finally, we show a clear effect on IN oligomerization of small molecule inhibitors of interactions of IN with its natural human cofactor LEDGF/p75, corroborating that IN oligomer enhancing drugs are active already at the level of the virus and strongly suggesting the presence of a dynamic, enhanceable equilibrium between the IN dimer and tetramer in viral particles. Although applied to the HIV-1 IN enzyme, our methodology for utilizing HIV virions as nanoscopic test tubes for probing PPIs is generic, i.e., other PPIs targeted into the HIV-1, or PPIs targeted into other viruses, can potentially be studied with a similar strategy. PMID:24654558

A novel integrase-containing element may interact with Laem-Singh virus (LSNV) to cause slow growth in giant tiger shrimp

PubMed Central

2011-01-01

Background From 2001-2003 monodon slow growth syndrome (MSGS) caused severe economic losses for Thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. In 2008, newly discovered Laem-Singh virus (LSNV) was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s) of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species. Results Using a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE) was found in giant tiger shrimp from MSGS ponds (GenBank accession number FJ498866). In situ hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV) occurred together in lymphoid organs (LO) of shrimp from MSGS ponds but not in shrimp from normal ponds. Tissue homogenates of shrimp from MSGS ponds yielded a fraction that gave positive RT-PCR reactions for both ICE and LSNV and showed viral-like particles by transmission electron microscopy (TEM). Bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of MSGS, and in situ hybridization assays revealed ICE and LSNV together in LO, eyes and gills. Viral-like particles similar to those seen in tissue extracts from natural infections were also seen by TEM. Conclusions ICE and LSNV were found together only in shrimp from MSGS ponds and only in shrimp showing gross signs of MSGS after injection with a preparation containing ICE and LSNV. ICE was never found in the absence of LSNV although LSNV was sometimes found in normal shrimp in the absence of ICE. The results suggest that ICE and LSNV may act together as component causes of MSGS, but this cannot be proven conclusively without single and combined bioassays

Systematic Prioritization and Integrative Analysis of Copy Number Variations in Schizophrenia Reveal Key Schizophrenia Susceptibility Genes

PubMed Central

Luo, Xiongjian; Huang, Liang; Han, Leng; Luo, Zhenwu; Hu, Fang; Tieu, Roger; Gan, Lin

2014-01-01

Schizophrenia is a common mental disorder with high heritability and strong genetic heterogeneity. Common disease-common variants hypothesis predicts that schizophrenia is attributable in part to common genetic variants. However, recent studies have clearly demonstrated that copy number variations (CNVs) also play pivotal roles in schizophrenia susceptibility and explain a proportion of missing heritability. Though numerous CNVs have been identified, many of the regions affected by CNVs show poor overlapping among different studies, and it is not known whether the genes disrupted by CNVs contribute to the risk of schizophrenia. By using cumulative scoring, we systematically prioritized the genes affected by CNVs in schizophrenia. We identified 8 top genes that are frequently disrupted by CNVs, including NRXN1, CHRNA7, BCL9, CYFIP1, GJA8, NDE1, SNAP29, and GJA5. Integration of genes affected by CNVs with known schizophrenia susceptibility genes (from previous genetic linkage and association studies) reveals that many genes disrupted by CNVs are also associated with schizophrenia. Further protein-protein interaction (PPI) analysis indicates that protein products of genes affected by CNVs frequently interact with known schizophrenia-associated proteins. Finally, systematic integration of CNVs prioritization data with genetic association and PPI data identifies key schizophrenia candidate genes. Our results provide a global overview of genes impacted by CNVs in schizophrenia and reveal a densely interconnected molecular network of de novo CNVs in schizophrenia. Though the prioritized top genes represent promising schizophrenia risk genes, further work with different prioritization methods and independent samples is needed to confirm these findings. Nevertheless, the identified key candidate genes may have important roles in the pathogenesis of schizophrenia, and further functional characterization of these genes may provide pivotal targets for future therapeutics and

Identification and analysis of integrons and cassette arrays in bacterial genomes.

PubMed

Cury, Jean; Jové, Thomas; Touchon, Marie; Néron, Bertrand; Rocha, Eduardo Pc

2016-06-02

Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program - IntegronFinder - to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Experimental single-strain mobilomics reveals events that shape pathogen emergence.

PubMed

Schoeniger, Joseph S; Hudson, Corey M; Bent, Zachary W; Sinha, Anupama; Williams, Kelly P

2016-08-19

Virulence genes on mobile DNAs such as genomic islands (GIs) and plasmids promote bacterial pathogen emergence. Excision is an early step in GI mobilization, producing a circular GI and a deletion site in the chromosome; circular forms are also known for some bacterial insertion sequences (ISs). The recombinant sequence at the junctions of such circles and deletions can be detected sensitively in high-throughput sequencing data, using new computational methods that enable empirical discovery of mobile DNAs. For the rich mobilome of a hospital Klebsiella pneumoniae strain, circularization junctions (CJs) were detected for six GIs and seven IS types. Our methods revealed differential biology of multiple mobile DNAs, imprecision of integrases and transposases, and differential activity among identical IS copies for IS26, ISKpn18 and ISKpn21 Using the resistance of circular dsDNA molecules to exonuclease, internally calibrated with the native plasmids, showed that not all molecules bearing GI CJs were circular. Transpositions were also detected, revealing replicon preference (ISKpn18 prefers a conjugative IncA/C2 plasmid), local action (IS26), regional preferences, selection (against capsule synthesis) and IS polarity inversion. Efficient discovery and global characterization of numerous mobile elements per experiment improves accounting for the new gene combinations that arise in emerging pathogens. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Quantitative proteomics reveals that peroxidases play key roles in post-flooding recovery in soybean roots.

PubMed

Khan, Mudassar Nawaz; Sakata, Katsumi; Hiraga, Susumu; Komatsu, Setsuko

2014-12-05

Soybean is an important legume crop that exhibits markedly reduced growth and yields under flooding conditions. To unravel the mechanisms involved in recovery after flooding in soybean root, gel-free proteomic analysis was performed. Morphological analysis revealed that growth suppression was more severe with increased flooding duration. Out of a total of 1645 and 1707 identified proteins, 73 and 21 proteins were changed significantly during the recovery stage following 2 and 4 days flooding, respectively. Based on the proteomic, clustering, and in silico protein-protein interaction analyses, six key enzymes were analyzed at the mRNA level. Lipoxygenase 1, which was increased at the protein level during the recovery period, was steadily down-regulated at the mRNA level. The peroxidase superfamily protein continuously increased in abundance during the course of recovery and was up-regulated at the mRNA level. HAD acid phosphatase was decreased at the protein level and down-regulated at the transcript level, while isoflavone reductase and an unknown protein were increased at both the protein and mRNA levels. Consistent with these findings, the enzymatic activity of peroxidase was decreased under flooding stress but increased significantly during the recovery sage. These results suggest that peroxidases might play key roles in post-flooding recovery in soybean roots through the scavenging of toxic radicals.

Molecular study on some antibiotic resistant genes in Salmonella spp. isolates

NASA Astrophysics Data System (ADS)

Nabi, Ari Q.

2017-09-01

Studying the genes related with antimicrobial resistance in Salmonella spp. is a crucial step toward a correct and faster treatment of infections caused by the pathogen. In this work Integron mediated antibiotic resistant gene IntI1 (Class I Integrase IntI1) and some plasmid mediated antibiotic resistance genes (Qnr) were scanned among the isolated non-Typhoid Salmonellae strains with known resistance to some important antimicrobial drugs using Sybr Green real time PCR. The aim of the study was to correlate the multiple antibiotics and antimicrobial resistance of Salmonella spp. with the presence of integrase (IntI1) gene and plasmid mediated quinolone resistant genes. Results revealed the presence of Class I Integrase gene in 76% of the isolates with confirmed multiple antibiotic resistances. Moreover, about 32% of the multiple antibiotic resistant serotypes showed a positive R-PCR for plasmid mediated qnrA gene encoding for nalidixic acid and ciprofloxacin resistance. No positive results could be revealed form R-PCRs targeting qnrB or qnrS. In light of these results we can conclude that the presence of at least one of the qnr genes and/or the presence of Integrase Class I gene were responsible for the multiple antibiotic resistance to for nalidixic acid and ciprofloxacin from the studied Salmonella spp. and further studies required to identify the genes related with multiple antibiotic resistance of the pathogen.

Prediction of the binding mode and resistance profile for a dual-target pyrrolyl diketo acid scaffold against HIV-1 integrase and reverse-transcriptase-associated ribonuclease H.

PubMed

Yang, Fengyuan; Zheng, Guoxun; Fu, Tingting; Li, Xiaofeng; Tu, Gao; Li, Ying Hong; Yao, Xiaojun; Xue, Weiwei; Zhu, Feng

2018-06-27

The rapid emergence of drug-resistant variants is one of the most common causes of highly active antiretroviral therapeutic (HAART) failure in patients infected with HIV-1. Compared with the existing HAART, the recently developed pyrrolyl diketo acid scaffold targeting both HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) is an efficient approach to counteract the failure of anti-HIV treatment due to drug resistance. However, the binding mode and potential resistance profile of these inhibitors with important mechanistic principles remain poorly understood. To address this issue, an integrated computational method was employed to investigate the binding mode of inhibitor JMC6F with HIV-1 IN and RNase H. By using per-residue binding free energy decomposition analysis, the following residues: Asp64, Thr66, Leu68, Asp116, Tyr143, Gln148 and Glu152 in IN, Asp443, Glu478, Trp536, Lys541 and Asp549 in RNase H were identified as key residues for JMC6F binding. And then computational alanine scanning was carried to further verify the key residues. Moreover, the resistance profile of the currently known major mutations in HIV-1 IN and 2 mutations in RNase H against JMC6F was predicted by in silico mutagenesis studies. The results demonstrated that only three mutations in HIV-1 IN (Y143C, Q148R and N155H) and two mutations in HIV-1 RNase H (Y501R and Y501W) resulted in a reduction of JMC6F potency, thus indicating their potential role in providing resistance to JMC6F. These data provided important insights into the binding mode and resistance profile of the inhibitors with a pyrrolyl diketo acid scaffold in HIV-1 IN and RNase H, which would be helpful for the development of more effective dual HIV-1 IN and RNase H inhibitors.

Three-party authenticated key agreements for optimal communication

PubMed Central

Lee, Tian-Fu; Hwang, Tzonelih

2017-01-01

Authenticated key agreements enable users to determine session keys, and to securely communicate with others over an insecure channel via the session keys. This study investigates the lower bounds on communications for three-party authenticated key agreements and considers whether or not the sub-keys for generating a session key can be revealed in the channel. Since two clients do not share any common secret key, they require the help of the server to authenticate their identities and exchange confidential and authenticated information over insecure networks. However, if the session key security is based on asymmetric cryptosystems, then revealing the sub-keys cannot compromise the session key. The clients can directly exchange the sub-keys and reduce the transmissions. In addition, authenticated key agreements were developed by using the derived results of the lower bounds on communications. Compared with related approaches, the proposed protocols had fewer transmissions and realized the lower bounds on communications. PMID:28355253

A QSAR study of integrase strand transfer inhibitors based on a large set of pyrimidine, pyrimidone, and pyridopyrazine carboxamide derivatives

NASA Astrophysics Data System (ADS)

de Campos, Luana Janaína; de Melo, Eduardo Borges

2017-08-01

In the present study, 199 compounds derived from pyrimidine, pyrimidone and pyridopyrazine carboxamides with inhibitory activity against HIV-1 integrase were modeled. Subsequently, a multivariate QSAR study was conducted with 54 molecules employed by Ordered Predictors Selection (OPS) and Partial Least Squares (PLS) for the selection of variables and model construction, respectively. Topological, electrotopological, geometric, and molecular descriptors were used. The selected real model was robust and free from chance correlation; in addition, it demonstrated favorable internal and external statistical quality. Once statistically validated, the training model was used to predict the activity of a second data set (n = 145). The root mean square deviation (RMSD) between observed and predicted values was 0.698. Although it is a value outside of the standards, only 15 (10.34%) of the samples exhibited higher residual values than 1 log unit, a result considered acceptable. Results of Williams and Euclidean applicability domains relative to the prediction showed that the predictions did not occur by extrapolation and that the model is representative of the chemical space of test compounds.

Analysis of the site-specific integration system of the Streptomyces aureofaciens phage μ1/6.

PubMed

Farkašovská, Jarmila; Godány, Andrej

2012-03-01

The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

A quantum mechanic/molecular mechanic study of the wild-type and N155S mutant HIV-1 integrase complexed with diketo acid.

PubMed

Alves, Cláudio Nahum; Martí, Sergio; Castillo, Raquel; Andrés, Juan; Moliner, Vicent; Tuñón, Iñaki; Silla, Estanislao

2008-04-01

Integrase (IN) is one of the three human immunodeficiency virus type 1 (HIV-1) enzymes essential for effective viral replication. Recently, mutation studies have been reported that have shown that a certain degree of viral resistance to diketo acids (DKAs) appears when some amino acid residues of the IN active site are mutated. Mutations represent a fascinating experimental challenge, and we invite theoretical simulations for the disclosure of still unexplored features of enzyme reactions. The aim of this work is to understand the molecular mechanisms of HIV-1 IN drug resistance, which will be useful for designing anti-HIV inhibitors with unique resistance profiles. In this study, we use molecular dynamics simulations, within the hybrid quantum mechanics/molecular mechanics (QM/MM) approach, to determine the protein-ligand interaction energy for wild-type and N155S mutant HIV-1 IN, both complexed with a DKA. This hybrid methodology has the advantage of the inclusion of quantum effects such as ligand polarization upon binding, which can be very important when highly polarizable groups are embedded in anisotropic environments, for example in metal-containing active sites. Furthermore, an energy terms decomposition analysis was performed to determine contributions of individual residues to the enzyme-inhibitor interactions. The results reveal that there is a strong interaction between the Lys-159, Lys-156, and Asn-155 residues and Mg(2+) cation and the DKA inhibitor. Our calculations show that the binding energy is higher in wild-type than in the N155S mutant, in accordance with the experimental results. The role of the mutated residue has thus been checked as maintaining the structure of the ternary complex formed by the protein, the Mg(2+) cation, and the inhibitor. These results might be useful to design compounds with more interesting anti-HIV-1 IN activity on the basis of its three-dimensional structure.

Substitutions at Amino Acid Positions 143, 148, and 155 of HIV-1 Integrase Define Distinct Genetic Barriers to Raltegravir Resistance In Vivo

PubMed Central

Fransen, Signe; Gupta, Soumi; Frantzell, Arne; Petropoulos, Christos J.

2012-01-01

Mutations at amino acids 143, 148, and 155 in HIV-1 integrase (IN) define primary resistance pathways in subjects failing raltegravir (RAL)-containing treatments. Although each pathway appears to be genetically distinct, shifts in the predominant resistant virus population have been reported under continued drug pressure. To better understand this dynamic, we characterized the RAL susceptibility of 200 resistant viruses, and we performed sequential clonal analysis for selected cases. Patient viruses containing Y143R, Q148R, or Q148H mutations consistently exhibited larger reductions in RAL susceptibility than patient viruses containing N155H mutations. Sequential analyses of virus populations from three subjects revealed temporal shifts in subpopulations representing N155H, Y143R, or Q148H escape pathways. Evaluation of molecular clones isolated from different time points demonstrated that Y143R and Q148H variants exhibited larger reductions in RAL susceptibility and higher IN-mediated replication capacity (RC) than N155H variants within the same subject. Furthermore, shifts from the N155H pathway to either the Q148R or H pathway or the Y143R pathway were dependent on the amino acid substitution at position 148 and the secondary mutations in Y143R- or Q148R- or H-containing variants and correlated with reductions in RAL susceptibility and restorations in RC. Our observations in patient viruses were confirmed by analyzing site-directed mutations. In summary, viruses that acquire mutations defining the 143 or 148 escape pathways are less susceptible to RAL and exhibit greater RC than viruses containing 155 pathway mutations. These selective pressures result in the displacement of N155H variants by 143 or 148 variants under continued drug exposure. PMID:22553340

Striking Plasticity of CRISPR-Cas9 and Key Role of Non-target DNA, as Revealed by Molecular Simulations.

PubMed

Palermo, Giulia; Miao, Yinglong; Walker, Ross C; Jinek, Martin; McCammon, J Andrew

2016-10-26

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the "closure" of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and-more importantly-call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications.

Switching regimens in virologically suppressed HIV-1-infected patients: evidence base and rationale for integrase strand transfer inhibitor (INSTI)-containing regimens.

PubMed

Raffi, F; Esser, S; Nunnari, G; Pérez-Valero, I; Waters, L

2016-10-01

In an era when most individuals with treated HIV infection can expect to live into old age, clinicians should proactively review their patients' current and future treatment needs and challenges. Clinical guidelines acknowledge that, in the setting of virological suppression, treatment switch may yield benefits in terms of tolerability, regimen simplification, adherence, convenience and long-term health considerations, particularly in the context of ageing. In this paper, we review evidence from six key clinical studies on switching virologically suppressed patients to regimens based on integrase strand transfer inhibitors (INSTIs), the antiretroviral class increasingly preferred as initial therapy in clinical guidelines. We review these studies and focus on the virological efficacy, safety, and tolerability of switching to INSTI-based regimens in suppressed HIV-positive individuals. We review the early switch studies SWITCHMRK and SPIRAL [assessing a switch from a ritonavir-boosted protease inhibitor (PI/r) to raltegravir (RAL)-containing regimens], together with data from STRATEGY-PI [assessing a switch to elvitegravir (EVG)-containing regimens; EVG/cobicistat (COBI)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF) vs. remaining on a PI/r-containing regimen], STRATEGY-NNRTI [assessing a switch to EVG/COBI/FTC/TDF vs. continuation of a nonnucleoside reverse transcriptase inhibitor (NNRTI) and two nucleoside reverse transcriptase inhibitors (NRTIs)], STRIIVING [assessing a switch to a dolutegravir (DTG)-containing regimen (abacavir (ABC)/lamivudine (3TC)/DTG) vs. staying on the background regimen], and GS study 109 [assessing a switch to EVG/COBI/FTC/tenofovir alafenamide fumarate (TAF) vs. continuation of FTC/TDF-based regimens]. Switching to INSTI-containing regimens has been shown to support good virological efficacy, with evidence from two studies demonstrating superior virological efficacy for a switch to EVG-containing regimens. In addition, switching

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation.

PubMed

Jurado, Kellie A; Wang, Hao; Slaughter, Alison; Feng, Lei; Kessl, Jacques J; Koh, Yasuhiro; Wang, Weifeng; Ballandras-Colas, Allison; Patel, Pratiq A; Fuchs, James R; Kvaratskhelia, Mamuka; Engelman, Alan

2013-05-21

Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development.

Virtual screening of integrase inhibitors by large scale binding free energy calculations: the SAMPL4 challenge

PubMed Central

Gallicchio, Emilio; Deng, Nanjie; He, Peng; Wickstrom, Lauren; Perryman, Alexander L.; Santiago, Daniel N.; Forli, Stefano; Olson, Arthur J.; Levy, Ronald M.

2014-01-01

As part of the SAMPL4 blind challenge, filtered AutoDock Vina ligand docking predictions and large scale binding energy distribution analysis method binding free energy calculations have been applied to the virtual screening of a focused library of candidate binders to the LEDGF site of the HIV integrase protein. The computational protocol leveraged docking and high level atomistic models to improve enrichment. The enrichment factor of our blind predictions ranked best among all of the computational submissions, and second best overall. This work represents to our knowledge the first example of the application of an all-atom physics-based binding free energy model to large scale virtual screening. A total of 285 parallel Hamiltonian replica exchange molecular dynamics absolute protein-ligand binding free energy simulations were conducted starting from docked poses. The setup of the simulations was fully automated, calculations were distributed on multiple computing resources and were completed in a 6-weeks period. The accuracy of the docked poses and the inclusion of intramolecular strain and entropic losses in the binding free energy estimates were the major factors behind the success of the method. Lack of sufficient time and computing resources to investigate additional protonation states of the ligands was a major cause of mispredictions. The experiment demonstrated the applicability of binding free energy modeling to improve hit rates in challenging virtual screening of focused ligand libraries during lead optimization. PMID:24504704

Efficacy and site-specificity of adenoviral vector integration mediated by the phage φC31 integrase.

PubMed

Robert, Marc-André; Zeng, Yue; Raymond, Benoît; Desfossé, Laurie; Mairey, Emilie; Tremblay, Jacques P; Massie, Bernard; Gilbert, Rénald

2012-12-01

Adenoviral vectors deleted of all their viral genes (helper-dependent [HD]) are efficient gene-transfer vehicles. Because transgene expression is rapidly lost in actively dividing cells, we investigated the feasibility of using phage φC31 integrase (φC31-Int) to integrate an HD carrying an attB site and the puromycin resistance gene into human cells (HeLa) and murine myoblasts (C2C12) by co-infection with a second HD-expressing φC31-Int. Because the HD genome is linear, we also investigated whether its circularization, through expression of Cre using a third HD, affects integration. Efficacy and specificity were determined by scoring the number of puromycin-resistant colonies and by sequencing integration sites. Unexpectedly, circularization of HD was unnecessary and it even reduced the integration efficacy. The maximum integration efficacy achieved was 0.5% in HeLa cells and 0.1% in C2C12 myoblasts. Up to 76% of the integration events occurred at pseudo attP sites and previously characterized hotspots were found. A small (two- to three-fold) increase in the number of γ-H2AX positive foci, accompanied by no noticeable change in γ-H2AX expression, indicated the low genotoxicity of φC31-Int. In conclusion, integration of HD mediated by φC31-Int is an attractive alternative to engineer cells, because it permits site-specific integration of large DNA fragments with low genotoxicity.

[Isolation and identification of the temperate bacteriophage from isolated strains of Streptococcus suis serotype 2].

PubMed

Ma, Yuling; Lu, Chengping; Fan, Hongjie

2008-04-01

A PCR assay was developed to study the distributional characteristics of phage integrase gene in Streptococcus suis serotype 2 (SS2). A 323bp distinct DNA target can be amplified in 25 strains of virulent SS2, while can not be amplified in avirulent strain T15, 5 strains of other serotypes (SS1, SS7, SS9) and strains of group C Streptococcus strains from pigs, which suggested that the phage integrase gene may be related to the pathogenicity of SS2 and can be consider as a detection factor of the virulent gene of SS2. The sequencing and restriction endonuclease analysis of the PCR products were also done. Comparisons between the sequences of phage integrase gene with that of SS2 strain, showed a high homology with SS2 China strains 98HAH33, 05ZYH33 and North American strain 89-1591. Complete cell lysis was observed with SS2 virulent strains but not with avirulent strain T15 after the induction by mitomycin C. Electron microscopy analysis of the lysate from SS2 virulent strains HA9801 and ZY05719 revealed the presence of phage particles. The induced phage, named SS2-HA and SS2-ZY, both have a small isometric nucleocapsid approximately 50 nm in diameter and have no tail and is therefore a member of the Tectiviridae family. The phage integrase gene sequence of phage SS2-HA and SS2-ZY shared high homologue identities with virulent SS2 strains, which suggested that the phage integrase gene of SS2 has high specify. The temperate phage and phage integrase gene can only detected from SS2 virulent strains but not from avirulent strain, and the detection of phage integrase gene was related to the virulence-associate factors of SS2, such as the muramidase-released protein gene (mrp), which suggested that the temperate phage of SS2 may be related to the pathogenicity of SS2.

The Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug Resistance

PubMed Central

Wainberg, Mark A.

2012-01-01

The use of highly active antiretroviral therapy (HAART) involves combinations of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. There are two broad classes of reverse transcriptase inhibitors, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first classes of such compounds were developed, viral resistance against them has necessitated the continuous development of novel compounds within each class. This paper considers the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second line therapy and describes the patterns of resistance associated with their use, as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with low genetic barrier are more commonly used in resource-limited settings. Their use results to the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings. More recently, the advent of integrase strand transfer inhibitors represents another major step forward toward control of HIV infection, but these compounds are also susceptible to problems of HIV drug resistance. PMID:24278679

Mechanism of inhibition of HIV-1 integrase by G-tetrad-forming oligonucleotides in Vitro.

PubMed

Jing, N; Marchand, C; Liu, J; Mitra, R; Hogan, M E; Pommier, Y

2000-07-14

The G-tetrad-forming oligonucleotides and have been identified as potent inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN) activity (Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGrath, M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754-1760; Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and Pommier, Y. (1996) Biochemistry 35, 13762-13771; Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). To understand the inhibition of HIV-1 IN activity by the G-quartet inhibitors, we have designed the oligonucleotides and, composed of three and four G-quartets with stem lengths of 19 and 24 A, respectively. The fact that increasing the G-quartet stem length from 15 to 24 A kept inhibition of HIV-1 IN activity unchanged suggests that the binding interaction occurs between a GTGT loop domain of the G-quartet inhibitors and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. Docking the NMR structure of (Jing and Hogan (1998)) into the x-ray structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (Maignan, S., Guilloteau, J.-P. , Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998) J. Mol. Biol. 282, 359-368), was performed using the GRAMM program. The statistical distributions of hydrogen bonding between HIV-1 IN and were obtained from the analyses of 1000 random docking structures. The docking results show a high probability of interaction between the GTGT loop residues of the G-quartet inhibitors and the catalytic site of HIV-1 IN, in agreement with the experimental observation.

The S230R Integrase Substitution Associated with Viral Rebound during DTG Monotherapy Confers Low Levels INSTI Drug Resistance.

PubMed

Pham, Hanh T; Labrie, Lydia; Wijting, Ingeborg E A; Hassounah, Said; Lok, Ka Yee; Portna, Inna; Goring, Mark; Han, Yingshan; Lungu, Cynthia; van der Ende, Marchina E; Brenner, Bluma G; Boucher, Charles A; Rijnders, Bart J A; van Kampen, Jeroen J A; Mesplède, Thibault; Wainberg, Mark A

2018-03-29

Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of HIV-infected individuals. Due to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virological failure while using DTG monotherapy. We evaluated the effect of S230R substitution in regard to IN enzyme activity, viral infectivity, replicative capacity and susceptibility to different INSTIs by biochemical and cell-based assays. S230R substitution conferred 63% reduction in enzyme efficiency. The S230R virus was 1.29-fold less infectious than wildtype (WT), but could replicate in PM1 cells without significant delay. Resistance levels against DTG, CAB, RAL and EVG in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively. Our data indicate that the S230R substitution is comparable to the previously reported R263K in some respects. Virological failure under DTG monotherapy can occur through the development of such S230R or R263K mutations without the need for high levels DTG resistance.

A long-acting integrase inhibitor protects female macaques from repeated high-dose intravaginal SHIV challenge.

PubMed

Andrews, Chasity D; Yueh, Yun Lan; Spreen, William R; St Bernard, Leslie; Boente-Carrera, Mar; Rodriguez, Kristina; Gettie, Agegnehu; Russell-Lodrigue, Kasi; Blanchard, James; Ford, Susan; Mohri, Hiroshi; Cheng-Mayer, Cecilia; Hong, Zhi; Ho, David D; Markowitz, Martin

2015-01-14

Long-acting GSK1265744 (GSK744 LA) is a strand transfer inhibitor of the HIV/SIV (simian immunodeficiency virus) integrase and was shown to be an effective preexposure prophylaxis (PrEP) agent in a low-dose intrarectal SHIV (simian-human immunodeficiency virus) rhesus macaque challenge model. We examined the pharmacokinetics and efficacy of GSK744 LA as PrEP against repeat high-dose intravaginal SHIV challenge in female rhesus macaques treated with Depo-Provera (depot medroxyprogesterone acetate), which promotes viral transmission vaginally. When Depo-Provera-treated female rhesus macaques were dosed with GSK744 LA (50 mg/kg) monthly, systemic and tissue drug concentrations were lower than previously observed in male rhesus macaques. GSK744 concentrations were fivefold lower on average in cervical tissues than in rectal tissues. Eight female rhesus macaques were treated with GSK744 LA at week 0, and four female rhesus macaques served as controls. All animals received a high-dose challenge of SHIV162P3 at week 1. No infection was detected in GSK744 LA-treated rhesus macaques, whereas viremia was detected 1 to 2 weeks after SHIV challenge in all control animals. The GSK744 LA-treated rhesus macaques were given a second administration of drug at week 4 and further challenged at weeks 5 and 7. GSK744 LA treatment protected six of eight female rhesus macaques against three high-dose SHIV challenges, whereas all control animals became infected after the first challenge (P = 0.0003, log-rank test). These results support further clinical development of GSK744 LA for PrEP. Copyright © 2015, American Association for the Advancement of Science.

Multiple Genetic Pathways Involving Amino Acid Position 143 of HIV-1 Integrase Are Preferentially Associated with Specific Secondary Amino Acid Substitutions and Confer Resistance to Raltegravir and Cross-Resistance to Elvitegravir

PubMed Central

Frantzell, Arne; Fransen, Signe; Petropoulos, Christos J.

2013-01-01

Y143C,R substitutions in HIV-1 integrase define one of three primary raltegravir (RAL) resistance pathways. Here we describe clinical isolates with alternative substitutions at position 143 (Y143A, Y143G, Y143H, and Y143S [Y143A,G,H,S]) that emerge less frequently, and we compare the genotypic and phenotypic profiles of these viruses to Y143C,R viruses to reconcile the preferential selection of Y143C,R variants during RAL treatment. Integrase amino acid sequences and RAL susceptibility were characterized in 117 patient isolates submitted for drug resistance testing and contained Y143 amino acid changes. The influence of specific Y143 substitutions on RAL susceptibility and their preferential association with particular secondary substitutions were further defined by evaluating the composition of patient virus populations along with a large panel of site-directed mutants. Our observations demonstrate that the RAL resistance profiles of Y143A,G,H,S viruses and their association with specific secondary substitutions are similar to the well-established Y143C profile but distinct from the Y143R profile. Y143R viruses differ from Y143A,C,G,H,S viruses in that Y143R confers a greater reduction in RAL susceptibility as a single substitution, consistent with a lower resistance barrier. Among Y143A,C,G,H,S viruses, the higher prevalence of Y143C viruses is the result of a lower genetic barrier than that of the Y143A,G,S viruses and a lower resistance barrier than that of the Y143H viruses. In addition, Y143A,C,G,H,S viruses require multiple secondary substitutions to develop large reductions in RAL susceptibility. Patient-derived viruses containing Y143 substitutions exhibit cross-resistance to elvitegravir. PMID:23733474

Human Immunodeficiency Virus Type 1 cDNA Integration: New Aromatic Hydroxylated Inhibitors and Studies of the Inhibition Mechanism

PubMed Central

Farnet, C. M.; Wang, B.; Hansen, M.; Lipford, J. R.; Zalkow, L.; Robinson, W. E.; Siegel, J.; Bushman, F.

1998-01-01

Integration of the human immunodeficiency virus type 1 (HIV-1) cDNA is a required step for viral replication. Integrase, the virus-encoded enzyme important for integration, has not yet been exploited as a target for clinically useful inhibitors. Here we report on the identification of new polyhydroxylated aromatic inhibitors of integrase including ellagic acid, purpurogallin, 4,8,12-trioxatricornan, and hypericin, the last of which is known to inhibit viral replication. These compounds and others were characterized in assays with subviral preintegration complexes (PICs) isolated from HIV-1-infected cells. Hypericin was found to inhibit PIC assays, while the other compounds tested were inactive. Counterscreening of these and other integrase inhibitors against additional DNA-modifying enzymes revealed that none of the polyhydroxylated aromatic compounds are active against enzymes that do not require metals (methylases, a pox virus topoisomerase). However, all were cross-reactive with metal-requiring enzymes (restriction enzymes, a reverse transcriptase), implicating metal atoms in the inhibitory mechanism. In mechanistic studies, we localized binding of some inhibitors to the catalytic domain of integrase by assaying competition of binding by labeled nucleotides. These findings help elucidate the mechanism of action of the polyhydroxylated aromatic inhibitors and provide practical guidance for further inhibitor development. PMID:9736543

Integrase inhibitors in late pregnancy and rapid HIV viral load reduction.

PubMed

Rahangdale, Lisa; Cates, Jordan; Potter, JoNell; Badell, Martina L; Seidman, Dominika; Miller, Emilly S; Coleman, Jenell S; Lazenby, Gweneth B; Levison, Judy; Short, William R; Yawetz, Sigal; Ciaranello, Andrea; Livingston, Elizabeth; Duthely, Lunthita; Rimawi, Bassam H; Anderson, Jean R; Stringer, Elizabeth M

2016-03-01

Minimizing time to HIV viral suppression is critical in pregnancy. Integrase strand transfer inhibitors (INSTIs), like raltegravir, are known to rapidly suppress plasma HIV RNA in nonpregnant adults. There are limited data in pregnant women. We describe time to clinically relevant reduction in HIV RNA in pregnant women using INSTI-containing and non-INSTI-containing antiretroviral therapy (ART) options. We conducted a retrospective cohort study of pregnant HIV-infected women in the United States from 2009 through 2015. We included women who initiated ART, intensified their regimen, or switched to a new regimen due to detectable viremia (HIV RNA >40 copies/mL) at ≥20 weeks gestation. Among women with a baseline HIV RNA permitting 1-log reduction, we estimated time to 1-log RNA reduction using the Kaplan-Meier estimator comparing women starting/adding an INSTI in their regimen vs other ART. To compare groups with similar follow-up time, we also conducted a subgroup analysis limited to women with ≤14 days between baseline and follow-up RNA data. This study describes 101 HIV-infected pregnant women from 11 US clinics. In all, 75% (76/101) of women were not taking ART at baseline; 24 were taking non-INSTI containing ART, and 1 received zidovudine monotherapy. In all, 39% (39/101) of women started an INSTI-containing regimen or added an INSTI to their ART regimen. Among 90 women with a baseline HIV RNA permitting 1-log reduction, the median time to 1-log RNA reduction was 8 days (interquartile range [IQR], 7-14) in the INSTI group vs 35 days (IQR, 20-53) in the non-INSTI ART group (P < .01). In a subgroup of 39 women with first and last RNA measurements ≤14 days apart, median time to 1-log reduction was 7 days (IQR, 6-10) in the INSTI group vs 11 days (IQR, 10-14) in the non-INSTI group (P < .01). ART that includes INSTIs appears to induce more rapid viral suppression than other ART regimens in pregnancy. Inclusion of an INSTI may play a role in optimal reduction

Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

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

Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira

2012-01-06

Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors,more » therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.« less

Mutations in Nonconserved Domains of Ty3 Integrase Affect Multiple Stages of the Ty3 Life Cycle

PubMed Central

Nymark-McMahon, M. Henrietta; Sandmeyer, Suzanne B.

1999-01-01

Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3′ ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3′ ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle. PMID:9847351

Many-objective optimization and visual analytics reveal key trade-offs for London's water supply

NASA Astrophysics Data System (ADS)

Matrosov, Evgenii S.; Huskova, Ivana; Kasprzyk, Joseph R.; Harou, Julien J.; Lambert, Chris; Reed, Patrick M.

2015-12-01

In this study, we link a water resource management simulator to multi-objective search to reveal the key trade-offs inherent in planning a real-world water resource system. We consider new supplies and demand management (conservation) options while seeking to elucidate the trade-offs between the best portfolios of schemes to satisfy projected water demands. Alternative system designs are evaluated using performance measures that minimize capital and operating costs and energy use while maximizing resilience, engineering and environmental metrics, subject to supply reliability constraints. Our analysis shows many-objective evolutionary optimization coupled with state-of-the art visual analytics can help planners discover more diverse water supply system designs and better understand their inherent trade-offs. The approach is used to explore future water supply options for the Thames water resource system (including London's water supply). New supply options include a new reservoir, water transfers, artificial recharge, wastewater reuse and brackish groundwater desalination. Demand management options include leakage reduction, compulsory metering and seasonal tariffs. The Thames system's Pareto approximate portfolios cluster into distinct groups of water supply options; for example implementing a pipe refurbishment program leads to higher capital costs but greater reliability. This study highlights that traditional least-cost reliability constrained design of water supply systems masks asset combinations whose benefits only become apparent when more planning objectives are considered.

Strand transfer inhibitors of HIV-1 integrase: bringing IN a new era of antiretroviral therapy.

PubMed

McColl, Damian J; Chen, Xiaowu

2010-01-01

HIV-1 integrase (IN) is one of three essential enzymes (along with reverse transcriptase and protease) encoded by the viral pol gene. IN mediates two critical reactions during viral replication; firstly 3'-end processing (3'EP) of the double-stranded viral DNA ends and then strand transfer (STF) which joins the viral DNA to the host chromosomal DNA forming a functional integrated proviral DNA. IN is a 288 amino acid protein containing three functional domains, the N-terminal domain (NTD), catalytic core domain (CCD) and the C-terminal domain (CTD). The CCD contains three conserved catalytic residues, Asp64, Asp116 and Glu152, which coordinate divalent metal ions essential for the STF reaction. Intensive research over the last two decades has led to the discovery and development of small molecule inhibitors of the IN STF reaction (INSTIs). INSTIs are catalytic inhibitors of IN, and act to chelate the divalent metal ions in the CCD. One INSTI, raltegravir (RAL, Merck Inc.) was approved in late 2007 for the treatment of HIV-1 infection in patients with prior antiretroviral (ARV) treatment experience and was recently approved also for first line therapy. A second INSTI, elvitegravir (EVG, Gilead Sciences, Inc.) is currently undergoing phase 3 studies in ARV treatment-experienced patients and phase 2 studies in ARV naïve patients as part of a novel fixed dose combination. Several additional INSTIs are in early stage clinical development. This review will discuss the discovery and development of this novel class of antiretrovirals. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, Vol 85, issue 1, 2010. Copyright 2009. Published by Elsevier B.V.

Experimental single-strain mobilomics reveals events that shape pathogen emergence

DOE PAGES

Schoeniger, Joseph S.; Hudson, Corey M.; Bent, Zachary W.; ...

2016-07-04

Virulence and resistance genes carried on mobile DNAs such as genomic islands (GIs) and plasmids promote bacterial pathogen emergence. An early step in the mobilization of GIs is their excision, which produces both a circular form of the GI and a deletion site in the chromosome; circular forms have also been described for some bacterial insertion sequences (ISs). We demonstrate that the recombinant sequence produced at the junction of such circles, and their corresponding deletion sites, can be detected sensitively in high throughput sequencing data, using new computational methods that enable empirical discovery of new mobile DNAs. Applied to themore » rich mobilome of a single strain (Kpn2146) of the emerging multidrug-resistant pathogen Klebsiella pneumoniae, our approach detected circular junctions for six GIs and seven IS types (several of the latter not previously known to circularize). Our methods further revealed differential biology of multiple mobile DNAs, imprecision of integrases and transposases, and differential activity among identical IS copies for IS26, ISKpn18 and ISKpn21. Exonuclease was used to enrich for circular dsDNA molecules, and internal calibration with the native Kpn2146 plasmids showed that not all molecules bearing GI and IS circular junctions were circular dsDNAs. Transposition events were also detected, revealing replicon preference (ISKpn18 preferring a conjugative IncA/C2 plasmid), local action (IS26), regional preferences, selection (against capsule synthesis), and left-right IS end swapping. Efficient discovery and global characterization of numerous mobile elements per experiment will allow detailed accounting of bacterial evolution, explaining the new gene combinations that arise in emerging pathogens.« less

Site-specific recombination in the cyanobacterium Anabaena sp. strain PCC 7120 catalyzed by the integrase of coliphage HK022.

PubMed

Melnikov, Olga; Zaritsky, Arieh; Zarka, Aliza; Boussiba, Sammy; Malchin, Natalia; Yagil, Ezra; Kolot, Mikhail

2009-07-01

The integrase (Int) of the lambda-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP x attB and attL x attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate P(glnA)-attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter P(glnA). The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.

A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads.

PubMed

He, Hong-qiu; Ma, Xiao-hui; Liu, Bin; Chen, Wei-zu; Wang, Cun-xin; Cheng, Shao-hui

2008-03-01

To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs. The donor DNA duplex, with a sequence identical to the U5 end of HIV-1 long terminal repeats, is labeled at its 5' end with biotin (BIO). The target DNA duplex is labeled at its 3' end with digoxin (DIG). IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product. Streptavidin-coated magnetic beads were used to capture the product, and the amount of DIG was measured as the ST reaction product. The assay was optimized in 96-well microplate format for high-throughput screening purpose. Moreover, the assay was applied in a ST reaction character study, and the efficiency of the assay in the identification of antiviral compounds was tested. The end-point values, measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings. The ST reaction character and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays. The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9. The assay presented here has been proven to be rapid, sensitive, and specific for the detection of IN ST activity, the reaction character study, as well as for the identification of antiviral drugs targeting IN.

Histone deacetylase inhibition rescues gene knockout levels achieved with integrase-defective lentiviral vectors encoding zinc-finger nucleases.

PubMed

Pelascini, Laetitia P L; Maggio, Ignazio; Liu, Jin; Holkers, Maarten; Cathomen, Toni; Gonçalves, Manuel A F V

2013-12-01

Zinc-finger nucleases (ZFNs) work as dimers to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. In doing so, they constitute powerful triggers to edit and to interrogate the function of genomic sequences in higher eukaryotes. A preferred route to introduce ZFNs into somatic cells relies on their cotransduction with two integrase-defective lentiviral vectors (IDLVs) each encoding a monomer of a functional heterodimeric pair. The episomal nature of IDLVs diminishes the risk of genotoxicity and ensures the strict transient expression profile necessary to minimize deleterious effects associated with long-term ZFN activity. However, by deploying IDLVs and conventional lentiviral vectors encoding HPRT1- or eGFP-specific ZFNs, we report that DSB formation at target alleles is limited after IDLV-mediated ZFN transfer. This IDLV-specific underperformance stems, to a great extent, from the activity of chromatin-remodeling histone deacetylases (HDACs). Importantly, the prototypic and U.S. Food and Drug Administration-approved inhibitors of metal-dependent HDACs, trichostatin A and vorinostat, respectively, did not hinder illegitimate recombination-mediated repair of targeted chromosomal DSBs. This allowed rescuing IDLV-mediated site-directed mutagenesis to levels approaching those achieved by using their isogenic chromosomally integrating counterparts. Hence, HDAC inhibition constitutes an efficacious expedient to incorporate in genome-editing strategies based on transient IDLV-mediated ZFN expression. Finally, we compared two of the most commonly used readout systems to measure targeted gene knockout activities based on restriction and mismatch-sensitive endonucleases. These experiments indicate that these enzymatic assays display a similar performance.

Comparison of Multiple Molecular Dynamics Trajectories Calculated for the Drug-Resistant HIV-1 Integrase T66I/M154I Catalytic Domain

PubMed Central

Brigo, Alessandro; Lee, Keun Woo; Iurcu Mustata, Gabriela; Briggs, James M.

2005-01-01

HIV-1 integrase (IN) is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multidrug therapy of AIDS. Single and multiple mutations of IN at residues T66, S153, or M154 confer degrees of resistance to several inhibitors that prevent the enzyme from performing its normal strand transfer activity. Four different conformations of IN were chosen from a prior molecular dynamics (MD) simulation on the modeled IN T66I/M154I catalytic core domain as starting points for additional MD studies. The aim of this article is to understand the dynamic features that may play roles in the catalytic activity of the double mutant enzyme in the absence of any inhibitor. Moreover, we want to verify the influence of using different starting points on the MD trajectories and associated dynamical properties. By comparison of the trajectories obtained from these MD simulations we have demonstrated that the starting point does not affect the conformational space explored by this protein and that the time of the simulation is long enough to achieve convergence for this system. PMID:15764656

On the security of a simple three-party key exchange protocol without server's public keys.

PubMed

Nam, Junghyun; Choo, Kim-Kwang Raymond; Park, Minkyu; Paik, Juryon; Won, Dongho

2014-01-01

Authenticated key exchange protocols are of fundamental importance in securing communications and are now extensively deployed for use in various real-world network applications. In this work, we reveal major previously unpublished security vulnerabilities in the password-based authenticated three-party key exchange protocol according to Lee and Hwang (2010): (1) the Lee-Hwang protocol is susceptible to a man-in-the-middle attack and thus fails to achieve implicit key authentication; (2) the protocol cannot protect clients' passwords against an offline dictionary attack; and (3) the indistinguishability-based security of the protocol can be easily broken even in the presence of a passive adversary. We also propose an improved password-based authenticated three-party key exchange protocol that addresses the security vulnerabilities identified in the Lee-Hwang protocol.

On the Security of a Simple Three-Party Key Exchange Protocol without Server's Public Keys

PubMed Central

Nam, Junghyun; Choo, Kim-Kwang Raymond; Park, Minkyu; Paik, Juryon; Won, Dongho

2014-01-01

Authenticated key exchange protocols are of fundamental importance in securing communications and are now extensively deployed for use in various real-world network applications. In this work, we reveal major previously unpublished security vulnerabilities in the password-based authenticated three-party key exchange protocol according to Lee and Hwang (2010): (1) the Lee-Hwang protocol is susceptible to a man-in-the-middle attack and thus fails to achieve implicit key authentication; (2) the protocol cannot protect clients' passwords against an offline dictionary attack; and (3) the indistinguishability-based security of the protocol can be easily broken even in the presence of a passive adversary. We also propose an improved password-based authenticated three-party key exchange protocol that addresses the security vulnerabilities identified in the Lee-Hwang protocol. PMID:25258723

The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein.

PubMed

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L; Levin, Henry L

2008-06-06

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.

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

Yin, Zhiqi; Shi, Ke; Banerjee, Surajit

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNAmore » remained elusive. In this paper, we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Finally, our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.« less

Crystal structure of the Rous sarcoma virus intasome

DOE PAGES

Yin, Zhiqi; Shi, Ke; Banerjee, Surajit; ...

2016-02-17

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNAmore » remained elusive. In this paper, we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Finally, our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.« less

Dissecting the mechanism of histone deacetylase inhibitors to enhance the activity of zinc finger nucleases delivered by integrase-defective lentiviral vectors.

PubMed

Joglekar, Alok V; Stein, Libby; Ho, Michelle; Hoban, Megan D; Hollis, Roger P; Kohn, Donald B

2014-07-01

Integrase-defective lentiviral vectors (IDLVs) have been of limited success in the delivery of zinc finger nucleases (ZFNs) to human cells, due to low expression. A reason for reduced gene expression has been proposed to involve the epigenetic silencing of vector genomes, carried out primarily by histone deacetylases (HDACs). In this study, we tested valproic acid (VPA), a known HDAC inhibitor (HDACi), for its ability to increase transgene expression from IDLVs, especially in the context of ZFN delivery. Using ZFNs targeting the human adenosine deaminase (ADA) gene in K562 cells, we demonstrated that treatment with VPA enhanced ZFN expression by up to 3-fold, resulting in improved allelic disruption at the ADA locus. Furthermore, three other U.S. Food and Drug Administration-approved HDACis (vorinostat, givinostat, and trichostatin-A) exhibited a similar effect on the activity of ZFN-IDLVs in K562 cells. In primary human CD34(+) cells, VPA- and vorinostat-treated cells showed higher levels of expression of both green fluorescent protein (GFP) as well as ZFNs from IDLVs. A major mechanism for the effects of HDAC inhibitors on improving expression was from their modulation of the cell cycle, and the influence of heterochromatinization was determined to be a lesser contributing factor.

Multi-Omics Reveals that Lead Exposure Disturbs Gut Microbiome Development, Key Metabolites, and Metabolic Pathways.

PubMed

Gao, Bei; Chi, Liang; Mahbub, Ridwan; Bian, Xiaoming; Tu, Pengcheng; Ru, Hongyu; Lu, Kun

2017-04-17

Lead exposure remains a global public health issue, and the recent Flint water crisis has renewed public concern about lead toxicity. The toxicity of lead has been well established in a variety of systems and organs. The gut microbiome has been shown to be highly involved in many critical physiological processes, including food digestion, immune system development, and metabolic homeostasis. However, despite the key role of the gut microbiome in human health, the functional impact of lead exposure on the gut microbiome has not been studied. The aim of this study is to define gut microbiome toxicity induced by lead exposure in C57BL/6 mice using multiomics approaches, including 16S rRNA sequencing, whole genome metagenomics sequencing, and gas chromatography-mass spectrometry (GC-MS) metabolomics. 16S rRNA sequencing revealed that lead exposure altered the gut microbiome trajectory and phylogenetic diversity. Metagenomics sequencing and metabolomics profiling showed that numerous metabolic pathways, including vitamin E, bile acids, nitrogen metabolism, energy metabolism, oxidative stress, and the defense/detoxification mechanism, were significantly disturbed by lead exposure. These perturbed molecules and pathways may have important implications for lead toxicity in the host. Taken together, these results demonstrated that lead exposure not only altered the gut microbiome community structures/diversity but also greatly affected metabolic functions, leading to gut microbiome toxicity.

Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance.

PubMed

Fun, Axel; Leitner, Thomas; Vandekerckhove, Linos; Däumer, Martin; Thielen, Alexander; Buchholz, Bernd; Hoepelman, Andy I M; Gisolf, Elizabeth H; Schipper, Pauline J; Wensing, Annemarie M J; Nijhuis, Monique

2018-01-05

Emergence of resistance against integrase inhibitor raltegravir in human immunodeficiency virus type 1 (HIV-1) patients is generally associated with selection of one of three signature mutations: Y143C/R, Q148K/H/R or N155H, representing three distinct resistance pathways. The mechanisms that drive selection of a specific pathway are still poorly understood. We investigated the impact of the HIV-1 genetic background and population dynamics on the emergence of raltegravir resistance. Using deep sequencing we analyzed the integrase coding sequence (CDS) in longitudinal samples from five patients who initiated raltegravir plus optimized background therapy at viral loads > 5000 copies/ml. To investigate the role of the HIV-1 genetic background we created recombinant viruses containing the viral integrase coding region from pre-raltegravir samples from two patients in whom raltegravir resistance developed through different pathways. The in vitro selections performed with these recombinant viruses were designed to mimic natural population bottlenecks. Deep sequencing analysis of the viral integrase CDS revealed that the virological response to raltegravir containing therapy inversely correlated with the relative amount of unique sequence variants that emerged suggesting diversifying selection during drug pressure. In 4/5 patients multiple signature mutations representing different resistance pathways were observed. Interestingly, the resistant population can consist of a single resistant variant that completely dominates the population but also of multiple variants from different resistance pathways that coexist in the viral population. We also found evidence for increased diversification after stronger bottlenecks. In vitro selections with low viral titers, mimicking population bottlenecks, revealed that both recombinant viruses and HXB2 reference virus were able to select mutations from different resistance pathways, although typically only one resistance pathway

Pathogenicity Island Cross Talk Mediated by Recombination Directionality Factors Facilitates Excision from the Chromosome.

PubMed

Carpenter, Megan R; Rozovsky, Sharon; Boyd, E Fidelma

2015-12-14

Pathogenicity islands (PAIs) are mobile integrated genetic elements (MIGEs) that contain a diverse range of virulence factors and are essential in the evolution of pathogenic bacteria. PAIs are widespread among bacteria and integrate into the host genome, commonly at a tRNA locus, via integrase-mediated site-specific recombination. The excision of PAIs is the first step in the horizontal transfer of these elements and is not well understood. In this study, we examined the role of recombination directionality factors (RDFs) and their relationship with integrases in the excision of two PAIs essential for Vibrio cholerae host colonization: Vibrio pathogenicity island 1 (VPI-1) and VPI-2. VPI-1 does not contain an RDF, which allowed us to answer the question of whether RDFs are an absolute requirement for excision. We found that an RDF was required for efficient excision of VPI-2 but not VPI-1 and that RDFs can induce excision of both islands. Expression data revealed that the RDFs act as transcriptional repressors to both VPI-1- and VPI-2-encoded integrases. We demonstrated that the RDFs Vibrio excision factor A (VefA) and VefB bind at the attachment sites (overlapping the int promoter region) of VPI-1 and VPI-2, thus supporting this mode of integrase repression. In addition, V. cholerae RDFs are promiscuous due to their dual functions of promoting excision of both VPI-1 and VPI-2 and acting as negative transcriptional regulators of the integrases. This is the first demonstration of cross talk between PAIs mediated via RDFs which reveals the complex interactions that occur between separately acquired MIGEs. Deciphering the mechanisms of pathogenicity island excision is necessary for understanding the evolution and spread of these elements to their nonpathogenic counterparts. Such mechanistic insight would assist in predicting the mobility of uncharacterized genetic elements. This study identified extensive RDF-mediated cross talk between two nonhomologous VPIs and

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein*S⃞

PubMed Central

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L.; Levin, Henry L.

2008-01-01

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the γ-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining. PMID:18397885

New insights into the interaction between pyrrolyl diketoacids and HIV-1 integrase active site and comparison with RNase H.

PubMed

Corona, Angela; di Leva, Francesco Saverio; Rigogliuso, Giuseppe; Pescatori, Luca; Madia, Valentina Noemi; Subra, Frederic; Delelis, Olivier; Esposito, Francesca; Cadeddu, Marta; Costi, Roberta; Cosconati, Sandro; Novellino, Ettore; di Santo, Roberto; Tramontano, Enzo

2016-10-01

HIV-1 integrase (IN) inhibitors are one of the most recent innovations in the treatment of HIV infection. The selection of drug resistance viral strains is however a still open issue requiring constant efforts to identify new anti-HIV-1 drugs. Pyrrolyl diketo acid (DKA) derivatives inhibit HIV-1 replication by interacting with the Mg 2+ cofactors within the HIV-1 IN active site or within the HIV-1 reverse-transcriptase associated ribonuclease H (RNase H) active site. While the interaction mode of pyrrolyl DKAs with the RNase H active site has been recently reported and substantiated by mutagenesis experiments, their interaction within the IN active site still lacks a detailed understanding. In this study, we investigated the binding mode of four pyrrolyl DKAs to the HIV-1 IN active site by molecular modeling coupled with site-directed mutagenesis studies showing that the DKA pyrrolyl scaffold primarily interacts with the IN amino residues P145, Q146 and Q148. Importantly, the tested DKAs demonstrated good effectiveness against HIV-1 Raltegravir resistant Y143A and N155H INs, thus showing an interaction pattern with relevant differences if compared with the first generation IN inhibitors. These data provide precious insights for the design of new HIV inhibitors active on clinically selected Raltegravir resistant variants. Furthermore, this study provides new structural information to modulate IN and RNase H inhibitory activities for development of dual-acting anti-HIV agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of dolutegravir functional monotherapy on HIV-1 virological response in integrase strand transfer inhibitor resistant patients.

PubMed

Naeger, Lisa K; Harrington, Patrick; Komatsu, Takashi; Deming, Damon

2016-01-01

VIKING-4 assessed the safety and efficacy of dolutegravir in heavily antiretroviral treatment-experienced patients who had documented integrase strand transfer inhibitor (INSTI) resistance-associated substitutions in their HIV. VIKING-4 had a placebo-controlled 7-day dolutegravir functional monotherapy phase followed by dolutegravir plus an optimized background regimen for 48 weeks. Independent resistance analyses evaluated week 48 virological responses in the VIKING-4 trial based on the presence of baseline INSTI resistance-associated substitutions and baseline dolutegravir phenotypic susceptibility. Response rates at week 48 based on baseline dolutegravir resistance subgroups were compared for the 7-day dolutegravir functional monotherapy arm and placebo-control arm. Additionally, genotypic and phenotypic resistance at day 8 and time of failure was analysed for the virological failures from both arms. Week 48 response rates for VIKING-4 were 23% (3/13) in the 7-day dolutegravir functional monotherapy arm compared with 60% (9/15) in the 7-day placebo arm. Response rates were consistently lower in the dolutegravir functional monotherapy arm across baseline INSTI genotypic and phenotypic subgroups. There was a higher proportion of virological failures in the 7-day dolutegravir functional monotherapy arm (n=6/13; 46%) compared with the 7-day placebo arm (n=3/15; 20%). Additionally, five virological failures in the dolutegravir arm had virus expressing emergent INSTI resistance-associated substitutions compared with two in the placebo arm. Analysis of response rates and resistance emergence in VIKING-4 suggests careful consideration should be given to the duration of functional monotherapy in future studies of highly treatment-experienced patients to reduce the risk of resistance and virological failure.

Autoshaping of key pecking in pigeons with negative reinforcement.

PubMed

Rachlin, H

1969-07-01

Pigeons exposed to gradually increasing intensities of pulsing electric shock pecked a key and thereby reduced the intensity of shock to zero for 2 min. Acquisition of key pecking was brought about through an autoshaping process in which periodic brief keylight presentations immediately preceded automatic reduction of the shock. On the occasions of such automatic reduction of shock preceding the first measured key peck, little or no orientation to the key was observed. Observations of pigeons with autoshaping of positive reinforcement also revealed little evidence of orientation toward the key.

New Insights into the Classification and Integration Specificity of Streptococcus Integrative Conjugative Elements through Extensive Genome Exploration.

PubMed

Ambroset, Chloé; Coluzzi, Charles; Guédon, Gérard; Devignes, Marie-Dominique; Loux, Valentin; Lacroix, Thomas; Payot, Sophie; Leblond-Bourget, Nathalie

2015-01-01

Recent genome analyses suggest that integrative and conjugative elements (ICEs) are widespread in bacterial genomes and therefore play an essential role in horizontal transfer. However, only a few of these elements are precisely characterized and correctly delineated within sequenced bacterial genomes. Even though previous analysis showed the presence of ICEs in some species of Streptococci, the global prevalence and diversity of ICEs was not analyzed in this genus. In this study, we searched for ICEs in the completely sequenced genomes of 124 strains belonging to 27 streptococcal species. These exhaustive analyses revealed 105 putative ICEs and 26 slightly decayed elements whose limits were assessed and whose insertion site was identified. These ICEs were grouped in seven distinct unrelated or distantly related families, according to their conjugation modules. Integration of these streptococcal ICEs is catalyzed either by a site-specific tyrosine integrase, a low-specificity tyrosine integrase, a site-specific single serine integrase, a triplet of site-specific serine integrases or a DDE transposase. Analysis of their integration site led to the detection of 18 target-genes for streptococcal ICE insertion including eight that had not been identified previously (ftsK, guaA, lysS, mutT, rpmG, rpsI, traG, and ebfC). It also suggests that all specificities have evolved to minimize the impact of the insertion on the host. This overall analysis of streptococcal ICEs emphasizes their prevalence and diversity and demonstrates that exchanges or acquisitions of conjugation and recombination modules are frequent.

Transcriptome analysis reveals key roles of AtLBR-2 in LPS-induced defense responses in plants.

PubMed

Iizasa, Sayaka; Iizasa, Ei'ichi; Watanabe, Keiichi; Nagano, Yukio

2017-12-29

Lipopolysaccharide (LPS) from Gram-negative bacteria cause innate immune responses in animals and plants. The molecules involved in LPS signaling in animals are well studied, whereas those in plants are not yet as well documented. Recently, we identified Arabidopsis AtLBR-2, which binds to LPS from Pseudomonas aeruginosa (pLPS) directly and regulates pLPS-induced defense responses, such as pathogenesis-related 1 (PR1) expression and reactive oxygen species (ROS) production. In this study, we investigated the pLPS-induced transcriptomic changes in wild-type (WT) and the atlbr-2 mutant Arabidopsis plants using RNA-Seq technology. RNA-Seq data analysis revealed that pLPS treatment significantly altered the expression of 2139 genes, with 605 up-regulated and 1534 down-regulated genes in WT. Gene ontology (GO) analysis on these genes showed that GO terms, "response to bacterium", "response to salicylic acid (SA) stimulus", and "response to abscisic acid (ABA) stimulus" were enriched amongst only in up-regulated genes, as compared to the genes that were down-regulated. Comparative analysis of differentially expressed genes between WT and the atlbr-2 mutant revealed that 65 genes were up-regulated in WT but not in the atlbr-2 after pLPS treatment. Furthermore, GO analysis on these 65 genes demonstrated their importance for the enrichment of several defense-related GO terms, including "response to bacterium", "response to SA stimulus", and "response to ABA stimulus". We also found reduced levels of pLPS-induced conjugated SA glucoside (SAG) accumulation in atlbr-2 mutants, and no differences were observed in the gene expression levels in SA-treated WT and the atlbr-2 mutants. These 65 AtLBR-2-dependent up-regulated genes appear to be important for the enrichment of some defense-related GO terms. Moreover, AtLBR-2 might be a key molecule that is indispensable for the up-regulation of defense-related genes and for SA signaling pathway, which is involved in defense against

Improving the Transduction of Bone Marrow–Derived Cells with an Integrase-Defective Lentiviral Vector

PubMed Central

Pay, S. Louise; Qi, Xiaoping; Willard, Jeffrey F.; Godoy, Juliana; Sankhavaram, Kavya; Horton, Ranier; Mitter, Sayak K.; Quigley, Judith L.; Chang, Lung-Ji; Grant, Maria B.; Boulton, Michael E.

2018-01-01

In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow–derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe.

PubMed

Fogg, Paul C M; Haley, Joshua A; Stark, W Marshall; Smith, Margaret C M

2017-03-01

Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces , most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor , by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different

Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe

PubMed Central

Haley, Joshua A.; Stark, W. Marshall

2016-01-01

ABSTRACT Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In Streptomyces, most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new Streptomyces phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest Streptomyces phage cluster (R4-like) and encodes a serine integrase. The attB site from Streptomyces venezuelae was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe int-attP locus. The attB site for ϕJoe integrase was occupied in several Streptomyces genomes, including that of S. coelicolor, by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed in vivo. Both the integrase and RDF were active in in vitro recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. IMPORTANCE Streptomyces spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the Streptomyces chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with

miR-142-3p is associated with aberrant Wingless/Integrase I (WNT) signaling during airway remodeling in asthma.

PubMed

Bartel, Sabine; Carraro, Gianni; Alessandrini, Francesca; Krauss-Etschmann, Susanne; Ricciardolo, Fabio L M; Bellusci, Saverio

2018-05-03

Asthma is characterized by a chronic inflammation and remodeling of the airways. While inflammation can be controlled, therapeutic options to revert remodeling do not exist. Thus, there is a large and unmet need to understand the underlying molecular mechanisms in order to develop novel therapies. we previously identified a pivotal role for miR-142-3p in regulating airway smooth muscle precursor (ASM) cell proliferation during lung development by fine-tuning the Wingless/Integrase I (WNT) signaling. Thus, we here aimed to investigate the relevance of this interaction in asthma. We performed qRT-PCR and immune-staining in a murine model for ovalbumin-induced allergic airway inflammation and in bronchial biopsies from patients with asthma and isolated primary fibroblasts thereof. miR-142-3p was increased in hyper-proliferative regions of lung in murine and human asthma, while this miRNA was excluded from regions with differentiated ASM cells. Increases in miR-142-3p were associated with a decrease of its known target Adenomatous polyposis coli (Apc). Further, we observed a differential expression of miR-142-3p in bronchial biopsies from patients with early or late onset severe asthma, which coincided with a differential WNT signature. Our data suggest that miR-142-3p is involved in regulating the balance between proliferation and differentiation of ASM cells in asthma, possibly via controlling WNT signaling. Thus, this miRNA might be an interesting target to prevent airway smooth muscle hyper-proliferation in asthma.

1-Hydroxypyrido[2,3-d]pyrimidin-2(1H)-ones as novel selective HIV integrase inhibitors obtained via privileged substructure-based compound libraries.

PubMed

Gao, Ping; Zhang, Lingzi; Sun, Lin; Huang, Tianguang; Tan, Jing; Zhang, Jian; Zhou, Zhongxia; Zhao, Tong; Menéndez-Arias, Luis; Pannecouque, Christophe; Clercq, Erik De; Zhan, Peng; Liu, Xinyong

2017-10-15

A small library containing 3-hydroxyquinazoline-2,4(1H,3H)-dione and 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one scaffolds was obtained via the copper(I)-catalyzed azidealkyne cycloaddition (CuAAC) reaction and evaluated for their anti-HIV activity in MT-4 cells. Among the synthesized compounds, several 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-one derivatives showed remarkable anti-HIV potency with EC 50 values ranging from 0.92 to 26.85µM. The most active one, IIA-2, also showed remarkable and selective potency against HIV type 1 integrase (IN). To the best of our knowledge, this is the first report showing that 1-hydroxypyrido[2,3-d]pyrimidin-2(1H)-ones are selective HIV IN inhibitors. Preliminary structure-activity relationship (SAR) studies suggested that the divalent metal ion chelators and the nature and position of substituents around the core are important for antiviral potency. Molecular modeling has been used to predict the binding site of the pyrido[2,3-d]pyrimidin-2(1H)-one core in HIV type 1 IN and suggestions are made for improvement of its inhibitory activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Valuing snorkeling visits to the Florida Keys with stated and revealed preference models

Treesearch

Timothy Park; J. Michael Bowker; Vernon R. Leeworthy

2002-01-01

Coastal coral reefs, especially in the Florida Keys, are declining at a disturbing rate. Marine ecologists and reef scientists have emphasized the importance of establishing nonmarket values of coral reefs to assess the cost effectiveness of coral reef management and remediation programs. The purpose of this paper is to develop a travel cost--contingent valuation model...

Changing the spatial pattern of TFL1 expression reveals its key role in the shoot meristem in controlling Arabidopsis flowering architecture

PubMed Central

Baumann, Kim; Venail, Julien; Berbel, Ana; Domenech, Maria Jose; Money, Tracy; Conti, Lucio; Hanzawa, Yoshie; Madueno, Francisco; Bradley, Desmond

2015-01-01

Models for the control of above-ground plant architectures show how meristems can be programmed to be either shoots or flowers. Molecular, genetic, transgenic, and mathematical studies have greatly refined these models, suggesting that the phase of the shoot reflects different genes contributing to its repression of flowering, its vegetativeness (‘veg’), before activators promote flower development. Key elements of how the repressor of flowering and shoot meristem gene TFL1 acts have now been tested, by changing its spatiotemporal pattern. It is shown that TFL1 can act outside of its normal expression domain in leaf primordia or floral meristems to repress flower identity. These data show how the timing and spatial pattern of TFL1 expression affect overall plant architecture. This reveals that the underlying pattern of TFL1 interactors is complex and that they may be spatially more widespread than TFL1 itself, which is confined to shoots. However, the data show that while TFL1 and floral genes can both act and compete in the same meristem, it appears that the main shoot meristem is more sensitive to TFL1 rather than floral genes. This spatial analysis therefore reveals how a difference in response helps maintain the ‘veg’ state of the shoot meristem. PMID:26019254

Expression Profiling of Regulatory and Biosynthetic Genes in Contrastingly Anthocyanin Rich Strawberry (Fragaria × ananassa) Cultivars Reveals Key Genetic Determinants of Fruit Color.

PubMed

Hossain, Mohammad Rashed; Kim, Hoy-Taek; Shanmugam, Ashokraj; Nath, Ujjal Kumar; Goswami, Gayatri; Song, Jae-Young; Park, Jong-In; Nou, Ill-Sup

2018-02-26

Anthocyanins are the resultant end-point metabolites of phenylapropanoid/flavonoid (F/P) pathway which is regulated at transcriptional level via a series of structural genes. Identifying the key genes and their potential interactions can provide us with the clue for novel points of intervention for improvement of the trait in strawberry. We profiled the expressions of putative regulatory and biosynthetic genes of cultivated strawberry in three developmental and characteristically colored stages of fruits of contrastingly anthocyanin rich cultivars: Tokun, Maehyang and Soelhyang. Besides FaMYB10, a well-characterized positive regulator, FaMYB5 , FabHLH3 and FabHLH3-delta might also act as potential positive regulators, while FaMYB11 , FaMYB9 , FabHLH33 and FaWD44-1 as potential negative regulators of anthocyanin biosynthesis in these high-anthocyanin cultivars. Among the early BGs, Fa4CL7 , FaF3H , FaCHI1 , FaCHI3 , and FaCHS, and among the late BGs, FaDFR4-3 , FaLDOX , and FaUFGT2 showed significantly higher expression in ripe fruits of high anthocyanin cultivars Maehyang and Soelhyang. Multivariate analysis revealed the association of these genes with total anthocyanins. Increasingly higher expressions of the key genes along the pathway indicates the progressive intensification of pathway flux leading to final higher accumulation of anthocyanins. Identification of these key genetic determinants of anthocyanin regulation and biosynthesis in Korean cultivars will be helpful in designing crop improvement programs.

Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

PubMed

Kawabe, Yoshinori; Shimomura, Takuya; Huang, Shuohao; Imanishi, Suguru; Ito, Akira; Kamihira, Masamichi

2016-07-01

Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre

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

Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae

EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengthsmore » of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.« less

Developmental transcriptional profiling reveals key insights into Triticeae reproductive development.

PubMed

Tran, Frances; Penniket, Carolyn; Patel, Rohan V; Provart, Nicholas J; Laroche, André; Rowland, Owen; Robert, Laurian S

2013-06-01

Despite their importance, there remains a paucity of large-scale gene expression-based studies of reproductive development in species belonging to the Triticeae. As a first step to address this deficiency, a gene expression atlas of triticale reproductive development was generated using the 55K Affymetrix GeneChip(®) wheat genome array. The global transcriptional profiles of the anther/pollen, ovary and stigma were analyzed at concurrent developmental stages, and co-expressed as well as preferentially expressed genes were identified. Data analysis revealed both novel and conserved regulatory factors underlying Triticeae floral development and function. This comprehensive resource rests upon detailed gene annotations, and the expression profiles are readily accessible via a web browser. © 2013 Her Majesty the Queen in Right of Canada as represented by the Minister of Agriculture and Agri-Food Canada.

New Insights into the Classification and Integration Specificity of Streptococcus Integrative Conjugative Elements through Extensive Genome Exploration

PubMed Central

Ambroset, Chloé; Coluzzi, Charles; Guédon, Gérard; Devignes, Marie-Dominique; Loux, Valentin; Lacroix, Thomas; Payot, Sophie; Leblond-Bourget, Nathalie

2016-01-01

Recent genome analyses suggest that integrative and conjugative elements (ICEs) are widespread in bacterial genomes and therefore play an essential role in horizontal transfer. However, only a few of these elements are precisely characterized and correctly delineated within sequenced bacterial genomes. Even though previous analysis showed the presence of ICEs in some species of Streptococci, the global prevalence and diversity of ICEs was not analyzed in this genus. In this study, we searched for ICEs in the completely sequenced genomes of 124 strains belonging to 27 streptococcal species. These exhaustive analyses revealed 105 putative ICEs and 26 slightly decayed elements whose limits were assessed and whose insertion site was identified. These ICEs were grouped in seven distinct unrelated or distantly related families, according to their conjugation modules. Integration of these streptococcal ICEs is catalyzed either by a site-specific tyrosine integrase, a low-specificity tyrosine integrase, a site-specific single serine integrase, a triplet of site-specific serine integrases or a DDE transposase. Analysis of their integration site led to the detection of 18 target-genes for streptococcal ICE insertion including eight that had not been identified previously (ftsK, guaA, lysS, mutT, rpmG, rpsI, traG, and ebfC). It also suggests that all specificities have evolved to minimize the impact of the insertion on the host. This overall analysis of streptococcal ICEs emphasizes their prevalence and diversity and demonstrates that exchanges or acquisitions of conjugation and recombination modules are frequent. PMID:26779141

Improvement of the activity of the anti-HIV-1 integrase aptamer T30175 by introducing a modified thymidine into the loops.

PubMed

Virgilio, Antonella; Amato, Teresa; Petraccone, Luigi; Esposito, Francesca; Grandi, Nicole; Tramontano, Enzo; Romero, Raquel; Haider, Shozeb; Gomez-Monterrey, Isabel; Novellino, Ettore; Mayol, Luciano; Esposito, Veronica; Galeone, Aldo

2018-05-10

In this paper, we report our investigations on analogues of the anti-human immunodeficiency virus type 1 (HIV-1) integrase (IN) aptamer T30175 in which the individual thymidines forming the loops were replaced by 5-hydroxymethyl-2'-deoxyuridine residues (H). Circular dichroism, nuclear magnetic resonance and gel electrophoresis investigations clearly indicated that all the modified aptamers preserve the ability to form the original 5'-5' end-stacked head-to-head dimeric G-quadruplex structure, in which each G-quadruplex adopts a parallel arrangement and is characterized by three G-tetrads, three propeller loops and one bulge-loop. All the modified aptamers were tested in an IN inhibition LEDGF-independent assay. While the modified aptamers INTB-H13 and INTB-H17 showed IC 50 values comparable with that of the parent aptamer (INTB-nat), analogues INTB-H2, INTB-H5 and, to a lesser extent, INTB-H9 showed a higher ability to inhibit the HIV IN than the unmodified aptamer. Molecular modelling studies evaluating the aptamer/HIV IN interaction highlighted the ability of the modified thymidines to establish several contacts with the target protein. All the data point to the importance of loops in the aptamer/target interaction and suggest that the site-specific replacement of loop residues with commercially available analogues can be considered a straightforward strategy to improve the biological activities of several G-quadruplex aptamers.

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

PubMed

Atwood, A; Lin, J H; Levin, H L

1996-01-01

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Experimental realization of equiangular three-state quantum key distribution

PubMed Central

Schiavon, Matteo; Vallone, Giuseppe; Villoresi, Paolo

2016-01-01

Quantum key distribution using three states in equiangular configuration combines a security threshold comparable with the one of the Bennett-Brassard 1984 protocol and a quantum bit error rate (QBER) estimation that does not need to reveal part of the key. We implement an entanglement-based version of the Renes 2004 protocol, using only passive optic elements in a linear scheme for the positive-operator valued measure (POVM), generating an asymptotic secure key rate of more than 10 kbit/s, with a mean QBER of 1.6%. We then demonstrate its security in the case of finite key and evaluate the key rate for both collective and general attacks. PMID:27465643

Autoshaping of key pecking in pigeons with negative reinforcement1

PubMed Central

Rachlin, Howard

1969-01-01

Pigeons exposed to gradually increasing intensities of pulsing electric shock pecked a key and thereby reduced the intensity of shock to zero for 2 min. Acquisition of key pecking was brought about through an autoshaping process in which periodic brief keylight presentations immediately preceded automatic reduction of the shock. On the occasions of such automatic reduction of shock preceding the first measured key peck, little or no orientation to the key was observed. Observations of pigeons with autoshaping of positive reinforcement also revealed little evidence of orientation toward the key. ImagesFig. 3.Fig. 4. PMID:16811371

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crystallographic Fragment Screening of the Catalytic Core Domain.

PubMed

Patel, Disha; Antwi, Janet; Koneru, Pratibha C; Serrao, Erik; Forli, Stefano; Kessl, Jacques J; Feng, Lei; Deng, Nanjie; Levy, Ronald M; Fuchs, James R; Olson, Arthur J; Engelman, Alan N; Bauman, Joseph D; Kvaratskhelia, Mamuka; Arnold, Eddy

2016-11-04

HIV-1 integrase (IN) is essential for virus replication and represents an important multifunctional therapeutic target. Recently discovered quinoline-based allosteric IN inhibitors (ALLINIs) potently impair HIV-1 replication and are currently in clinical trials. ALLINIs exhibit a multimodal mechanism of action by inducing aberrant IN multimerization during virion morphogenesis and by competing with IN for binding to its cognate cellular cofactor LEDGF/p75 during early steps of HIV-1 infection. However, quinoline-based ALLINIs impose a low genetic barrier for the evolution of resistant phenotypes, which highlights a need for discovery of second-generation inhibitors. Using crystallographic screening of a library of 971 fragments against the HIV-1 IN catalytic core domain (CCD) followed by a fragment expansion approach, we have identified thiophenecarboxylic acid derivatives that bind at the CCD-CCD dimer interface at the principal lens epithelium-derived growth factor (LEDGF)/p75 binding pocket. The most active derivative (5) inhibited LEDGF/p75-dependent HIV-1 IN activity in vitro with an IC 50 of 72 μm and impaired HIV-1 infection of T cells at an EC 50 of 36 μm The identified lead compound, with a relatively small molecular weight (221 Da), provides an optimal building block for developing a new class of inhibitors. Furthermore, although structurally distinct thiophenecarboxylic acid derivatives target a similar pocket at the IN dimer interface as the quinoline-based ALLINIs, the lead compound, 5, inhibited IN mutants that confer resistance to quinoline-based compounds. Collectively, our findings provide a plausible path for structure-based development of second-generation ALLINIs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The history of antiretrovirals: key discoveries over the past 25 years.

PubMed

De Clercq, Erik

2009-09-01

Within 25 years after zidovudine (3'-azido-2',3'-dideoxythymidine, AZT) was first described as an inhibitor of HIV replication, 25 anti-HIV drugs have been formally approved for clinical use in the treatment of HIV infections: seven nucleoside reverse transcriptase inhibitors (NRTIs): zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir and emtricitabine; one nucleotide reverse transcriptase inhibitor (NtRTI): tenofovir [in its oral prodrug form: tenofovir disoproxil fumarate (TDF)]; four non-nucleoside reverse transcriptase inhibitors (NNRTIs): nevirapine, delavirdine, efavirenz and etravirine; ten protease inhibitors (PIs): saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir; one fusion inhibitor (FI): enfuvirtide; one co-receptor inhibitor (CRI): maraviroc and one integrase inhibitor (INI): raltegravir. These compounds are used in various drug combination (some at fixed dose) regimens so as to achieve the highest possible benefit and tolerability, and to diminish the risk of virus-drug resistance development. (c) 2009 John Wiley & Sons, Ltd.

Revealing of photon-number splitting attack on quantum key distribution system by photon-number resolving devices

NASA Astrophysics Data System (ADS)

Gaidash, A. A.; Egorov, V. I.; Gleim, A. V.

2016-08-01

Quantum cryptography allows distributing secure keys between two users so that any performed eavesdropping attempt would be immediately discovered. However, in practice an eavesdropper can obtain key information from multi-photon states when attenuated laser radiation is used as a source of quantum states. In order to prevent actions of an eavesdropper, it is generally suggested to implement special cryptographic protocols, like decoy states or SARG04. In this paper, we describe an alternative method based on monitoring photon number statistics after detection. We provide a useful rule of thumb to estimate approximate order of difference of expected distribution and distribution in case of attack. Formula for calculating a minimum value of total pulses or time-gaps to resolve attack is shown. Also formulas for actual fraction of raw key known to Eve were derived. This method can therefore be used with any system and even combining with mentioned special protocols.

Secure remote synchronization and secure key distribution in electro-optic networks revealed by symmetries

NASA Astrophysics Data System (ADS)

Xu, Mingfeng; Pan, Wei; Zhang, Liyue

2018-07-01

Despite the intuition that synchronization of different nodes in coupled oscillator networks results from information exchange between them, it has recently been shown that remote nodes could be partially synchronous even when they are separated by intermediately unsynchronized nodes. Here based on electro-optic system, we report on a more stronger form of such synchronization pattern that is termed as secure remote synchronization, in which two remotely separated nodes could have identically synchronized dynamical behaviors while the rest of the network are both statistically and information-theoretically incoherent relative to the two synchronized nodes. The generalized form of mirror symmetry in the network structure is identified to be a key mechanism allowing for secure remote synchronization. Moreover, this synchronization mode is robust against a wild range of system parameters and noise perturbing the intermediary dynamics. The lack of information about the synchronized dynamics in the rest of the network suggests that our results could potentially lead to network-based solutions for secure key distribution and secure communication.

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

Chen, Qi; Cheng, Xiaolin; Univ. of Tennessee, Knoxville, TN

Although Elvitegravir (EVG) is a newly developed antiretrovirals drug to treat the acquired immunodeficiency syndrome (AIDS), drug resistance has already been found in clinic, such as E92Q/N155H and Q148H/G140S. Several structural investigations have already been reported to reveal the molecular mechanism of the drug resistance. As full length crystal structure for HIV-1 integrase is still unsolved, we use in this paper the crystal structure of the full length prototype foamy virus (PFV) in complex with virus DNA and inhibitor Elvitegravir as a template to construct the wild type and E92Q/N155H mutant system of HIV-1 integrase. Molecular dynamic simulations was usedmore » to revel the binding mode and the drug resistance of the EVG ligand in E92Q/N155H. Several important interactions were discovered between the mutated residues and the residues in the active site of the E92Q/N155H double mutant pattern, and cross correlation and clustering methods were used for detailed analysis. The results from the MD simulation studies will be used to guide the experimental efforts of developing novel inhibitors against drug-resistant HIV integrase mutants.« less

Hologram quantitative structure-activity relationship and comparative molecular field analysis studies within a series of tricyclic phthalimide HIV-1 integrase inhibitors.

PubMed

Magalhães, Uiaran de Oliveira; Souza, Alessandra Mendonça Teles de; Albuquerque, Magaly Girão; Brito, Monique Araújo de; Bello, Murilo Lamim; Cabral, Lucio Mendes; Rodrigues, Carlos Rangel

2013-01-01

Acquired immunodeficiency syndrome is a public health problem worldwide caused by the Human immunodeficiency virus (HIV). Treatment with antiretroviral drugs is the best option for viral suppression, reducing morbidity and mortality. However, viral resistance in HIV-1 therapy has been reported. HIV-1 integrase (IN) is an essential enzyme for effective viral replication and an attractive target for the development of new inhibitors. In the study reported here, two- and three-dimensional quantitative structure-activity relationship (2D/3D-QSAR) studies, applying hologram quantitative structure-activity relationship (HQSAR) and comparative molecular field analysis (CoMFA) methods, respectively, were performed on a series of tricyclic phthalimide HIV-1 IN inhibitors. The best HQSAR model (q (2) = 0.802, r (2) = 0.972) was obtained using atoms, bonds, and connectivity as the fragment distinction, a fragment size of 2-5 atoms, hologram length of 61 bins, and six components. The best CoMFA model (q (2) = 0.748, r (2) = 0.974) was obtained with alignment of all atoms of the tricyclic phthalimide moiety (alignment II). The HQSAR contribution map identified that the carbonyl-hydroxy-aromatic nitrogen motif made a positive contribution to the activity of the compounds. Furthermore, CoMFA contour maps suggested that bulky groups in meta and para positions in the phenyl ring would increase the biological activity of this class. The conclusions of this work may lead to a better understanding of HIV-1 IN inhibition and contribute to the design of new and more potent derivatives.

Hologram quantitative structure–activity relationship and comparative molecular field analysis studies within a series of tricyclic phthalimide HIV-1 integrase inhibitors

PubMed Central

de Oliveira Magalhães, Uiaran; de Souza, Alessandra Mendonça Teles; Albuquerque, Magaly Girão; de Brito, Monique Araújo; Bello, Murilo Lamim; Cabral, Lucio Mendes; Rodrigues, Carlos Rangel

2013-01-01

Acquired immunodeficiency syndrome is a public health problem worldwide caused by the Human immunodeficiency virus (HIV). Treatment with antiretroviral drugs is the best option for viral suppression, reducing morbidity and mortality. However, viral resistance in HIV-1 therapy has been reported. HIV-1 integrase (IN) is an essential enzyme for effective viral replication and an attractive target for the development of new inhibitors. In the study reported here, two- and three-dimensional quantitative structure–activity relationship (2D/3D-QSAR) studies, applying hologram quantitative structure–activity relationship (HQSAR) and comparative molecular field analysis (CoMFA) methods, respectively, were performed on a series of tricyclic phthalimide HIV-1 IN inhibitors. The best HQSAR model (q2 = 0.802, r2 = 0.972) was obtained using atoms, bonds, and connectivity as the fragment distinction, a fragment size of 2–5 atoms, hologram length of 61 bins, and six components. The best CoMFA model (q2 = 0.748, r2 = 0.974) was obtained with alignment of all atoms of the tricyclic phthalimide moiety (alignment II). The HQSAR contribution map identified that the carbonyl-hydroxy-aromatic nitrogen motif made a positive contribution to the activity of the compounds. Furthermore, CoMFA contour maps suggested that bulky groups in meta and para positions in the phenyl ring would increase the biological activity of this class. The conclusions of this work may lead to a better understanding of HIV-1 IN inhibition and contribute to the design of new and more potent derivatives. PMID:24039405

Transcriptome Analysis of Three Sheep Intestinal Regions reveals Key Pathways and Hub Regulatory Genes of Large Intestinal Lipid Metabolism.

PubMed

Chao, Tianle; Wang, Guizhi; Ji, Zhibin; Liu, Zhaohua; Hou, Lei; Wang, Jin; Wang, Jianmin

2017-07-13

The large intestine, also known as the hindgut, is an important part of the animal digestive system. Recent studies on digestive system development in ruminants have focused on the rumen and the small intestine, but the molecular mechanisms underlying sheep large intestine metabolism remain poorly understood. To identify genes related to intestinal metabolism and to reveal molecular regulation mechanisms, we sequenced and compared the transcriptomes of mucosal epithelial tissues among the cecum, proximal colon and duodenum. A total of 4,221 transcripts from 3,254 genes were identified as differentially expressed transcripts. Between the large intestine and duodenum, differentially expressed transcripts were found to be significantly enriched in 6 metabolism-related pathways, among which PPAR signaling was identified as a key pathway. Three genes, CPT1A, LPL and PCK1, were identified as higher expression hub genes in the large intestine. Between the cecum and colon, differentially expressed transcripts were significantly enriched in 5 lipid metabolism related pathways, and CEPT1 and MBOAT1 were identified as hub genes. This study provides important information regarding the molecular mechanisms of intestinal metabolism in sheep and may provide a basis for further study.

Redefinition and unification of the SXT/R391 family of integrative and conjugative elements.

PubMed

Bioteau, Audrey; Durand, Romain; Burrus, Vincent

2018-04-13

Integrative and conjugative elements (ICEs) of the SXT/R391 family are key drivers of the spread of antibiotic resistance in Vibrio cholerae , the infectious agent of cholera, and other pathogenic bacteria. The SXT/R391 family of ICEs was defined based on the conservation of a core set of 52 genes and site-specific integration into the 5' end of the chromosomal gene prfC Hence, the integrase gene int has been intensively used as a marker to detect SXT/R391 ICEs in clinical isolates. ICEs sharing most core genes but differing by their integration site and integrase gene have been recently reported and excluded from the SXT/R391 family. Here we explored the prevalence and diversity of atypical ICEs in Genbank databases and their relationship with typical SXT/R391 ICEs. We found atypical ICEs in V. cholerae isolates that predate the emergence and expansion of typical SXT/R391 ICEs in the mid-1980s in seventh pandemic toxigenic V. cholerae O1 and O139 strains. Our analyses revealed that while atypical ICEs are not associated with antibiotic resistance genes, they often carry cation efflux pumps suggesting heavy metal resistance. Atypical ICEs constitute a polyphyletic group likely because of occasional recombination events with typical ICEs. Furthermore, we show that the alternative integration and excision genes of atypical ICEs remain under the control of SetCD, the main activator of the conjugative functions of SXT/R391 ICEs. Together these observations indicate that substitution of the integration/excision module and change of specificity of integration do not preclude atypical ICEs from inclusion into the SXT/R391 family. Importance Vibrio cholerae is the causative agent of cholera, an acute intestinal infection that remains to this day a world public health threat. Integrative and conjugative elements (ICEs) of the SXT/R391 family have played a major role in spreading antimicrobial resistance in seventh pandemic V. cholerae but also in several species of

In vivo phosphoproteome characterization reveals key starch granule-binding phosphoproteins involved in wheat water-deficit response.

PubMed

Chen, Guan-Xing; Zhen, Shou-Min; Liu, Yan-Lin; Yan, Xing; Zhang, Ming; Yan, Yue-Ming

2017-10-23

Drought stress during grain development causes significant yield loss in cereal production. The phosphorylated modification of starch granule-binding proteins (SGBPs) is an important mechanism regulating wheat starch biosynthesis. In this study, we performed the first proteomics and phosphoproteomics analyses of SGBPs in elite Chinese bread wheat (Triticum aestivum L.) cultivar Jingdong 17 under well-watered and water-stress conditions. Water stress treatment caused significant reductions in spike grain numbers and weight, total starch and amylopectin content, and grain yield. Two-dimensional gel electrophoresis revealed that the quantity of SGBPs was reduced significantly by water-deficit treatment. Phosphoproteome characterization of SGBPs under water-deficit treatment demonstrated a reduced level of phosphorylation of main starch synthesis enzymes, particularly for granule-bound starch synthase (GBSS I), starch synthase II-a (SS II-a), and starch synthase III (SS III). Specifically, the Ser34 site of the GBSSI protein, the Tyr358 site of SS II-a, and the Ser837 site of SS III-a exhibited significant less phosphorylation under water-deficit treatment than well-watered treatment. Furthermore, the expression levels of several key genes related with starch biosynthesis detected by qRT-PCR were decreased significantly at 15 days post-anthesis under water-deficit treatment. Immunolocalization showed a clear movement of GBSS I from the periphery to the interior of starch granules during grain development, under both water-deficit and well-watered conditions. Our results demonstrated that the reduction in gene expression or transcription level, protein expression and phosphorylation levels of starch biosynthesis related enzymes under water-deficit conditions is responsible for the significant decrease in total starch content and grain yield.

Phage-host interactions analysis of newly characterized Oenococcus oeni bacteriophages: Implications for malolactic fermentation in wine.

PubMed

Costantini, Antonella; Doria, Francesca; Saiz, Juan-Carlos; Garcia-Moruno, Emilia

2017-04-04

Nowadays, only few phages infecting Oenococcus oeni, the principal lactic acid bacteria (LAB) species responsible for malolactic fermentation (MLF) in wine, have been characterized. In the present study, to better understanding the factors affecting the lytic activity of Oenococcus phages, fifteen O. oeni bacteriophages have been studied in detail, both with molecular and microbiological methods. No correlations were found between genome sizes, type of integrase genes, or morphology and the lytic activity of the 15 tested phages. Interestingly, though phage attack in a wine at the end of alcoholic fermentation seems not to be a problem, it can indeed represent a risk factor for MLF when the alcohol content is low, feature that may be a key point for choosing the appropriate time for malolactic starter inoculation. Additionally, it was observed that some phages genomes bear 2 or 3 types of integrase genes, which point to horizontal gene transfer between O. oeni bacteriophages. Copyright © 2017. Published by Elsevier B.V.

Clinical Improvement by Switching to an Integrase Strand Transfer Inhibitor in Hemophiliac Patients with HIV: The Japan Cohort Study of HIV Patients Infected through Blood Products.

PubMed

Kawado, Miyuki; Hashimoto, Shuji; Oka, Shin-Ichi; Fukutake, Katsuyuki; Higasa, Satoshi; Yatsuhashi, Hiroshi; Ogane, Miwa; Okamoto, Manabu; Shirasaka, Takuma

2017-01-01

This study aimed to determine improvement in HIV RNA levels and the CD4 cell count by switching to an antiretroviral regimen with an integrase strand transfer inhibitor (INSTI) in patients with HIV. This study was conducted on Japanese patients with HIV who were infected by blood products in the 1980s. Data were collected between 2007 and 2014. Data of 564 male hemophiliac patients with HIV from the Japan Cohort Study of HIV Patients Infected through Blood Products were available. Changes in antiretroviral regimen use, HIV RNA levels, and the CD4 cell count between 2007 and 2014 were examined. From 2007 to 2014, the proportion of use of a regimen with an INSTI increased from 0.0% to 41.0%. For patients with HIV who used a regimen, including an INSTI, the proportion of HIV RNA levels <50 copies/mL significantly increased from 58.3% in 2007 to 90.6% in 2014. Additionally, the median CD4 cell count significantly increased from 380/μL to 438/μL. There is a large effect of switching to an antiretroviral regimen with an INSTI for Japanese patients with HIV who are infected by blood products. This suggests that performing this switch in clinical practice will lead to favorable effects.

Structure-Based Mutational Analysis of the C-Terminal DNA-Binding Domain of Human Immunodeficiency Virus Type 1 Integrase: Critical Residues for Protein Oligomerization and DNA Binding

PubMed Central

Lutzke, Ramon A. Puras; Plasterk, Ronald H. A.

1998-01-01

The C-terminal domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a dimer that binds to DNA in a nonspecific manner. The structure of the minimal region required for DNA binding (IN220–270) has been solved by nuclear magnetic resonance spectroscopy. The overall fold of the C-terminal domain of HIV-1 IN is similar to those of Src homology region 3 domains. Based on the structure of IN220–270, we studied the role of 15 amino acid residues potentially involved in DNA binding and oligomerization by mutational analysis. We found that two amino acid residues, arginine 262 and leucine 234, contribute to DNA binding in the context of IN220–270, as indicated by protein-DNA UV cross-link analysis. We also analyzed mutant proteins representing portions of the full-length IN protein. Amino acid substitution of residues located in the hydrophobic dimer interface, such as L241A and L242A, results in the loss of oligomerization of IN; consequently, the levels of 3′ processing, DNA strand transfer, and intramolecular disintegration are strongly reduced. These results suggest that dimerization of the C-terminal domain of IN is important for correct multimerization of IN. PMID:9573250

A giant sediment trap in the Florida keys

USGS Publications Warehouse

Shinn, E.A.; Reich, C.D.; Locker, S.D.; Hine, A.C.

1996-01-01

Aerial photography, high-resolution seismic profiling, coring and jet probing have revealed a large sediment-filled sinkhole in the Key Largo National Marine Sanctuary off Key Largo, Florida. The 600-m-diameter feature straddles coral reef and carbonate-sand facies and contains >55 m of marine lime sand and aragonite mud. Bulk 14C age determinations of mud from a 30- m sediment core indicate infilling rates exceeding 20 m/ka between 3 and 5.6 ka. The total thickness and nature of the sediment near the base of the sinkhole are not known.

Linear indices of the "molecular pseudograph's atom adjacency matrix": definition, significance-interpretation, and application to QSAR analysis of flavone derivatives as HIV-1 integrase inhibitors.

PubMed

Marrero-Ponce, Yovani

2004-01-01

process of a set of flavone derivatives inhibitors of HIV-1 integrase. Quantitative models found are significant from a statistical point of view (R of 0.965, 0.902, and 0.927, respectively) and permit a clear interpretation of the studied properties in terms of the structural features of molecules. A LOO cross-validation procedure revealed that the regression models had a fairly good predictability (q2 of 0.679, 0.543, and 0.721, respectively). The comparison with other approaches reveals good behavior of the method proposed. The approach described in this paper appears to be an excellent alternative or guides for discovery and optimization of new lead compounds.

Genome Alignment Spanning Major Poaceae Lineages Reveals Heterogeneous Evolutionary Rates and Alters Inferred Dates for Key Evolutionary Events.

PubMed

Wang, Xiyin; Wang, Jingpeng; Jin, Dianchuan; Guo, Hui; Lee, Tae-Ho; Liu, Tao; Paterson, Andrew H

2015-06-01

Multiple comparisons among genomes can clarify their evolution, speciation, and functional innovations. To date, the genome sequences of eight grasses representing the most economically important Poaceae (grass) clades have been published, and their genomic-level comparison is an essential foundation for evolutionary, functional, and translational research. Using a formal and conservative approach, we aligned these genomes. Direct comparison of paralogous gene pairs all duplicated simultaneously reveal striking variation in evolutionary rates among whole genomes, with nucleotide substitution slowest in rice and up to 48% faster in other grasses, adding a new dimension to the value of rice as a grass model. We reconstructed ancestral genome contents for major evolutionary nodes, potentially contributing to understanding the divergence and speciation of grasses. Recent fossil evidence suggests revisions of the estimated dates of key evolutionary events, implying that the pan-grass polyploidization occurred ∼96 million years ago and could not be related to the Cretaceous-Tertiary mass extinction as previously inferred. Adjusted dating to reflect both updated fossil evidence and lineage-specific evolutionary rates suggested that maize subgenome divergence and maize-sorghum divergence were virtually simultaneous, a coincidence that would be explained if polyploidization directly contributed to speciation. This work lays a solid foundation for Poaceae translational genomics. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Quantitative Analysis of HIV-1 Preintegration Complexes

PubMed Central

Engelman, Alan; Oztop, Ilker; Vandegraaff, Nick; Raghavendra, Nidhanapati K.

2009-01-01

Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3′ processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3′ processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3′-OHs to the 5′-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3′ processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3′ processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3′ processing and DNA strand transfer activities. PMID:19233280

Proteomic analyses reveal the key roles of BrlA and AbaA in biogenesis of gliotoxin in Aspergillus fumigatus

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

Shin, Kwang-Soo, E-mail: shinks@dju.kr; Kim, Young Hwan; Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764

2015-07-31

The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found thatmore » the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus. - Highlights: • Proteome analyses of WT and mutants reveal 13 differentially expressed proteins. • The GliT and GliM proteins are significantly down-regulated by ΔabaA and ΔbrlA. • Expression of other gliotoxin biosynthetic genes is lowered by ΔabaA and ΔbrlA. • Growth of ΔbrlA strain lacking GliT is completely inhibited by exogenous gliotoxin. • BrlA and AbaA play key roles in biogenesis of gliotoxin in Aspergillus fumigatus.« less

Rapid activity prediction of HIV-1 integrase inhibitors: harnessing docking energetic components for empirical scoring by chemometric and artificial neural network approaches

NASA Astrophysics Data System (ADS)

Thangsunan, Patcharapong; Kittiwachana, Sila; Meepowpan, Puttinan; Kungwan, Nawee; Prangkio, Panchika; Hannongbua, Supa; Suree, Nuttee

2016-06-01

Improving performance of scoring functions for drug docking simulations is a challenging task in the modern discovery pipeline. Among various ways to enhance the efficiency of scoring function, tuning of energetic component approach is an attractive option that provides better predictions. Herein we present the first development of rapid and simple tuning models for predicting and scoring inhibitory activity of investigated ligands docked into catalytic core domain structures of HIV-1 integrase (IN) enzyme. We developed the models using all energetic terms obtained from flexible ligand-rigid receptor dockings by AutoDock4, followed by a data analysis using either partial least squares (PLS) or self-organizing maps (SOMs). The models were established using 66 and 64 ligands of mercaptobenzenesulfonamides for the PLS-based and the SOMs-based inhibitory activity predictions, respectively. The models were then evaluated for their predictability quality using closely related test compounds, as well as five different unrelated inhibitor test sets. Weighting constants for each energy term were also optimized, thus customizing the scoring function for this specific target protein. Root-mean-square error (RMSE) values between the predicted and the experimental inhibitory activities were determined to be <1 (i.e. within a magnitude of a single log scale of actual IC50 values). Hence, we propose that, as a pre-functional assay screening step, AutoDock4 docking in combination with these subsequent rapid weighted energy tuning methods via PLS and SOMs analyses is a viable approach to predict the potential inhibitory activity and to discriminate among small drug-like molecules to target a specific protein of interest.

Transcriptome Analysis of Tomato Flower Pedicel Tissues Reveals Abscission Zone-Specific Modulation of Key Meristem Activity Genes

PubMed Central

Sun, Xiuli; Zhang, Rongzhi; Wu, Liang; Liang, Yanchun; Mao, Long

2013-01-01

Tomato flower abscises at the anatomically distinct abscission zone that separates the pedicel into basal and apical portions. During abscission, cell separation occurs only at the abscission zone indicating distinctive molecular regulation in its cells. We conducted a transcriptome analysis of tomato pedicel tissues during ethylene promoted abscission. We found that the abscission zone was the most active site with the largest set of differentially expressed genes when compared with basal and apical portions. Gene Ontology analyses revealed enriched transcription regulation and hydrolase activities in the abscission zone. We also demonstrate coordinated responses of hormone and cell wall related genes. Besides, a number of ESTs representing homologs of key Arabidopsis shoot apical meristem activity genes were found to be preferentially expressed in the abscission zone, including WUSCHEL (WUS), KNAT6, LATERAL ORGAN BOUNDARIES DOMAIN PROTEIN 1(LBD1), and BELL-like homeodomain protein 1 (BLH1), as well as tomato axillary meristem genes BLIND (Bl) and LATERAL SUPPRESSOR (Ls). More interestingly, the homologs of WUS and the potential functional partner OVATE FAMILIY PROTEIN (OFP) were subsequently down regulated during abscission while Bl and AGL12 were continuously and specifically induced in the abscission zone. The expression patterns of meristem activity genes corroborate the idea that cells of the abscission zone confer meristem-like nature and coincide with the course of abscission and post-abscission cell differentiation. Our data therefore propose a possible regulatory scheme in tomato involving meristem genes that may be required not only for the abscission zone development, but also for abscission. PMID:23390523

Transcriptomic and Proteomic Analyses Reveal Key Innate Immune Signatures in the Host Response to the Gastrointestinal Pathogen Campylobacter concisus

PubMed Central

Deshpande, Nandan P.; Man, Si Ming; Burgos-Portugal, Jose A.; Khattak, Faisal A.; Raftery, Mark J.; Wilkins, Marc R.; Mitchell, Hazel M.

2014-01-01

Pathogenic species within the genus Campylobacter are responsible for a considerable burden on global health. Campylobacter concisus is an emergent pathogen that plays a role in acute and chronic gastrointestinal disease. Despite ongoing research on Campylobacter virulence mechanisms, little is known regarding the immunological profile of the host response to Campylobacter infection. In this study, we describe a comprehensive global profile of innate immune responses to C. concisus infection in differentiated THP-1 macrophages infected with an adherent and invasive strain of C. concisus. Using RNA sequencing (RNA-seq), quantitative PCR (qPCR), mass spectrometry, and confocal microscopy, we observed differential expression of pattern recognition receptors and robust upregulation of DNA- and RNA-sensing molecules. In particular, we observed IFI16 inflammasome assembly in C. concisus-infected macrophages. Global profiling of the transcriptome revealed the significant regulation of a total of 8,343 transcripts upon infection with C. concisus, which included the activation of key inflammatory pathways involving CREB1, NF-κB, STAT, and interferon regulatory factor signaling. Thirteen microRNAs and 333 noncoding RNAs were significantly regulated upon infection, including MIR221, which has been associated with colorectal carcinogenesis. This study represents a major advance in our understanding of host recognition and innate immune responses to infection by C. concisus. PMID:25486993

Logic programming reveals alteration of key transcription factors in multiple myeloma.

PubMed

Miannay, Bertrand; Minvielle, Stéphane; Roux, Olivier; Drouin, Pierre; Avet-Loiseau, Hervé; Guérin-Charbonnel, Catherine; Gouraud, Wilfried; Attal, Michel; Facon, Thierry; Munshi, Nikhil C; Moreau, Philippe; Campion, Loïc; Magrangeas, Florence; Guziolowski, Carito

2017-08-23

Innovative approaches combining regulatory networks (RN) and genomic data are needed to extract biological information for a better understanding of diseases, such as cancer, by improving the identification of entities and thereby leading to potential new therapeutic avenues. In this study, we confronted an automatically generated RN with gene expression profiles (GEP) from a cohort of multiple myeloma (MM) patients and normal individuals using global reasoning on the RN causality to identify key-nodes. We modeled each patient by his or her GEP, the RN and the possible automatically detected repairs needed to establish a coherent flow of the information that explains the logic of the GEP. These repairs could represent cancer mutations leading to GEP variability. With this reasoning, unmeasured protein states can be inferred, and we can simulate the impact of a protein perturbation on the RN behavior to identify therapeutic targets. We showed that JUN/FOS and FOXM1 activities are altered in almost all MM patients and identified two survival markers for MM patients. Our results suggest that JUN/FOS-activation has a strong impact on the RN in view of the whole GEP, whereas FOXM1-activation could be an interesting way to perturb an MM subgroup identified by our method.

Quantum key distribution with an entangled light emitting diode

NASA Astrophysics Data System (ADS)

Dzurnak, B.; Stevenson, R. M.; Nilsson, J.; Dynes, J. F.; Yuan, Z. L.; Skiba-Szymanska, J.; Farrer, I.; Ritchie, D. A.; Shields, A. J.

2015-12-01

Measurements performed on entangled photon pairs shared between two parties can allow unique quantum cryptographic keys to be formed, creating secure links between users. An advantage of using such entangled photon links is that they can be adapted to propagate entanglement to end users of quantum networks with only untrusted nodes. However, demonstrations of quantum key distribution with entangled photons have so far relied on sources optically excited with lasers. Here, we realize a quantum cryptography system based on an electrically driven entangled-light-emitting diode. Measurement bases are passively chosen and we show formation of an error-free quantum key. Our measurements also simultaneously reveal Bell's parameter for the detected light, which exceeds the threshold for quantum entanglement.

Key Strengths of an Innovative Volunteer Training Workshop

ERIC Educational Resources Information Center

Sellick, Angelika; Bournot-Trites, Monique; Reeder, Ken; Scales, Andrew; Smith, Mark; Zappa-Hollman, Sandra

2011-01-01

The study involved 14 volunteer facilitators, four UBC staff members, and the researcher as participant; the data collected were observation notes, questionnaires, results from focus groups, and interviews. The study revealed that the key strengths of the training workshop lay in its approach to training, its focus on confidence and capacity…

New hematological key for bovine leukemia virus-infected Japanese Black cattle.

PubMed

Mekata, Hirohisa; Yamamoto, Mari; Kirino, Yumi; Sekiguchi, Satoshi; Konnai, Satoru; Horii, Yoichiro; Norimine, Junzo

2018-02-20

The European Community's (EC) Key, which is also called Bendixen's Key, is a well-established bovine leukemia virus (BLV) diagnostic method that classifies cattle according to the absolute lymphocyte count and age. The EC Key was originally designed for dairy cattle and is not necessarily suitable for Japanese Black (JB) beef cattle. This study revealed the lymphocyte counts in the BLV-free and -infected JB cattle were significantly lower than those in the Holstein cattle. Therefore, applying the EC Key to JB cattle could result in a large number of undetected BLV-infected cattle. Our proposed hematological key, which was designed for JB cattle, improves the detection of BLV-infected cattle by approximately 20%. We believe that this study could help promote BLV control.

The γ Dor stars as revealed by Kepler: A key to reveal deep-layer rotation in A and F stars

NASA Astrophysics Data System (ADS)

Salmon, S. J. A. J.; Ouazzani, R.-M.; Antoci, V.; Bedding, T. R.; Murphy, S. J.

2017-09-01

The γ Dor pulsating stars present high-order gravity modes, which make them important targets in the intermediate-and low-mass main-sequence region of the Hertzsprung-Russell diagram. Whilst we have only access to rotation in the envelope of the Sun, the g modes of γ Dor stars can in principle deliver us constraints on the inner layers. With the puzzling discovery of unexpectedly low rotation rates in the core of red giants, the γ Dor stars appear now as unique targets to explore internal angular momentum transport in the progenitors of red giants. Yet, the γ Dor pulsations remain hard to detect from the ground for their periods are close to 1 day. While the CoRoT space mission first revealed intriguing frequency spectra, the almost uninterrupted 4-year photometry from the Kepler mission eventually shed a new light on them. It revealed regularities in the spectra, expected to bear signature of physical processes, including rotation, in the shear layers close to the convective core. We present here the first results of our effort to derive exploitable seismic diagnosis for mid- to fast rotators among γ Dor stars. We confirm their potential to explore the rotation history of this early phase of stellar evolution.

Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel β-barrel consisting of two Greek-key motifs

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

Geerds, Christina; Wohlmann, Jens; Haas, Albert

The structure of VapB, a member of the Vap protein family that is involved in virulence of the bacterial pathogen R. equi, was determined by SAD phasing and reveals an eight-stranded antiparallel β-barrel similar to avidin, suggestive of a binding function. Made up of two Greek-key motifs, the topology of VapB is unusual or even unique. Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively.more » To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 Å resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel β-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded β-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.« less

Quantum key distribution with an entangled light emitting diode

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

Dzurnak, B.; Stevenson, R. M.; Nilsson, J.

Measurements performed on entangled photon pairs shared between two parties can allow unique quantum cryptographic keys to be formed, creating secure links between users. An advantage of using such entangled photon links is that they can be adapted to propagate entanglement to end users of quantum networks with only untrusted nodes. However, demonstrations of quantum key distribution with entangled photons have so far relied on sources optically excited with lasers. Here, we realize a quantum cryptography system based on an electrically driven entangled-light-emitting diode. Measurement bases are passively chosen and we show formation of an error-free quantum key. Our measurementsmore » also simultaneously reveal Bell's parameter for the detected light, which exceeds the threshold for quantum entanglement.« less

Bimodal voltage dependence of TRPA1: mutations of a key pore helix residue reveal strong intrinsic voltage-dependent inactivation.

PubMed

Wan, Xia; Lu, Yungang; Chen, Xueqin; Xiong, Jian; Zhou, Yuanda; Li, Ping; Xia, Bingqing; Li, Min; Zhu, Michael X; Gao, Zhaobing

2014-07-01

Transient receptor potential A1 (TRPA1) is implicated in somatosensory processing and pathological pain sensation. Although not strictly voltage-gated, ionic currents of TRPA1 typically rectify outwardly, indicating channel activation at depolarized membrane potentials. However, some reports also showed TRPA1 inactivation at high positive potentials, implicating voltage-dependent inactivation. Here we report a conserved leucine residue, L906, in the putative pore helix, which strongly impacts the voltage dependency of TRPA1. Mutation of the leucine to cysteine (L906C) converted the channel from outward to inward rectification independent of divalent cations and irrespective to stimulation by allyl isothiocyanate. The mutant, but not the wild-type channel, displayed exclusively voltage-dependent inactivation at positive potentials. The L906C mutation also exhibited reduced sensitivity to inhibition by TRPA1 blockers, HC030031 and ruthenium red. Further mutagenesis of the leucine to all natural amino acids individually revealed that most substitutions at L906 (15/19) resulted in inward rectification, with exceptions of three amino acids that dramatically reduced channel activity and one, methionine, which mimicked the wild-type channel. Our data are plausibly explained by a bimodal gating model involving both voltage-dependent activation and inactivation of TRPA1. We propose that the key pore helix residue, L906, plays an essential role in responding to the voltage-dependent gating.

Key-value store with internal key-value storage interface

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

Bent, John M.; Faibish, Sorin; Ting, Dennis P. J.

A key-value store is provided having one or more key-value storage interfaces. A key-value store on at least one compute node comprises a memory for storing a plurality of key-value pairs; and an abstract storage interface comprising a software interface module that communicates with at least one persistent storage device providing a key-value interface for persistent storage of one or more of the plurality of key-value pairs, wherein the software interface module provides the one or more key-value pairs to the at least one persistent storage device in a key-value format. The abstract storage interface optionally processes one or moremore » batch operations on the plurality of key-value pairs. A distributed embodiment for a partitioned key-value store is also provided.« less

The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions

PubMed Central

Merchant, Sabeeha S.; Prochnik, Simon E.; Vallon, Olivier; Harris, Elizabeth H.; Karpowicz, Steven J.; Witman, George B.; Terry, Astrid; Salamov, Asaf; Fritz-Laylin, Lillian K.; Maréchal-Drouard, Laurence; Marshall, Wallace F.; Qu, Liang-Hu; Nelson, David R.; Sanderfoot, Anton A.; Spalding, Martin H.; Kapitonov, Vladimir V.; Ren, Qinghu; Ferris, Patrick; Lindquist, Erika; Shapiro, Harris; Lucas, Susan M.; Grimwood, Jane; Schmutz, Jeremy; Cardol, Pierre; Cerutti, Heriberto; Chanfreau, Guillaume; Chen, Chun-Long; Cognat, Valérie; Croft, Martin T.; Dent, Rachel; Dutcher, Susan; Fernández, Emilio; Ferris, Patrick; Fukuzawa, Hideya; González-Ballester, David; González-Halphen, Diego; Hallmann, Armin; Hanikenne, Marc; Hippler, Michael; Inwood, William; Jabbari, Kamel; Kalanon, Ming; Kuras, Richard; Lefebvre, Paul A.; Lemaire, Stéphane D.; Lobanov, Alexey V.; Lohr, Martin; Manuell, Andrea; Meier, Iris; Mets, Laurens; Mittag, Maria; Mittelmeier, Telsa; Moroney, James V.; Moseley, Jeffrey; Napoli, Carolyn; Nedelcu, Aurora M.; Niyogi, Krishna; Novoselov, Sergey V.; Paulsen, Ian T.; Pazour, Greg; Purton, Saul; Ral, Jean-Philippe; Riaño-Pachón, Diego Mauricio; Riekhof, Wayne; Rymarquis, Linda; Schroda, Michael; Stern, David; Umen, James; Willows, Robert; Wilson, Nedra; Zimmer, Sara Lana; Allmer, Jens; Balk, Janneke; Bisova, Katerina; Chen, Chong-Jian; Elias, Marek; Gendler, Karla; Hauser, Charles; Lamb, Mary Rose; Ledford, Heidi; Long, Joanne C.; Minagawa, Jun; Page, M. Dudley; Pan, Junmin; Pootakham, Wirulda; Roje, Sanja; Rose, Annkatrin; Stahlberg, Eric; Terauchi, Aimee M.; Yang, Pinfen; Ball, Steven; Bowler, Chris; Dieckmann, Carol L.; Gladyshev, Vadim N.; Green, Pamela; Jorgensen, Richard; Mayfield, Stephen; Mueller-Roeber, Bernd; Rajamani, Sathish; Sayre, Richard T.; Brokstein, Peter; Dubchak, Inna; Goodstein, David; Hornick, Leila; Huang, Y. Wayne; Jhaveri, Jinal; Luo, Yigong; Martínez, Diego; Ngau, Wing Chi Abby; Otillar, Bobby; Poliakov, Alexander; Porter, Aaron; Szajkowski, Lukasz; Werner, Gregory; Zhou, Kemin; Grigoriev, Igor V.; Rokhsar, Daniel S.; Grossman, Arthur R.

2010-01-01

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella. PMID:17932292

Stable integration and expression of heterologous genes in several lactobacilli using an integration vector constructed from the integrase and attP sequences of phage ΦAT3 isolated from Lactobacillus casei ATCC 393.

PubMed

Lin, Chao-Fen; Lo, Ta-Chun; Kuo, Yang-Cheng; Lin, Thy-Hou

2013-04-01

An integration vector capable of stably integrating and maintaining in the chromosomes of several lactobacilli over hundreds of generations has been constructed. The major integration machinery used is based on the ΦAT3 integrase (int) and attP sequences determined previously. A novel core sequence located at the 3' end of the tRNA(leu) gene is identified in Lactobacillus fermentum ATCC 14931 as the integration target by the integration vector though most of such sequences found in other lactobacilli are similar to that determined previously. Due to the lack of an appropriate attB site in Lactococcus lactis MG1363, the integration vector is found to be unable to integrate into the chromosome of the strain. However, such integration can be successfully restored by cotransforming the integration vector with a replicative one harboring both attB and erythromycin resistance sequences into the strain. Furthermore, the integration vector constructed carries a promoter region of placT from the chromosome of Lactobacillus rhamnosus TCELL-1 which is used to express green fluorescence and luminance protein genes in the lactobacilli studied.

Florida Keys

NASA Image and Video Library

2002-12-13

The Florida Keys are a chain of islands, islets and reefs extending from Virginia Key to the Dry Tortugas for about 309 kilometers (192 miles). The keys are chiefly limestone and coral formations. The larger islands of the group are Key West (with its airport), Key Largo, Sugarloaf Key, and Boca Chica Key. A causeway extends from the mainland to Key West. This image was acquired on October 28, 2001, by the Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) on NASA's Terra satellite. With its 14 spectral bands from the visible to the thermal infrared wavelength region, and its high spatial resolution of 15 to 90 meters (about 50 to 300 feet), ASTER images Earth to map and monitor the changing surface of our planet. http://photojournal.jpl.nasa.gov/catalog/PIA03890

Structure of the Trehalose-6-phosphate Phosphatase from Brugia malayi Reveals Key Design Principles for Anthelmintic Drugs

PubMed Central

Farelli, Jeremiah D.; Galvin, Brendan D.; Li, Zhiru; Liu, Chunliang; Aono, Miyuki; Garland, Megan; Hallett, Olivia E.; Causey, Thomas B.; Ali-Reynolds, Alana; Saltzberg, Daniel J.; Carlow, Clotilde K. S.; Dunaway-Mariano, Debra; Allen, Karen N.

2014-01-01

Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible. PMID:24992307

High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States.

PubMed

Zhang, Yi; Kitazawa, Soichiro; Peran, Ivan; Stenzoski, Natalie; McCallum, Scott A; Raleigh, Daniel P; Royer, Catherine A

2016-11-23

Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.

Function key and shortcut key use in airway facilities.

DOT National Transportation Integrated Search

2003-02-01

This document provides information on the function keys and shortcut keys used by systems in the Federal Aviation Administration : Airway Facilities (AF) work environment. It includes a catalog of the function keys and shortcut keys used by each syst...

Metaproteomic Analysis of a Chemosynthetic Hydrothermal Vent Community Reveals Insights into Key-Metabolic Processes

NASA Astrophysics Data System (ADS)

Steen, I.; Stokke, R.; Lanzen, A.; Pedersen, R.; Øvreås, L.; Urich, T.

2010-12-01

In 2005 researchers at the Centre for Geobiology, University of Bergen, Norway, discovered two active vent fields at the southwestern Mohns Ridge in the Norwegian-Greenland Sea. The fields harbours both low-temperature iron deposits and high-temperature white smoker vents. Distinct microbial mats were abundantly present and located in close vicinity to the hydrothermal vent sites. Characteristics of the mat environment were steep physical and chemical gradients with temperatures ranging from 10°C in the top layer to 90°C at 10 cm bsf and high concentrations of hydrogen sulfide and methane. The work presented here focus on the In situ community activities, and is part of an integrated strategy combining metagenomics, metatranscriptomics and metaproteomics to in-depth characterise these newly discovered hydrothermal vent communities. Extracted proteins were separated via SDS-PAGE. Peptides extracted after In-gel tryptic digest was injected into an Ultimate 3000 nanoLC system connected to a linear quadropole ion trap-orbitrap (LTQ-Orbitrap XL) mass spectrometer equipped with a nanoelectrospray ion source. A custom database of open reading frames (ORFs) from the combined metatranscriptome and metagenome datasets was implemented and searched against using Mascot 2.2; the IRMa tool box [1] was used in peptide validation. Validated ORFs were subjected to a Blastp search against Refseq with an E-value cut-off of 0.001. A total of 1097 proteins with ≥ 2 peptides were identified of which 921 gave a hit against Refseq, containing 519 unique proteins. Key enzymes of the sulfur oxidation pathway (sox) were found, which were taxonomically affiliated to Epsilonproteobacteria. In addition, this group actively expressed hydrogenases and membrane proteins involved in aerobic and anaerobic respiratory chains. Enzymes of dissimilatory sulfate-reduction (APS-reductase, AprAB and DsrA2) were found with closest hit to members of the Deltaproteobacteria. These findings indicate an

The 3D structures of G-quadruplexes of HIV-1 integrase inhibitors: molecular dynamics simulations in aqueous solution and in the gas phase.

PubMed

Li, Ming-Hui; Zhou, Yi-Han; Luo, Quan; Li, Ze-Sheng

2010-04-01

The unimolecular G-quadruplex structures of d(GGGTGGGTGGGTGGGT) (G1) and d(GTGGTGGGTGGGTGGGT) (G2) are known as the potent nanomolar HIV-1 integrase inhibitors, thus investigating the 3D structures of the two sequences is significant for structure-based rational anti-HIV drug design. In this research, based on the experimental data of circular dichroism (CD) spectropolarimetry and electrospray ionization mass spectrometry (ESI-MS), the initial models of G1 and G2 were constructed by molecular modeling method. The modeling structures of G1 and G2 are intramolecular parallel-stranded quadruplex conformation with three guanine tetrads. Particularly, the structure of G2 possesses a T loop residue between the first and the second G residues that are the component of two adjacent same-stranded G-tetrad planes. This structure proposed by us has a very novel geometry and is different from all reported G-quadruplexes. The extended (35 ns) molecular dynamic (MD) simulations for the models indicate that the G-quadruplexes maintain their structures very well in aqueous solution whether the existence of K(+) or NH (4) (+) in the central channel. Furthermore, we perform 500 ns MD simulations for the models in the gas phase. The results show that all the ion-G-quadruplex complexes are maintained during the whole simulations, despite the large magnitude of phosphate-phosphate repulsions. The gas phase MD simulations provide a good explanation to ESI-MS experiments. Our 3D structures for G1 and G2 will assist in understanding geometric formalism of G-quadruplex folding and may be helpful as a platform for rational anti-HIV drug design.

Representing Microbial Dormancy in Soil Decomposition Models Improves Model Performance and Reveals Key Ecosystem Controls on Microbial Activity

NASA Astrophysics Data System (ADS)

He, Y.; Yang, J.; Zhuang, Q.; Wang, G.; Liu, Y.

2014-12-01

Climate feedbacks from soils can result from environmental change and subsequent responses of plant and microbial communities and nutrient cycling. Explicit consideration of microbial life history traits and strategy may be necessary to predict climate feedbacks due to microbial physiology and community changes and their associated effect on carbon cycling. In this study, we developed an explicit microbial-enzyme decomposition model and examined model performance with and without representation of dormancy at six temperate forest sites with observed soil efflux ranged from 4 to 10 years across different forest types. We then extrapolated the model to all temperate forests in the Northern Hemisphere (25-50°N) to investigate spatial controls on microbial and soil C dynamics. Both models captured the observed soil heterotrophic respiration (RH), yet no-dormancy model consistently exhibited large seasonal amplitude and overestimation in microbial biomass. Spatially, the total RH from temperate forests based on dormancy model amounts to 6.88PgC/yr, and 7.99PgC/yr based on no-dormancy model. However, no-dormancy model notably overestimated the ratio of microbial biomass to SOC. Spatial correlation analysis revealed key controls of soil C:N ratio on the active proportion of microbial biomass, whereas local dormancy is primarily controlled by soil moisture and temperature, indicating scale-dependent environmental and biotic controls on microbial and SOC dynamics. These developments should provide essential support to modeling future soil carbon dynamics and enhance the avenue for collaboration between empirical soil experiment and modeling in the sense that more microbial physiological measurements are needed to better constrain and evaluate the models.

Integrated genomics of Mucorales reveals novel therapeutic targets

USDA-ARS?s Scientific Manuscript database

Mucormycosis is a life-threatening infection caused by Mucorales fungi. We sequenced 30 fungal genomes and performed transcriptomics with three representative Rhizopus and Mucor strains with human airway epithelial cells during fungal invasion to reveal key host and fungal determinants contributing ...

Antiviral activity of dolutegravir in subjects with failure on an integrase inhibitor-based regimen: week 24 phase 3 results from VIKING-3

PubMed Central

Nichols, G; Mills, A; Grossberg, R; Lazzarin, A; Maggiolo, F; Molina, J; Pialoux, G; Wright, D; Ait-Khaled, M; Huang, J; Vavro, C; Wynne, B; Yeo, J

2012-01-01

Background VIKING-3 aimed to examine efficacy and safety of dolutegravir (DTG) 50 mg twice daily in patients with resistance to multiple ARV classes, including integrase inhibitors (INI). Methods RAL and/or EVG-resistant (current or historical) adult subjects with screening plasma HIV-1 RNA ≥500 c/mL and resistance to ≥2 other ART classes received open-label DTG 50 mg BID while continuing their failing regimen (without RAL/EVG). At Day 8 the background regimen was optimised and DTG continued. Activity of the optimized background regimen (OBR) was determined by Monogram Net Assessment. Primary endpoints were antiviral efficacy at Day 8 and Week 24. Results 183 subjects enrolled, 124 with INI-resistance at screening and 59 with historical (but no screening) resistance. Population was advanced: at BL, median CD4 140, prior ART 13 yrs, 56% CDC Class C; 79% had >2 NRTI, 75% >1 NNRTI, and 70% >2 PI resistance-associated mutations, and 61% had non-R5 HIV detected. Of the 114 subjects who had the opportunity to complete 24 weeks on study before data cutoff, 72 (63%) had <50 c/mL RNA at Week 24 (SNAPSHOT algorithm). Mean HIV RNA declined by 1.4 log10 c/mL (95% CI: 1.3, 1.5; p < 0.001) at Day 8; response differed by genotype pathway (Table). Primary INI mutations at BL N Mean HIV RNA (log 10) Change from BL (SD) at Day 8 %>1 log HIV RNA decline of <50 c/mL at Day 8 TOTAL 183 −1.4 (0.61) 82% T66 1 −1.9 100% Y143 28 −1.7 (0.42) 96% N155 33 −1.4 (0.51) 82% Q148 + ≤1 secondary mutation# 32 −1.1 (0.51) 69% Q148 + ≥2 secondary mutations# 20 −1.0 (0.81) 48% ≥2 primary mutations 8 −1.4 (0.76) 75% No primary mutations 60 −1.6 (0.55) 95% # Key secondary mutations comprised G140_ACS, L741, E138_AKT. In subjects with Q148 pathway mutations, virologic response decreased with increasing number of secondary mutations. Background overall susceptibility score (OSS) was not associated with Wk 24 response: % <50 c/mL were 83%, 63%, 59% and 69% for OSS 0, 1, 2 and >2

Metal Binding by the D,DX35E Motif of Human Immunodeficiency Virus Type 1 Integrase: Selective Rescue of Cys Substitutions by Mn2+ In Vitro

PubMed Central

Gao, Kui; Wong, Steven; Bushman, Frederic

2004-01-01

The D,DX35E motif characteristic of retroviral integrase enzymes (INs) is expected to bind the required metal cofactors (Mg2+ or Mn2+), but direct evidence for a catalytic role has been lacking. Here we used a metal rescue strategy to investigate metal binding. We established conditions for analysis of an activity of IN, disintegration, in both Mg2+ and Mn2+, and tested IN mutants with cysteine substitutions in each acidic residue of the D,DX35E motif. Mn2+ but not Mg2+ can bind tightly to Cys, so if metal binding at the acidic residues is mechanistically important, it is expected that the Cys-substituted enzymes would be active in the presence of Mn2+ only. Of the three acidic residues, a strong metal rescue effect was obtained for D116C, a weaker rescue was seen for D64C, and no rescue was seen with E152C. Modest rescue could also be detected for D116C in normal integration in vitro. Comparison to Ser and Ala substitutions at D116 established that the rescue was selective for Cys. Further studies of the response to pH suggest that the metal cofactor may stabilize the deprotonated nucleophile active in catalysis, and studies of the response to NaCl titrations disclose an additional role for the metal cofactor in stabilizing the IN-DNA complex. PMID:15194746

Simple Web-based interactive key development software (WEBiKEY) and an example key for Kuruna (Poaceae: Bambusoideae).

PubMed

Attigala, Lakshmi; De Silva, Nuwan I; Clark, Lynn G

2016-04-01

Programs that are user-friendly and freely available for developing Web-based interactive keys are scarce and most of the well-structured applications are relatively expensive. WEBiKEY was developed to enable researchers to easily develop their own Web-based interactive keys with fewer resources. A Web-based multiaccess identification tool (WEBiKEY) was developed that uses freely available Microsoft ASP.NET technologies and an SQL Server database for Windows-based hosting environments. WEBiKEY was tested for its usability with a sample data set, the temperate woody bamboo genus Kuruna (Poaceae). WEBiKEY is freely available to the public and can be used to develop Web-based interactive keys for any group of species. The interactive key we developed for Kuruna using WEBiKEY enables users to visually inspect characteristics of Kuruna and identify an unknown specimen as one of seven possible species in the genus.

Structure of the CRISPR Interference Complex CSM Reveals Key Similarities with Cascade

PubMed Central

Rouillon, Christophe; Zhou, Min; Zhang, Jing; Politis, Argyris; Beilsten-Edmands, Victoria; Cannone, Giuseppe; Graham, Shirley; Robinson, Carol V.; Spagnolo, Laura; White, Malcolm F.

2013-01-01

Summary The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I–III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family. PMID:24119402

Microarray analysis reveals key genes and pathways in Tetralogy of Fallot

PubMed Central

He, Yue-E; Qiu, Hui-Xian; Jiang, Jian-Bing; Wu, Rong-Zhou; Xiang, Ru-Lian; Zhang, Yuan-Hai

2017-01-01

The aim of the present study was to identify key genes that may be involved in the pathogenesis of Tetralogy of Fallot (TOF) using bioinformatics methods. The GSE26125 microarray dataset, which includes cardiovascular tissue samples derived from 16 children with TOF and five healthy age-matched control infants, was downloaded from the Gene Expression Omnibus database. Differential expression analysis was performed between TOF and control samples to identify differentially expressed genes (DEGs) using Student's t-test, and the R/limma package, with a log2 fold-change of >2 and a false discovery rate of <0.01 set as thresholds. The biological functions of DEGs were analyzed using the ToppGene database. The ReactomeFIViz application was used to construct functional interaction (FI) networks, and the genes in each module were subjected to pathway enrichment analysis. The iRegulon plugin was used to identify transcription factors predicted to regulate the DEGs in the FI network, and the gene-transcription factor pairs were then visualized using Cytoscape software. A total of 878 DEGs were identified, including 848 upregulated genes and 30 downregulated genes. The gene FI network contained seven function modules, which were all comprised of upregulated genes. Genes enriched in Module 1 were enriched in the following three neurological disorder-associated signaling pathways: Parkinson's disease, Alzheimer's disease and Huntington's disease. Genes in Modules 0, 3 and 5 were dominantly enriched in pathways associated with ribosomes and protein translation. The Xbox binding protein 1 transcription factor was demonstrated to be involved in the regulation of genes encoding the subunits of cytoplasmic and mitochondrial ribosomes, as well as genes involved in neurodegenerative disorders. Therefore, dysfunction of genes involved in signaling pathways associated with neurodegenerative disorders, ribosome function and protein translation may contribute to the pathogenesis of TOF

The Power of Proofs-of-Possession: Securing Multiparty Signatures against Rogue-Key Attacks

NASA Astrophysics Data System (ADS)

Ristenpart, Thomas; Yilek, Scott

Multiparty signature protocols need protection against rogue-key attacks, made possible whenever an adversary can choose its public key(s) arbitrarily. For many schemes, provable security has only been established under the knowledge of secret key (KOSK) assumption where the adversary is required to reveal the secret keys it utilizes. In practice, certifying authorities rarely require the strong proofs of knowledge of secret keys required to substantiate the KOSK assumption. Instead, proofs of possession (POPs) are required and can be as simple as just a signature over the certificate request message. We propose a general registered key model, within which we can model both the KOSK assumption and in-use POP protocols. We show that simple POP protocols yield provable security of Boldyreva's multisignature scheme [11], the LOSSW multisignature scheme [28], and a 2-user ring signature scheme due to Bender, Katz, and Morselli [10]. Our results are the first to provide formal evidence that POPs can stop rogue-key attacks.

5-Hydroxypyrido[2,3-b]pyrazin-6(5H)-one derivatives as novel dual inhibitors of HIV-1 reverse transcriptase-associated ribonuclease H and integrase.

PubMed

Sun, Lin; Gao, Ping; Dong, Guanyu; Zhang, Xujie; Cheng, Xiqiang; Ding, Xiao; Wang, Xueshun; Daelemans, Dirk; De Clercq, Erik; Pannecouque, Christophe; Menéndez-Arias, Luis; Zhan, Peng; Liu, Xinyong

2018-06-18

We reported herein the design, synthesis and biological evaluation of a series of 5-hydroxypyrido[2,3-b]pyrazin-6(5H)-one derivatives as HIV-1 reverse transcriptase (RT) ribonuclease H (RNase H) inhibitors using a privileged structure-guided scaffold refining strategy. In view of the similarities between the pharmacophore model of RNase H and integrase (IN) inhibitors as well as their catalytic sites, we also performed IN inhibition assays. Notably, the majority of these derivatives inhibited RNase H and IN at micromolar concentrations. Among them, compound 7a exhibited similar inhibitory activity against RNase H and IN (IC 50 RNase H  = 1.77 μM, IC 50 IN  = 1.18 μM, ratio = 1.50). To the best of our knowledge, this is the first reported dual HIV-1 RNase H-IN inhibitor based on a 5-hydroxypyrido[2,3-b]pyrazin-6(5H)-one structure. Molecular modeling has been used to predict the binding mode of 7a in complex with the catalytic cores of HIV-1 RNase H and IN. Taken together these results strongly support the feasibility of developing HIV-1 dual inhibitors from analog-based optimization of divalent metal ion chelators. Recently, the identification of dual inhibitors proved to be a highly effective strategy for novel antivirals discovery. Therefore, these compounds appear to be useful leads that can be further modified to develop more valuable anti-HIV-1 molecules with suitable drug profiles. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

An investigation into the differentiating characteristics between car key burglars and regular burglars.

PubMed

Chapman, Rachel; Smith, Lisa L; Bond, John W

2012-07-01

Car key burglary has recently become the focus of empirical investigation as offenders, no longer able to steal vehicles without first obtaining their keys, resort to "burgling" target properties. Research surrounding the modus operandi of these offenses is beginning to emerge; however, little attention has been paid to investigating the characteristics of car key burglary offenders. Challenging the assumption that car key burglary offenses are perpetrated by regular burglars, this study aims to differentiate between offenders. Logistic regression analysis of 110 car key and 110 regular burglary offenders revealed that car key burglars are more likely to have previous vehicle theft convictions and are also more likely to be detected on information supplied to the police than regular burglars. Regular burglars are more likely to have previous shoplifting convictions. It was concluded that car key burglars are a distinct sample of offenders and the implications of these findings are discussed. © 2012 American Academy of Forensic Sciences.

Parallel comparative proteomics and phosphoproteomics reveal that cattle myostatin regulates phosphorylation of key enzymes in glycogen metabolism and glycolysis pathway

PubMed Central

Yang, Shuping; Li, Xin; Liu, Xinfeng; Ding, Xiangbin; Xin, Xiangbo; Jin, Congfei; Zhang, Sheng; Li, Guangpeng; Guo, Hong

2018-01-01

MSTN-encoded myostatin is a negative regulator of skeletal muscle development. Here, we utilized the gluteus tissues from MSTN gene editing and wild type Luxi beef cattle which are native breed of cattle in China, performed tandem mass tag (TMT) -based comparative proteomics and phosphoproteomics analyses to investigate the regulatory mechanism of MSTN related to cellular metabolism and signaling pathway in muscle development. Out of 1,315 proteins, 69 differentially expressed proteins (DEPs) were found in global proteomics analysis. Meanwhile, 149 differentially changed phosphopeptides corresponding to 76 unique phosphorylated proteins (DEPPs) were detected from 2,600 identified phosphopeptides in 702 phosphorylated proteins. Bioinformatics analyses suggested that majority of DEPs and DEPPs were closely related to glycolysis, glycogenolysis, and muscle contractile fibre processes. The global discovery results were validated by Multiple Reaction Monitoring (MRM)-based targeted peptide quantitation analysis, western blotting, and muscle glycogen content measurement. Our data revealed that increase in abundance of key enzymes and phosphorylation on their regulatory sites appears responsible for the enhanced glycogenolysis and glycolysis in MSTN−/−. The elevated glycogenolysis was assocaited with an enhanced phosphorylation of Ser1018 in PHKA1, and Ser641/Ser645 in GYS1, which were regulated by upstream phosphorylated AKT-GSK3β pathway and highly consistent with the lower glycogen content in gluteus of MSTN−/−. Collectively, this study provides new insights into the regulatory mechanisms of MSTN involved in energy metabolism and muscle growth. PMID:29541418

Evaluation of the effect of short-term treatment with the integrase inhibitor raltegravir (Isentress) on the course of progressive feline leukemia virus infection.

PubMed

Boesch, Andrea; Cattori, Valentino; Riond, Barbara; Willi, Barbara; Meli, Marina L; Rentsch, Katharina M; Hosie, Margaret J; Hofmann-Lehmann, Regina; Lutz, Hans

2015-02-25

Cats persistently infected with the gammaretrovirus feline leukemia virus (FeLV) are at risk to die within months to years from FeLV-associated disease, such as immunosuppression, anemia or lymphoma/leukemia. The integrase inhibitor raltegravir has been demonstrated to reduce FeLV replication in vitro. The aim of the present study was to investigate raltegravir in vivo for its safety and efficacy to suppress FeLV replication. The safety was tested in three naïve specified pathogen-free (SPF) cats during a 15 weeks treatment period (initially 20mg then 40mg orally b.i.d.). No adverse effects were noted. The efficacy was tested in seven persistently FeLV-infected SPF cats attained from 18 cats experimentally exposed to FeLV-A/Glasgow-1. The seven cats were treated during nine weeks (40mg then 80mg b.i.d.). Raltegravir was well tolerated even at the higher dose. A significant decrease in plasma viral RNA loads (∼5×) was found; however, after treatment termination a rebound effect was observed. Only one cat developed anti-FeLV antibodies and viral RNA loads remained decreased after treatment termination. Of note, one of the untreated FeLV-A infected cats developed fatal FeLV-C associated anemia within 5 weeks of FeLV-A infection. Moreover, progressive FeLV infection was associated with significantly lower enFeLV loads prior to infection supporting that FeLV susceptibility may be related to the genetic background of the cat. Overall, our data demonstrate the ability of raltegravir to reduce viral replication also in vivo. However, no complete control of viremia was achieved. Further investigations are needed to find an optimized treatment against FeLV. (250 words). Copyright © 2014 Elsevier B.V. All rights reserved.

Dolutegravir versus placebo in subjects harbouring HIV-1 with integrase inhibitor resistance associated substitutions: 48-week results from VIKING-4, a randomized study.

PubMed

Akil, Bisher; Blick, Gary; Hagins, Debbie P; Ramgopal, Moti N; Richmond, Gary J; Samuel, Rafik M; Givens, Naomi; Vavro, Cindy; Song, Ivy H; Wynne, Brian; Ait-Khaled, Mounir

2015-01-01

The Phase III VIKING-3 study demonstrated that dolutegravir (DTG) 50 mg twice daily was efficacious in antiretroviral therapy (ART)-experienced subjects harbouring raltegravir- and/or elvitegravir-resistant HIV-1. VIKING-4 (ING116529) included a placebo-controlled 7-day monotherapy phase to demonstrate that short-term antiviral activity was attributable to DTG. VIKING-4 is a Phase III randomized, double-blind study in therapy-experienced adults with integrase inhibitor (INI)-resistant virus randomized to DTG 50 mg twice daily or placebo while continuing their failing regimen (without raltegravir or elvitegravir) for 7 days (clinicaltrials.gov identifier NCT01568892). At day 8, all subjects switched to open-label DTG 50 mg twice daily and optimized background therapy including ≥1 fully active drug. The primary end point was change from baseline in plasma HIV-1 RNA at day 8. The study population (n=30) was highly ART-experienced with advanced HIV disease. Patients had extensive baseline resistance to all approved antiretroviral classes. Adjusted mean change in HIV-1 RNA at day 8 was 
-1.06 log10 copies/ml for the DTG arm and 0.10 log10 copies/ml for the placebo arm (treatment difference -1.16 log10 copies/ml [-1.52, -0.80]; P<0.001). Overall, 47% and 57% of subjects had plasma HIV-1 RNA <50 and <400 copies/ml at week 24, and 40% and 53% at week 48, respectively. No discontinuations due to drug-related adverse events occurred in the study. The observed day 8 antiviral activity in this highly treatment-experienced population with INI-resistant HIV-1 was attributable to DTG. Longer-term efficacy (after considering baseline ART resistance) and safety during the open-label phase were in-line with the results of the larger VIKING-3 study.

Simple Web-based interactive key development software (WEBiKEY) and an example key for Kuruna (Poaceae: Bambusoideae)1

PubMed Central

Attigala, Lakshmi; De Silva, Nuwan I.; Clark, Lynn G.

2016-01-01

Premise of the study: Programs that are user-friendly and freely available for developing Web-based interactive keys are scarce and most of the well-structured applications are relatively expensive. WEBiKEY was developed to enable researchers to easily develop their own Web-based interactive keys with fewer resources. Methods and Results: A Web-based multiaccess identification tool (WEBiKEY) was developed that uses freely available Microsoft ASP.NET technologies and an SQL Server database for Windows-based hosting environments. WEBiKEY was tested for its usability with a sample data set, the temperate woody bamboo genus Kuruna (Poaceae). Conclusions: WEBiKEY is freely available to the public and can be used to develop Web-based interactive keys for any group of species. The interactive key we developed for Kuruna using WEBiKEY enables users to visually inspect characteristics of Kuruna and identify an unknown specimen as one of seven possible species in the genus. PMID:27144109

Geometrically and conformationally restrained cinnamoyl compounds as inhibitors of HIV-1 integrase: synthesis, biological evaluation, and molecular modeling.

PubMed

Artico, M; Di Santo, R; Costi, R; Novellino, E; Greco, G; Massa, S; Tramontano, E; Marongiu, M E; De Montis, A; La Colla, P

1998-10-08

Various cinnammoyl-based structures were synthesized and tested in enzyme assays as inhibitors of the HIV-1 integrase (IN). The majority of compounds were designed as geometrically or conformationally constrained analogues of caffeic acid phenethyl ester (CAPE) and were characterized by a syn disposition of the carbonyl group with respect to the vinylic double bond. Since the cinnamoyl moiety present in flavones such as quercetin (inactive on HIV-1-infected cells) is frozen in an anti arrangement, it was hoped that fixing our compounds in a syn disposition could favor anti-HIV-1 activity in cell-based assays. Geometrical and conformational properties of the designed compounds were taken into account through analysis of X-ray structures available from the Cambridge Structural Database. The polyhydroxylated analogues were prepared by reacting 3,4-bis(tetrahydropyran-2-yloxy)benzaldehyde with various compounds having active methylene groups such as 2-propanone, cyclopentanone, cyclohexanone, 1,3-diacetylbenzene, 2, 4-dihydroxyacetophenone, 2,3-dihydro-1-indanone, 2,3-dihydro-1, 3-indandione, and others. While active against both 3'-processing and strand-transfer reactions, the new compounds, curcumin included, failed to inhibit the HIV-1 multiplication in acutely infected MT-4 cells. Nevertheless, they specifically inhibited the enzymatic reactions associated with IN, being totally inactive against other viral (HIV-1 reverse transcriptase) and cellular (RNA polymerase II) nucleic acid-processing enzymes. On the other hand, title compounds were endowed with remarkable antiproliferative activity, whose potency correlated neither with the presence of catechols (possible source of reactive quinones) nor with inhibition of topoisomerases. The SARs developed for our compounds led to novel findings concerning the molecular determinants of IN inhibitory activity within the class of cinnamoyl-based structures. We hypothesize that these compounds bind to IN featuring the

Key challenges in the development and implementation of telehealth projects.

PubMed

Joseph, Victor; West, Robert M; Shickle, Darren; Keen, Justin; Clamp, Susan

2011-01-01

A literature review was carried out to identify the key challenges in the implementation of telehealth. This was followed by a survey of organisations in England involved in telehealth projects in order to understand the challenges they faced. Ten of the 13 health or local authority organisations surveyed had telehealth projects and three were at the planning stage. The analysis revealed seven key challenges facing implementers of telehealth in England. Based on the findings from the literature review and the survey, a model was constructed and a checklist drawn up. The model contained the following elements: identifying issues, needs and partners; producing a strategy; securing funding; implementing changes; and monitoring and evaluating a telehealth project. The checklist was validated by using key informants from the organisations originally surveyed. The checklist may be useful to guide telehealth development and implementation in the future.

Quantum key management

DOEpatents

Hughes, Richard John; Thrasher, James Thomas; Nordholt, Jane Elizabeth

2016-11-29

Innovations for quantum key management harness quantum communications to form a cryptography system within a public key infrastructure framework. In example implementations, the quantum key management innovations combine quantum key distribution and a quantum identification protocol with a Merkle signature scheme (using Winternitz one-time digital signatures or other one-time digital signatures, and Merkle hash trees) to constitute a cryptography system. More generally, the quantum key management innovations combine quantum key distribution and a quantum identification protocol with a hash-based signature scheme. This provides a secure way to identify, authenticate, verify, and exchange secret cryptographic keys. Features of the quantum key management innovations further include secure enrollment of users with a registration authority, as well as credential checking and revocation with a certificate authority, where the registration authority and/or certificate authority can be part of the same system as a trusted authority for quantum key distribution.

Incorporation of aptamers in the terminal loop of shRNAs yields an effective and novel combinatorial targeting strategy.

PubMed

Pang, Ka Ming; Castanotto, Daniela; Li, Haitang; Scherer, Lisa; Rossi, John J

2018-01-09

Gene therapy by engineering patient's own blood cells to confer HIV resistance can potentially lead to a functional cure for AIDS. Toward this goal, we have previously developed an anti-HIV lentivirus vector that deploys a combination of shRNA, ribozyme and RNA decoy. To further improve this therapeutic vector against viral escape, we sought an additional reagent to target HIV integrase. Here, we report the development of a new strategy for selection and expression of aptamer for gene therapy. We developed a SELEX protocol (multi-tag SELEX) for selecting RNA aptamers against proteins with low solubility or stability, such as integrase. More importantly, we expressed these aptamers in vivo by incorporating them in the terminal loop of shRNAs. This novel strategy allowed efficient expression of the shRNA-aptamer fusions that targeted RNAs and proteins simultaneously. Expressed shRNA-aptamer fusions targeting HIV integrase or reverse transcriptase inhibited HIV replication in cell cultures. Viral inhibition was further enhanced by combining an anti-integrase aptamer with an anti-HIV Tat-Rev shRNA. This construct exhibited efficacy comparable to that of integrase inhibitor Raltegravir. Our strategy for the selection and expression of RNA aptamers can potentially extend to other gene therapy applications. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Gene Expression in Class 2 Integrons Is SOS-Independent and Involves Two Pc Promoters.

PubMed

Jové, Thomas; Da Re, Sandra; Tabesse, Aurore; Gassama-Sow, Amy; Ploy, Marie-Cécile

2017-01-01

Integrons are powerful bacterial genetic elements that permit the expression and dissemination of antibiotic-resistance gene cassettes. They contain a promoter Pc that allows the expression of gene cassettes captured through site-specific recombination catalyzed by IntI, the integron-encoded integrase. Class 1 and 2 integrons are found in both clinical and environmental settings. The regulation of intI and of Pc promoters has been extensively studied in class 1 integrons and the regulatory role of the SOS response on intI expression has been shown. Here we investigated class 2 integrons. We characterized the P intI2 promoter and showed that intI2 expression is not regulated via the SOS response. We also showed that, unlike class 1 integrons, class 2 integrons possess not one but two active Pc promoters that are located within the attI2 region that seem to contribute equally to gene cassette expression. Class 2 integrons mostly encode an inactive truncated integrase, but the rare class 2 integrons that encode an active integrase are associated with less efficient Pc2 promoter variants. We propose an evolutionary model for class 2 integrons in which the absence of repression of the integrase gene expression led to mutations resulting in either inactive integrase or Pc variants of weaker activity, thereby reducing the potential fitness cost of these integrons.

1,4-Bis(5-(naphthalen-1-yl)thiophen-2-yl)naphthalene, a small molecule, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular Lens epithelium-derived growth factor.

PubMed

Gu, Wan-gang; Ip, Denis Tsz-Ming; Liu, Si-jie; Chan, Joseph H; Wang, Yan; Zhang, Xuan; Zheng, Yong-tang; Wan, David Chi-Cheong

2014-04-25

Translocation of viral integrase (IN) into the nucleus is a critical precondition of integration during the life cycle of HIV, a causative agent of Acquired Immunodeficiency Syndromes (AIDS). As the first discovered cellular factor to interact with IN, Lens epithelium-derived growth factor (LEDGF/p75) plays an important role in the process of integration. Disruption of the LEDGF/p75-IN interaction has provided a great interest for anti-HIV agent discovery. In this work, we reported that one small molecular compound, 1,4-bis(5-(naphthalen-1-yl)thiophen-2-yl)naphthalene(Compound 15), potently inhibit the IN-LEDGF/p75 interaction and affect the HIV-1 IN nuclear distribution at 1 μM. The putative binding mode of Compound 15 was constructed by a molecular docking simulation to provide structural insights into the ligand-binding mechanism. Compound 15 suppressed viral replication by measuring p24 antigen production in HIV-1IIIB acute infected C8166 cells with EC50 value of 11.19 μM. Compound 15 might supply useful structural information for further anti-HIV agent discovery. Copyright © 2014. Published by Elsevier Ireland Ltd.

Hacking on decoy-state quantum key distribution system with partial phase randomization

NASA Astrophysics Data System (ADS)

Sun, Shi-Hai; Jiang, Mu-Sheng; Ma, Xiang-Chun; Li, Chun-Yan; Liang, Lin-Mei

2014-04-01

Quantum key distribution (QKD) provides means for unconditional secure key transmission between two distant parties. However, in practical implementations, it suffers from quantum hacking due to device imperfections. Here we propose a hybrid measurement attack, with only linear optics, homodyne detection, and single photon detection, to the widely used vacuum + weak decoy state QKD system when the phase of source is partially randomized. Our analysis shows that, in some parameter regimes, the proposed attack would result in an entanglement breaking channel but still be able to trick the legitimate users to believe they have transmitted secure keys. That is, the eavesdropper is able to steal all the key information without discovered by the users. Thus, our proposal reveals that partial phase randomization is not sufficient to guarantee the security of phase-encoding QKD systems with weak coherent states.

Hacking on decoy-state quantum key distribution system with partial phase randomization.

PubMed

Sun, Shi-Hai; Jiang, Mu-Sheng; Ma, Xiang-Chun; Li, Chun-Yan; Liang, Lin-Mei

2014-04-23

Quantum key distribution (QKD) provides means for unconditional secure key transmission between two distant parties. However, in practical implementations, it suffers from quantum hacking due to device imperfections. Here we propose a hybrid measurement attack, with only linear optics, homodyne detection, and single photon detection, to the widely used vacuum + weak decoy state QKD system when the phase of source is partially randomized. Our analysis shows that, in some parameter regimes, the proposed attack would result in an entanglement breaking channel but still be able to trick the legitimate users to believe they have transmitted secure keys. That is, the eavesdropper is able to steal all the key information without discovered by the users. Thus, our proposal reveals that partial phase randomization is not sufficient to guarantee the security of phase-encoding QKD systems with weak coherent states.

Comparative proteomics analysis of Bacillus amyloliquefaciens SQR9 revealed the key proteins involved in in situ root colonization.

PubMed

Qiu, Meihua; Xu, Zhihui; Li, Xingxing; Li, Qing; Zhang, Nan; Shen, Qirong; Zhang, Ruifu

2014-12-05

Bacillus Amyloliquefaciens SQR9 is a well-investigated plant growth-promoting rhizobacteria with strong root colonization capability. To identify the key proteins involved in in situ root colonization and biofilm formation, the proteomic profiles of planktonic and root colonized SQR9 cells were compared. A total of 755 proteins were identified, of which 78 and 95 proteins were significantly increased and deceased, respectively, when SQR9 was colonized on the root. The proteins that were closely affiliated with the root colonization belonged to the functional categories of biocontrol, detoxification, biofilm formation, cell motility and chemotaxis, transport, and degradation of plant polysaccharides. A two-component system protein ResE was increased 100-fold when compared to the planktonic status; impairment of the resE gene postponed the formation of cell biofilm and decreased the root colonization capability, which may be regulated through the spo0A-sinI-yqxM pathway. The SQR9 proteomic data provide valuable clues for screening key proteins in the plant-rhizobacteria interaction.

Practical private database queries based on a quantum-key-distribution protocol

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

Jakobi, Markus; Humboldt-Universitaet zu Berlin, D-10117 Berlin; Simon, Christoph

2011-02-15

Private queries allow a user, Alice, to learn an element of a database held by a provider, Bob, without revealing which element she is interested in, while limiting her information about the other elements. We propose to implement private queries based on a quantum-key-distribution protocol, with changes only in the classical postprocessing of the key. This approach makes our scheme both easy to implement and loss tolerant. While unconditionally secure private queries are known to be impossible, we argue that an interesting degree of security can be achieved by relying on fundamental physical principles instead of unverifiable security assumptions inmore » order to protect both the user and the database. We think that the scope exists for such practical private queries to become another remarkable application of quantum information in the footsteps of quantum key distribution.« less

TSLP: A Key Regulator of Asthma Pathogenesis.

PubMed

West, Erin E; Kashyap, Mohit; Leonard, Warren J

2012-12-01

Asthma is a complex disorder of the airways that is characterized by T helper type 2 (Th2) inflammation. The pleiotrophic cytokine TSLP has emerged as an important player involved in orchestrating the inflammation seen in asthma and other atopic diseases. Early research elucidated the role of TSLP on CD4 + T cells, and recent work has revealed the impact of TSLP on multiple cell types. Furthermore, TSLP plays an important role in the sequential progression of atopic dermatitis to asthma, clarifying the key role of TSLP in the pathogenesis of asthma, a finding with therapeutic implications.

Towards secure quantum key distribution protocol for wireless LANs: a hybrid approach

NASA Astrophysics Data System (ADS)

Naik, R. Lalu; Reddy, P. Chenna

2015-12-01

The primary goals of security such as authentication, confidentiality, integrity and non-repudiation in communication networks can be achieved with secure key distribution. Quantum mechanisms are highly secure means of distributing secret keys as they are unconditionally secure. Quantum key distribution protocols can effectively prevent various attacks in the quantum channel, while classical cryptography is efficient in authentication and verification of secret keys. By combining both quantum cryptography and classical cryptography, security of communications over networks can be leveraged. Hwang, Lee and Li exploited the merits of both cryptographic paradigms for provably secure communications to prevent replay, man-in-the-middle, and passive attacks. In this paper, we propose a new scheme with the combination of quantum cryptography and classical cryptography for 802.11i wireless LANs. Since quantum cryptography is premature in wireless networks, our work is a significant step forward toward securing communications in wireless networks. Our scheme is known as hybrid quantum key distribution protocol. Our analytical results revealed that the proposed scheme is provably secure for wireless networks.

Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesis.

PubMed

Hilton, Nicholas A; Sladewski, Thomas E; Perry, Jenna A; Pataki, Zemplen; Sinclair-Davis, Amy N; Muniz, Richard S; Tran, Holly L; Wurster, Jenna I; Seo, Jiwon; de Graffenried, Christopher L

2018-05-21

The protist parasite Trypanosoma brucei is an obligate extracellular pathogen that retains its highly-polarized morphology during cell division and has evolved a novel cytokinetic process independent of non-muscle myosin II. The polo-like kinase homolog TbPLK is essential for transmission of cell polarity during division and for cytokinesis. We previously identified a putative TbPLK substrate named Tip of the Extending FAZ 1 (TOEFAZ1) as an essential kinetoplastid-specific component of the T. brucei cytokinetic machinery. We performed a proximity-dependent biotinylation (BioID) screen using TOEFAZ1 as a means to identify additional proteins that are involved in cytokinesis. Using quantitative proteomic methods, we identified nearly 500 TOEFAZ1-proximal proteins and characterized 59 in further detail. Among the candidates, we identified an essential putative phosphatase that regulates the expression level and localization of both TOEFAZ1 and TbPLK, a previously uncharacterized protein that is necessary for the assembly of a new cell posterior, and a microtubule plus-end directed orphan kinesin that is required for completing cleavage furrow ingression. The identification of these proteins provides new insight into T. brucei cytokinesis and establishes TOEFAZ1 as a key component of this essential and uniquely-configured process in kinetoplastids. This article is protected by copyright. All rights reserved. © 2018 John Wiley & Sons Ltd.

Suboptimal Doses of Raltegravir Cause Aberrant HIV Integrations | Center for Cancer Research

Cancer.gov

When a cell is infected with HIV, a DNA copy of the HIV genome is inserted into that cell’s chromosomal DNA. This insertion reaction is carried out by the viral enzyme integrase (IN) and involves two distinct steps: removal of two nucleotides from each 3’ end of the viral DNA, followed by the strand transfer reaction, in which the viral DNA ends are inserted into the host chromosomal DNA. Integration is essential for viral replication, making it an important target for antiviral therapy. Raltegravir, and the other approved integrase inhibitor, Elvitegravir, are called integrase strand transfer inhibitors (INSTIs), because they bind to the active site of IN and block the strand transfer reaction.      

Retroviral DNA Integration

PubMed Central

2016-01-01

The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

Key management of the double random-phase-encoding method using public-key encryption

NASA Astrophysics Data System (ADS)

Saini, Nirmala; Sinha, Aloka

2010-03-01

Public-key encryption has been used to encode the key of the encryption process. In the proposed technique, an input image has been encrypted by using the double random-phase-encoding method using extended fractional Fourier transform. The key of the encryption process have been encoded by using the Rivest-Shamir-Adelman (RSA) public-key encryption algorithm. The encoded key has then been transmitted to the receiver side along with the encrypted image. In the decryption process, first the encoded key has been decrypted using the secret key and then the encrypted image has been decrypted by using the retrieved key parameters. The proposed technique has advantage over double random-phase-encoding method because the problem associated with the transmission of the key has been eliminated by using public-key encryption. Computer simulation has been carried out to validate the proposed technique.

Optical key system

DOEpatents

Hagans, Karla G.; Clough, Robert E.

2000-01-01

An optical key system comprises a battery-operated optical key and an isolated lock that derives both its operating power and unlock signals from the correct optical key. A light emitting diode or laser diode is included within the optical key and is connected to transmit a bit-serial password. The key user physically enters either the code-to-transmit directly, or an index to a pseudorandom number code, in the key. Such person identification numbers can be retained permanently, or ephemeral. When a send button is pressed, the key transmits a beam of light modulated with the password information. The modulated beam of light is received by a corresponding optical lock with a photovoltaic cell that produces enough power from the beam of light to operate a password-screen digital logic. In one application, an acceptable password allows a two watt power laser diode to pump ignition and timing information over a fiberoptic cable into a sealed engine compartment. The receipt of a good password allows the fuel pump, spark, and starter systems to each operate. Therefore, bypassing the lock mechanism as is now routine with automobile thieves is pointless because the engine is so thoroughly disabled.

Public-key quantum digital signature scheme with one-time pad private-key

NASA Astrophysics Data System (ADS)

Chen, Feng-Lin; Liu, Wan-Fang; Chen, Su-Gen; Wang, Zhi-Hua

2018-01-01

A quantum digital signature scheme is firstly proposed based on public-key quantum cryptosystem. In the scheme, the verification public-key is derived from the signer's identity information (such as e-mail) on the foundation of identity-based encryption, and the signature private-key is generated by one-time pad (OTP) protocol. The public-key and private-key pair belongs to classical bits, but the signature cipher belongs to quantum qubits. After the signer announces the public-key and generates the final quantum signature, each verifier can verify publicly whether the signature is valid or not with the public-key and quantum digital digest. Analysis results show that the proposed scheme satisfies non-repudiation and unforgeability. Information-theoretic security of the scheme is ensured by quantum indistinguishability mechanics and OTP protocol. Based on the public-key cryptosystem, the proposed scheme is easier to be realized compared with other quantum signature schemes under current technical conditions.

Low-Temperature Biodiesel Research Reveals Potential Key to Successful Blend Performance (Fact Sheet)

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

Not Available

Relatively low-cost solutions could improve reliability while making biodiesel blends an affordable option. While biodiesel has very low production costs and the potential to displace up to 10% of petroleum diesel, until now, issues with cold weather performance have prevented biodiesel blends from being widely adopted. Some biodiesel blends have exhibited unexplained low-temperature performance problems even at blend levels as low as 2% by volume. The most common low-temperature performance issue is vehicle stalling caused by fuel filter clogging, which prevents fuel from reaching the engine. Research at the National Renewable Energy Laboratory (NREL) reveals the properties responsible for thesemore » problems, clearing a path for the development of solutions and expanded use of energy-conserving and low-emissions alternative fuel. NREL researchers set out to study the unpredictable nature of biodiesel crystallization, the condition that impedes the flow of fuel in cold weather. Their research revealed for the first time that saturated monoglyceride impurities common to the biodiesel manufacturing process create crystals that can cause fuel filter clogging and other problems when cooling at slow rates. Biodiesel low-temperature operational problems are commonly referred to as 'precipitates above the cloud point (CP).' NREL's Advanced Biofuels team spiked distilled soy and animal fat-derived B100, as well as B20, B10, and B5 biodiesel blends with three saturated monoglycerides (SMGs) at concentration levels comparable to those of real-world fuels. Above a threshold or eutectic concentration, the SMGs (monomyristin, monopalmitin, and monostearin) were shown to significantly raise the biodiesel CP, and had an even greater impact on the final melting temperature. Researchers discovered that upon cooling, monoglyceride initially precipitates as a metastable crystal, but it transforms over time or upon slight heating into a more stable crystal with a much lower

Finite-key analysis for measurement-device-independent quantum key distribution.

PubMed

Curty, Marcos; Xu, Feihu; Cui, Wei; Lim, Charles Ci Wen; Tamaki, Kiyoshi; Lo, Hoi-Kwong

2014-04-29

Quantum key distribution promises unconditionally secure communications. However, as practical devices tend to deviate from their specifications, the security of some practical systems is no longer valid. In particular, an adversary can exploit imperfect detectors to learn a large part of the secret key, even though the security proof claims otherwise. Recently, a practical approach--measurement-device-independent quantum key distribution--has been proposed to solve this problem. However, so far its security has only been fully proven under the assumption that the legitimate users of the system have unlimited resources. Here we fill this gap and provide a rigorous security proof against general attacks in the finite-key regime. This is obtained by applying large deviation theory, specifically the Chernoff bound, to perform parameter estimation. For the first time we demonstrate the feasibility of long-distance implementations of measurement-device-independent quantum key distribution within a reasonable time frame of signal transmission.

Enhanced limonene production in cyanobacteria reveals photosynthesis limitations.

PubMed

Wang, Xin; Liu, Wei; Xin, Changpeng; Zheng, Yi; Cheng, Yanbing; Sun, Su; Li, Runze; Zhu, Xin-Guang; Dai, Susie Y; Rentzepis, Peter M; Yuan, Joshua S

2016-12-13

Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-d-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/bioproduct production in cyanobacteria.

Optical key system

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

Hagans, K.G.; Clough, R.E.

2000-04-25

An optical key system comprises a battery-operated optical key and an isolated lock that derives both its operating power and unlock signals from the correct optical key. A light emitting diode or laser diode is included within the optical key and is connected to transmit a bit-serial password. The key user physically enters either the code-to-transmit directly, or an index to a pseudorandom number code, in the key. Such person identification numbers can be retained permanently, or ephemeral. When a send button is pressed, the key transmits a beam of light modulated with the password information. The modulated beam ofmore » light is received by a corresponding optical lock with a photovoltaic cell that produces enough power from the beam of light to operate a password-screen digital logic. In one application, an acceptable password allows a two watt power laser diode to pump ignition and timing information over a fiberoptic cable into a sealed engine compartment. The receipt of a good password allows the fuel pump, spark, and starter systems to each operate. Therefore, bypassing the lock mechanism as is now routine with automobile thieves is pointless because the engine is so thoroughly disabled.« less

Poly(A) code analyses reveal key determinants for tissue-specific mRNA alternative polyadenylation

PubMed Central

Weng, Lingjie; Li, Yi; Xie, Xiaohui; Shi, Yongsheng

2016-01-01

mRNA alternative polyadenylation (APA) is a critical mechanism for post-transcriptional gene regulation and is often regulated in a tissue- and/or developmental stage-specific manner. An ultimate goal for the APA field has been to be able to computationally predict APA profiles under different physiological or pathological conditions. As a first step toward this goal, we have assembled a poly(A) code for predicting tissue-specific poly(A) sites (PASs). Based on a compendium of over 600 features that have known or potential roles in PAS selection, we have generated and refined a machine-learning algorithm using multiple high-throughput sequencing-based data sets of tissue-specific and constitutive PASs. This code can predict tissue-specific PASs with >85% accuracy. Importantly, by analyzing the prediction performance based on different RNA features, we found that PAS context, including the distance between alternative PASs and the relative position of a PAS within the gene, is a key feature for determining the susceptibility of a PAS to tissue-specific regulation. Our poly(A) code provides a useful tool for not only predicting tissue-specific APA regulation, but also for studying its underlying molecular mechanisms. PMID:27095026

Work Keys USA.

ERIC Educational Resources Information Center

Work Keys USA, 1998

1998-01-01

"Work Keys" is a comprehensive program for assessing and teaching workplace skills. This serial "special issue" features 18 first-hand reports on Work Keys projects in action in states across North America. They show how the Work Keys is helping businesses and educators solve the challenge of building a world-class work force.…

Key attributes of expert NRL referees.

PubMed

Morris, Gavin; O'Connor, Donna

2017-05-01

Experiential knowledge of elite National Rugby League (NRL) referees was investigated to determine the key attributes contributing to expert officiating performance. Fourteen current first-grade NRL referees were asked to identify the key attributes they believed contributed to their expert refereeing performance. The modified Delphi method involved a 3-round process of an initial semi-structured interview followed by 2 questionnaires to reach consensus of opinion. The data revealed 25 attributes that were rated as most important that underpin expert NRL refereeing performance. Results illustrate the significance of the cognitive category, with the top 6 ranked attributes all cognitive skills. Of these, the referees ranked decision-making accuracy as the most important attribute, followed by reading the game, communication, game understanding, game management and knowledge of the rules. Player rapport, positioning and teamwork were the top ranked game skill attributes underpinning performance excellence. Expert referees also highlighted a number of psychological attributes (e.g., concentration, composure and mental toughness) that were significant to performance. There were only 2 physiological attributes (fitness, aerobic endurance) that were identified as significant to elite officiating performance. In summary, expert consensus was attained which successfully provided a hierarchy of the most significant attributes of expert NRL refereeing performance.

High-capacity quantum key distribution via hyperentangled degrees of freedom

NASA Astrophysics Data System (ADS)

Simon, David S.; Sergienko, Alexander V.

2014-06-01

Quantum key distribution (QKD) has long been a promising area for the application of quantum effects in solving real-world problems. However, two major obstacles have stood in the way of its widespread application: low secure key generation rates and short achievable operating distances. In this paper, a new physical mechanism for dealing with the first of these problems is proposed: the interplay between different degrees of freedom in a hyperentangled system (parametric down-conversion) is used to increase the Hilbert space dimension available for key generation while maintaining security. Polarization-based Bell tests provide security checking, while orbital angular momentum (OAM) and total angular momentum (TAM) provide a higher key generation rate. Whether to measure TAM or OAM is decided randomly in each trial. The concurrent noncommutativity of TAM with OAM and polarization provides the physical basis for quantum security. TAM measurements link polarization to OAM, so that if the legitimate participants measure OAM while the eavesdropper measures TAM (or vice-versa), then polarization entanglement is lost, revealing the eavesdropper. In contrast to other OAM-based QKD methods, complex active switching between OAM bases is not required; instead, passive switching by beam splitters combined with much simpler active switching between polarization bases makes implementation at high OAM more practical.

Finite key analysis for symmetric attacks in quantum key distribution

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

Meyer, Tim; Kampermann, Hermann; Kleinmann, Matthias

2006-10-15

We introduce a constructive method to calculate the achievable secret key rate for a generic class of quantum key distribution protocols, when only a finite number n of signals is given. Our approach is applicable to all scenarios in which the quantum state shared by Alice and Bob is known. In particular, we consider the six state protocol with symmetric eavesdropping attacks, and show that for a small number of signals, i.e., below n{approx}10{sup 4}, the finite key rate differs significantly from the asymptotic value for n{yields}{infinity}. However, for larger n, a good approximation of the asymptotic value is found.more » We also study secret key rates for protocols using higher-dimensional quantum systems.« less

Partially Key Distribution with Public Key Cryptosystem Based on Error Control Codes

NASA Astrophysics Data System (ADS)

Tavallaei, Saeed Ebadi; Falahati, Abolfazl

Due to the low level of security in public key cryptosystems based on number theory, fundamental difficulties such as "key escrow" in Public Key Infrastructure (PKI) and a secure channel in ID-based cryptography, a new key distribution cryptosystem based on Error Control Codes (ECC) is proposed . This idea is done by some modification on McEliece cryptosystem. The security of ECC cryptosystem obtains from the NP-Completeness of block codes decoding. The capability of generating public keys with variable lengths which is suitable for different applications will be provided by using ECC. It seems that usage of these cryptosystems because of decreasing in the security of cryptosystems based on number theory and increasing the lengths of their keys would be unavoidable in future.

[Key morphofunctional transformations in the evolution of chiropterans (Bats, Chiroptera)].

PubMed

Kovaleva, I M

2014-01-01

Study on the morphology and morphogenesis of wing membranes in Bats has revealed some peculiarities in their structure and development. Understanding the embryogenesis of these animals, as well as attraction of data obtained on their molecular genetics and paleontology, allows one to single out some factors that could have initiated evolutionary modifications in development programs. A scenario of the key morphofunctional transformations in the forelimbs during the evolution of chiropterans is given.

Keys to Scholarship

ERIC Educational Resources Information Center

Hebert, Terri

2011-01-01

Up ahead, a foreboding wooden door showing wear from passage of earlier travelers is spotted. As the old porch light emits a pale yellow glow, a key ring emerges from deep inside the coat pocket. Searching for just the right key, the voyager settles on one that also shows age. As the key enters its receptacle and begins to turn, a clicking noise…

GC/MS-based metabolomic studies reveal key roles of glycine in regulating silk synthesis in silkworm, Bombyx mori.

PubMed

Chen, Quanmei; Liu, Xinyu; Zhao, Ping; Sun, Yanhui; Zhao, Xinjie; Xiong, Ying; Xu, Guowang; Xia, Qingyou

2015-02-01

Metabolic profiling of silkworm, especially the factors that affect silk synthesis at the metabolic level, is little known. Herein, metabolomic method based on gas chromatography-mass spectrometry was applied to identify key metabolic changes in silk synthesis deficient silkworms. Forty-six differential metabolites were identified in Nd group with the defect of silk synthesis. Significant changes in the levels of glycine and uric acid (up-regulation), carbohydrates and free fatty acids (down-regulation) were observed. The further metabolomics of silk synthesis deficient silkworms by decreasing silk proteins synthesis using knocking out fibroin heavy chain gene or extirpating silk glands operation showed that the changes of the metabolites were almost consistent with those of the Nd group. Furthermore, the increased silk yields by supplying more glycine or its related metabolite confirmed that glycine is a key metabolite to regulate silk synthesis. These findings provide important insights into the regulation between metabolic profiling and silk synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparative analysis of prophages in Streptococcus mutans genomes

PubMed Central

Fu, Tiwei; Fan, Xiangyu; Long, Quanxin; Deng, Wanyan; Song, Jinlin

2017-01-01

Prophages have been considered genetic units that have an intimate association with novel phenotypic properties of bacterial hosts, such as pathogenicity and genomic variation. Little is known about the genetic information of prophages in the genome of Streptococcus mutans, a major pathogen of human dental caries. In this study, we identified 35 prophage-like elements in S. mutans genomes and performed a comparative genomic analysis. Comparative genomic and phylogenetic analyses of prophage sequences revealed that the prophages could be classified into three main large clusters: Cluster A, Cluster B, and Cluster C. The S. mutans prophages in each cluster were compared. The genomic sequences of phismuN66-1, phismuNLML9-1, and phismu24-1 all shared similarities with the previously reported S. mutans phages M102, M102AD, and ϕAPCM01. The genomes were organized into seven major gene clusters according to the putative functions of the predicted open reading frames: packaging and structural modules, integrase, host lysis modules, DNA replication/recombination modules, transcriptional regulatory modules, other protein modules, and hypothetical protein modules. Moreover, an integrase gene was only identified in phismuNLML9-1 prophages. PMID:29158986

Automated secured cost effective key refreshing technique to enhance WiMAX privacy key management

NASA Astrophysics Data System (ADS)

Sridevi, B.; Sivaranjani, S.; Rajaram, S.

2013-01-01

In all walks of life the way of communication is transformed by the rapid growth of wireless communication and its pervasive use. A wireless network which is fixed and richer in bandwidth is specified as IEEE 802.16, promoted and launched by an industrial forum is termed as Worldwide Interoperability for Microwave Access (WiMAX). This technology enables seamless delivery of wireless broadband service for fixed and/or mobile users. The obscurity is the long delay which occurs during the handoff management in every network. Mobile WiMAX employs an authenticated key management protocol as a part of handoff management in which the Base Station (BS) controls the distribution of keying material to the Mobile Station (MS). The protocol employed is Privacy Key Management Version 2- Extensible Authentication Protocol (PKMV2-EAP) which is responsible for the normal and periodical authorization of MSs, reauthorization as well as key refreshing. Authorization key (AK) and Traffic Encryption key (TEK) plays a vital role in key exchange. When the lifetime of key expires, MS has to request for a new key to BS which in turn leads to repetition of authorization, authentication as well as key exchange. To avoid service interruption during reauthorization , two active keys are transmitted at the same time by BS to MS. The consequences of existing work are hefty amount of bandwidth utilization, time consumption and large storage. It is also endured by Man in the Middle attack and Impersonation due to lack of security in key exchange. This paper designs an automatic mutual refreshing of keys to minimize bandwidth utilization, key storage and time consumption by proposing Previous key and Iteration based Key Refreshing Function (PKIBKRF). By integrating PKIBKRF in key generation, the simulation results indicate that 21.8% of the bandwidth and storage of keys are reduced and PKMV2 mutual authentication time is reduced by 66.67%. The proposed work is simulated with Qualnet model and

Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera.

PubMed

Gallardo, Miguel; Barrio, Santiago; Fernandez, Marisol; Paradela, Alberto; Arenas, Alicia; Toldos, Oscar; Ayala, Rosa; Albizua, Enriqueta; Jimenez, Ana; Redondo, Santiago; Garcia-Martin, Rosa Maria; Gilsanz, Florinda; Albar, Juan Pablo; Martinez-Lopez, Joaquin

2013-11-19

JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

Adaptive Control Model Reveals Systematic Feedback and Key Molecules in Metabolic Pathway Regulation

PubMed Central

Moffitt, Richard A.; Merrill, Alfred H.; Wang, May D.

2011-01-01

Abstract Robust behavior in metabolic pathways resembles stabilized performance in systems under autonomous control. This suggests we can apply control theory to study existing regulation in these cellular networks. Here, we use model-reference adaptive control (MRAC) to investigate the dynamics of de novo sphingolipid synthesis regulation in a combined theoretical and experimental case study. The effects of serine palmitoyltransferase over-expression on this pathway are studied in vitro using human embryonic kidney cells. We report two key results from comparing numerical simulations with observed data. First, MRAC simulations of pathway dynamics are comparable to simulations from a standard model using mass action kinetics. The root-sum-square (RSS) between data and simulations in both cases differ by less than 5%. Second, MRAC simulations suggest systematic pathway regulation in terms of adaptive feedback from individual molecules. In response to increased metabolite levels available for de novo sphingolipid synthesis, feedback from molecules along the main artery of the pathway is regulated more frequently and with greater amplitude than from other molecules along the branches. These biological insights are consistent with current knowledge while being new that they may guide future research in sphingolipid biology. In summary, we report a novel approach to study regulation in cellular networks by applying control theory in the context of robust metabolic pathways. We do this to uncover potential insight into the dynamics of regulation and the reverse engineering of cellular networks for systems biology. This new modeling approach and the implementation routines designed for this case study may be extended to other systems. Supplementary Material is available at www.liebertonline.com/cmb. PMID:21314456

Expanding the Definition of the Classical Bipartite Nuclear Localization Signal

PubMed Central

Lange, Allison; McLane, Laura M.; Mills, Ryan E.; Devine, Scott E.; Corbett, Anita H.

2010-01-01

Nuclear localization signals (NLSs) are amino acid sequences that target cargo proteins into the nucleus. Rigorous characterization of NLS motifs is essential to understanding and predicting pathways for nuclear import. The best-characterized NLS is the classical NLS (cNLS), which is recognized by the cNLS receptor, importin-α. cNLSs are conventionally defined as having one (monopartite) or two clusters of basic amino acids separated by a 9-12 amino acid linker (bipartite). Motivated by the finding that Ty1 integrase, which contains an unconventional putative bipartite cNLS with a 29 amino acid linker, exploits the classical nuclear import machinery, we assessed the functional boundaries for linker length within a bipartite cNLS. We confirmed that the integrase cNLS is a bona fide bipartite cNLS, then carried out a systematic analysis of linker length in an obligate bipartite cNLS cargo, which revealed that some linkers longer than conventionally defined can function in nuclear import. Linker function is dependent on the sequence and likely the inherent flexibility of the linker. Subsequently, we interrogated the Saccharomyces cerevisiae proteome to identify cellular proteins containing putative long bipartite cNLSs. We experimentally confirmed that Rrp4 contains a bipartite cNLS with a 25 amino acid linker. Our studies reveal that the traditional definition of bipartite cNLSs is too restrictive and linker length can vary depending on amino acid composition PMID:20028483

Human Immunodeficiency Virus Type 1 Employs the Cellular Dynein Light Chain 1 Protein for Reverse Transcription through Interaction with Its Integrase Protein

PubMed Central

Jayappa, Kallesh Danappa; Ao, Zhujun; Wang, Xiaoxia; Mouland, Andrew J.; Shekhar, Sudhanshu; Yang, Xi

2015-01-01

ABSTRACT In this study, we examined the requirement for host dynein adapter proteins such as dynein light chain 1 (DYNLL1), dynein light chain Tctex-type 1 (DYNLT1), and p150Glued in early steps of human immunodeficiency virus type 1 (HIV-1) replication. We found that the knockdown (KD) of DYNLL1, but not DYNLT1 or p150Glued, resulted in significantly lower levels of HIV-1 reverse transcription in cells. Following an attempt to determine how DYNLL1 could impact HIV-1 reverse transcription, we detected the DYNLL1 interaction with HIV-1 integrase (IN) but not with capsid (CA), matrix (MA), or reverse transcriptase (RT) protein. Furthermore, by mutational analysis of putative DYNLL1 interaction motifs in IN, we identified the motifs 52GQVD and 250VIQD in IN as essential for DYNLL1 interaction. The DYNLL1 interaction-defective IN mutant HIV-1 (HIV-1INQ53A/Q252A) exhibited impaired reverse transcription. Through further investigations, we have also detected relatively smaller amounts of particulate CA in DYNLL1-KD cells or in infections with HIV-1INQ53A/Q252A mutant virus. Overall, our study demonstrates the novel interaction between HIV-1 IN and cellular DYNLL1 proteins and suggests the requirement of this virus-cell interaction for proper uncoating and efficient reverse transcription of HIV-1. IMPORTANCE Host cellular DYNLL1, DYNLT1, and p150Glued proteins have been implicated in the replication of several viruses. However, their roles in HIV-1 replication have not been investigated. For the first time, we demonstrated that during viral infection, HIV-1 IN interacts with DYNLL1, and their interaction was found to have a role in proper uncoating and efficient reverse transcription of HIV-1. Thus, interaction of IN and DYNLL1 may be a potential target for future anti-HIV therapy. Moreover, while our study has evaluated the involvement of IN in HIV-1 uncoating and reverse transcription, it also predicts a possible mechanism by which IN contributes to these early viral

Same pattern, different mechanism: Locking onto the role of key species in seafloor ecosystem process

PubMed Central

Woodin, Sarah Ann; Volkenborn, Nils; Pilditch, Conrad A.; Lohrer, Andrew M.; Wethey, David S.; Hewitt, Judi E.; Thrush, Simon F.

2016-01-01

Seafloor biodiversity is a key mediator of ecosystem functioning, but its role is often excluded from global budgets or simplified to black boxes in models. New techniques allow quantification of the behavior of animals living below the sediment surface and assessment of the ecosystem consequences of complex interactions, yielding a better understanding of the role of seafloor animals in affecting key processes like primary productivity. Combining predictions based on natural history, behavior of key benthic species and environmental context allow assessment of differences in functioning and process, even when the measured ecosystem property in different systems is similar. Data from three sedimentary systems in New Zealand illustrate this. Analysis of the behaviors of the infaunal ecosystem engineers in each system revealed three very different mechanisms driving ecosystem function: density and excretion, sediment turnover and surface rugosity, and hydraulic activities and porewater bioadvection. Integrative metrics of ecosystem function in some cases differentiate among the systems (gross primary production) and in others do not (photosynthetic efficiency). Analyses based on behaviors and activities revealed important ecosystem functional differences and can dramatically improve our ability to model the impact of stressors on ecosystem and global processes. PMID:27230562

Same pattern, different mechanism: Locking onto the role of key species in seafloor ecosystem process.

PubMed

Woodin, Sarah Ann; Volkenborn, Nils; Pilditch, Conrad A; Lohrer, Andrew M; Wethey, David S; Hewitt, Judi E; Thrush, Simon F

2016-05-27

Seafloor biodiversity is a key mediator of ecosystem functioning, but its role is often excluded from global budgets or simplified to black boxes in models. New techniques allow quantification of the behavior of animals living below the sediment surface and assessment of the ecosystem consequences of complex interactions, yielding a better understanding of the role of seafloor animals in affecting key processes like primary productivity. Combining predictions based on natural history, behavior of key benthic species and environmental context allow assessment of differences in functioning and process, even when the measured ecosystem property in different systems is similar. Data from three sedimentary systems in New Zealand illustrate this. Analysis of the behaviors of the infaunal ecosystem engineers in each system revealed three very different mechanisms driving ecosystem function: density and excretion, sediment turnover and surface rugosity, and hydraulic activities and porewater bioadvection. Integrative metrics of ecosystem function in some cases differentiate among the systems (gross primary production) and in others do not (photosynthetic efficiency). Analyses based on behaviors and activities revealed important ecosystem functional differences and can dramatically improve our ability to model the impact of stressors on ecosystem and global processes.

Improved key-rate bounds for practical decoy-state quantum-key-distribution systems

NASA Astrophysics Data System (ADS)

Zhang, Zhen; Zhao, Qi; Razavi, Mohsen; Ma, Xiongfeng

2017-01-01

The decoy-state scheme is the most widely implemented quantum-key-distribution protocol in practice. In order to account for the finite-size key effects on the achievable secret key generation rate, a rigorous statistical fluctuation analysis is required. Originally, a heuristic Gaussian-approximation technique was used for this purpose, which, despite its analytical convenience, was not sufficiently rigorous. The fluctuation analysis has recently been made rigorous by using the Chernoff bound. There is a considerable gap, however, between the key-rate bounds obtained from these techniques and that obtained from the Gaussian assumption. Here we develop a tighter bound for the decoy-state method, which yields a smaller failure probability. This improvement results in a higher key rate and increases the maximum distance over which secure key exchange is possible. By optimizing the system parameters, our simulation results show that our method almost closes the gap between the two previously proposed techniques and achieves a performance similar to that of conventional Gaussian approximations.

Enhanced limonene production in cyanobacteria reveals photosynthesis limitations

PubMed Central

Wang, Xin; Liu, Wei; Xin, Changpeng; Zheng, Yi; Cheng, Yanbing; Sun, Su; Li, Runze; Zhu, Xin-Guang; Dai, Susie Y.; Rentzepis, Peter M.; Yuan, Joshua S.

2016-01-01

Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-d-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/bioproduct production in cyanobacteria. PMID:27911807

Traffic at the tmRNA Gene

PubMed Central

Williams, Kelly P.

2003-01-01

A partial screen for genetic elements integrated into completely sequenced bacterial genomes shows more significant bias in specificity for the tmRNA gene (ssrA) than for any type of tRNA gene. Horizontal gene transfer, a major avenue of bacterial evolution, was assessed by focusing on elements using this single attachment locus. Diverse elements use ssrA; among enterobacteria alone, at least four different integrase subfamilies have independently evolved specificity for ssrA, and almost every strain analyzed presents a unique set of integrated elements. Even elements using essentially the same integrase can be very diverse, as is a group with an ssrA-specific integrase of the P4 subfamily. This same integrase appears to promote damage routinely at attachment sites, which may be adaptive. Elements in arrays can recombine; one such event mediated by invertible DNA segments within neighboring elements likely explains the monophasic nature of Salmonella enterica serovar Typhi. One of a limited set of conserved sequences occurs at the attachment site of each enterobacterial element, apparently serving as a transcriptional terminator for ssrA. Elements were usually found integrated into tRNA-like sequence at the 3′ end of ssrA, at subsites corresponding to those used in tRNA genes; an exception was found at the non-tRNA-like 3′ end produced by ssrA gene permutation in cyanobacteria, suggesting that, during the evolution of new site specificity by integrases, tropism toward a conserved 3′ end of an RNA gene may be as strong as toward a tRNA-like sequence. The proximity of ssrA and smpB, which act in concert, was also surveyed. PMID:12533482

[Efficacy of dolutegravir in treatment-experienced patients: the SAILING and VIKING trials].

PubMed

Moreno, Santiago; Berenguer, Juan

2015-03-01

Dolutegravir is an HIV integrase inhibitor with a high genetic barrier to resistance and is active against raltegravir- and/or elvitegravir-resistant strains. The clinical development of dolutegravir for HIV infection rescue therapy is based on 3 clinical trials. In the SAILING trial, dolutegravir (5 mg once daily) in combination with 2 other antiretroviral agents was well tolerated and showed greater virological effect than raltegravir (400 mg twice daily) in the treatment of integrase inhibitor-naïve adults with virological failure infected with HIV strains with at least two-class drug resistance. The VIKING studies were designed to evaluate the efficacy of dolutegravir as rescue therapy in treatment-experienced patients infected with HIV strains with resistance mutations to raltegravir and/or elvitegravir. VIKING-1-2 was a dose-ranging phase IIb trial. VIKING-3 was a phase III trial in which dolutegravir (50 mg twice daily) formed part of an optimized regimen and proved safe and effective in this difficult-to-treat group of patients. Dolutegravir is the integrase inhibitor of choice for rescue therapy in multiresistant HIV infection, both in integrase inhibitor-naïve patients and in those previously treated with raltegravir or elvitegravir. Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

Structure-based virtual screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75).

PubMed

Panwar, Umesh; Singh, Sanjeev Kumar

2017-10-23

HIV-1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure-based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory, and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine-Z742267384, Maybridge-HTS02400, and Specs-AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behavior and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.

A Simple Exercise Reveals the Way Students Think about Scientific Modeling

ERIC Educational Resources Information Center

Ruebush, Laura; Sulikowski, Michelle; North, Simon

2009-01-01

Scientific modeling is an integral part of contemporary science, yet many students have little understanding of how models are developed, validated, and used to predict and explain phenomena. A simple modeling exercise led to significant gains in understanding key attributes of scientific modeling while revealing some stubborn misconceptions.…

Public Key Cryptography.

ERIC Educational Resources Information Center

Tapson, Frank

1996-01-01

Describes public key cryptography, also known as RSA, which is a system using two keys, one used to put a message into cipher and another used to decipher the message. Presents examples using small prime numbers. (MKR)

Population Genetics of Two Key Mosquito Vectors of Rift Valley Fever Virus Reveals New Insights into the Changing Disease Outbreak Patterns in Kenya

PubMed Central

Tchouassi, David P.; Bastos, Armanda D. S.; Sole, Catherine L.; Diallo, Mawlouth; Lutomiah, Joel; Mutisya, James; Mulwa, Francis; Borgemeister, Christian; Sang, Rosemary; Torto, Baldwyn

2014-01-01

Rift Valley fever (RVF) outbreaks in Kenya have increased in frequency and range to include northeastern Kenya where viruses are increasingly being isolated from known (Aedes mcintoshi) and newly-associated (Ae. ochraceus) vectors. The factors contributing to these changing outbreak patterns are unclear and the population genetic structure of key vectors and/or specific virus-vector associations, in particular, are under-studied. By conducting mitochondrial and nuclear DNA analyses on >220 Kenyan specimens of Ae. mcintoshi and Ae. ochraceus, we uncovered high levels of vector complexity which may partly explain the disease outbreak pattern. Results indicate that Ae. mcintoshi consists of a species complex with one of the member species being unique to the newly-established RVF outbreak-prone northeastern region of Kenya, whereas Ae. ochraceus is a homogeneous population that appears to be undergoing expansion. Characterization of specimens from a RVF-prone site in Senegal, where Ae. ochraceus is a primary vector, revealed direct genetic links between the two Ae. ochraceus populations from both countries. Our data strongly suggest that unlike Ae. mcintoshi, Ae. ochraceus appears to be a relatively recent, single 'introduction' into Kenya. These results, together with increasing isolations from this vector, indicate that Ae. ochraceus will likely be of greater epidemiological importance in future RVF outbreaks in Kenya. Furthermore, the overall vector complexity calls into question the feasibility of mosquito population control approaches reliant on genetic modification. PMID:25474018

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages.

PubMed

Cheng, Lucy; Marinelli, Laura J; Grosset, Noël; Fitz-Gibbon, Sorel T; Bowman, Charles A; Dang, Brian Q; Russell, Daniel A; Jacobs-Sera, Deborah; Shi, Baochen; Pellegrini, Matteo; Miller, Jeff F; Gautier, Michel; Hatfull, Graham F; Modlin, Robert L

2018-03-01

A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity.

PubMed

Nancolas, Bethany; Sessions, Richard B; Halestrap, Andrew P

2015-02-15

The proton-linked monocarboxylate transporters (MCTs) are required for lactic acid transport into and out of all mammalian cells. Thus, they play an essential role in tumour cells that are usually highly glycolytic and are promising targets for anti-cancer drugs. AR-C155858 is a potent MCT1 inhibitor (Ki ~2 nM) that also inhibits MCT2 when associated with basigin but not MCT4. Previous work [Ovens, M.J. et al. (2010) Biochem. J. 425, 523-530] revealed that AR-C155858 binding to MCT1 occurs from the intracellular side and involves transmembrane helices (TMs) 7-10. In the present paper, we generate a molecular model of MCT4 based on our previous models of MCT1 and identify residues in the intracellular substrate-binding cavity that differ significantly between MCT4 and MCT1/MCT2 and so might account for differences in inhibitor binding. We tested their involvement using site-directed mutagenesis (SDM) of MCT1 to change residues individually or in combination with their MCT4 equivalent and determined inhibitor sensitivity following expression in Xenopus oocytes. Phe360 and Ser364 were identified as important for AR-C155858 binding with the F360Y/S364G mutant exhibiting >100-fold reduction in inhibitor sensitivity. To refine the binding site further, we used molecular dynamics (MD) simulations and additional SDM. This approach implicated six more residues whose involvement was confirmed by both transport studies and [3H]-AR-C155858 binding to oocyte membranes. Taken together, our data imply that Asn147, Arg306 and Ser364 are important for directing AR-C155858 to its final binding site which involves interaction of the inhibitor with Lys38, Asp302 and Phe360 (residues that also play key roles in the translocation cycle) and also Leu274 and Ser278.

75 FR 17463 - Key West Bank, Key West, Florida; Notice of Appointment of Receiver

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-06

... DEPARTMENT OF THE TREASURY Office of Thrift Supervision Key West Bank, Key West, Florida; Notice of Appointment of Receiver Notice is hereby given that, pursuant to the authority contained in... Federal Deposit Insurance Corporation as sole Receiver for Key West Bank, Key West, Florida, (OTS No...

Key Objectives Bank: Year 9. Key Stage 3: National Strategy.

ERIC Educational Resources Information Center

Department for Education and Skills, London (England).

In each sub-section of the "Framework for Teaching English: Years 7, 8 and 9," certain key objectives are identified in boldface print. These objectives are key because they signify skills or understanding which are crucial to pupils' language development. They are challenging for the age group and are important markers of progress. This…

Experimental quantum key distribution with finite-key security analysis for noisy channels.

PubMed

Bacco, Davide; Canale, Matteo; Laurenti, Nicola; Vallone, Giuseppe; Villoresi, Paolo

2013-01-01

In quantum key distribution implementations, each session is typically chosen long enough so that the secret key rate approaches its asymptotic limit. However, this choice may be constrained by the physical scenario, as in the perspective use with satellites, where the passage of one terminal over the other is restricted to a few minutes. Here we demonstrate experimentally the extraction of secure keys leveraging an optimal design of the prepare-and-measure scheme, according to recent finite-key theoretical tight bounds. The experiment is performed in different channel conditions, and assuming two distinct attack models: individual attacks or general quantum attacks. The request on the number of exchanged qubits is then obtained as a function of the key size and of the ambient quantum bit error rate. The results indicate that viable conditions for effective symmetric, and even one-time-pad, cryptography are achievable.

Identification of key regulators of pancreatic cancer progression through multidimensional systems-level analysis.

PubMed

Rajamani, Deepa; Bhasin, Manoj K

2016-05-03

Pancreatic cancer is an aggressive cancer with dismal prognosis, urgently necessitating better biomarkers to improve therapeutic options and early diagnosis. Traditional approaches of biomarker detection that consider only one aspect of the biological continuum like gene expression alone are limited in their scope and lack robustness in identifying the key regulators of the disease. We have adopted a multidimensional approach involving the cross-talk between the omics spaces to identify key regulators of disease progression. Multidimensional domain-specific disease signatures were obtained using rank-based meta-analysis of individual omics profiles (mRNA, miRNA, DNA methylation) related to pancreatic ductal adenocarcinoma (PDAC). These domain-specific PDAC signatures were integrated to identify genes that were affected across multiple dimensions of omics space in PDAC (genes under multiple regulatory controls, GMCs). To further pin down the regulators of PDAC pathophysiology, a systems-level network was generated from knowledge-based interaction information applied to the above identified GMCs. Key regulators were identified from the GMC network based on network statistics and their functional importance was validated using gene set enrichment analysis and survival analysis. Rank-based meta-analysis identified 5391 genes, 109 miRNAs and 2081 methylation-sites significantly differentially expressed in PDAC (false discovery rate ≤ 0.05). Bimodal integration of meta-analysis signatures revealed 1150 and 715 genes regulated by miRNAs and methylation, respectively. Further analysis identified 189 altered genes that are commonly regulated by miRNA and methylation, hence considered GMCs. Systems-level analysis of the scale-free GMCs network identified eight potential key regulator hubs, namely E2F3, HMGA2, RASA1, IRS1, NUAK1, ACTN1, SKI and DLL1, associated with important pathways driving cancer progression. Survival analysis on individual key regulators revealed

Nonmarket economic user values of the Florida Keys/Key West

Treesearch

Vernon R. Leeworthy; J. Michael Bowker

1997-01-01

This report provides estimates of the nonmarket economic user values for recreating visitors to the Florida Keys/Key West that participated in natural resource-based activities. Results from estimated travel cost models are presented, including visitor’s responses to prices and estimated per person-trip user values. Annual user values are also calculated and presented...

Network analysis of ChIP-Seq data reveals key genes in prostate cancer.

PubMed

Zhang, Yu; Huang, Zhen; Zhu, Zhiqiang; Liu, Jianwei; Zheng, Xin; Zhang, Yuhai

2014-09-03

Prostate cancer (PC) is the second most common cancer among men in the United States, and it imposes a considerable threat to human health. A deep understanding of its underlying molecular mechanisms is the premise for developing effective targeted therapies. Recently, deep transcriptional sequencing has been used as an effective genomic assay to obtain insights into diseases and may be helpful in the study of PC. In present study, ChIP-Seq data for PC and normal samples were compared, and differential peaks identified, based upon fold changes (with P-values calculated with t-tests). Annotations of these peaks were performed. Protein-protein interaction (PPI) network analysis was performed with BioGRID and constructed with Cytoscape, following which the highly connected genes were screened. We obtained a total of 5,570 differential peaks, including 3,726 differentially enriched peaks in tumor samples and 1,844 differentially enriched peaks in normal samples. There were eight significant regions of the peaks. The intergenic region possessed the highest score (51%), followed by intronic (31%) and exonic (11%) regions. The analysis revealed the top 35 highly connected genes, which comprised 33 differential genes (such as YWHAQ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein and θ polypeptide) from ChIP-Seq data and 2 differential genes retrieved from the PPI network: UBA52 (ubiquitin A-52 residue ribosomal protein fusion product (1) and SUMO2 (SMT3 suppressor of mif two 3 homolog (2) . Our findings regarding potential PC-related genes increase the understanding of PC and provides direction for future research.

Cellular and molecular mechanisms of HIV-1 integration targeting.

PubMed

Engelman, Alan N; Singh, Parmit K

2018-07-01

Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.

Secure key storage and distribution

DOEpatents

Agrawal, Punit

2015-06-02

This disclosure describes a distributed, fault-tolerant security system that enables the secure storage and distribution of private keys. In one implementation, the security system includes a plurality of computing resources that independently store private keys provided by publishers and encrypted using a single security system public key. To protect against malicious activity, the security system private key necessary to decrypt the publication private keys is not stored at any of the computing resources. Rather portions, or shares of the security system private key are stored at each of the computing resources within the security system and multiple security systems must communicate and share partial decryptions in order to decrypt the stored private key.

Fundamental finite key limits for one-way information reconciliation in quantum key distribution

NASA Astrophysics Data System (ADS)

Tomamichel, Marco; Martinez-Mateo, Jesus; Pacher, Christoph; Elkouss, David

2017-11-01

The security of quantum key distribution protocols is guaranteed by the laws of quantum mechanics. However, a precise analysis of the security properties requires tools from both classical cryptography and information theory. Here, we employ recent results in non-asymptotic classical information theory to show that one-way information reconciliation imposes fundamental limitations on the amount of secret key that can be extracted in the finite key regime. In particular, we find that an often used approximation for the information leakage during information reconciliation is not generally valid. We propose an improved approximation that takes into account finite key effects and numerically test it against codes for two probability distributions, that we call binary-binary and binary-Gaussian, that typically appear in quantum key distribution protocols.

A zebrafish model of PINK1 deficiency reveals key pathway dysfunction including HIF signaling.

PubMed

Priyadarshini, M; Tuimala, J; Chen, Y C; Panula, P

2013-06-01

The PTEN induced putative kinase 1 (PINK1) gene is mutated in patients with hereditary early onset Parkinson's disease (PD). The targets of PINK1 and the mechanisms in PD are still not fully understood. Here, we carried out a high-throughput and unbiased microarray study to identify novel functions and pathways for PINK1. In larval zebrafish, the function of pink1 was inhibited using splice-site morpholino oligonucleotides and the samples were hybridized on a two-color gene expression array. We found 177 significantly altered genes in pink1 morphants compared with the uninjected wildtype controls (log fold change values from -1.6 to +0.9). The five most prominent pathways based on critical biological processes and key toxicological responses were hypoxia-inducible factor (HIF) signaling, TGF-β signaling, mitochondrial dysfunction, RAR activation, and biogenesis of mitochondria. Furthermore, we verified that potentially important genes such as hif1α, catalase, SOD3, and atp1a2a were downregulated in pink1 morphants, whereas genes such as fech, pax2a, and notch1a were upregulated. Some of these genes have been found to play important roles in HIF signaling pathways. The pink1 morphants were found to have heart dysfunction, increased erythropoiesis, increased expression of vascular endothelial growth factors, and increased ROS. Our findings suggest that a lack of pink1 in zebrafish alters many vital and critical pathways in addition to the HIF signaling pathway. Copyright © 2013 Elsevier Inc. All rights reserved.

Physical Unclonable Function Hardware Keys Utilizing Kirchhoff-Law Secure Key Exchange and Noise-Based Logic

NASA Astrophysics Data System (ADS)

Kish, Laszlo B.; Kwan, Chiman

Weak unclonable function (PUF) encryption key means that the manufacturer of the hardware can clone the key but not anybody else. Strong unclonable function (PUF) encryption key means that even the manufacturer of the hardware is unable to clone the key. In this paper, first we introduce an "ultra" strong PUF with intrinsic dynamical randomness, which is not only unclonable but also gets renewed to an independent key (with fresh randomness) during each use via the unconditionally secure key exchange. The solution utilizes the Kirchhoff-law-Johnson-noise (KLJN) method for dynamical key renewal and a one-time-pad secure key for the challenge/response process. The secure key is stored in a flash memory on the chip to provide tamper-resistance and nonvolatile storage with zero power requirements in standby mode. Simplified PUF keys are shown: a strong PUF utilizing KLJN protocol during the first run and noise-based logic (NBL) hyperspace vector string verification method for the challenge/response during the rest of its life or until it is re-initialized. Finally, the simplest PUF utilizes NBL without KLJN thus it can be cloned by the manufacturer but not by anybody else.

Museum specimens reveal loss of pollen host plants as key factor driving wild bee decline in The Netherlands.

PubMed

Scheper, Jeroen; Reemer, Menno; van Kats, Ruud; Ozinga, Wim A; van der Linden, Giel T J; Schaminée, Joop H J; Siepel, Henk; Kleijn, David

2014-12-09

Evidence for declining populations of both wild and managed bees has raised concern about a potential global pollination crisis. Strategies to mitigate bee loss generally aim to enhance floral resources. However, we do not really know whether loss of preferred floral resources is the key driver of bee decline because accurate assessment of host plant preferences is difficult, particularly for species that have become rare. Here we examine whether population trends of wild bees in The Netherlands can be explained by trends in host plants, and how this relates to other factors such as climate change. We determined host plant preference of bee species using pollen loads on specimens in entomological collections that were collected before the onset of their decline, and used atlas data to quantify population trends of bee species and their host plants. We show that decline of preferred host plant species was one of two main factors associated with bee decline. Bee body size, the other main factor, was negatively related to population trend, which, because larger bee species have larger pollen requirements than smaller species, may also point toward food limitation as a key factor driving wild bee loss. Diet breadth and other potential factors such as length of flight period or climate change sensitivity were not important in explaining twentieth century bee population trends. These results highlight the species-specific nature of wild bee decline and indicate that mitigation strategies will only be effective if they target the specific host plants of declining species.

Museum specimens reveal loss of pollen host plants as key factor driving wild bee decline in The Netherlands

PubMed Central

Scheper, Jeroen; Reemer, Menno; van Kats, Ruud; Ozinga, Wim A.; van der Linden, Giel T. J.; Schaminée, Joop H. J.; Siepel, Henk; Kleijn, David

2014-01-01

Evidence for declining populations of both wild and managed bees has raised concern about a potential global pollination crisis. Strategies to mitigate bee loss generally aim to enhance floral resources. However, we do not really know whether loss of preferred floral resources is the key driver of bee decline because accurate assessment of host plant preferences is difficult, particularly for species that have become rare. Here we examine whether population trends of wild bees in The Netherlands can be explained by trends in host plants, and how this relates to other factors such as climate change. We determined host plant preference of bee species using pollen loads on specimens in entomological collections that were collected before the onset of their decline, and used atlas data to quantify population trends of bee species and their host plants. We show that decline of preferred host plant species was one of two main factors associated with bee decline. Bee body size, the other main factor, was negatively related to population trend, which, because larger bee species have larger pollen requirements than smaller species, may also point toward food limitation as a key factor driving wild bee loss. Diet breadth and other potential factors such as length of flight period or climate change sensitivity were not important in explaining twentieth century bee population trends. These results highlight the species-specific nature of wild bee decline and indicate that mitigation strategies will only be effective if they target the specific host plants of declining species. PMID:25422416

Expression profiling reveals Spot 42 small RNA as a key regulator in the central metabolism of Aliivibrio salmonicida

PubMed Central

2012-01-01

Background Spot 42 was discovered in Escherichia coli nearly 40 years ago as an abundant, small and unstable RNA. Its biological role has remained obscure until recently, and is today implicated in having broader roles in the central and secondary metabolism. Spot 42 is encoded by the spf gene. The gene is ubiquitous in the Vibrionaceae family of gamma-proteobacteria. One member of this family, Aliivibrio salmonicida, causes cold-water vibriosis in farmed Atlantic salmon. Its genome encodes Spot 42 with 84% identity to E. coli Spot 42. Results We generated a A. salmonicida spf deletion mutant. We then used microarray and Northern blot analyses to monitor global effects on the transcriptome in order to provide insights into the biological roles of Spot 42 in this bacterium. In the presence of glucose, we found a surprisingly large number of ≥ 2X differentially expressed genes, and several major cellular processes were affected. A gene encoding a pirin-like protein showed an on/off expression pattern in the presence/absence of Spot 42, which suggests that Spot 42 plays a key regulatory role in the central metabolism by regulating the switch between fermentation and respiration. Interestingly, we discovered an sRNA named VSsrna24, which is encoded immediately downstream of spf. This new sRNA has an expression pattern opposite to that of Spot 42, and its expression is repressed by glucose. Conclusions We hypothesize that Spot 42 plays a key role in the central metabolism, in part by regulating the pyruvat dehydrogenase enzyme complex via pirin. PMID:22272603

Quantum key distribution with finite resources: Secret key rates via Renyi entropies

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

Abruzzo, Silvestre; Kampermann, Hermann; Mertz, Markus

A realistic quantum key distribution (QKD) protocol necessarily deals with finite resources, such as the number of signals exchanged by the two parties. We derive a bound on the secret key rate which is expressed as an optimization problem over Renyi entropies. Under the assumption of collective attacks by an eavesdropper, a computable estimate of our bound for the six-state protocol is provided. This bound leads to improved key rates in comparison to previous results.

Identification of Key Odorants in Withering-Flavored Green Tea by Aroma Extract Dilution Analysis

NASA Astrophysics Data System (ADS)

Mizukami, Yuzo; Yamaguchi, Yuichi

This research aims to identify key odorants in withering-flavored green tea. Application of the aroma extract dilution analysis using the volatile fraction of green tea and withering-flavored green tea revealed 25 and 35 odor-active peaks with the flavor dilution factors of≥4, respectively. 4-mercapto-4-methylpentan-2-one, (E)-2-nonenal, linalool, (E,Z)-2,6-nonadienal and 3-methylnonane-2,4-dione were key odorants in green tea with the flavor dilution factor of≥16. As well as these 5 odorants, 1-octen-3-one, β-damascenone, geraniol, β-ionone, (Z)-methyljasmonate, indole and coumarine contributed to the withering flavor of green tea.

Quantum dense key distribution

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

Degiovanni, I.P.; Ruo Berchera, I.; Castelletto, S.

2004-03-01

This paper proposes a protocol for quantum dense key distribution. This protocol embeds the benefits of a quantum dense coding and a quantum key distribution and is able to generate shared secret keys four times more efficiently than the Bennet-Brassard 1984 protocol. We hereinafter prove the security of this scheme against individual eavesdropping attacks, and we present preliminary experimental results, showing its feasibility.

33 CFR 110.189a - Key West Harbor, Key West, Fla., naval explosives anchorage area.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Key West Harbor, Key West, Fla..., DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Anchorage Grounds § 110.189a Key West Harbor, Key West, Fla., naval explosives anchorage area. (a) The anchorage ground. A circular area with its...

33 CFR 110.189a - Key West Harbor, Key West, Fla., naval explosives anchorage area.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Key West Harbor, Key West, Fla..., DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Anchorage Grounds § 110.189a Key West Harbor, Key West, Fla., naval explosives anchorage area. (a) The anchorage ground. A circular area with its...

33 CFR 110.189a - Key West Harbor, Key West, Fla., naval explosives anchorage area.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Key West Harbor, Key West, Fla..., DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Anchorage Grounds § 110.189a Key West Harbor, Key West, Fla., naval explosives anchorage area. (a) The anchorage ground. A circular area with its...

33 CFR 110.189a - Key West Harbor, Key West, Fla., naval explosives anchorage area.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Key West Harbor, Key West, Fla..., DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Anchorage Grounds § 110.189a Key West Harbor, Key West, Fla., naval explosives anchorage area. (a) The anchorage ground. A circular area with its...

33 CFR 110.189a - Key West Harbor, Key West, Fla., naval explosives anchorage area.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Key West Harbor, Key West, Fla..., DEPARTMENT OF HOMELAND SECURITY ANCHORAGES ANCHORAGE REGULATIONS Anchorage Grounds § 110.189a Key West Harbor, Key West, Fla., naval explosives anchorage area. (a) The anchorage ground. A circular area with its...

The HIV-1 integrase-LEDGF allosteric inhibitor MUT-A: resistance profile, impairment of virus maturation and infectivity but without influence on RNA packaging or virus immunoreactivity.

PubMed

Amadori, Céline; van der Velden, Yme Ubeles; Bonnard, Damien; Orlov, Igor; van Bel, Nikki; Le Rouzic, Erwann; Miralles, Laia; Brias, Julie; Chevreuil, Francis; Spehner, Daniele; Chasset, Sophie; Ledoussal, Benoit; Mayr, Luzia; Moreau, François; García, Felipe; Gatell, José; Zamborlini, Alessia; Emiliani, Stéphane; Ruff, Marc; Klaholz, Bruno P; Moog, Christiane; Berkhout, Ben; Plana, Montserrat; Benarous, Richard

2017-11-09

HIV-1 Integrase (IN) interacts with the cellular co-factor LEDGF/p75 and tethers the HIV preintegration complex to the host genome enabling integration. Recently a new class of IN inhibitors was described, the IN-LEDGF allosteric inhibitors (INLAIs). Designed to interfere with the IN-LEDGF interaction during integration, the major impact of these inhibitors was surprisingly found on virus maturation, causing a reverse transcription defect in target cells. Here we describe the MUT-A compound as a genuine INLAI with an original chemical structure based on a new type of scaffold, a thiophene ring. MUT-A has all characteristics of INLAI compounds such as inhibition of IN-LEDGF/p75 interaction, IN multimerization, dual antiretroviral (ARV) activities, normal packaging of genomic viral RNA and complete Gag protein maturation. MUT-A has more potent ARV activity compared to other INLAIs previously reported, but similar profile of resistance mutations and absence of ARV activity on SIV. HIV-1 virions produced in the presence of MUT-A were non-infectious with the formation of eccentric condensates outside of the core. In studying the immunoreactivity of these non-infectious virions, we found that inactivated HIV-1 particles were captured by anti-HIV-specific neutralizing and non-neutralizing antibodies (b12, 2G12, PGT121, 4D4, 10-1074, 10E8, VRC01) with efficiencies comparable to non-treated virus. Autologous CD4 + T lymphocyte proliferation and cytokine induction by monocyte-derived dendritic cells (MDDC) pulsed either with MUT-A-inactivated HIV or non-treated HIV were also comparable. Although strongly defective in infectivity, HIV-1 virions produced in the presence of the MUT-A INLAI have a normal protein and genomic RNA content as well as B and T cell immunoreactivities comparable to non-treated HIV-1. These inactivated viruses might form an attractive new approach in vaccine research in an attempt to study if this new type of immunogen could elicit an immune response

Image encryption using fingerprint as key based on phase retrieval algorithm and public key cryptography

NASA Astrophysics Data System (ADS)

Zhao, Tieyu; Ran, Qiwen; Yuan, Lin; Chi, Yingying; Ma, Jing

2015-09-01

In this paper, a novel image encryption system with fingerprint used as a secret key is proposed based on the phase retrieval algorithm and RSA public key algorithm. In the system, the encryption keys include the fingerprint and the public key of RSA algorithm, while the decryption keys are the fingerprint and the private key of RSA algorithm. If the users share the fingerprint, then the system will meet the basic agreement of asymmetric cryptography. The system is also applicable for the information authentication. The fingerprint as secret key is used in both the encryption and decryption processes so that the receiver can identify the authenticity of the ciphertext by using the fingerprint in decryption process. Finally, the simulation results show the validity of the encryption scheme and the high robustness against attacks based on the phase retrieval technique.

Limitations on quantum key repeaters.

PubMed

Bäuml, Stefan; Christandl, Matthias; Horodecki, Karol; Winter, Andreas

2015-04-23

A major application of quantum communication is the distribution of entangled particles for use in quantum key distribution. Owing to noise in the communication line, quantum key distribution is, in practice, limited to a distance of a few hundred kilometres, and can only be extended to longer distances by use of a quantum repeater, a device that performs entanglement distillation and quantum teleportation. The existence of noisy entangled states that are undistillable but nevertheless useful for quantum key distribution raises the question of the feasibility of a quantum key repeater, which would work beyond the limits of entanglement distillation, hence possibly tolerating higher noise levels than existing protocols. Here we exhibit fundamental limits on such a device in the form of bounds on the rate at which it may extract secure key. As a consequence, we give examples of states suitable for quantum key distribution but unsuitable for the most general quantum key repeater protocol.

Comparative genomic analysis of clinical and environmental Vibrio vulnificus isolates revealed biotype 3 evolutionary relationships.

PubMed

Koton, Yael; Gordon, Michal; Chalifa-Caspi, Vered; Bisharat, Naiel

2014-01-01

In 1996 a common-source outbreak of severe soft tissue and bloodstream infections erupted among Israeli fish farmers and fish consumers due to changes in fish marketing policies. The causative pathogen was a new strain of Vibrio vulnificus, named biotype 3, which displayed a unique biochemical and genotypic profile. Initial observations suggested that the pathogen erupted as a result of genetic recombination between two distinct populations. We applied a whole genome shotgun sequencing approach using several V. vulnificus strains from Israel in order to study the pan genome of V. vulnificus and determine the phylogenetic relationship of biotype 3 with existing populations. The core genome of V. vulnificus based on 16 draft and complete genomes consisted of 3068 genes, representing between 59 and 78% of the whole genome of 16 strains. The accessory genome varied in size from 781 to 2044 kbp. Phylogenetic analysis based on whole, core, and accessory genomes displayed similar clustering patterns with two main clusters, clinical (C) and environmental (E), all biotype 3 strains formed a distinct group within the E cluster. Annotation of accessory genomic regions found in biotype 3 strains and absent from the core genome yielded 1732 genes, of which the vast majority encoded hypothetical proteins, phage-related proteins, and mobile element proteins. A total of 1916 proteins (including 713 hypothetical proteins) were present in all human pathogenic strains (both biotype 3 and non-biotype 3) and absent from the environmental strains. Clustering analysis of the non-hypothetical proteins revealed 148 protein clusters shared by all human pathogenic strains; these included transcriptional regulators, arylsulfatases, methyl-accepting chemotaxis proteins, acetyltransferases, GGDEF family proteins, transposases, type IV secretory system (T4SS) proteins, and integrases. Our study showed that V. vulnificus biotype 3 evolved from environmental populations and formed a genetically

Extending key sharing: how to generate a key tightly coupled to a network security policy

NASA Astrophysics Data System (ADS)

Kazantzidis, Matheos

2006-04-01

Current state of the art security policy technologies, besides the small scale limitation and largely manual nature of accompanied management methods, are lacking a) in real-timeliness of policy implementation and b) vulnerabilities and inflexibility stemming from the centralized policy decision making; even if, for example, a policy description or access control database is distributed, the actual decision is often a centralized action and forms a system single point of failure. In this paper we are presenting a new fundamental concept that allows implement a security policy by a systematic and efficient key distribution procedure. Specifically, we extend the polynomial Shamir key splitting. According to this, a global key is split into n parts, any k of which can re-construct the original key. In this paper we present a method that instead of having "any k parts" be able to re-construct the original key, the latter can only be reconstructed if keys are combined as any access control policy describes. This leads into an easily deployable key generation procedure that results a single key per entity that "knows" its role in the specific access control policy from which it was derived. The system is considered efficient as it may be used to avoid expensive PKI operations or pairwise key distributions as well as provides superior security due to its distributed nature, the fact that the key is tightly coupled to the policy, and that policy change may be implemented easier and faster.

Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity

PubMed Central

Nancolas, Bethany; Sessions, Richard B.; Halestrap, Andrew P.

2014-01-01

The proton-linked monocarboxylate transporters (MCTs) are required for lactic acid transport into and out of all mammalian cells. Thus, they play an essential role in tumour cells that are usually highly glycolytic and are promising targets for anti-cancer drugs. AR-C155858 is a potent MCT1 inhibitor (Ki ~2 nM) that also inhibits MCT2 when associated with basigin but not MCT4. Previous work [Ovens, M.J. et al. (2010) Biochem. J. 425, 523–530] revealed that AR-C155858 binding to MCT1 occurs from the intracellular side and involves transmembrane helices (TMs) 7–10. In the present paper, we generate a molecular model of MCT4 based on our previous models of MCT1 and identify residues in the intracellular substrate-binding cavity that differ significantly between MCT4 and MCT1/MCT2 and so might account for differences in inhibitor binding. We tested their involvement using site-directed mutagenesis (SDM) of MCT1 to change residues individually or in combination with their MCT4 equivalent and determined inhibitor sensitivity following expression in Xenopus oocytes. Phe360 and Ser364 were identified as important for AR-C155858 binding with the F360Y/S364G mutant exhibiting >100-fold reduction in inhibitor sensitivity. To refine the binding site further, we used molecular dynamics (MD) simulations and additional SDM. This approach implicated six more residues whose involvement was confirmed by both transport studies and [3H]-AR-C155858 binding to oocyte membranes. Taken together, our data imply that Asn147, Arg306 and Ser364 are important for directing AR-C155858 to its final binding site which involves interaction of the inhibitor with Lys38, Asp302 and Phe360 (residues that also play key roles in the translocation cycle) and also Leu274 and Ser278. PMID:25437897

Safety, tolerability, and pharmacokinetics of the HIV integrase inhibitor dolutegravir given twice daily with rifampin or once daily with rifabutin: results of a phase 1 study among healthy subjects.

PubMed

Dooley, Kelly E; Sayre, Patrick; Borland, Julie; Purdy, Elizabeth; Chen, Shuguang; Song, Ivy; Peppercorn, Amanda; Everts, Stephanie; Piscitelli, Stephen; Flexner, Charles

2013-01-01

Cotreatment of tuberculosis (TB) and HIV among coinfected patients is now the standard of care. Rifampin (RIF) is a standard part of TB treatment but is a potent inducer of drug metabolizing enzymes. This study evaluated the effect of RIF or rifabutin (RBT) on the pharmacokinetics of the investigational HIV integrase inhibitor, dolutegravir (DTG). Phase I pharmacokinetic drug interaction study. In arm 1, healthy subjects received 50 mg of DTG once daily for 7 days (period 1), then 50 mg of DTG twice daily for 7 days (period 2), then 50 mg of DTG twice daily together with 600 mg of RIF once daily for 14 days (period 3). In arm 2, subjects received 50 mg of DTG once daily for 7 days (period 1) then 50 mg of DTG once daily together with 300 mg of RBT once daily for 14 days (period 2). PK sampling was performed at the end of each period. In arm 1, comparing period 3 to period 1, the geometric mean ratio (GMR) for the 24-hour area under the time-concentration curve (AUC0-24) was 1.33 [90% confidence interval (CI): 1.14 to 1.53], and the GMR for the trough (Cτ) was 1.22 (90% CI: 1.01 to 1.48). Comparing period 2 to period 1 in arm 2, the GMR for the AUC0-24 was 0.95 (90% CI: 0.82 to 1.10), and the GMR for the Cτ was 0.70 (90% CI: 0.57 to 0.87). Regimens including twice-daily DTG and RIF or once-daily DTG and RBT may represent a new treatment option for patients who require concomitant treatment of HIV and TB.

Epoxide pathways improve model predictions of isoprene markers and reveal key role of acidity in aerosol formation.

PubMed

Pye, Havala O T; Pinder, Robert W; Piletic, Ivan R; Xie, Ying; Capps, Shannon L; Lin, Ying-Hsuan; Surratt, Jason D; Zhang, Zhenfa; Gold, Avram; Luecken, Deborah J; Hutzell, William T; Jaoui, Mohammed; Offenberg, John H; Kleindienst, Tadeusz E; Lewandowski, Michael; Edney, Edward O

2013-10-01

Isoprene significantly contributes to organic aerosol in the southeastern United States where biogenic hydrocarbons mix with anthropogenic emissions. In this work, the Community Multiscale Air Quality model is updated to predict isoprene aerosol from epoxides produced under both high- and low-NOx conditions. The new aqueous aerosol pathways allow for explicit predictions of two key isoprene-derived species, 2-methyltetrols and 2-methylglyceric acid, that are more consistent with observations than estimates based on semivolatile partitioning. The new mechanism represents a significant source of organic carbon in the lower 2 km of the atmosphere and captures the abundance of 2-methyltetrols relative to organosulfates during the simulation period. For the parametrization considered here, a 25% reduction in SOx emissions effectively reduces isoprene aerosol, while a similar reduction in NOx leads to small increases in isoprene aerosol.

46 CFR 7.100 - Florida Reefs and Keys from Miami, FL to Marquesas Keys, FL.

Code of Federal Regulations, 2014 CFR

2014-10-01

... latitude 24°27.7′ N. longitude 81°48.1′ W. (Key West Entrance Lighted Whistle Buoy); thence to Cosgrove... 46 Shipping 1 2014-10-01 2014-10-01 false Florida Reefs and Keys from Miami, FL to Marquesas Keys... TO THE PUBLIC BOUNDARY LINES Atlantic Coast § 7.100 Florida Reefs and Keys from Miami, FL to...

46 CFR 7.100 - Florida Reefs and Keys from Miami, FL to Marquesas Keys, FL.

Code of Federal Regulations, 2012 CFR

2012-10-01

... latitude 24°27.7′ N. longitude 81°48.1′ W. (Key West Entrance Lighted Whistle Buoy); thence to Cosgrove... 46 Shipping 1 2012-10-01 2012-10-01 false Florida Reefs and Keys from Miami, FL to Marquesas Keys... TO THE PUBLIC BOUNDARY LINES Atlantic Coast § 7.100 Florida Reefs and Keys from Miami, FL to...

46 CFR 7.100 - Florida Reefs and Keys from Miami, FL to Marquesas Keys, FL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... latitude 24°27.7′ N. longitude 81°48.1′ W. (Key West Entrance Lighted Whistle Buoy); thence to Cosgrove... 46 Shipping 1 2010-10-01 2010-10-01 false Florida Reefs and Keys from Miami, FL to Marquesas Keys... TO THE PUBLIC BOUNDARY LINES Atlantic Coast § 7.100 Florida Reefs and Keys from Miami, FL to...

46 CFR 7.100 - Florida Reefs and Keys from Miami, FL to Marquesas Keys, FL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... latitude 24°27.7′ N. longitude 81°48.1′ W. (Key West Entrance Lighted Whistle Buoy); thence to Cosgrove... 46 Shipping 1 2011-10-01 2011-10-01 false Florida Reefs and Keys from Miami, FL to Marquesas Keys... TO THE PUBLIC BOUNDARY LINES Atlantic Coast § 7.100 Florida Reefs and Keys from Miami, FL to...

46 CFR 7.100 - Florida Reefs and Keys from Miami, FL to Marquesas Keys, FL.

Code of Federal Regulations, 2013 CFR

2013-10-01

... latitude 24°27.7′ N. longitude 81°48.1′ W. (Key West Entrance Lighted Whistle Buoy); thence to Cosgrove... 46 Shipping 1 2013-10-01 2013-10-01 false Florida Reefs and Keys from Miami, FL to Marquesas Keys... TO THE PUBLIC BOUNDARY LINES Atlantic Coast § 7.100 Florida Reefs and Keys from Miami, FL to...

Identification of the Key Fields and Their Key Technical Points of Oncology by Patent Analysis.

PubMed

Zhang, Ting; Chen, Juan; Jia, Xiaofeng

2015-01-01

This paper aims to identify the key fields and their key technical points of oncology by patent analysis. Patents of oncology applied from 2006 to 2012 were searched in the Thomson Innovation database. The key fields and their key technical points were determined by analyzing the Derwent Classification (DC) and the International Patent Classification (IPC), respectively. Patent applications in the top ten DC occupied 80% of all the patent applications of oncology, which were the ten fields of oncology to be analyzed. The number of patent applications in these ten fields of oncology was standardized based on patent applications of oncology from 2006 to 2012. For each field, standardization was conducted separately for each of the seven years (2006-2012) and the mean of the seven standardized values was calculated to reflect the relative amount of patent applications in that field; meanwhile, regression analysis using time (year) and the standardized values of patent applications in seven years (2006-2012) was conducted so as to evaluate the trend of patent applications in each field. Two-dimensional quadrant analysis, together with the professional knowledge of oncology, was taken into consideration in determining the key fields of oncology. The fields located in the quadrant with high relative amount or increasing trend of patent applications are identified as key ones. By using the same method, the key technical points in each key field were identified. Altogether 116,820 patents of oncology applied from 2006 to 2012 were retrieved, and four key fields with twenty-nine key technical points were identified, including "natural products and polymers" with nine key technical points, "fermentation industry" with twelve ones, "electrical medical equipment" with four ones, and "diagnosis, surgery" with four ones. The results of this study could provide guidance on the development direction of oncology, and also help researchers broaden innovative ideas and discover new

Genetic variability of psychrotolerant Acidithiobacillus ferrivorans revealed by (meta)genomic analysis.

PubMed

González, Carolina; Yanquepe, María; Cardenas, Juan Pablo; Valdes, Jorge; Quatrini, Raquel; Holmes, David S; Dopson, Mark

2014-11-01

Acidophilic microorganisms inhabit low pH environments such as acid mine drainage that is generated when sulfide minerals are exposed to air. The genome sequence of the psychrotolerant Acidithiobacillus ferrivorans SS3 was compared to a metagenome from a low temperature acidic stream dominated by an A. ferrivorans-like strain. Stretches of genomic DNA characterized by few matches to the metagenome, termed 'metagenomic islands', encoded genes associated with metal efflux and pH homeostasis. The metagenomic islands were enriched in mobile elements such as phage proteins, transposases, integrases and in one case, predicted to be flanked by truncated tRNAs. Cus gene clusters predicted to be involved in copper efflux and further Cus-like RND systems were predicted to be located in metagenomic islands and therefore, constitute part of the flexible gene complement of the species. Phylogenetic analysis of Cus clusters showed both lineage specificity within the Acidithiobacillus genus as well as niche specificity associated with an acidic environment. The metagenomic islands also contained a predicted copper efflux P-type ATPase system and a polyphosphate kinase potentially involved in polyphosphate mediated copper resistance. This study identifies genetic variability of low temperature acidophiles that likely reflects metal resistance selective pressures in the copper rich environment. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A study revealing the key aroma compounds of steamed bread made by Chinese traditional sourdough*

PubMed Central

Zhang, Guo-hua; Wu, Tao; Sadiq, Faizan A.; Yang, Huan-yi; Liu, Tong-jie; Ruan, Hui; He, Guo-qing

2016-01-01

Aroma of Chinese steamed bread (CSB) is one of the important parameters that determines the overall quality attributes and consumer acceptance. However, the aroma profile of CSB still remains poorly understood, mainly because of relying on only a single method for aroma extraction in previous studies. Therefore, the objective of this study was to determine the volatile aroma compounds of five different samples of CSB using three different aroma extraction methods, namely solid-phase microextraction (SPME), simultaneous distillation–extraction (SDE), and purge and trap (P&T). All samples showed a unique aroma profile, which could be attributed to their unique microbial consortia. (E)-2-Nonenal and (E,E)-2,4-decadienal were the most prevalent aromatic compounds revealed by SDE, which have not been reported previously, while ethanol and acetic acid proved to be the most dominant compounds by both SPME and P&T. Our approach of combining three different aroma extraction methods provided better insights into the aroma profile of CSB, which had remained largely unknown in previous studies. PMID:27704748

Integrated metagenomic analysis of the rumen microbiome of cattle reveals key biological mechanisms associated with methane traits.

PubMed

Wang, Haiying; Zheng, Huiru; Browne, Fiona; Roehe, Rainer; Dewhurst, Richard J; Engel, Felix; Hemmje, Matthias; Lu, Xiangwu; Walsh, Paul

2017-07-15

Methane is one of the major contributors to global warming. The rumen microbiota is directly involved in methane production in cattle. The link between variation in rumen microbial communities and host genetics has important applications and implications in bioscience. Having the potential to reveal the full extent of microbial gene diversity and complex microbial interactions, integrated metagenomics and network analysis holds great promise in this endeavour. This study investigates the rumen microbial community in cattle through the integration of metagenomic and network-based approaches. Based on the relative abundance of 1570 microbial genes identified in a metagenomics analysis, the co-abundance network was constructed and functional modules of microbial genes were identified. One of the main contributions is to develop a random matrix theory-based approach to automatically determining the correlation threshold used to construct the co-abundance network. The resulting network, consisting of 549 microbial genes and 3349 connections, exhibits a clear modular structure with certain trait-specific genes highly over-represented in modules. More specifically, all the 20 genes previously identified to be associated with methane emissions are found in a module (hypergeometric test, p<10 -11 ). One third of genes are involved in methane metabolism pathways. The further examination of abundance profiles across 8 samples of genes highlights that the revealed pattern of metagenomics abundance has a strong association with methane emissions. Furthermore, the module is significantly enriched with microbial genes encoding enzymes that are directly involved in methanogenesis (hypergeometric test, p<10 -9 ). Copyright © 2017 Elsevier Inc. All rights reserved.

HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor.

PubMed

Wang, Hao; Jurado, Kellie A; Wu, Xiaolin; Shun, Ming-Chieh; Li, Xiang; Ferris, Andrea L; Smith, Steven J; Patel, Pratiq A; Fuchs, James R; Cherepanov, Peter; Kvaratskhelia, Mamuka; Hughes, Stephen H; Engelman, Alan

2012-12-01

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.

Functional genomics of fuzzless-lintless mutant of Gossypium hirsutum L. cv. MCU5 reveal key genes and pathways involved in cotton fibre initiation and elongation

PubMed Central

2012-01-01

fl mutant revealed key genes and pathways involved at various stages of fibre development. Our data implicated the significant role of mitochondria mediated energy metabolism during fibre elongation process. The delayed expression of genes involved in phytohormone signalling and stress responsive TFs in the fl mutant suggests the need for a coordinated expression of regulatory mechanisms in fibre cell initiation and differentiation. PMID:23151214

NASA's Chandra Reveals Origin of Key Cosmic Explosions

NASA Astrophysics Data System (ADS)

2010-02-01

WASHINGTON -- New findings from NASA's Chandra X-ray Observatory have provided a major advance in understanding a type of supernova critical for studying the dark energy that astronomers think pervades the universe. The results show mergers of two dense stellar remnants are the likely cause of many of the supernovae that have been used to measure the accelerated expansion of the universe. These supernovae, called Type Ia, serve as cosmic mile markers to measure expansion of the universe because they can be seen at large distances, and they follow a reliable pattern of brightness. However, until now, scientists have been unsure what actually causes the explosions. "These are such critical objects in understanding the universe," said Marat Gilfanov of the Max Planck Institute for Astrophysics in Germany and lead author of the study that appears in the Feb. 18 edition of the journal Nature. "It was a major embarrassment that we did not know how they worked. Now we are beginning to understand what lights the fuse of these explosions." Most scientists agree a Type Ia supernova occurs when a white dwarf star -- a collapsed remnant of an elderly star -- exceeds its weight limit, becomes unstable and explodes. Scientists have identified two main possibilities for pushing the white dwarf over the edge: two white dwarfs merging or accretion, a process in which the white dwarf pulls material from a sun-like companion star until it exceeds its weight limit. "Our results suggest the supernovae in the galaxies we studied almost all come from two white dwarfs merging," said co-author Akos Bogdan, also of Max Planck. "This is probably not what many astronomers would expect." The difference between these two scenarios may have implications for how these supernovae can be used as "standard candles" -- objects of a known brightness -- to track vast cosmic distances. Because white dwarfs can come in a range of masses, the merger of two could result in explosions that vary somewhat in brightness. Because these two scenarios would generate different amounts of X-ray emission, Gilfanov and Bogdan used Chandra to observe five nearby elliptical galaxies and the central region of the Andromeda galaxy. A Type 1a supernova caused by accreting material produces significant X- ray emission prior to the explosion. A supernova from a merger of two white dwarfs, on the other hand, would create significantly less X-ray emission than the accretion scenario. The scientists found the observed X-ray emission was a factor of 30 to 50 times smaller than expected from the accretion scenario, effectively ruling it out. This implies that white dwarf mergers dominate in these galaxies. An open question remains whether these white dwarf mergers are the primary catalyst for Type Ia supernovae in spiral galaxies. Further studies are required to know if supernovae in spiral galaxies are caused by mergers or a mixture of the two processes. Another intriguing consequence of this result is that a pair of white dwarfs is relatively hard to spot, even with the best telescopes. "To many astrophysicists, the merger scenario seemed to be less likely because too few double-white-dwarf systems appeared to exist," said Gilfanov. "Now this path to supernovae will have to be investigated in more detail." In addition to the X-rays observed with Chandra, other data critical for this result came from NASA's Spitzer Space Telescope and the ground-based, infrared Two Micron All Sky Survey. The infrared brightness of the galaxies allowed the team to estimate how many supernovae should occur. NASA's Marshall Space Flight Center in Huntsville, Ala., manages the Chandra program for NASA's Science Mission Directorate in Washington. The Smithsonian Astrophysical Observatory controls Chandra's science and flight operations from Cambridge, Mass. More information, including images and other multimedia, can be found at: http://chandra.harvard.edu and http://chandra.nasa.gov

Principles of visual key construction-with a visual identification key to the Fagaceae of the southeastern United States.

PubMed

Kirchoff, Bruce K; Leggett, Roxanne; Her, Va; Moua, Chue; Morrison, Jessica; Poole, Chamika

2011-01-01

Advances in digital imaging have made possible the creation of completely visual keys. By a visual key we mean a key based primarily on images, and that contains a minimal amount of text. Characters in visual keys are visually, not verbally defined. In this paper we create the first primarily visual key to a group of taxa, in this case the Fagaceae of the southeastern USA. We also modify our recently published set of best practices for image use in illustrated keys to make them applicable to visual keys. Photographs of the Fagaceae were obtained from internet and herbarium databases or were taken specifically for this project. The images were printed and then sorted into hierarchical groups. These hierarchical groups of images were used to create the 'couplets' in the key. A reciprocal process of key creation and testing was used to produce the final keys. Four keys were created, one for each of the parts-leaves, buds, fruits and bark. Species description pages consisting of multiple images were also created for each of the species in the key. Creation and testing of the key resulted in a modified list of best practices for image use visual keys. The inclusion of images into paper and electronic keys has greatly increased their ease of use. However, virtually all of these keys are still based upon verbally defined, atomistic characters. The creation of primarily visual keys allows us to overcome the well-known limitations of linguistic-based characters and create keys that are much easier to use, especially for botanical novices.

Crystal and electronic structures of magnesium(II), copper(II), and mixed magnesium(II)-copper(II) complexes of the quinoline half of styrylquinoline-type HIV-1 integrase inhibitors.

PubMed

Courcot, B; Firley, D; Fraisse, B; Becker, P; Gillet, J-M; Pattison, P; Chernyshov, D; Sghaier, M; Zouhiri, F; Desmaële, D; d'Angelo, J; Bonhomme, F; Geiger, S; Ghermani, N E

2007-05-31

A new target in AIDS therapy development is HIV-1 integrase (IN). It was proven that HIV-1 IN required divalent metal cations to achieve phosphodiester bond cleavage of DNA. Accordingly, all newly investigated potent IN inhibitors contain chemical fragments possessing a high ability to chelate metal cations. One of the promising leads in the polyhydroxylated styrylquinolines (SQLs) series is (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-methoxyphenyl)-ethenyl]-7-quinoline carboxylic acid (1). The present study focuses on the quinoline-based progenitor (2), which is actually the most probable chelating part of SQLs. Conventional and synchrotron low-temperature X-ray crystallographic studies were used to investigate the chelating power of progenitor 2. Mg2+ and Cu2+ cations were selected for this purpose, and three types of metal complexes of 2 were obtained: Mg(II) complex (4), Cu(II) complex (5) and mixed Mg(II)-Cu(II) complexes (6 and 7). The analysis of the crystal structure of complex 4 indicates that two tridentate ligands coordinate two Mg2+ cations, both in octahedral geometry. The Mg-Mg distance was found equal to 3.221(1) A, in agreement with the metal-metal distance of 3.9 A encountered in the crystal structure of Escherichia coli DNA polymerase I. In 5, the complex is formed by two bidentate ligands coordinating one copper ion in tetrahedral geometry. Both mixed Mg(II)-Cu(II) complexes, 6 and 7 exhibit an original arrangement of four ligands linked to a central heterometallic cluster consisting of three octahedrally coordinated magnesium ions and one tetrahedrally coordinated copper ion. Quantum mechanics calculations were also carried out in order to display the electrostatic potential generated by the dianionic ligand 2 and complex 4 and to quantify the binding energy (BE) during the formation of the magnesium complex of progenitor 2. A comparison of the binding energies of two hypothetical monometallic Mg(II) complexes with that found in the bimetallic magnesium

Identification of the Key Fields and Their Key Technical Points of Oncology by Patent Analysis

PubMed Central

Zhang, Ting; Chen, Juan; Jia, Xiaofeng

2015-01-01

Background This paper aims to identify the key fields and their key technical points of oncology by patent analysis. Methodology/Principal Findings Patents of oncology applied from 2006 to 2012 were searched in the Thomson Innovation database. The key fields and their key technical points were determined by analyzing the Derwent Classification (DC) and the International Patent Classification (IPC), respectively. Patent applications in the top ten DC occupied 80% of all the patent applications of oncology, which were the ten fields of oncology to be analyzed. The number of patent applications in these ten fields of oncology was standardized based on patent applications of oncology from 2006 to 2012. For each field, standardization was conducted separately for each of the seven years (2006–2012) and the mean of the seven standardized values was calculated to reflect the relative amount of patent applications in that field; meanwhile, regression analysis using time (year) and the standardized values of patent applications in seven years (2006–2012) was conducted so as to evaluate the trend of patent applications in each field. Two-dimensional quadrant analysis, together with the professional knowledge of oncology, was taken into consideration in determining the key fields of oncology. The fields located in the quadrant with high relative amount or increasing trend of patent applications are identified as key ones. By using the same method, the key technical points in each key field were identified. Altogether 116,820 patents of oncology applied from 2006 to 2012 were retrieved, and four key fields with twenty-nine key technical points were identified, including “natural products and polymers” with nine key technical points, “fermentation industry” with twelve ones, “electrical medical equipment” with four ones, and “diagnosis, surgery” with four ones. Conclusions/Significance The results of this study could provide guidance on the development

Key Health Information Technologies and Related Issues for Iran: A Qualitative Study.

PubMed

Hemmat, Morteza; Ayatollahi, Haleh; Maleki, Mohammadreza; Saghafi, Fatemeh

2018-01-01

Planning for the future of Health Information Technology (HIT) requires applying a systematic approach when conducting foresight studies. The aim of this study was to identify key health information technologies and related issues for Iran until 2025. This was a qualitative study and the participants included experts and policy makers in the field of health information technology. In-depth semi-structured interviews were conducted and data were analyzed by using framework analysis and MAXQDA software. The findings revealed that the development of national health information network, electronic health records, patient health records, a cloud-based service center, interoperability standards, patient monitoring technologies, telehealth, mhealth, clinical decision support systems, health information technology and mhealth infrastructure were found to be the key technologies for the future. These technologies could influence the economic, organizational and individual levels. To achieve them, the economic and organizational obstacles need to be overcome. In this study, a number of key technologies and related issues were identified. This approach can help to focus on the most important technologies in the future and to priorities these technologies for better resource allocation and policy making.

Key Health Information Technologies and Related Issues for Iran: A Qualitative Study

PubMed Central

Hemmat, Morteza; Ayatollahi, Haleh; Maleki, Mohammadreza; Saghafi, Fatemeh

2018-01-01

Background and Objective: Planning for the future of Health Information Technology (HIT) requires applying a systematic approach when conducting foresight studies. The aim of this study was to identify key health information technologies and related issues for Iran until 2025. Methods: This was a qualitative study and the participants included experts and policy makers in the field of health information technology. In-depth semi-structured interviews were conducted and data were analyzed by using framework analysis and MAXQDA software. Results: The findings revealed that the development of national health information network, electronic health records, patient health records, a cloud-based service center, interoperability standards, patient monitoring technologies, telehealth, mhealth, clinical decision support systems, health information technology and mhealth infrastructure were found to be the key technologies for the future. These technologies could influence the economic, organizational and individual levels. To achieve them, the economic and organizational obstacles need to be overcome. Conclusion: In this study, a number of key technologies and related issues were identified. This approach can help to focus on the most important technologies in the future and to priorities these technologies for better resource allocation and policy making. PMID:29854016

Key Exchange Trust Evaluation in Peer-to-Peer Sensor Networks With Unconditionally Secure Key Exchange

NASA Astrophysics Data System (ADS)

Gonzalez, Elias; Kish, Laszlo B.

2016-03-01

As the utilization of sensor networks continue to increase, the importance of security becomes more profound. Many industries depend on sensor networks for critical tasks, and a malicious entity can potentially cause catastrophic damage. We propose a new key exchange trust evaluation for peer-to-peer sensor networks, where part of the network has unconditionally secure key exchange. For a given sensor, the higher the portion of channels with unconditionally secure key exchange the higher the trust value. We give a brief introduction to unconditionally secured key exchange concepts and mention current trust measures in sensor networks. We demonstrate the new key exchange trust measure on a hypothetical sensor network using both wired and wireless communication channels.

Nearest private query based on quantum oblivious key distribution

NASA Astrophysics Data System (ADS)

Xu, Min; Shi, Run-hua; Luo, Zhen-yu; Peng, Zhen-wan

2017-12-01

Nearest private query is a special private query which involves two parties, a user and a data owner, where the user has a private input (e.g., an integer) and the data owner has a private data set, and the user wants to query which element in the owner's private data set is the nearest to his input without revealing their respective private information. In this paper, we first present a quantum protocol for nearest private query, which is based on quantum oblivious key distribution (QOKD). Compared to the classical related protocols, our protocol has the advantages of the higher security and the better feasibility, so it has a better prospect of applications.

The emotional importance of key: do Beatles songs written in different keys convey different emotional tones?

PubMed

Whissel, R; Whissel, C

2000-12-01

Lyrics from 155 songs written by the Lennon-McCartney team were scored using the Dictionary of Affect in Language. Resultant scores (pleasantness, activation, and imagery of words) were compared across key signatures using one way analyses of variance. Words from songs written in minor keys were less pleasant and less active than those from songs written in major keys. Words from songs written in the key of F scored extremely low on all three measures. Lyrics from the keys of C, D, and G were relatively active in tone. Results from Dictionary scoring were compared with assignments of character to keys made more than one century ago and with current musicians' opinions.

A Recommended Set of Key Arctic Indicators

NASA Astrophysics Data System (ADS)

Stanitski, D.; Druckenmiller, M.; Fetterer, F. M.; Gerst, M.; Intrieri, J. M.; Kenney, M. A.; Meier, W.; Overland, J. E.; Stroeve, J.; Trainor, S.

2017-12-01

The Arctic is an interconnected and environmentally sensitive system of ice, ocean, land, atmosphere, ecosystems, and people. From local to pan-Arctic scales, the area has already undergone major changes in physical and societal systems and will continue at a pace that is greater than twice the global average. Key Arctic indicators can quantify these changes. Indicators serve as the bridge between complex information and policy makers, stakeholders, and the general public, revealing trends and information people need to make important socioeconomic decisions. This presentation evaluates and compiles more than 70 physical, biological, societal and economic indicators into an approachable summary that defines the changing Arctic. We divided indicators into "existing," "in development," "possible," and "aspirational". In preparing a paper on Arctic Indicators for a special issue of the journal Climatic Change, our group established a set of selection criteria to identify indicators to specifically guide decision-makers in their responses to climate change. A goal of the analysis is to select a manageable composite list of recommended indicators based on sustained, reliable data sources with known user communities. The selected list is also based on the development of a conceptual model that identifies components and processes critical to our understanding of the Arctic region. This list of key indicators is designed to inform the plans and priorities of multiple groups such as the U.S. Global Change Research Program (USGCRP), Interagency Arctic Research Policy Committee (IARPC), and the Arctic Council.

Key on demand (KoD) for software-defined optical networks secured by quantum key distribution (QKD).

PubMed

Cao, Yuan; Zhao, Yongli; Colman-Meixner, Carlos; Yu, Xiaosong; Zhang, Jie

2017-10-30

Software-defined optical networking (SDON) will become the next generation optical network architecture. However, the optical layer and control layer of SDON are vulnerable to cyberattacks. While, data encryption is an effective method to minimize the negative effects of cyberattacks, secure key interchange is its major challenge which can be addressed by the quantum key distribution (QKD) technique. Hence, in this paper we discuss the integration of QKD with WDM optical networks to secure the SDON architecture by introducing a novel key on demand (KoD) scheme which is enabled by a novel routing, wavelength and key assignment (RWKA) algorithm. The QKD over SDON with KoD model follows two steps to provide security: i) quantum key pools (QKPs) construction for securing the control channels (CChs) and data channels (DChs); ii) the KoD scheme uses RWKA algorithm to allocate and update secret keys for different security requirements. To test our model, we define a security probability index which measures the security gain in CChs and DChs. Simulation results indicate that the security performance of CChs and DChs can be enhanced by provisioning sufficient secret keys in QKPs and performing key-updating considering potential cyberattacks. Also, KoD is beneficial to achieve a positive balance between security requirements and key resource usage.

Emerging contaminants and nutrients synergistically affect the spread of class 1 integron-integrase (intI1) and sul1 genes within stable streambed bacterial communities.

PubMed

Subirats, Jèssica; Timoner, Xisca; Sànchez-Melsió, Alexandre; Balcázar, José Luis; Acuña, Vicenç; Sabater, Sergi; Borrego, Carles M

2018-07-01

Wastewater effluents increase the nutrient load of receiving streams while introducing a myriad of anthropogenic chemical pollutants that challenge the resident aquatic (micro)biota. Disentangling the effects of both kind of stressors and their potential interaction on the dissemination of antibiotic resistance genes in bacterial communities requires highly controlled manipulative experiments. In this work, we investigated the effects of a combined regime of nutrients (at low, medium and high concentrations) and a mixture of emerging contaminants (ciprofloxacin, erythromycin, sulfamethoxazole, diclofenac, and methylparaben) on the bacterial composition, abundance and antibiotic resistance profile of biofilms grown in artificial streams. In particular, we investigated the effect of this combined stress on genes encoding resistance to ciprofloxacin (qnrS), erythromycin (ermB), sulfamethoxazole (sul1 and sul2) as well as the class 1 integron-integrase gene (intI1). Only genes conferring resistance to sulfonamides (sul1 and sul2) and intI1 gene were detected in all treatments during the study period. Besides, bacterial communities exposed to emerging contaminants showed higher copy numbers of sul1 and intI1 genes than those not exposed, whereas nutrient amendments did not affect their abundance. However, bacterial communities exposed to both emerging contaminants and a high nutrient concentration (1, 25 and 1 mg L -1 of phosphate, nitrate and ammonium, respectively) showed the highest increase on the abundance of sul1 and intI1 genes thus suggesting a factors synergistic effect of both stressors. Since none of the treatments caused a significant change on the composition of bacterial communities, the enrichment of sul1 and intI1 genes within the community was caused by their dissemination under the combined pressure exerted by nutrients and emerging contaminants. To the best of our knowledge, this is the first study demonstrating the contribution of nutrients on

Gene expression profiles reveal key genes for early diagnosis and treatment of adamantinomatous craniopharyngioma.

PubMed

Yang, Jun; Hou, Ziming; Wang, Changjiang; Wang, Hao; Zhang, Hongbing

2018-04-23

Adamantinomatous craniopharyngioma (ACP) is an aggressive brain tumor that occurs predominantly in the pediatric population. Conventional diagnosis method and standard therapy cannot treat ACPs effectively. In this paper, we aimed to identify key genes for ACP early diagnosis and treatment. Datasets GSE94349 and GSE68015 were obtained from Gene Expression Omnibus database. Consensus clustering was applied to discover the gene clusters in the expression data of GSE94349 and functional enrichment analysis was performed on gene set in each cluster. The protein-protein interaction (PPI) network was built by the Search Tool for the Retrieval of Interacting Genes, and hubs were selected. Support vector machine (SVM) model was built based on the signature genes identified from enrichment analysis and PPI network. Dataset GSE94349 was used for training and testing, and GSE68015 was used for validation. Besides, RT-qPCR analysis was performed to analyze the expression of signature genes in ACP samples compared with normal controls. Seven gene clusters were discovered in the differentially expressed genes identified from GSE94349 dataset. Enrichment analysis of each cluster identified 25 pathways that highly associated with ACP. PPI network was built and 46 hubs were determined. Twenty-five pathway-related genes that overlapped with the hubs in PPI network were used as signatures to establish the SVM diagnosis model for ACP. The prediction accuracy of SVM model for training, testing, and validation data were 94, 85, and 74%, respectively. The expression of CDH1, CCL2, ITGA2, COL8A1, COL6A2, and COL6A3 were significantly upregulated in ACP tumor samples, while CAMK2A, RIMS1, NEFL, SYT1, and STX1A were significantly downregulated, which were consistent with the differentially expressed gene analysis. SVM model is a promising classification tool for screening and early diagnosis of ACP. The ACP-related pathways and signature genes will advance our knowledge of ACP pathogenesis

Mechanical unfolding reveals stable 3-helix intermediates in talin and α-catenin

PubMed Central

2018-01-01

Mechanical stability is a key feature in the regulation of structural scaffolding proteins and their functions. Despite the abundance of α-helical structures among the human proteome and their undisputed importance in health and disease, the fundamental principles of their behavior under mechanical load are poorly understood. Talin and α-catenin are two key molecules in focal adhesions and adherens junctions, respectively. In this study, we used a combination of atomistic steered molecular dynamics (SMD) simulations, polyprotein engineering, and single-molecule atomic force microscopy (smAFM) to investigate unfolding of these proteins. SMD simulations revealed that talin rod α-helix bundles as well as α-catenin α-helix domains unfold through stable 3-helix intermediates. While the 5-helix bundles were found to be mechanically stable, a second stable conformation corresponding to the 3-helix state was revealed. Mechanically weaker 4-helix bundles easily unfolded into a stable 3-helix conformation. The results of smAFM experiments were in agreement with the findings of the computational simulations. The disulfide clamp mutants, designed to protect the stable state, support the 3-helix intermediate model in both experimental and computational setups. As a result, multiple discrete unfolding intermediate states in the talin and α-catenin unfolding pathway were discovered. Better understanding of the mechanical unfolding mechanism of α-helix proteins is a key step towards comprehensive models describing the mechanoregulation of proteins. PMID:29698481

Direct visualization reveals kinetics of meiotic chromosome synapsis

DOE PAGES

Rog, Ofer; Dernburg, Abby  F.

2015-03-17

The synaptonemal complex (SC) is a conserved protein complex that stabilizes interactions along homologous chromosomes (homologs) during meiosis. The SC regulates genetic exchanges between homologs, thereby enabling reductional division and the production of haploid gametes. Here, we directly observe SC assembly (synapsis) by optimizing methods for long-term fluorescence recording in C. elegans. We report that synapsis initiates independently on each chromosome pair at or near pairing centers—specialized regions required for homolog associations. Once initiated, the SC extends rapidly and mostly irreversibly to chromosome ends. Quantitation of SC initiation frequencies and extension rates reveals that initiation is a rate-limiting step inmore » homolog interactions. Eliminating the dynein-driven chromosome movements that accompany synapsis severely retards SC extension, revealing a new role for these conserved motions. This work provides the first opportunity to directly observe and quantify key aspects of meiotic chromosome interactions and will enable future in vivo analysis of germline processes.« less

The distinction between key ideas in teaching school physics and key ideas in the discipline of physics

NASA Astrophysics Data System (ADS)

Deng, Zongyi

2001-05-01

The distinction between key ideas in teaching a high school science and key ideas in the corresponding discipline of science has been largely ignored in scholarly discourse about what science teachers should teach and about what they should know. This article clarifies this distinction through exploring how and why key ideas in teaching high school physics differ from key ideas in the discipline of physics. Its theoretical underpinnings include Dewey's (1902/1990) distinction between the psychological and the logical and Harré's (1986) epistemology of science. It analyzes how and why the key ideas in teaching color, the speed of light, and light interference at the high school level differ from the key ideas at the disciplinary level. The thesis is that key ideas in teaching high school physics can differ from key ideas in the discipline in some significant ways, and that the differences manifest Dewey's distinction. As a result, the article challenges the assumption of equating key ideas in teaching a high school science with key ideas in the corresponding discipline of science, and the assumption that having a college degree in science is sufficient to teach high school science. Furthermore, the article expands the concept of pedagogical content knowledge by arguing that key ideas in teaching high school physics constitute an essential component.

76 FR 68314 - Special Local Regulations; Key West World Championship, Atlantic Ocean; Key West, FL

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-04

...-AA08 Special Local Regulations; Key West World Championship, Atlantic Ocean; Key West, FL AGENCY: Coast... World Championship, a series of high-speed boat races. The event is scheduled to take place on Wednesday... Key West World Championship, a series of high-speed boat races. The event will be held on the waters...

Symmetric Key Services Markup Language (SKSML)

NASA Astrophysics Data System (ADS)

Noor, Arshad

Symmetric Key Services Markup Language (SKSML) is the eXtensible Markup Language (XML) being standardized by the OASIS Enterprise Key Management Infrastructure Technical Committee for requesting and receiving symmetric encryption cryptographic keys within a Symmetric Key Management System (SKMS). This protocol is designed to be used between clients and servers within an Enterprise Key Management Infrastructure (EKMI) to secure data, independent of the application and platform. Building on many security standards such as XML Signature, XML Encryption, Web Services Security and PKI, SKSML provides standards-based capability to allow any application to use symmetric encryption keys, while maintaining centralized control. This article describes the SKSML protocol and its capabilities.

Key Reconciliation for High Performance Quantum Key Distribution

PubMed Central

Martinez-Mateo, Jesus; Elkouss, David; Martin, Vicente

2013-01-01

Quantum Key Distribution is carving its place among the tools used to secure communications. While a difficult technology, it enjoys benefits that set it apart from the rest, the most prominent is its provable security based on the laws of physics. QKD requires not only the mastering of signals at the quantum level, but also a classical processing to extract a secret-key from them. This postprocessing has been customarily studied in terms of the efficiency, a figure of merit that offers a biased view of the performance of real devices. Here we argue that it is the throughput the significant magnitude in practical QKD, specially in the case of high speed devices, where the differences are more marked, and give some examples contrasting the usual postprocessing schemes with new ones from modern coding theory. A good understanding of its implications is very important for the design of modern QKD devices. PMID:23546440

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses.

PubMed

Pritham, Ellen J; Putliwala, Tasneem; Feschotte, Cédric

2007-04-01

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we

Metabolic characteristics of dominant microbes and key rare species from an acidic hot spring in Taiwan revealed by metagenomics

DOE PAGES

Lin, Kuei -Han; Liao, Ben -Yang; Chang, Hao -Wei; ...

2015-12-03

Microbial diversity and community structures in acidic hot springs have been characterized by 16S rRNA gene-based diversity surveys. However, our understanding regarding the interactions among microbes, or between microbes and environmental factors, remains limited. In the present study, a metagenomic approach, followed by bioinformatics analyses, were used to predict interactions within the microbial ecosystem in Shi-Huang-Ping (SHP), an acidic hot spring in northern Taiwan. Characterizing environmental parameters and potential metabolic pathways highlighted the importance of carbon assimilatory pathways. Four distinct carbon assimilatory pathways were identified in five dominant genera of bacteria. Of those dominant carbon fixers, Hydrogenobaculum bacteria outcompeted othermore » carbon assimilators and dominated the SHP, presumably due to their ability to metabolize hydrogen and to withstand an anaerobic environment with fluctuating temperatures. Furthermore, most dominant microbes were capable of metabolizing inorganic sulfur-related compounds (abundant in SHP). However, Acidithiobacillus ferrooxidans was the only species among key rare microbes with the capability to fix nitrogen, suggesting a key role in nitrogen cycling. In addition to potential metabolic interactions, based on the 16S rRNAs gene sequence of Nanoarchaeum-related and its potential host Ignicoccus-related archaea, as well as sequences of viruses and CRISPR arrays, we inferred that there were complex microbe-microbe interactions. In conclusion, our study provided evidence that there were numerous microbe-microbe and microbe-environment interactions within the microbial community in an acidic hot spring. We proposed that Hydrogenobaculum bacteria were the dominant microbial genus, as they were able to metabolize hydrogen, assimilate carbon and live in an anaerobic environment with fluctuating temperatures.« less

Metabolic characteristics of dominant microbes and key rare species from an acidic hot spring in Taiwan revealed by metagenomics

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

Lin, Kuei -Han; Liao, Ben -Yang; Chang, Hao -Wei

Microbial diversity and community structures in acidic hot springs have been characterized by 16S rRNA gene-based diversity surveys. However, our understanding regarding the interactions among microbes, or between microbes and environmental factors, remains limited. In the present study, a metagenomic approach, followed by bioinformatics analyses, were used to predict interactions within the microbial ecosystem in Shi-Huang-Ping (SHP), an acidic hot spring in northern Taiwan. Characterizing environmental parameters and potential metabolic pathways highlighted the importance of carbon assimilatory pathways. Four distinct carbon assimilatory pathways were identified in five dominant genera of bacteria. Of those dominant carbon fixers, Hydrogenobaculum bacteria outcompeted othermore » carbon assimilators and dominated the SHP, presumably due to their ability to metabolize hydrogen and to withstand an anaerobic environment with fluctuating temperatures. Furthermore, most dominant microbes were capable of metabolizing inorganic sulfur-related compounds (abundant in SHP). However, Acidithiobacillus ferrooxidans was the only species among key rare microbes with the capability to fix nitrogen, suggesting a key role in nitrogen cycling. In addition to potential metabolic interactions, based on the 16S rRNAs gene sequence of Nanoarchaeum-related and its potential host Ignicoccus-related archaea, as well as sequences of viruses and CRISPR arrays, we inferred that there were complex microbe-microbe interactions. In conclusion, our study provided evidence that there were numerous microbe-microbe and microbe-environment interactions within the microbial community in an acidic hot spring. We proposed that Hydrogenobaculum bacteria were the dominant microbial genus, as they were able to metabolize hydrogen, assimilate carbon and live in an anaerobic environment with fluctuating temperatures.« less

Studies on the key aroma compounds in raw (unheated) and heated Japanese soy sauce.

PubMed

Kaneko, Shu; Kumazawa, Kenji; Nishimura, Osamu

2013-04-10

An investigation using the aroma extract dilution analysis (AEDA) technique of the aroma concentrate from a raw Japanese soy sauce and the heated soy sauce revealed 40 key aroma compounds including 7 newly identified compounds. Among them, 5(or 2)-ethyl-4-hydroxy-2(or 5)-methyl-3(2H)-furanone and 3-hydroxy-4,5-dimethyl-2(5H)-furanone exhibited the highest flavor dilution (FD) factor of 2048, followed by 3-(methylthio)propanal, 4-ethyl-2-methoxyphenol, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone having FD factors from 128 to 512 in the raw soy sauce. Furthermore, comparative AEDAs, a quantitative analysis, and a sensory analysis demonstrated that whereas most of the key aroma compounds in the raw soy sauce were common in the heated soy sauce, some of the Strecker aldehydes and 4-vinylphenols contributed less to the raw soy sauce aroma. The model decarboxylation reactions of the phenolic acids during heating of the raw soy sauce revealed that although all reactions resulted in low yields, the hydroxycinnamic acid derivatives were much more reactive than the hydroxybenzoic acid derivatives due to the stable reaction intermediates. Besides the quantitative analyses of the soy sauces, the estimation of the reaction yields of the phenolic compounds in the heated soy sauce revealed that although only the 4-vinylphenols increased during heating of the raw soy sauce, they might not mainly be formed as decarboxylation products from the corresponding hydroxycinnamic acids but from the other proposed precursors, such as lignin, shakuchirin, and esters with arabinoxylan.

Origins of the Xylella fastidiosa Prophage-Like Regions and Their Impact in Genome Differentiation

PubMed Central

de Mello Varani, Alessandro; Souza, Rangel Celso; Nakaya, Helder I.; de Lima, Wanessa Cristina; Paula de Almeida, Luiz Gonzaga; Kitajima, Elliot Watanabe; Chen, Jianchi; Civerolo, Edwin; Vasconcelos, Ana Tereza Ribeiro; Van Sluys, Marie-Anne

2008-01-01

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes. PMID:19116666

Origins of the Xylella fastidiosa prophage-like regions and their impact in genome differentiation.

PubMed

de Mello Varani, Alessandro; Souza, Rangel Celso; Nakaya, Helder I; de Lima, Wanessa Cristina; Paula de Almeida, Luiz Gonzaga; Kitajima, Elliot Watanabe; Chen, Jianchi; Civerolo, Edwin; Vasconcelos, Ana Tereza Ribeiro; Van Sluys, Marie-Anne

2008-01-01

Xylella fastidiosa is a Gram negative plant pathogen causing many economically important diseases, and analyses of completely sequenced X. fastidiosa genome strains allowed the identification of many prophage-like elements and possibly phage remnants, accounting for up to 15% of the genome composition. To better evaluate the recent evolution of the X. fastidiosa chromosome backbone among distinct pathovars, the number and location of prophage-like regions on two finished genomes (9a5c and Temecula1), and in two candidate molecules (Ann1 and Dixon) were assessed. Based on comparative best bidirectional hit analyses, the majority (51%) of the predicted genes in the X. fastidiosa prophage-like regions are related to structural phage genes belonging to the Siphoviridae family. Electron micrograph reveals the existence of putative viral particles with similar morphology to lambda phages in the bacterial cell in planta. Moreover, analysis of microarray data indicates that 9a5c strain cultivated under stress conditions presents enhanced expression of phage anti-repressor genes, suggesting switches from lysogenic to lytic cycle of phages under stress-induced situations. Furthermore, virulence-associated proteins and toxins are found within these prophage-like elements, thus suggesting an important role in host adaptation. Finally, clustering analyses of phage integrase genes based on multiple alignment patterns reveal they group in five lineages, all possessing a tyrosine recombinase catalytic domain, and phylogenetically close to other integrases found in phages that are genetic mosaics and able to perform generalized and specialized transduction. Integration sites and tRNA association is also evidenced. In summary, we present comparative and experimental evidence supporting the association and contribution of phage activity on the differentiation of Xylella genomes.

A novel site-specific recombination system derived from bacteriophage phiMR11.

PubMed

Rashel, Mohammad; Uchiyama, Jumpei; Ujihara, Takako; Takemura, Iyo; Hoshiba, Hiroshi; Matsuzaki, Shigenobu

2008-04-04

We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.

Islander: A database of precisely mapped genomic islands in tRNA and tmRNA genes

DOE PAGES

Hudson, Corey M.; Lau, Britney Y.; Williams, Kelly P.

2014-11-05

Genomic islands are mobile DNAs that are major agents of bacterial and archaeal evolution. Integration into prokaryotic chromosomes usually occurs site-specifically at tRNA or tmRNA gene (together, tDNA) targets, catalyzed by tyrosine integrases. This splits the target gene, yet sequences within the island restore the disrupted gene; the regenerated target and its displaced fragment precisely mark the endpoints of the island. We applied this principle to search for islands in genomic DNA sequences. Our algorithm identifies tDNAs, finds fragments of those tDNAs in the same replicon and removes unlikely candidate islands through a series of filters. A search for islandsmore » in 2168 whole prokaryotic genomes produced 3919 candidates. The website Islander (recently moved to http://bioinformatics.sandia.gov/islander/) presents these precisely mapped candidate islands, the gene content and the island sequence. The algorithm further insists that each island encode an integrase, and attachment site sequence identity is carefully noted; therefore, the database also serves in the study of integrase site-specificity and its evolution.« less

25 CFR 502.14 - Key employee.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 25 Indians 2 2010-04-01 2010-04-01 false Key employee. 502.14 Section 502.14 Indians NATIONAL....14 Key employee. Key employee means: (a) A person who performs one or more of the following functions... gaming operation. (d) Any other person designated by the tribe as a key employee. [57 FR 12392, Apr. 9...

Known plaintext attack on double random phase encoding using fingerprint as key and a method for avoiding the attack.

PubMed

Tashima, Hideaki; Takeda, Masafumi; Suzuki, Hiroyuki; Obi, Takashi; Yamaguchi, Masahiro; Ohyama, Nagaaki

2010-06-21

We have shown that the application of double random phase encoding (DRPE) to biometrics enables the use of biometrics as cipher keys for binary data encryption. However, DRPE is reported to be vulnerable to known-plaintext attacks (KPAs) using a phase recovery algorithm. In this study, we investigated the vulnerability of DRPE using fingerprints as cipher keys to the KPAs. By means of computational experiments, we estimated the encryption key and restored the fingerprint image using the estimated key. Further, we propose a method for avoiding the KPA on the DRPE that employs the phase retrieval algorithm. The proposed method makes the amplitude component of the encrypted image constant in order to prevent the amplitude component of the encrypted image from being used as a clue for phase retrieval. Computational experiments showed that the proposed method not only avoids revealing the cipher key and the fingerprint but also serves as a sufficiently accurate verification system.

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

PubMed Central

González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred

2017-01-01

ABSTRACT Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site

Quantum Key Distribution

NASA Astrophysics Data System (ADS)

Hughes, Richard

2004-05-01

Quantum key distribution (QKD) uses single-photon communications to generate the shared, secret random number sequences that are used to encrypt and decrypt secret communications. The unconditional security of QKD is based on the interplay between fundamental principles of quantum physics and information theory. An adversary can neither successfully tap the transmissions, nor evade detection (eavesdropping raises the key error rate above a threshold value). QKD could be particularly attractive for free-space optical communications, both ground-based and for satellites. I will describe a QKD experiment performed over multi-kilometer line-of-sight paths, which serves as a model for a satellite-to-ground key distribution system. The system uses single-photon polarization states, without active polarization switching, and for the first time implements the complete BB84 QKD protocol including, reconciliation, privacy amplification and the all-important authentication stage. It is capable of continuous operation throughout the day and night, achieving the self-sustaining production of error-free, shared, secret bits. I will also report on the results of satellite-to-ground QKD modeling.

Expression profiles of key phenylpropanoid genes during Vanilla planifolia pod development reveal a positive correlation between PAL gene expression and vanillin biosynthesis.

PubMed

Fock-Bastide, Isabelle; Palama, Tony Lionel; Bory, Séverine; Lécolier, Aurélie; Noirot, Michel; Joët, Thierry

2014-01-01

In Vanilla planifolia pods, development of flavor precursors is dependent on the phenylpropanoid pathway. The distinctive vanilla aroma is produced by numerous phenolic compounds of which vanillin is the most important. Because of the economic importance of vanilla, vanillin biosynthetic pathways have been extensively studied but agreement has not yet been reached on the processes leading to its accumulation. In order to explore the transcriptional control exerted on these pathways, five key phenylpropanoid genes expressed during pod development were identified and their mRNA accumulation profiles were evaluated during pod development and maturation using quantitative real-time PCR. As a prerequisite for expression analysis using qRT-PCR, five potential reference genes were tested, and two genes encoding Actin and EF1 were shown to be the most stable reference genes for accurate normalization during pod development. For the first time, genes encoding a phenylalanine ammonia-lyase (VpPAL1) and a cinnamate 4-hydroxylase (VpC4H1) were identified in vanilla pods and studied during maturation. Among phenylpropanoid genes, differential regulation was observed from 3 to 8 months after pollination. VpPAL1 was gradually up-regulated, reaching the maximum expression level at maturity. In contrast, genes encoding 4HBS, C4H, OMT2 and OMT3 did not show significant increase in expression levels after the fourth month post-pollination. Expression profiling of these key phenylpropanoid genes is also discussed in light of accumulation patterns for key phenolic compounds. Interestingly, VpPAL1 gene expression was shown to be positively correlated to maturation and vanillin accumulation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

The neural bases of key competencies of emotional intelligence.

PubMed

Krueger, Frank; Barbey, Aron K; McCabe, Kevin; Strenziok, Maren; Zamboni, Giovanna; Solomon, Jeffrey; Raymont, Vanessa; Grafman, Jordan

2009-12-29

Emotional intelligence (EI) refers to a set of competencies that are essential features of human social life. Although the neural substrates of EI are virtually unknown, it is well established that the prefrontal cortex (PFC) plays a crucial role in human social-emotional behavior. We studied a unique sample of combat veterans from the Vietnam Head Injury Study, which is a prospective, long-term follow-up study of veterans with focal penetrating head injuries. We administered the Mayer-Salovey-Caruso Emotional Intelligence Test as a valid standardized psychometric measure of EI behavior to examine two key competencies of EI: (i) Strategic EI as the competency to understand emotional information and to apply it for the management of the self and of others and (ii) Experiential EI as the competency to perceive emotional information and to apply it for the integration into thinking. The results revealed that key competencies underlying EI depend on distinct neural PFC substrates. First, ventromedial PFC damage diminishes Strategic EI, and therefore, hinders the understanding and managing of emotional information. Second, dorsolateral PFC damage diminishes Experiential EI, and therefore, hinders the perception and integration of emotional information. In conclusion, EI should be viewed as complementary to cognitive intelligence and, when considered together, provide a more complete understanding of human intelligence.

The neural bases of key competencies of emotional intelligence

PubMed Central

Krueger, Frank; Barbey, Aron K.; McCabe, Kevin; Strenziok, Maren; Zamboni, Giovanna; Solomon, Jeffrey; Raymont, Vanessa; Grafman, Jordan

2009-01-01

Emotional intelligence (EI) refers to a set of competencies that are essential features of human social life. Although the neural substrates of EI are virtually unknown, it is well established that the prefrontal cortex (PFC) plays a crucial role in human social-emotional behavior. We studied a unique sample of combat veterans from the Vietnam Head Injury Study, which is a prospective, long-term follow-up study of veterans with focal penetrating head injuries. We administered the Mayer-Salovey-Caruso Emotional Intelligence Test as a valid standardized psychometric measure of EI behavior to examine two key competencies of EI: (i) Strategic EI as the competency to understand emotional information and to apply it for the management of the self and of others and (ii) Experiential EI as the competency to perceive emotional information and to apply it for the integration into thinking. The results revealed that key competencies underlying EI depend on distinct neural PFC substrates. First, ventromedial PFC damage diminishes Strategic EI, and therefore, hinders the understanding and managing of emotional information. Second, dorsolateral PFC damage diminishes Experiential EI, and therefore, hinders the perception and integration of emotional information. In conclusion, EI should be viewed as complementary to cognitive intelligence and, when considered together, provide a more complete understanding of human intelligence. PMID:20080795

Secure key from bound entanglement.

PubMed

Horodecki, Karol; Horodecki, Michał; Horodecki, Paweł; Oppenheim, Jonathan

2005-04-29

We characterize the set of shared quantum states which contain a cryptographically private key. This allows us to recast the theory of privacy as a paradigm closely related to that used in entanglement manipulation. It is shown that one can distill an arbitrarily secure key from bound entangled states. There are also states that have less distillable private keys than the entanglement cost of the state. In general, the amount of distillable key is bounded from above by the relative entropy of entanglement. Relationships between distillability and distinguishability are found for a class of states which have Bell states correlated to separable hiding states. We also describe a technique for finding states exhibiting irreversibility in entanglement distillation.

78 FR 33221 - Special Local Regulation; Annual Swim Around Key West, Atlantic Ocean and Gulf of Mexico; Key...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-04

... Fort Zach State Park, North through Key West Harbor, East through Flemming Cut, South on Cow Key... State Park, North through Key West Harbor, East through Flemming Cut, South on Cow Key Channel and West...

Finite-key security analysis of quantum key distribution with imperfect light sources

DOE PAGES

Mizutani, Akihiro; Curty, Marcos; Lim, Charles Ci Wen; ...

2015-09-09

In recent years, the gap between theory and practice in quantum key distribution (QKD) has been significantly narrowed, particularly for QKD systems with arbitrarily flawed optical receivers. The status for QKD systems with imperfect light sources is however less satisfactory, in the sense that the resulting secure key rates are often overly dependent on the quality of state preparation. This is especially the case when the channel loss is high. Very recently, to overcome this limitation, Tamaki et al proposed a QKD protocol based on the so-called 'rejected data analysis', and showed that its security in the limit of infinitelymore » long keys is almost independent of any encoding flaw in the qubit space, being this protocol compatible with the decoy state method. Here, as a step towards practical QKD, we show that a similar conclusion is reached in the finite-key regime, even when the intensity of the light source is unstable. More concretely, we derive security bounds for a wide class of realistic light sources and show that the bounds are also efficient in the presence of high channel loss. Our results strongly suggest the feasibility of long distance provably secure communication with imperfect light sources.« less

Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation

PubMed Central

Pashley, Clare L.; Hewitt, Eric W.; Radford, Sheena E.

2016-01-01

The mouse and human β2-microglobulin protein orthologs are 70 % identical in sequence and share 88 % sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH 2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3 M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hβ2m and mβ2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation. PMID:26780548

Comparison is key.

PubMed

Stone, Mark H; Stenner, A Jackson

2014-01-01

Several concepts from Georg Rasch's last papers are discussed. The key one is comparison because Rasch considered the method of comparison fundamental to science. From the role of comparison stems scientific inference made operational by a properly developed frame of reference producing specific objectivity. The exact specifications Rasch outlined for making comparisons are explicated from quotes, and the role of causality derived from making comparisons is also examined. Understanding causality has implications for what can and cannot be produced via Rasch measurement. His simple examples were instructive, but the implications are far reaching upon first establishing the key role of comparison.

Effects of Combined CCR5/Integrase Inhibitors-Based Regimen on Mucosal Immunity in HIV-Infected Patients Naïve to Antiretroviral Therapy: A Pilot Randomized Trial.

PubMed

Serrano-Villar, Sergio; Sainz, Talia; Ma, Zhong-Min; Utay, Netanya S; Chun, Tae-Wook; Wook-Chun, Tae; Mann, Surinder; Kashuba, Angela D; Siewe, Basile; Albanese, Anthony; Troia-Cancio, Paolo; Sinclair, Elizabeth; Somasunderam, Anoma; Yotter, Tammy; Deeks, Steven G; Landay, Alan; Pollard, Richard B; Miller, Christopher J; Moreno, Santiago; Asmuth, David M

2016-01-01

Whether initiation of antiretroviral therapy (ART) regimens aimed at achieving greater concentrations within gut associated lymphoid tissue (GALT) impacts the level of mucosal immune reconstitution, inflammatory markers and the viral reservoir remains unknown. We included 12 HIV- controls and 32 ART-naïve HIV patients who were randomized to efavirenz, maraviroc or maraviroc+raltegravir, each with fixed-dose tenofovir disoproxil fumarate/emtricitabine. Rectal and duodenal biopsies were obtained at baseline and at 9 months of ART. We performed a comprehensive assay of T-cell subsets by flow cytometry, T-cell density in intestinal biopsies, plasma and tissue concentrations of antiretroviral drugs by high-performance liquid chromatography/mass spectroscopy, and plasma interleukin-6 (IL-6), lipoteichoic acid (LTA), soluble CD14 (sCD14) and zonulin-1 each measured by ELISA. Total cell-associated HIV DNA was measured in PBMC and rectal and duodenal mononuclear cells. Twenty-six HIV-infected patients completed the follow-up. In the duodenum, the quadruple regimen resulted in greater CD8+ T-cell density decline, greater normalization of mucosal CCR5+CD4+ T-cells and increase of the naïve/memory CD8+ T-cell ratio, and a greater decline of sCD14 levels and duodenal HIV DNA levels (P = 0.004 and P = 0.067, respectively), with no changes in HIV RNA in plasma or tissue. Maraviroc showed the highest drug distribution to the gut tissue, and duodenal concentrations correlated well with other T-cell markers in duodenum, i.e., the CD4/CD8 ratio, %CD4+ and %CD8+ HLA-DR+CD38+ T-cells. Maraviroc use elicited greater activation of the mucosal naïve CD8+ T-cell subset, ameliorated the distribution of the CD8+ T-cell maturational subsets and induced higher improvement of zonulin-1 levels. These data suggest that combined CCR5 and integrase inhibitor based combination therapy in ART treatment naïve patients might more effectively reconstitute duodenal immunity, decrease inflammatory

ChlamyNET: a Chlamydomonas gene co-expression network reveals global properties of the transcriptome and the early setup of key co-expression patterns in the green lineage.

PubMed

Romero-Campero, Francisco J; Perez-Hurtado, Ignacio; Lucas-Reina, Eva; Romero, Jose M; Valverde, Federico

2016-03-12

interconnecting key biological processes such as light response with carbon and nitrogen metabolism. Our study reveals that key elements in the regulation of carbon and nitrogen metabolism, light response and cell cycle identified in higher plants were already established in Chlamydomonas. These conserved elements are not only limited to transcription factors, regulators and their targets, but also include the cis-regulatory elements recognized by them.

An improved authenticated key agreement protocol for telecare medicine information system.

PubMed

Liu, Wenhao; Xie, Qi; Wang, Shengbao; Hu, Bin

2016-01-01

In telecare medicine information systems (TMIS), identity authentication of patients plays an important role and has been widely studied in the research field. Generally, it is realized by an authenticated key agreement protocol, and many such protocols were proposed in the literature. Recently, Zhang et al. pointed out that Islam et al.'s protocol suffers from the following security weaknesses: (1) Any legal but malicious patient can reveal other user's identity; (2) An attacker can launch off-line password guessing attack and the impersonation attack if the patient's identity is compromised. Zhang et al. also proposed an improved authenticated key agreement scheme with privacy protection for TMIS. However, in this paper, we point out that Zhang et al.'s scheme cannot resist off-line password guessing attack, and it fails to provide the revocation of lost/stolen smartcard. In order to overcome these weaknesses, we propose an improved protocol, the security and authentication of which can be proven using applied pi calculus based formal verification tool ProVerif.

Feasibility of continuous-variable quantum key distribution with noisy coherent states

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

Usenko, Vladyslav C.; Department of Optics, Palacky University, CZ-772 07 Olomouc; Filip, Radim

2010-02-15

We address security of the quantum key distribution scheme based on the noisy modulation of coherent states and investigate how it is robust against noise in the modulation regardless of the particular technical implementation. As the trusted preparation noise is shown to be security breaking even for purely lossy channels, we reveal the essential difference between two types of trusted noise, namely sender-side preparation noise and receiver-side detection noise, the latter being security preserving. We consider the method of sender-side state purification to compensate the preparation noise and show its applicability in the realistic conditions of channel loss, untrusted channelmore » excess noise, and trusted detection noise. We show that purification makes the scheme robust to the preparation noise (i.e., even the arbitrary noisy coherent states can in principle be used for the purpose of quantum key distribution). We also take into account the effect of realistic reconciliation and show that the purification method is still efficient in this case up to a limited value of preparation noise.« less

Quantum key distribution network for multiple applications

NASA Astrophysics Data System (ADS)

Tajima, A.; Kondoh, T.; Ochi, T.; Fujiwara, M.; Yoshino, K.; Iizuka, H.; Sakamoto, T.; Tomita, A.; Shimamura, E.; Asami, S.; Sasaki, M.

2017-09-01

The fundamental architecture and functions of secure key management in a quantum key distribution (QKD) network with enhanced universal interfaces for smooth key sharing between arbitrary two nodes and enabling multiple secure communication applications are proposed. The proposed architecture consists of three layers: a quantum layer, key management layer and key supply layer. We explain the functions of each layer, the key formats in each layer and the key lifecycle for enabling a practical QKD network. A quantum key distribution-advanced encryption standard (QKD-AES) hybrid system and an encrypted smartphone system were developed as secure communication applications on our QKD network. The validity and usefulness of these systems were demonstrated on the Tokyo QKD Network testbed.

Algorithms for Lightweight Key Exchange.

PubMed

Alvarez, Rafael; Caballero-Gil, Cándido; Santonja, Juan; Zamora, Antonio

2017-06-27

Public-key cryptography is too slow for general purpose encryption, with most applications limiting its use as much as possible. Some secure protocols, especially those that enable forward secrecy, make a much heavier use of public-key cryptography, increasing the demand for lightweight cryptosystems that can be implemented in low powered or mobile devices. This performance requirements are even more significant in critical infrastructure and emergency scenarios where peer-to-peer networks are deployed for increased availability and resiliency. We benchmark several public-key key-exchange algorithms, determining those that are better for the requirements of critical infrastructure and emergency applications and propose a security framework based on these algorithms and study its application to decentralized node or sensor networks.

Key Largo Limestone revisited: Pleistocene shelf-edge facies, Florida Keys, USA

USGS Publications Warehouse

Gray, Multer H.; Gischler, E.; Lundberg, J.; Simmons, K.R.; Shinn, E.A.

2002-01-01

New dates and analysis of 12 deep and 57 shallow cores allow a more detailed interpretation of the Pleistocene shelf edge of the Florida Platform as found in various facies of the Key Largo Limestone beneath the Florida Keys. In this study a three-phase evolution of the Quaternary units (Q1-Q5) of the Key Largo is presented with new subdivision of the Q5. (1) In the first phase, the Q1 and Q2 (perhaps deposited during oxygen-isotope stage 11) deep-water quartz-rich environment evolved into a shallow carbonate phase. (2) Subsequently, a Q3 (presumably corresponding to oxygen-isotope stage 9) flourishing reef and productive high-platform sediment phase developed. (3) Finally, a Q4 and Q5 (corresponding to oxygen-isotope stages 7 and 5) stabilization phase occured with reefs and leeward productive lagoons, followed by lower sea levels presenting a sequence of younger (isotope substages 5c, 5a) shelf-margin wedges, sediment veneers and outlier reefs. The Key Largo Limestone provides an accessible model of a carbonate shelf edge with fluctuating water depth, bordering a deep seaward basin for a period of at least 300 ka. During this time, at least four onlaps/offlaps, often separated by periods of karst development with associated diagenetic alterations, took place. The story presented by this limestone not only allows a better understanding of the history of south Florida but also aids in the interpretation of similar persistent shelf-edge sites bordering deep basins in other areas.

Finite-key analysis for quantum key distribution with weak coherent pulses based on Bernoulli sampling

NASA Astrophysics Data System (ADS)

Kawakami, Shun; Sasaki, Toshihiko; Koashi, Masato

2017-07-01

An essential step in quantum key distribution is the estimation of parameters related to the leaked amount of information, which is usually done by sampling of the communication data. When the data size is finite, the final key rate depends on how the estimation process handles statistical fluctuations. Many of the present security analyses are based on the method with simple random sampling, where hypergeometric distribution or its known bounds are used for the estimation. Here we propose a concise method based on Bernoulli sampling, which is related to binomial distribution. Our method is suitable for the Bennett-Brassard 1984 (BB84) protocol with weak coherent pulses [C. H. Bennett and G. Brassard, Proceedings of the IEEE Conference on Computers, Systems and Signal Processing (IEEE, New York, 1984), Vol. 175], reducing the number of estimated parameters to achieve a higher key generation rate compared to the method with simple random sampling. We also apply the method to prove the security of the differential-quadrature-phase-shift (DQPS) protocol in the finite-key regime. The result indicates that the advantage of the DQPS protocol over the phase-encoding BB84 protocol in terms of the key rate, which was previously confirmed in the asymptotic regime, persists in the finite-key regime.

Temporal steering and security of quantum key distribution with mutually unbiased bases against individual attacks

NASA Astrophysics Data System (ADS)

Bartkiewicz, Karol; Černoch, Antonín; Lemr, Karel; Miranowicz, Adam; Nori, Franco

2016-06-01

Temporal steering, which is a temporal analog of Einstein-Podolsky-Rosen steering, refers to temporal quantum correlations between the initial and final state of a quantum system. Our analysis of temporal steering inequalities in relation to the average quantum bit error rates reveals the interplay between temporal steering and quantum cloning, which guarantees the security of quantum key distribution based on mutually unbiased bases against individual attacks. The key distributions analyzed here include the Bennett-Brassard 1984 protocol and the six-state 1998 protocol by Bruss. Moreover, we define a temporal steerable weight, which enables us to identify a kind of monogamy of temporal correlation that is essential to quantum cryptography and useful for analyzing various scenarios of quantum causality.

Single-lineage transcriptome analysis reveals key regulatory pathways in primitive erythroid progenitors in the mouse embryo

PubMed Central

Isern, Joan; He, Zhiyong; Fraser, Stuart T.; Nowotschin, Sonja; Ferrer-Vaquer, Anna; Moore, Rebecca; Hadjantonakis, Anna-Katerina; Schulz, Vincent; Tuck, David; Gallagher, Patrick G.

2011-01-01

Primitive erythroid (EryP) progenitors are the first cell type specified from the mesoderm late in gastrulation. We used a transgenic reporter to image and purify the earliest blood progenitors and their descendants from developing mouse embryos. EryP progenitors exhibited remarkable proliferative capacity in the yolk sac immediately before the onset of circulation, when these cells comprise nearly half of all cells of the embryo. Global expression profiles generated at 24-hour intervals from embryonic day 7.5 through 2.5 revealed 2 abrupt changes in transcript diversity that coincided with the entry of EryPs into the circulation and with their late maturation and enucleation, respectively. These changes were paralleled by the expression of critical regulatory factors. Experiments designed to test predictions from these data demonstrated that the Wnt-signaling pathway is active in EryP progenitors, which display an aerobic glycolytic profile and the numbers of which are regulated by transforming growth factor-β1 and hypoxia. This is the first transcriptome assembled for a single hematopoietic lineage of the embryo over the course of its differentiation. PMID:21263157

Optimizing Requirements Decisions with KEYS

NASA Technical Reports Server (NTRS)

Jalali, Omid; Menzies, Tim; Feather, Martin

2008-01-01

Recent work with NASA's Jet Propulsion Laboratory has allowed for external access to five of JPL's real-world requirements models, anonymized to conceal proprietary information, but retaining their computational nature. Experimentation with these models, reported herein, demonstrates a dramatic speedup in the computations performed on them. These models have a well defined goal: select mitigations that retire risks which, in turn, increases the number of attainable requirements. Such a non-linear optimization is a well-studied problem. However identification of not only (a) the optimal solution(s) but also (b) the key factors leading to them is less well studied. Our technique, called KEYS, shows a rapid way of simultaneously identifying the solutions and their key factors. KEYS improves on prior work by several orders of magnitude. Prior experiments with simulated annealing or treatment learning took tens of minutes to hours to terminate. KEYS runs much faster than that; e.g for one model, KEYS ran 13,000 times faster than treatment learning (40 minutes versus 0.18 seconds). Processing these JPL models is a non-linear optimization problem: the fewest mitigations must be selected while achieving the most requirements. Non-linear optimization is a well studied problem. With this paper, we challenge other members of the PROMISE community to improve on our results with other techniques.

SLAR image interpretation keys for geographic analysis

NASA Technical Reports Server (NTRS)

Coiner, J. C.

1972-01-01

A means for side-looking airborne radar (SLAR) imagery to become a more widely used data source in geoscience and agriculture is suggested by providing interpretation keys as an easily implemented interpretation model. Interpretation problems faced by the researcher wishing to employ SLAR are specifically described, and the use of various types of image interpretation keys to overcome these problems is suggested. With examples drawn from agriculture and vegetation mapping, direct and associate dichotomous image interpretation keys are discussed and methods of constructing keys are outlined. Initial testing of the keys, key-based automated decision rules, and the role of the keys in an information system for agriculture are developed.

Comparison of the aggregation of homologous β2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation.

PubMed

Pashley, Clare L; Hewitt, Eric W; Radford, Sheena E

2016-02-13

The mouse and human β2-microglobulin protein orthologs are 70% identical in sequence and share 88% sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human β2m (hβ2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH2.5) in the absence of NaCl, mouse β2m (mβ2m) requires the addition of 0.3M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hβ2m and mβ2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

In vitro model of production of antibodies; a new approach to reveal the presence of key bacteria in polymicrobial environments.

PubMed

Wu, Chongcong; Nakka, Sravya; Mansouri, Sepahdar; Bengtsson, Torbjörn; Nayeri, Tayeb; Nayeri, Fariba

2016-09-09

There is a rapid emergence of multiple resistant gram-negative bacteria due to overuse of antibiotics in the treatment of infections. Biofilms consist of polymicrobial communities that survive the host's defense system. The key bacteria in biofilms are slow growing and support an attachment and rapid growth of other microorganisms. Current antimicrobial strategies often fail due to poor diagnosis of key pathogens in biofilms. The study aims to develop anti-bacterial human antibodies in vitro from patients who had recently undergone a systemic infection by pathogenic bacteria and to use these antibodies as a tool for detecting bacteria in biofilms. Lymphocytes were separated from whole blood of patients (n = 10) and stimulated with heat-killed bacteria to produce antibodies in vitro. The specificity of antibodies in recognizing the bacteria against which they were directed was evaluated by surface plasmon resonance system (SPR) and electron microscopy. The ulcer secretions from patients with chronic and acute leg ulcers and healthy controls were analyzed by the SPR system and the results were compared with culture studies. The produced antibodies recognized bacteria with high sensitivity (SPR). The antibodies against Enterococcus fecalis bound specifically to the microorganism in a bacterial co-culture that was visualized by electron microscopy. In the present work, a method for producing specific antibodies against bacteria is introduced to recognize bacterial components in body fluids of patients suffering from pathogenic biofilms. This diagnostic technique may be most useful in clinical microbiology and in the choice of antibiotics in the treatment of serious infections.

The Geology of the Florida Keys.

ERIC Educational Resources Information Center

Shinn, Eugene A.

1988-01-01

Describes some of the ancient geologic history of the Florida Keys from Key Largo to Key West including the effects of glaciers, sea level rise, reef distribution, spurs and grooves, backstepping and ecological zonation, growth rates and erosion. Predicts future changes in this area. (CW)

No Child Left Behind in Art Education Policy: A Review of Key Recommendations for Arts Language Revisions

ERIC Educational Resources Information Center

Grey, Anne C.

2010-01-01

From bipartisan origins and a laudable intent, the No Child Left Behind (Act) of 2001 has profoundly altered the condition of art education. A historical vantage point and review of literature reveals the current status of pending arts language revisions to the NCLB Act, as well as a pressing need to examine the key recommendations and to consider…

Widespread dissemination of class 1 integron components in soils and related ecosystems as revealed by cultivation-independent analysis.

PubMed

Jechalke, Sven; Schreiter, Susanne; Wolters, Birgit; Dealtry, Simone; Heuer, Holger; Smalla, Kornelia

2013-01-01

Class 1 integrons contribute to the emerging problem of antibiotic resistance in human medicine by acquisition, exchange, and expression of resistance genes embedded within gene cassettes. Besides the clinical setting they were recently reported from environmental habitats and often located on plasmids and transposons, facilitating their transfer and spread within bacterial communities. In this study we aimed to provide insights into the occurrence of genes typically associated with the class 1 integrons in previously not studied environments with or without human impact and their association with IncP-1 plasmids. Total community DNA was extracted from manure-treated and untreated soils, lettuce and potato rhizosphere, digestates, and an on-farm biopurification system and screened by PCR with subsequent Southern blot hybridization for the presence of the class 1 integrase gene intI1 as well as qacE and qacEΔ 1 resistance genes. The results revealed a widespread dissemination of class 1 integrons in the environments analyzed, mainly related to the presence of qacEΔ 1 genes. All 28 IncP-1ε plasmids carrying class 1 integrons, which were captured exogenously in a recent study from piggery manure and soils treated with manure, carried qacEΔ 1 genes. Based on the strong hybridization signals in the rhizosphere of lettuce compared to the potato rhizosphere, the abundances of intI1, qacE/qacEΔ 1, and sul1 genes were quantified relative to the 16S rRNA gene abundance by real-time PCR in the rhizosphere of lettuce planted in three different soils and in the corresponding bulk soil. A significant enrichment of intI1 and qacE/qacEΔ 1 genes was confirmed in the rhizosphere of lettuce compared to bulk soil. Additionally, the relative abundance of korB genes specific for IncP-1 plasmids was enriched in the rhizosphere and correlated to the intI1 gene abundance indicating that IncP-1 plasmids might have contributed to the spread of class 1 integrons in the analyzed soils.

Captured key electrical safety lockout system

DOEpatents

Darimont, Daniel E.

1995-01-01

A safety lockout apparatus for an electrical circuit includes an electrical switch, a key, a lock and a blocking mechanism. The electrical switch is movable between an ON position at which the electrical circuit is energized and an OFF position at which the electrical circuit is deactivated. The lock is adapted to receive the key and is rotatable among a plurality of positions by the key. The key is only insertable and removable when the lock is at a preselected position. The lock is maintained in the preselected position when the key is removed from the lock. The blocking mechanism physically maintains the switch in its OFF position when the key is removed from the lock. The blocking mechanism preferably includes a member driven by the lock between a first position at which the electrical switch is movable between its ON and OFF positions and a second position at which the member physically maintains the electrical switch in its OFF position. Advantageously, the driven member's second position corresponds to the preselected position at which the key can be removed from and inserted into the lock.

Captured key electrical safety lockout system

DOEpatents

Darimont, D.E.

1995-10-31

A safety lockout apparatus for an electrical circuit includes an electrical switch, a key, a lock and a blocking mechanism. The electrical switch is movable between an ON position at which the electrical circuit is energized and an OFF position at which the electrical circuit is deactivated. The lock is adapted to receive the key and is rotatable among a plurality of positions by the key. The key is only insertable and removable when the lock is at a preselected position. The lock is maintained in the preselected position when the key is removed from the lock. The blocking mechanism physically maintains the switch in its OFF position when the key is removed from the lock. The blocking mechanism preferably includes a member driven by the lock between a first position at which the electrical switch is movable between its ON and OFF positions and a second position at which the member physically maintains the electrical switch in its OFF position. Advantageously, the driven member`s second position corresponds to the preselected position at which the key can be removed from and inserted into the lock. 7 figs.

Membrane protein properties revealed through data-rich electrostatics calculations

PubMed Central

Guerriero, Christopher J.; Brodsky, Jeffrey L.; Grabe, Michael

2015-01-01

SUMMARY The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem including: full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane induced pKa shifts, calculation of non-polar energies, and command-line scripting for large scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane potentially revealing interesting functional information. PMID:26118532

Key handling in wireless sensor networks

NASA Astrophysics Data System (ADS)

Li, Y.; Newe, T.

2007-07-01

With the rapid growth of Wireless Sensor Networks (WSNs), many advanced application areas have received significant attention. However, security will be an important factor for their full adoption. Wireless sensor nodes pose unique challenges and as such traditional security protocols, used in traditional networks cannot be applied directly. Some new protocols have been published recently with the goal of providing both privacy of data and authentication of sensor nodes for WSNs. Such protocols can employ private-key and/or public key cryptographic algorithms. Public key algorithms hold the promise of simplifying the network infrastructure required to provide security services such as: privacy, authentication and non-repudiation, while symmetric algorithms require less processing power on the lower power wireless node. In this paper a selection of key establishment/agreement protocols are reviewed and they are broadly divided into two categories: group key agreement protocols and pair-wise key establishment protocols. A summary of the capabilities and security related services provided by each protocol is provided.

Secret-key expansion from covert communication

NASA Astrophysics Data System (ADS)

Arrazola, Juan Miguel; Amiri, Ryan

2018-02-01

Covert communication allows the transmission of messages in such a way that it is not possible for adversaries to detect that the communication is occurring. This provides protection in situations where knowledge that two parties are talking to each other may be incriminating to them. In this work, we study how covert communication can be used for a different purpose: secret key expansion. First, we show that any message transmitted in a secure covert protocol is also secret and therefore unknown to an adversary. We then propose a covert communication protocol where the amount of key consumed in the protocol is smaller than the transmitted key, thus leading to secure secret key expansion. We derive precise conditions for secret key expansion to occur, showing that it is possible when there are sufficiently low levels of noise for a given security level. We conclude by examining how secret key expansion from covert communication can be performed in a computational security model.

Network theory inspired analysis of time-resolved expression data reveals key players guiding P. patens stem cell development.

PubMed

Busch, Hauke; Boerries, Melanie; Bao, Jie; Hanke, Sebastian T; Hiss, Manuel; Tiko, Theodhor; Rensing, Stefan A

2013-01-01

Transcription factors (TFs) often trigger developmental decisions, yet, their transcripts are often only moderately regulated and thus not easily detected by conventional statistics on expression data. Here we present a method that allows to determine such genes based on trajectory analysis of time-resolved transcriptome data. As a proof of principle, we have analysed apical stem cells of filamentous moss (P. patens) protonemata that develop from leaflets upon their detachment from the plant. By our novel correlation analysis of the post detachment transcriptome kinetics we predict five out of 1,058 TFs to be involved in the signaling leading to the establishment of pluripotency. Among the predicted regulators is the basic helix loop helix TF PpRSL1, which we show to be involved in the establishment of apical stem cells in P. patens. Our methodology is expected to aid analysis of key players of developmental decisions in complex plant and animal systems.

High-efficiency Gaussian key reconciliation in continuous variable quantum key distribution

NASA Astrophysics Data System (ADS)

Bai, ZengLiang; Wang, XuYang; Yang, ShenShen; Li, YongMin

2016-01-01

Efficient reconciliation is a crucial step in continuous variable quantum key distribution. The progressive-edge-growth (PEG) algorithm is an efficient method to construct relatively short block length low-density parity-check (LDPC) codes. The qua-sicyclic construction method can extend short block length codes and further eliminate the shortest cycle. In this paper, by combining the PEG algorithm and qua-si-cyclic construction method, we design long block length irregular LDPC codes with high error-correcting capacity. Based on these LDPC codes, we achieve high-efficiency Gaussian key reconciliation with slice recon-ciliation based on multilevel coding/multistage decoding with an efficiency of 93.7%.

Continuous-variable quantum key distribution with 1 Mbps secure key rate.

PubMed

Huang, Duan; Lin, Dakai; Wang, Chao; Liu, Weiqi; Fang, Shuanghong; Peng, Jinye; Huang, Peng; Zeng, Guihua

2015-06-29

We report the first continuous-variable quantum key distribution (CVQKD) experiment to enable the creation of 1 Mbps secure key rate over 25 km standard telecom fiber in a coarse wavelength division multiplexers (CWDM) environment. The result is achieved with two major technological advances: the use of a 1 GHz shot-noise-limited homodyne detector and the implementation of a 50 MHz clock system. The excess noise due to noise photons from local oscillator and classical data channels in CWDM is controlled effectively. We note that the experimental verification of high-bit-rate CVQKD in the multiplexing environment is a significant step closer toward large-scale deployment in fiber networks.

Identification and Characterization of Putative Integron-Like Elements of the Heavy-Metal-Hypertolerant Strains of Pseudomonas spp.

PubMed

Ciok, Anna; Adamczuk, Marcin; Bartosik, Dariusz; Dziewit, Lukasz

2016-11-28

Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas .

Continuous variable quantum key distribution: finite-key analysis of composable security against coherent attacks.

PubMed

Furrer, F; Franz, T; Berta, M; Leverrier, A; Scholz, V B; Tomamichel, M; Werner, R F

2012-09-07

We provide a security analysis for continuous variable quantum key distribution protocols based on the transmission of two-mode squeezed vacuum states measured via homodyne detection. We employ a version of the entropic uncertainty relation for smooth entropies to give a lower bound on the number of secret bits which can be extracted from a finite number of runs of the protocol. This bound is valid under general coherent attacks, and gives rise to keys which are composably secure. For comparison, we also give a lower bound valid under the assumption of collective attacks. For both scenarios, we find positive key rates using experimental parameters reachable today.

A Public-Key Based Authentication and Key Establishment Protocol Coupled with a Client Puzzle.

ERIC Educational Resources Information Center

Lee, M. C.; Fung, Chun-Kan

2003-01-01

Discusses network denial-of-service attacks which have become a security threat to the Internet community and suggests the need for reliable authentication protocols in client-server applications. Presents a public-key based authentication and key establishment protocol coupled with a client puzzle protocol and validates it through formal logic…

Secret Key Crypto Implementations

NASA Astrophysics Data System (ADS)

Bertoni, Guido Marco; Melzani, Filippo

This chapter presents the algorithm selected in 2001 as the Advanced Encryption Standard. This algorithm is the base for implementing security and privacy based on symmetric key solutions in almost all new applications. Secret key algorithms are used in combination with modes of operation to provide different security properties. The most used modes of operation are presented in this chapter. Finally an overview of the different techniques of software and hardware implementations is given.

Human Activity Determines the Presence of Integron-Associated and Antibiotic Resistance Genes in Southwestern British Columbia.

PubMed

Uyaguari-Díaz, Miguel I; Croxen, Matthew A; Luo, Zhiyao; Cronin, Kirby I; Chan, Michael; Baticados, Waren N; Nesbitt, Matthew J; Li, Shaorong; Miller, Kristina M; Dooley, Damion; Hsiao, William; Isaac-Renton, Judith L; Tang, Patrick; Prystajecky, Natalie

2018-01-01

The dissemination of antibiotic resistant bacteria from anthropogenic sources into the environment poses an emerging public health threat. Antibiotic resistance genes (ARGs) and gene-capturing systems such as integron-associated integrase genes ( intI ) play a key role in alterations of microbial communities and the spread of antibiotic resistant bacteria into the environment. In order to assess the effect of anthropogenic activities on watersheds in southwestern British Columbia, the presence of putative antibiotic resistance and integrase genes was analyzed in the microbiome of agricultural, urban influenced, and protected watersheds. A metagenomics approach and high-throughput quantitative PCR (HT qPCR) were used to screen for elements of resistance including ARGs and intI . Metagenomic sequencing of bacterial genomic DNA was used to characterize the resistome of microbial communities present in watersheds over a 1-year period. There was a low prevalence of ARGs relative to the microbial population (<1%). Analysis of the metagenomic sequences detected a total of 60 elements of resistance including 46 ARGs, intI1 , and groEL/ intI1 genes and 12 quaternary ammonium compounds ( qac ) resistance genes across all watershed locations. The relative abundance and richness of ARGs was found to be highest in agriculture impacted watersheds compared to urban and protected watersheds. A downstream transport pattern was observed in the impacted watersheds (urban and agricultural) during dry months. Similar to other reports, this study found a strong association between intI1 and ARGs (e.g., sul1 ), an association which may be used as a proxy for anthropogenic activities. Chemical analysis of water samples for three major groups of antibiotics was below the detection limit. However, the high richness and gene copy numbers (GCNs) of ARGs in impacted sites suggest that the effects of effluents on microbial communities are occurring even at low concentrations of antimicrobials

Human Activity Determines the Presence of Integron-Associated and Antibiotic Resistance Genes in Southwestern British Columbia

PubMed Central

Uyaguari-Díaz, Miguel I.; Croxen, Matthew A.; Luo, Zhiyao; Cronin, Kirby I.; Chan, Michael; Baticados, Waren N.; Nesbitt, Matthew J.; Li, Shaorong; Miller, Kristina M.; Dooley, Damion; Hsiao, William; Isaac-Renton, Judith L.; Tang, Patrick; Prystajecky, Natalie

2018-01-01

The dissemination of antibiotic resistant bacteria from anthropogenic sources into the environment poses an emerging public health threat. Antibiotic resistance genes (ARGs) and gene-capturing systems such as integron-associated integrase genes (intI) play a key role in alterations of microbial communities and the spread of antibiotic resistant bacteria into the environment. In order to assess the effect of anthropogenic activities on watersheds in southwestern British Columbia, the presence of putative antibiotic resistance and integrase genes was analyzed in the microbiome of agricultural, urban influenced, and protected watersheds. A metagenomics approach and high-throughput quantitative PCR (HT qPCR) were used to screen for elements of resistance including ARGs and intI. Metagenomic sequencing of bacterial genomic DNA was used to characterize the resistome of microbial communities present in watersheds over a 1-year period. There was a low prevalence of ARGs relative to the microbial population (<1%). Analysis of the metagenomic sequences detected a total of 60 elements of resistance including 46 ARGs, intI1, and groEL/intI1 genes and 12 quaternary ammonium compounds (qac) resistance genes across all watershed locations. The relative abundance and richness of ARGs was found to be highest in agriculture impacted watersheds compared to urban and protected watersheds. A downstream transport pattern was observed in the impacted watersheds (urban and agricultural) during dry months. Similar to other reports, this study found a strong association between intI1 and ARGs (e.g., sul1), an association which may be used as a proxy for anthropogenic activities. Chemical analysis of water samples for three major groups of antibiotics was below the detection limit. However, the high richness and gene copy numbers (GCNs) of ARGs in impacted sites suggest that the effects of effluents on microbial communities are occurring even at low concentrations of antimicrobials in the

Key rate for calibration robust entanglement based BB84 quantum key distribution protocol

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

Gittsovich, O.; Moroder, T.

2014-12-04

We apply the approach of verifying entanglement, which is based on the sole knowledge of the dimension of the underlying physical system to the entanglement based version of the BB84 quantum key distribution protocol. We show that the familiar one-way key rate formula holds already if one assumes the assumption that one of the parties is measuring a qubit and no further assumptions about the measurement are needed.

Investigating on the fermentation behavior of six lactic acid bacteria strains in barley malt wort reveals limitation in key amino acids and buffer capacity.

PubMed

Nsogning, Sorelle Dongmo; Fischer, Susann; Becker, Thomas

2018-08-01

Understanding lactic acid bacteria (LAB) fermentation behavior in malt wort is a milestone towards flavor improvement of lactic acid fermented malt beverages. Therefore, this study aims to outline deficiencies that may exist in malt wort fermentation. First, based on six LAB strains, cell viability and vitality were evaluated. Second, sugars, organic acids, amino acids, pH value and buffering capacity (BC) were monitored. Finally, the implication of key amino acids, fructose and wort BC on LAB growth was determined. Short growth phase coupled with prompt cell death and a decrease in metabolic activity was observed. Low wort BC caused rapid pH drop with lactic acid accumulation, which conversely increased the BC leading to less pH change at late-stage fermentation. Lactic acid content (≤3.9 g/L) was higher than the reported inhibitory concentration (1.8 g/L). Furthermore, sugars were still available but fructose and key amino acids lysine, arginine and glutamic acid were considerably exhausted (≤98%). Wort supplementations improved cell growth and viability leading to conclude that key amino acid depletion coupled with low BC limits LAB growth in malt wort. Then, a further increase in organic acid reduces LAB viability. This knowledge opens doors for LAB fermentation process optimization in malt wort. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Power of Key: Celebrating 20 Years of Innovation at the Key Learning Community

ERIC Educational Resources Information Center

Kunkel, Christine

2007-01-01

The Key Learning Community in Indianapolis was the first school in the world to base its approach on the theory of multiple intelligences. Ms. Kunkel, Key's principal, reflects on the school's continuing growth and success--even in the face of pressures to standardize--and shares the history of its founding. (Contains 5 endnotes.)

Topological robustness analysis of protein interaction networks reveals key targets for overcoming chemotherapy resistance in glioma

NASA Astrophysics Data System (ADS)

Azevedo, Hátylas; Moreira-Filho, Carlos Alberto

2015-11-01

Biological networks display high robustness against random failures but are vulnerable to targeted attacks on central nodes. Thus, network topology analysis represents a powerful tool for investigating network susceptibility against targeted node removal. Here, we built protein interaction networks associated with chemoresistance to temozolomide, an alkylating agent used in glioma therapy, and analyzed their modular structure and robustness against intentional attack. These networks showed functional modules related to DNA repair, immunity, apoptosis, cell stress, proliferation and migration. Subsequently, network vulnerability was assessed by means of centrality-based attacks based on the removal of node fractions in descending orders of degree, betweenness, or the product of degree and betweenness. This analysis revealed that removing nodes with high degree and high betweenness was more effective in altering networks’ robustness parameters, suggesting that their corresponding proteins may be particularly relevant to target temozolomide resistance. In silico data was used for validation and confirmed that central nodes are more relevant for altering proliferation rates in temozolomide-resistant glioma cell lines and for predicting survival in glioma patients. Altogether, these results demonstrate how the analysis of network vulnerability to topological attack facilitates target prioritization for overcoming cancer chemoresistance.

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

PubMed Central

Gibson, Richard M.; Meyer, Ashley M.; Winner, Dane; Archer, John; Feyertag, Felix; Ruiz-Mateos, Ezequiel; Leal, Manuel; Robertson, David L.; Schmotzer, Christine L.

2014-01-01

With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new

KEY COMPARISON: CCQM-K21 Key Comparison Determination of pp’-DDT in fish oil

NASA Astrophysics Data System (ADS)

Webb, K. S.; Carter, D.; Wolff Briche, C. S. J.

2003-01-01

A key comparison on the determination of (pp'-dichlorodiphenyl) trichloroethane (pp'-DDT) in a fish oil matrix has been successfully completed. Nine NMIs participated in this key comparison and used the technique of isotope dilution gas-chromatography mass spectrometry (ID/GC/MS) for the determinations. Two samples (A and B) of fish oil were distributed to participants, each gravimetrically spiked with pp'-DDT. The KCRV for Sample A is 0.0743 +/- 0.0020 µg g-1 and that of Sample B is 0.1655 +/- 0.0014 µg g-1 of pp'-DDT in fish oil. The results for Sample A showed a RSD of 3.5%, the RSD for Sample B was within 1%. These results were an improvement over those of the corresponding pilot study (CCQM-P21), where at a mass fraction of pp'-DDT in fish oil of 0.311 µg g-1 the RSD was 2.6%. The compound pp'-DDT is a typical organochlorine pesticide and this key comparison has shown that NMIs have the ability to measure such compounds at levels typically found in the environment. The compound (pp'-dichlorodiphenyl) dichloroethylene (pp'-DDE), a metabolite of pp'-DDT, was the subject of a previous key comparison (CCQM-K5). The compound pp'-DDT is technically more challenging than that of pp'-DDE since it can decompose during the measurement procedure. Consequently the success of this key comparison, combined with that of CCQM-K5 demonstrates a broad measurement capability by NMIs for organochlorine compounds in the environment. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the Mutual Recognition Arrangement (MRA).

Computational Modeling Reveals Key Contributions of KCNQ and hERG Currents to the Malleability of Uterine Action Potentials Underpinning Labor

PubMed Central

Tong, Wing-Chiu; Tribe, Rachel M.; Smith, Roger; Taggart, Michael J.

2014-01-01

The electrical excitability of uterine smooth muscle cells is a key determinant of the contraction of the organ during labor and is manifested by spontaneous, periodic action potentials (APs). Near the end of term, APs vary in shape and size reflecting an ability to change the frequency, duration and amplitude of uterine contractions. A recent mathematical model quantified several ionic features of the electrical excitability in uterine smooth muscle cells. It replicated many of the experimentally recorded uterine AP configurations but its limitations were evident when trying to simulate the long-duration bursting APs characteristic of labor. A computational parameter search suggested that delayed rectifying K+ currents could be a key model component requiring improvement to produce the longer-lasting bursting APs. Of the delayed rectifying K+ currents family it is of interest that KCNQ and hERG channels have been reported to be gestationally regulated in the uterus. These currents exhibit features similar to the broadly defined uterine I K1 of the original mathematical model. We thus formulated new quantitative descriptions for several I KCNQ and I hERG. Incorporation of these currents into the uterine cell model enabled simulations of the long-lasting bursting APs. Moreover, we used this modified model to simulate the effects of different contributions of I KCNQ and I hERG on AP form. Our findings suggest that the alterations in expression of hERG and KCNQ channels can potentially provide a mechanism for fine tuning of AP forms that lends a malleability for changing between plateau-like and long-lasting bursting-type APs as uterine cells prepare for parturition. PMID:25474527

Performance of device-independent quantum key distribution

NASA Astrophysics Data System (ADS)

Cao, Zhu; Zhao, Qi; Ma, Xiongfeng

2016-07-01

Quantum key distribution provides information-theoretically-secure communication. In practice, device imperfections may jeopardise the system security. Device-independent quantum key distribution solves this problem by providing secure keys even when the quantum devices are untrusted and uncharacterized. Following a recent security proof of the device-independent quantum key distribution, we improve the key rate by tightening the parameter choice in the security proof. In practice where the system is lossy, we further improve the key rate by taking into account the loss position information. From our numerical simulation, our method can outperform existing results. Meanwhile, we outline clear experimental requirements for implementing device-independent quantum key distribution. The maximal tolerable error rate is 1.6%, the minimal required transmittance is 97.3%, and the minimal required visibility is 96.8 % .

Harry Potter and the Dichotomous Key

ERIC Educational Resources Information Center

Crowther, David T.

2003-01-01

In this lesson, students use Bertie Bott's Every Flavor Beans--a "wild" candy written about in the Harry Potter books and now available in stores--to learn about classification and dichotomous keys. In these activities, students sort jelly beans according to a key and then construct a key for a "new" flavor of beans. Students then build on their…

"Key Concepts in ELT": Taking Stock

ERIC Educational Resources Information Center

Hall, Graham

2012-01-01

This article identifies patterns and trends within "Key Concepts in ELT", both since the inception of the feature in ELT Journal in 1993 and during the 17 years of the current editorship. After outlining the aims of the series, the article identifies key themes that have emerged over time, exploring the links between "Key Concepts" pieces and the…

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution.

PubMed

Shivange, Amol V; Roccatano, Danilo; Schwaneberg, Ulrich

2016-01-01

Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

Key Nutrients.

ERIC Educational Resources Information Center

Federal Extension Service (USDA), Washington, DC.

Lessons written to help trainer agents prepare aides for work with families in the Food and Nutrition Program are presented in this booklet. The key nutrients discussed in the 10 lessons are protein, carbohydrates, fat, calcium, iron, iodine, and Vitamins A, B, C, and D. the format of each lesson is as follows: Purpose, Presentation, Application…

Viral fitness cost prevents HIV-1 from evading dolutegravir drug pressure

PubMed Central

2013-01-01

Background Clinical studies have shown that integrase strand transfer inhibitors can be used to treat HIV-1 infection. Although the first-generation integrase inhibitors are susceptible to the emergence of resistance mutations that impair their efficacy in therapy, such resistance has not been identified to date in drug-naïve patients who have been treated with the second-generation inhibitor dolutegravir. During previous in vitro selection study, we identified a R263K mutation as the most common substitution to arise in the presence of dolutegravir with H51Y arising as a secondary mutation. Additional experiments reported here provide a plausible explanation for the absence of reported dolutegravir resistance among integrase inhibitor-naïve patients to date. Results We now show that H51Y in combination with R263K increases resistance to dolutegravir but is accompanied by dramatic decreases in both enzymatic activity and viral replication. Conclusions Since H51Y and R263K may define a unique resistance pathway to dolutegravir, our results are consistent with the absence of resistance mutations in antiretroviral drug-naive patients treated with this drug. PMID:23432922

KEY COMPARISON: Final report on CCQM-K69 key comparison: Testosterone glucuronide in human urine

NASA Astrophysics Data System (ADS)

Liu, Fong-Ha; Mackay, Lindsey; Murby, John

2010-01-01

The CCQM-K69 key comparison of testosterone glucuronide in human urine was organized under the auspices of the CCQM Organic Analysis Working Group (OAWG). The National Measurement Institute Australia (NMIA) acted as the coordinating laboratory for the comparison. The samples distributed for the key comparison were prepared at NMIA with funding from the World Anti-Doping Agency (WADA). WADA granted the approval for this material to be used for the intercomparison provided the distribution and handling of the material were strictly controlled. Three national metrology institutes (NMIs)/designated institutes (DIs) developed reference methods and submitted data for the key comparison along with two other laboratories who participated in the parallel pilot study. A good selection of analytical methods and sample workup procedures was displayed in the results submitted considering the complexities of the matrix involved. The comparability of measurement results was successfully demonstrated by the participating NMIs. Only the key comparison data were used to estimate the key comparison reference value (KCRV), using the arithmetic mean approach. The reported expanded uncertainties for results ranged from 3.7% to 6.7% at the 95% level of confidence and all results agreed within the expanded uncertainty of the KCRV. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

Practical challenges in quantum key distribution

DOE PAGES

Diamanti, Eleni; Lo, Hoi -Kwong; Qi, Bing; ...

2016-11-08

Here, quantum key distribution (QKD) promises unconditional security in data communication and is currently being deployed in commercial applications. Nonetheless, before QKD can be widely adopted, it faces a number of important challenges such as secret key rate, distance, size, cost and practical security. Here, we survey those key challenges and the approaches that are currently being taken to address them.

Practical challenges in quantum key distribution

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

Diamanti, Eleni; Lo, Hoi -Kwong; Qi, Bing

Here, quantum key distribution (QKD) promises unconditional security in data communication and is currently being deployed in commercial applications. Nonetheless, before QKD can be widely adopted, it faces a number of important challenges such as secret key rate, distance, size, cost and practical security. Here, we survey those key challenges and the approaches that are currently being taken to address them.

High key rate continuous-variable quantum key distribution with a real local oscillator.

PubMed

Wang, Tao; Huang, Peng; Zhou, Yingming; Liu, Weiqi; Ma, Hongxin; Wang, Shiyu; Zeng, Guihua

2018-02-05

Continuous-variable quantum key distribution (CVQKD) with a real local oscillator (LO) has been extensively studied recently due to its security and simplicity. In this paper, we propose a novel implementation of a high-key-rate CVQKD with a real LO. Particularly, with the help of the simultaneously generated reference pulse, the phase drift of the signal is tracked in real time and then compensated. By utilizing the time and polarization multiplexing techniques to isolate the reference pulse and controlling the intensity of it, not only the contamination from it is suppressed, but also a high accuracy of the phase compensation can be guaranteed. Besides, we employ homodyne detection on the signal to ensure the high quantum efficiency and heterodyne detection on the reference pulse to acquire the complete phase information of it. In order to suppress the excess noise, a theoretical noise model for our scheme is established. According to this model, the impact of the modulation variance and the intensity of the reference pulse are both analysed theoretically and then optimized according to the experimental data. By measuring the excess noise in the 25km optical fiber transmission system, a 3.14Mbps key rate in the asymptotic regime proves to be achievable. This work verifies the feasibility of the high-key-rate CVQKD with a real LO within the metropolitan area.

Social patterns revealed through random matrix theory

NASA Astrophysics Data System (ADS)

Sarkar, Camellia; Jalan, Sarika

2014-11-01

Despite the tremendous advancements in the field of network theory, very few studies have taken weights in the interactions into consideration that emerge naturally in all real-world systems. Using random matrix analysis of a weighted social network, we demonstrate the profound impact of weights in interactions on emerging structural properties. The analysis reveals that randomness existing in particular time frame affects the decisions of individuals rendering them more freedom of choice in situations of financial security. While the structural organization of networks remains the same throughout all datasets, random matrix theory provides insight into the interaction pattern of individuals of the society in situations of crisis. It has also been contemplated that individual accountability in terms of weighted interactions remains as a key to success unless segregation of tasks comes into play.

Key volatile compounds in red koji-shochu, a Monascus-fermented product, and their formation steps during fermentation.

PubMed

Rahayu, Yen Yen Sally; Yoshizaki, Yumiko; Yamaguchi, Keiko; Okutsu, Kayu; Futagami, Taiki; Tamaki, Hisanori; Sameshima, Yoshihiro; Takamine, Kazunori

2017-06-01

Red koji, which refers the solid culture grown koji mold on the steamed rice, is one of the ingredients of Asian fermented foods including the Japanese spirit shochu. This study was aimed at elucidating the characteristic flavor and key volatile compounds of red koji-shochu as well as the mechanism of their formation. Sensory evaluation showed that red koji-shochu has the distinctive flavors cheese, sour, milky, and oily. Fifteen key volatile compounds of red koji-shochu were identified by gas chromatography-mass spectrometry and high-performance liquid chromatography, and by comparison between red koji-shochu and white koji-shochu, as another typical shochu. The mash analysis revealed that ketone compounds and short-chain acids derive from red koji. Furthermore, although other key compounds were produced by yeast, it is highly likely that their concentrations were affected directly or indirectly by the high activities of protease and lipase in red koji. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

PubMed

Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

2015-04-24

Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by

48 CFR 2452.237-70 - Key personnel.

Code of Federal Regulations, 2010 CFR

2010-10-01

... contracts when it is necessary for contract performance to identify the contractor's key personnel: Key... perform as follows: [List Key Personnel and/or positions, and tasks, percentage of effort, number of hours...

A Hybrid Key Management Scheme for WSNs Based on PPBR and a Tree-Based Path Key Establishment Method

PubMed Central

Zhang, Ying; Liang, Jixing; Zheng, Bingxin; Chen, Wei

2016-01-01

With the development of wireless sensor networks (WSNs), in most application scenarios traditional WSNs with static sink nodes will be gradually replaced by Mobile Sinks (MSs), and the corresponding application requires a secure communication environment. Current key management researches pay less attention to the security of sensor networks with MS. This paper proposes a hybrid key management schemes based on a Polynomial Pool-based key pre-distribution and Basic Random key pre-distribution (PPBR) to be used in WSNs with MS. The scheme takes full advantages of these two kinds of methods to improve the cracking difficulty of the key system. The storage effectiveness and the network resilience can be significantly enhanced as well. The tree-based path key establishment method is introduced to effectively solve the problem of communication link connectivity. Simulation clearly shows that the proposed scheme performs better in terms of network resilience, connectivity and storage effectiveness compared to other widely used schemes. PMID:27070624

Quantum key distribution without the wavefunction

NASA Astrophysics Data System (ADS)

Niestegge, Gerd

A well-known feature of quantum mechanics is the secure exchange of secret bit strings which can then be used as keys to encrypt messages transmitted over any classical communication channel. It is demonstrated that this quantum key distribution allows a much more general and abstract access than commonly thought. The results include some generalizations of the Hilbert space version of quantum key distribution, but are based upon a general nonclassical extension of conditional probability. A special state-independent conditional probability is identified as origin of the superior security of quantum key distribution; this is a purely algebraic property of the quantum logic and represents the transition probability between the outcomes of two consecutive quantum measurements.

Device-independent quantum key distribution

NASA Astrophysics Data System (ADS)

Hänggi, Esther

2010-12-01

In this thesis, we study two approaches to achieve device-independent quantum key distribution: in the first approach, the adversary can distribute any system to the honest parties that cannot be used to communicate between the three of them, i.e., it must be non-signalling. In the second approach, we limit the adversary to strategies which can be implemented using quantum physics. For both approaches, we show how device-independent quantum key distribution can be achieved when imposing an additional condition. In the non-signalling case this additional requirement is that communication is impossible between all pairwise subsystems of the honest parties, while, in the quantum case, we demand that measurements on different subsystems must commute. We give a generic security proof for device-independent quantum key distribution in these cases and apply it to an existing quantum key distribution protocol, thus proving its security even in this setting. We also show that, without any additional such restriction there always exists a successful joint attack by a non-signalling adversary.

Entropy driven key-lock assembly.

PubMed

Odriozola, G; Jiménez-Angeles, F; Lozada-Cassou, M

2008-09-21

The effective interaction between a sphere with an open cavity (lock) and a spherical macroparticle (key), both immersed in a hard sphere fluid, is studied by means of Monte Carlo simulations. As a result, a two-dimensional map of the key-lock effective interaction potential is constructed, which leads to the proposal of a self-assembling mechanism: There exists trajectories through which the key-lock pair could assemble avoiding trespassing potential barriers. Hence, solely the entropic contribution can induce their self-assembling even in the absence of attractive forces. This study points out the solvent contribution within the underlying mechanisms of substrate-protein assemblydisassembly processes, which are important steps of the enzyme catalysis and protein mediated transport.

Entropy driven key-lock assembly

NASA Astrophysics Data System (ADS)

Odriozola, G.; Jiménez-Ángeles, F.; Lozada-Cassou, M.

2008-09-01

The effective interaction between a sphere with an open cavity (lock) and a spherical macroparticle (key), both immersed in a hard sphere fluid, is studied by means of Monte Carlo simulations. As a result, a two-dimensional map of the key-lock effective interaction potential is constructed, which leads to the proposal of a self-assembling mechanism: There exists trajectories through which the key-lock pair could assemble avoiding trespassing potential barriers. Hence, solely the entropic contribution can induce their self-assembling even in the absence of attractive forces. This study points out the solvent contribution within the underlying mechanisms of substrate-protein assembly/disassembly processes, which are important steps of the enzyme catalysis and protein mediated transport.

KeySlinger and StarSlinger: Secure Key Exchange and Encrypted File Transfer on Smartphones

DTIC Science & Technology

2011-05-01

format data to exchange because contact information can be exported to V- Cards using existing APIs. For these reasons it was chosen as the medium to... Card format allows customization of this field. The service provider field serves to identify the app the key is for and the username field stores the...public key data. A sample V- Card field looks like Listing 1 below. IMPP;TextSecure

Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

PubMed

Marcoline, Frank V; Bethel, Neville; Guerriero, Christopher J; Brodsky, Jeffrey L; Grabe, Michael

2015-08-04

The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

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

Endsley, Mark A., E-mail: maendsle@utmb.edu; Somasunderam, Anoma D., E-mail: asomasun@utmb.edu; Li, Guangyu, E-mail: LIG001@mail.etsu.edu

Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4{sup +} T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizesmore » with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4{sup +} T lymphocytes.« less

Algorithms for Lightweight Key Exchange †

PubMed Central

Santonja, Juan; Zamora, Antonio

2017-01-01

Public-key cryptography is too slow for general purpose encryption, with most applications limiting its use as much as possible. Some secure protocols, especially those that enable forward secrecy, make a much heavier use of public-key cryptography, increasing the demand for lightweight cryptosystems that can be implemented in low powered or mobile devices. This performance requirements are even more significant in critical infrastructure and emergency scenarios where peer-to-peer networks are deployed for increased availability and resiliency. We benchmark several public-key key-exchange algorithms, determining those that are better for the requirements of critical infrastructure and emergency applications and propose a security framework based on these algorithms and study its application to decentralized node or sensor networks. PMID:28654006

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.

PubMed

González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred; Llosa, Matxalen

2017-06-15

Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site

Pressure–Temperature Phase Diagram Reveals Spin–Lattice Interactions in Co[N(CN) 2 ] 2

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

Musfeldt, J. L.; O’Neal, K. R.; Brinzari, T. V.

2017-04-07

Diamond anvil cell techniques, synchrotron-based infrared and Raman spectroscopies, and lattice dynamics calculations are combined with prior magnetic property work to reveal the pressure–temperature phase diagram of Co[N(CN)2]2. The second-order structural boundaries converge on key areas of activity involving the spin state exposing how the pressure-induced local lattice distortions trigger the ferromagnetic → antiferromagnetic transition in this quantum material.

An Empirical Analysis of the Cascade Secret Key Reconciliation Protocol for Quantum Key Distribution

DTIC Science & Technology

2011-09-01

performance with the parity checks within each pass increasing and as a result, the processing time is expected to increase as well. A conclusion is drawn... timely manner has driven efforts to develop new key distribution methods. The most promising method is Quantum Key Distribution (QKD) and is...thank the QKD Project Team for all of the insight and support they provided in such a short time period. Thanks are especially in order for my

Fully device-independent conference key agreement

NASA Astrophysics Data System (ADS)

Ribeiro, Jérémy; Murta, Gláucia; Wehner, Stephanie

2018-02-01

We present a security analysis of conference key agreement (CKA) in the most adversarial model of device independence (DI). Our protocol can be implemented by any experimental setup that is capable of performing Bell tests [specifically, the Mermin-Ardehali-Belinskii-Klyshko (MABK) inequality], and security can in principle be obtained for any violation of the MABK inequality that detects genuine multipartite entanglement among the N parties involved in the protocol. As our main tool, we derive a direct physical connection between the N -partite MABK inequality and the Clauser-Horne-Shimony-Holt (CHSH) inequality, showing that certain violations of the MABK inequality correspond to a violation of the CHSH inequality between one of the parties and the other N -1 . We compare the asymptotic key rate for device-independent conference key agreement (DICKA) to the case where the parties use N -1 device-independent quantum key distribution protocols in order to generate a common key. We show that for some regime of noise the DICKA protocol leads to better rates.

Drug resistance in B and non-B subtypes amongst subjects recently diagnosed as primary/recent or chronic HIV-infected over the period 2013-2016: Impact on susceptibility to first-line strategies including integrase strand-transfer inhibitors.

PubMed

Andreis, Samantha; Basso, Monica; Scaggiante, Renzo; Cruciani, Mario; Ferretto, Roberto; Manfrin, Vinicio; Panese, Sandro; Rossi, Maria Cristina; Francavilla, Ermenegildo; Boldrin, Caterina; Alvarez, Mario; Dal Bello, Federico; Mengoli, Carlo; Turriziani, Ombretta; Sarmati, Loredana; Antonelli, Guido; Andreoni, Massimo; Palù, Giorgio; Parisi, Saverio Giuseppe

2017-09-01

To characterize the prevalence of transmitted drug resistance mutations (TDRMs) by plasma analysis of 750 patients at the time of HIV diagnosis from January 1, 2013 to November 16, 2016 in the Veneto region (Italy), where all drugs included in the recommended first line therapies were prescribed, included integrase strand transfer inhibitors (InNSTI). TDRMs were defined according to the Stanford HIV database algorithm. Subtype B was the most prevalent HIV clade (67.3%). A total of 92 patients (12.3%) were expected to be resistant to one drug at least, most with a single class mutation (60/68-88.2% in subtype B infected subjectsand 23/24-95.8% in non-B subjects) and affecting mainly NNRTIs. No significant differences were observed between the prevalence rates of TDRMs involving one or more drugs, except for the presence of E138A quite only in patients with B subtype and other NNRTI in subjects with non-B infection. The diagnosis of primary/recent infection was made in 73 patients (9.7%): they had almost only TDRMs involving a single class. Resistance to InSTI was studied in 484 subjects (53 with primary-recent infection), one patient had 143C in 2016, a total of thirteen 157Q mutations were detected (only one in primary/recent infection). Only one major InSTI-TDRM was identified but monitoring of TDRMs should continue in the light of continuing presence of NNRTI-related mutation amongst newly diagnosed subjects, sometime impacting also to modern NNRTI drugs recommended in first-line therapy. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Anomalous charge transport in conjugated polymers reveals underlying mechanisms of trapping and percolation

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

Mollinger, Sonya A.; Salleo, Alberto; Spakowitz, Andrew J.

While transport in conjugated polymers has many similarities to that in crystalline inorganic materials, several key differences reveal the unique relationship between the morphology of polymer films and the charge mobility. We develop a model that directly incorporates the molecular properties of the polymer film and correctly predicts these unique transport features. At low degree of polymerization, the increase of the mobility with the polymer chain length reveals trapping at chain ends, and saturation of the mobility at high degree of polymerization results from conformational traps within the chains. Similarly, the inverse field dependence of the mobility reveals that transportmore » on single polymer chains is characterized by the ability of the charge to navigate around kinks and loops in the chain. Lastly, these insights emphasize the connection between the polymer conformations and the transport and thereby offer a route to designing improved device morphologies through molecular design and materials processing.« less

Anomalous charge transport in conjugated polymers reveals underlying mechanisms of trapping and percolation

DOE PAGES

Mollinger, Sonya A.; Salleo, Alberto; Spakowitz, Andrew J.

2016-11-10

While transport in conjugated polymers has many similarities to that in crystalline inorganic materials, several key differences reveal the unique relationship between the morphology of polymer films and the charge mobility. We develop a model that directly incorporates the molecular properties of the polymer film and correctly predicts these unique transport features. At low degree of polymerization, the increase of the mobility with the polymer chain length reveals trapping at chain ends, and saturation of the mobility at high degree of polymerization results from conformational traps within the chains. Similarly, the inverse field dependence of the mobility reveals that transportmore » on single polymer chains is characterized by the ability of the charge to navigate around kinks and loops in the chain. Lastly, these insights emphasize the connection between the polymer conformations and the transport and thereby offer a route to designing improved device morphologies through molecular design and materials processing.« less

Asymmetry of perceived key movement in chorale sequences: converging evidence from a probe-tone analysis.

PubMed

Cuddy, L L; Thompson, W F

1992-01-01

In a probe-tone experiment, two groups of listeners--one trained, the other untrained, in traditional music theory--rated the goodness of fit of each of the 12 notes of the chromatic scale to four-voice harmonic sequences. Sequences were 12 simplified excerpts from Bach chorales, 4 nonmodulating, and 8 modulating. Modulations occurred either one or two steps in either the clockwise or the counterclockwise direction on the cycle of fifths. A consistent pattern of probe-tone ratings was obtained for each sequence, with no significant differences between listener groups. Two methods of analysis (Fourier analysis and regression analysis) revealed a directional asymmetry in the perceived key movement conveyed by modulating sequences. For a given modulation distance, modulations in the counterclockwise direction effected a clearer shift in tonal organization toward the final key than did clockwise modulations. The nature of the directional asymmetry was consistent with results reported for identification and rating of key change in the sequences (Thompson & Cuddy, 1989a). Further, according to the multiple-regression analysis, probe-tone ratings did not merely reflect the distribution of tones in the sequence. Rather, ratings were sensitive to the temporal structure of the tonal organization in the sequence.

LACIE analyst interpretation keys

NASA Technical Reports Server (NTRS)

Baron, J. G.; Payne, R. W.; Palmer, W. F. (Principal Investigator)

1979-01-01

Two interpretation aids, 'The Image Analysis Guide for Wheat/Small Grains Inventories' and 'The United States and Canadian Great Plains Regional Keys', were developed during LACIE phase 2 and implemented during phase 3 in order to provide analysts with a better understanding of the expected ranges in color variation of signatures for individual biostages and of the temporal sequences of LANDSAT signatures. The keys were tested using operational LACIE data, and the results demonstrate that their use provides improved labeling accuracy in all analyst experience groupings, in all geographic areas within the U.S. Great Plains, and during all periods of crop development.

Evaluating the use of key performance indicators to evidence the patient experience.

PubMed

McCance, Tanya; Hastings, Jack; Dowler, Hilda

2015-11-01

To test eight person-centred key performance indicators and the feasibility of an appropriate measurement framework as an approach to evidencing the patient experience. The value of measuring the quality of patient care is undisputed in the international literature, however, the type of measures that can be used to generate data that is meaningful for practice continues to be debated. This paper offers a different perspective to the 'measurement' of the nursing and midwifery contribution to the patient experience. Fourth generation evaluation was the methodological approach used to evaluate the implementation of the key performance indicators and measurement framework across three participating organisations involving nine practice settings. Data were collected by repeated use of claims, concerns and issues with staff working across nine participating sites (n = 18) and the senior executives from the three partner organisations (n = 12). Data were collected during the facilitated sessions with stakeholders and analysed in conjunction with the data generated from the measurement framework. The data reveal the inherent value placed on the evidence generated from the implementation of the key performance indicators as reflected in the following themes: measuring what matters; evidencing the patient experience; engaging staff; a focus for improving practice; and articulating and demonstrating the positive contribution of nursing and midwifery. The implementation of the key performance indicators and the measurement framework has been effective in generating evidence that demonstrates the patient experience. The nature of the data generated not only privileges the patient voice but also offers feedback to nurses and midwives that can inform the development of person-centred cultures. The use of these indicators will produce evidence of patient experience that can be used by nurse and midwives to celebrate and further inform person-centred practice. © 2015 John

Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases

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

Mayes, Heather B.; Knott, Brandon C.; Crowley, Michael F.

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase ( TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleavingmore » the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2S O ring configuration as it reaches its binding site. Lastly, this work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.« less

Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases

DOE PAGES

Mayes, Heather B.; Knott, Brandon C.; Crowley, Michael F.; ...

2016-06-01

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase ( TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleavingmore » the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2S O ring configuration as it reaches its binding site. Lastly, this work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.« less

Teamwork situated in multiteam systems: Key lessons learned and future opportunities.

PubMed

Shuffler, Marissa L; Carter, Dorothy R

2018-01-01

Many important contexts requiring teamwork, including health care, space exploration, national defense, and scientific discovery, present important challenges that cannot be addressed by a single team working independently. Instead, the complex goals these contexts present often require effectively coordinated efforts of multiple specialized teams working together as a multiteam system (MTS). For almost 2 decades, researchers have endeavored to understand the novelties and nuances for teamwork and collaboration that ensue when teams operate together as "component teams" in these interdependent systems. In this special issue on the settings of teamwork, we aim to synthesize what is known thus far regarding teamwork situated in MTS contexts and offer new directions and considerations for developing, maintaining, and sustaining effective collaboration in MTSs. Our review of extant research on MTSs reveals 7 key lessons learned regarding teamwork situated in MTSs, but also reveals that much is left to learn about the science and practice of ensuring effective multiteam functioning. We elaborate these lessons and delineate 4 major opportunities for advancing the science of MTSs as a critical embedding context for collaboration and teamwork, now and in the future. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Server-Controlled Identity-Based Authenticated Key Exchange

NASA Astrophysics Data System (ADS)

Guo, Hua; Mu, Yi; Zhang, Xiyong; Li, Zhoujun

We present a threshold identity-based authenticated key exchange protocol that can be applied to an authenticated server-controlled gateway-user key exchange. The objective is to allow a user and a gateway to establish a shared session key with the permission of the back-end servers, while the back-end servers cannot obtain any information about the established session key. Our protocol has potential applications in strong access control of confidential resources. In particular, our protocol possesses the semantic security and demonstrates several highly-desirable security properties such as key privacy and transparency. We prove the security of the protocol based on the Bilinear Diffie-Hellman assumption in the random oracle model.

Ebola virus infection kinetics in chimeric mice reveal a key role of T cells as barriers for virus dissemination

PubMed Central

Lüdtke, Anja; Ruibal, Paula; Wozniak, David M.; Pallasch, Elisa; Wurr, Stephanie; Bockholt, Sabrina; Gómez-Medina, Sergio; Qiu, Xiangguo; Kobinger, Gary P.; Rodríguez, Estefanía; Günther, Stephan; Krasemann, Susanne; Idoyaga, Juliana; Oestereich, Lisa; Muñoz-Fontela, César

2017-01-01

Ebola virus (EBOV) causes severe systemic disease in humans and non-human primates characterized by high levels of viremia and virus titers in peripheral organs. The natural portals of virus entry are the mucosal surfaces and the skin where macrophages and dendritic cells (DCs) are primary EBOV targets. Due to the migratory properties of DCs, EBOV infection of these cells has been proposed as a necessary step for virus dissemination via draining lymph nodes and blood. Here we utilize chimeric mice with competent hematopoietic-driven immunity, to show that EBOV primarily infects CD11b+ DCs in non-lymphoid and lymphoid tissues, but spares the main cross-presenting CD103+ DC subset. Furthermore, depletion of CD8 and CD4 T cells resulted in loss of early control of virus replication, viremia and fatal Ebola virus disease (EVD). Thus, our findings point out at T cell function as a key determinant of EVD progress and outcome. PMID:28256637

[Mechanisms of action, pharmacology and interactions of dolutegravir].

PubMed

Ribera, Esteban; Podzamczer, Daniel

2015-03-01

Dolutegravir is a second-generation integrase strand transfer inhibitor (INSTI), whose potential and binding half-life in the integrase are far superior to those of raltegravir and elvitegravir, conferring it with unique characteristics in terms of its genetic barrier to resistance and activity against viruses with one or more mutations in the integrase. The pharmacokinetic properties of dolutegravir allow once-daily dosing (50 mg), with or without food, maintaining concentrations far above those effective against wild-type viruses. If integrase resistance mutations are present, the recommended dosing regimen is 50 mg/12 h. The distribution of dolutegravir in cerebrospinal fluid is good and effective concentrations are also reached in the male and female genital tracts. Dolutegravir is metabolized by UGT1A1 and, to a lesser extent, by CYP3A4, without being an inducer or inhibitor of the usual metabolic systems. It has a very low potential for drug interactions and can be administered in routine doses with most drugs. Dose adjustment is not required, even in patients with renal insufficiency or mild or moderate liver failure. Increasing the dose of dolutegravir (50 mg/12 h) is only recommended when administered with efavirenz, nevirapine, fosamprenavir/r, tipranavir/r, rifampicin, carbamazepine, phenytoin and phenobarbital. Coadministration of dolutegravir with etravirine is not recommended without a protease inhibitor or with Hypericum perforatum. Dolutegravir should be administered 2 h before or 6 h after antacids or products with polyvalent cations. Dolutegravir can reduce renal tubule secretion of substances excreted via OCT2, with a slight initial increase in creatinine, with no risk of renal toxicity. The drug can also increase metformin concentrations and consequently monitoring is recommended in case dose adjustment is required. In summary, dolutegravir has excellent pharmacokinetic and drug interaction profiles. Copyright © 2015 Elsevier España, S

Geology and hydrogeology of the Florida Keys

USGS Publications Warehouse

Halley, Robert B.; Vacher, H. L.; Shinn,

1997-01-01

This chapter discusses the geology and hydrogeology of the Florida Keys, and focuses on the islands formed of Pleistocene limestone. These islands, which are crossed when driving from Miami to Key West, are typically regarded as "the Florida Keys." The outstanding and fragile character of ecosystems on and around the Florida Keys has prompted State and Federal efforts to protect and preserve the remaining public portions of the region. The Florida Keys were largely ignored during the sixteenth, seventeenth, and eighteenth centuries, although the waters just offshore provided a major shipping thoroughfare to and from the New World. The Florida Keys are now recognized as one of the great recreational and environmental resources of the United States. The islands are outposts of a laid-back, tropical resort culture that has as its foundation warmth and clear water. A significant part of the attraction is fishing, diving, and boating around the area's coral reefs, which the islands protect. But the reefs were not always so highly valued. The Florida Keys that have protected the reefs for millennia, may now be the source of the agents that may accomplish what Agassiz thought was beyond man's power a century ago.

Numerical approach for unstructured quantum key distribution

PubMed Central

Coles, Patrick J.; Metodiev, Eric M.; Lütkenhaus, Norbert

2016-01-01

Quantum key distribution (QKD) allows for communication with security guaranteed by quantum theory. The main theoretical problem in QKD is to calculate the secret key rate for a given protocol. Analytical formulas are known for protocols with symmetries, since symmetry simplifies the analysis. However, experimental imperfections break symmetries, hence the effect of imperfections on key rates is difficult to estimate. Furthermore, it is an interesting question whether (intentionally) asymmetric protocols could outperform symmetric ones. Here we develop a robust numerical approach for calculating the key rate for arbitrary discrete-variable QKD protocols. Ultimately this will allow researchers to study ‘unstructured' protocols, that is, those that lack symmetry. Our approach relies on transforming the key rate calculation to the dual optimization problem, which markedly reduces the number of parameters and hence the calculation time. We illustrate our method by investigating some unstructured protocols for which the key rate was previously unknown. PMID:27198739

Multiparty quantum key agreement with single particles

NASA Astrophysics Data System (ADS)

Liu, Bin; Gao, Fei; Huang, Wei; Wen, Qiao-yan

2013-04-01

Two conditions must be satisfied in a secure quantum key agreement (QKA) protocol: (1) outside eavesdroppers cannot gain the generated key without introducing any error; (2) the generated key cannot be determined by any non-trivial subset of the participants. That is, a secure QKA protocol can not only prevent the outside attackers from stealing the key, but also resist the attack from inside participants, i.e. some dishonest participants determine the key alone by illegal means. How to resist participant attack is an aporia in the design of QKA protocols, especially the multi-party ones. In this paper we present the first secure multiparty QKA protocol against both outside and participant attacks. Further more, we have proved its security in detail.

Method for encryption and transmission of digital keying data

DOEpatents

Mniszewski, Susan M.; Springer, Edward A.; Brenner, David P.

1988-01-01

A method for the encryption, transmission, and subsequent decryption of digital keying data. The method utilizes the Data Encryption Standard and is implemented by means of a pair of apparatus, each of which is selectable to operate as either a master unit or remote unit. Each unit contains a set of key encryption keys which are indexed by a common indexing system. The master unit operates upon command from the remote unit to generate a data encryption key and encrypt the data encryption key using a preselected key encryption key. The encrypted data encryption key and an index designator are then downloaded to the remote unit, where the data encryption key is decrypted for subsequent use in the encryption and transmission data. Downloading of the encrypted data encryption key enables frequent change of keys without requiring manual entry or storage of keys at the remote unit.

48 CFR 952.215-70 - Key Personnel.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false Key Personnel. 952.215-70... SOLICITATION PROVISIONS AND CONTRACT CLAUSES Text of Provisions and Clauses 952.215-70 Key Personnel. As prescribed in 915.408-70, the contracting officer shall insert the following clause: Key Personnel (DEC 2000...

48 CFR 952.215-70 - Key Personnel.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false Key Personnel. 952.215-70... SOLICITATION PROVISIONS AND CONTRACT CLAUSES Text of Provisions and Clauses 952.215-70 Key Personnel. As prescribed in 915.408-70, the contracting officer shall insert the following clause: Key Personnel (DEC 2000...

48 CFR 952.215-70 - Key Personnel.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 48 Federal Acquisition Regulations System 5 2011-10-01 2011-10-01 false Key Personnel. 952.215-70... SOLICITATION PROVISIONS AND CONTRACT CLAUSES Text of Provisions and Clauses 952.215-70 Key Personnel. As prescribed in 915.408-70, the contracting officer shall insert the following clause: Key Personnel (DEC 2000...

48 CFR 952.215-70 - Key Personnel.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false Key Personnel. 952.215-70... SOLICITATION PROVISIONS AND CONTRACT CLAUSES Text of Provisions and Clauses 952.215-70 Key Personnel. As prescribed in 915.408-70, the contracting officer shall insert the following clause: Key Personnel (DEC 2000...

48 CFR 952.215-70 - Key Personnel.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Key Personnel. 952.215-70... SOLICITATION PROVISIONS AND CONTRACT CLAUSES Text of Provisions and Clauses 952.215-70 Key Personnel. As prescribed in 915.408-70, the contracting officer shall insert the following clause: Key Personnel (DEC 2000...

Secure image retrieval with multiple keys

NASA Astrophysics Data System (ADS)

Liang, Haihua; Zhang, Xinpeng; Wei, Qiuhan; Cheng, Hang

2018-03-01

This article proposes a secure image retrieval scheme under a multiuser scenario. In this scheme, the owner first encrypts and uploads images and their corresponding features to the cloud; then, the user submits the encrypted feature of the query image to the cloud; next, the cloud compares the encrypted features and returns encrypted images with similar content to the user. To find the nearest neighbor in the encrypted features, an encryption with multiple keys is proposed, in which the query feature of each user is encrypted by his/her own key. To improve the key security and space utilization, global optimization and Gaussian distribution are, respectively, employed to generate multiple keys. The experiments show that the proposed encryption can provide effective and secure image retrieval for each user and ensure confidentiality of the query feature of each user.

An SSH key management system: easing the pain of managing key/user/account associations

NASA Astrophysics Data System (ADS)

Arkhipkin, D.; Betts, W.; Lauret, J.; Shiryaev, A.

2008-07-01

Cyber security requirements for secure access to computing facilities often call for access controls via gatekeepers and the use of two-factor authentication. Using SSH keys to satisfy the two factor authentication requirement has introduced a potentially challenging task of managing the keys and their associations with individual users and user accounts. Approaches for a facility with the simple model of one remote user corresponding to one local user would not work at facilities that require a many-to-many mapping between users and accounts on multiple systems. We will present an SSH key management system we developed, tested and deployed to address the many-to-many dilemma in the environment of the STAR experiment. We will explain its use in an online computing context and explain how it makes possible the management and tracing of group account access spread over many sub-system components (data acquisition, slow controls, trigger, detector instrumentation, etc.) without the use of shared passwords for remote logins.

Music performance and the perception of key.

PubMed

Thompson, W F; Cuddy, L L

1997-02-01

The effect of music performance on perceived key movement was examined. Listeners judged key movement in sequences presented without performance expression (mechanical) in Experiment 1 and with performance expression in Experiment 2. Modulation distance varied. Judgments corresponded to predictions based on the cycle of fifths and toroidal models of key relatedness, with the highest correspondence for performed versions with the toroidal model. In Experiment 3, listeners compared mechanical sequences with either performed sequences or modifications of performed sequences. Modifications preserved expressive differences between chords, but not between voices. Predictions from Experiments 1 and 2 held only for performed sequences, suggesting that differences between voices are informative of key movement. Experiment 4 confirmed that modifications did not disrupt musicality. Analyses of performances further suggested a link between performance expression and key.

New and investigational antiretroviral drugs for HIV infection: mechanisms of action and early research findings.

PubMed

Saag, Michael S

2012-12-01

Numerous investigational antiretroviral agents are in clinical development. Among them are festinavir (BMS986001), a thymidine analogue similar to stavudine with reduced potential for toxicity; GS-7340, a prodrug of tenofovir that achieves greater intracellular concentrations; MK-1439, a nonnucleoside analogue reverse transcriptase inhibitor (NNRTI) that retains activity against common NNRTI-associated resistance mutations; and albuvirtide, a long-acting parenteral fusion inhibitor. Investigational integrase strand transfer inhibitors (InSTIs) include elvitegravir, recently approved by the US Food and Drug Administration (FDA) as part of a once-daily, single-tablet formulation with cobicistat/tenofovir/emtricitabine; dolutegravir, which maintains some activity against raltegravir- and elvitegravir-resistant mutants; and S/GSK1265744, which also maintains some activity against resistance mutations in the integrase gene and is being developed as a long-lasting parenteral agent. Novel 2-(quinolin-3-yl)acetic acid derivatives (LEDGINs), agents that were originally thought to inhibit the interaction of integrase with its cofactor lens epithelium-derived growth factor p75 (LEDGF/p75), be active against InSTI-resistant mutants and to have additive activity when combined with InSTIs. This article summarizes a presentation by Michael S. Saag, MD, at the IAS-USA live Improving the Management of HCV Disease continuing medical education program held in New York in October 2012.

Flipping chromosomes in deep-sea archaea

PubMed Central

Catchpole, Ryan; Gadelle, Danièle; Marguet, Evelyne; Barbe, Valérie; Forterre, Patrick

2017-01-01

One of the major mechanisms driving the evolution of all organisms is genomic rearrangement. In hyperthermophilic Archaea of the order Thermococcales, large chromosomal inversions occur so frequently that even closely related genomes are difficult to align. Clearly not resulting from the native homologous recombination machinery, the causative agent of these inversions has remained elusive. We present a model in which genomic inversions are catalyzed by the integrase enzyme encoded by a family of mobile genetic elements. We characterized the integrase from Thermococcus nautili plasmid pTN3 and showed that besides canonical site-specific reactions, it catalyzes low sequence specificity recombination reactions with the same outcome as homologous recombination events on DNA segments as short as 104bp both in vitro and in vivo, in contrast to other known tyrosine recombinases. Through serial culturing, we showed that the integrase-mediated divergence of T. nautili strains occurs at an astonishing rate, with at least four large-scale genomic inversions appearing within 60 generations. Our results and the ubiquitous distribution of pTN3-like integrated elements suggest that a major mechanism of evolution of an entire order of Archaea results from the activity of a selfish mobile genetic element. PMID:28628615

Autoimmune disease: A role for new anti-viral therapies?

PubMed

Dreyfus, David H

2011-12-01

Many chronic human diseases may have an underlying autoimmune mechanism. In this review, the author presents a case of autoimmune CIU (chronic idiopathic urticaria) in stable remission after therapy with a retroviral integrase inhibitor, raltegravir (Isentress). Previous reports located using the search terms "autoimmunity" and "anti-viral" and related topics in the pubmed data-base are reviewed suggesting that novel anti-viral agents such as retroviral integrase inhibitors, gene silencing therapies and eventually vaccines may provide new options for anti-viral therapy of autoimmune diseases. Cited epidemiologic and experimental evidence suggests that increased replication of epigenomic viral pathogens such as Epstein-Barr Virus (EBV) in chronic human autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus Erythematosus (SLE), and multiple sclerosis (MS) may activate endogenous human retroviruses (HERV) as a pathologic mechanism. Memory B cells are the reservoir of infection of EBV and also express endogenous retroviruses, thus depletion of memory b-lymphocytes by monoclonal antibodies (Rituximab) may have therapeutic anti-viral effects in addition to effects on B-lymphocyte presentation of both EBV and HERV superantigens. Other novel anti-viral therapies of chronic autoimmune diseases, such as retroviral integrase inhibitors, could be effective, although not without risk. Copyright © 2011 Elsevier B.V. All rights reserved.

Chromodomains direct integration of retrotransposons to heterochromatin

PubMed Central

Gao, Xiang; Hou, Yi; Ebina, Hirotaka; Levin, Henry L.; Voytas, Daniel F.

2008-01-01

The enrichment of mobile genetic elements in heterochromatin may be due, in part, to targeted integration. The chromoviruses are Ty3/gypsy retrotransposons with chromodomains at their integrase C termini. Chromodomains are logical determinants for targeting to heterochromatin, because the chromodomain of heterochromatin protein 1 (HP1) typically recognizes histone H3 K9 methylation, an epigenetic mark characteristic of heterochromatin. We describe three groups of chromoviruses based on amino acid sequence relationships of their integrase C termini. Genome sequence analysis indicates that representative chromoviruses from each group are enriched in gene-poor regions of the genome relative to other retrotransposons, and when fused to fluorescent marker proteins, the chromodomains target proteins to specific subnuclear foci coincident with heterochromatin. The chromodomain of the fungal element, MAGGY, interacts with histone H3 dimethyl- and trimethyl-K9, and when the MAGGY chromodomain is fused to integrase of the Schizosaccharomyces pombe Tf1 retrotransposon, new Tf1 insertions are directed to sites of H3 K9 methylation. Repetitive sequences such as transposable elements trigger the RNAi pathway resulting in their epigenetic modification. Our results suggest a dynamic interplay between retrotransposons and heterochromatin, wherein mobile elements recognize heterochromatin at the time of integration and then perpetuate the heterochromatic mark by triggering epigenetic modification. PMID:18256242

Molecular mechanisms of retroviral integration site selection

PubMed Central

Kvaratskhelia, Mamuka; Sharma, Amit; Larue, Ross C.; Serrao, Erik; Engelman, Alan

2014-01-01

Retroviral replication proceeds through an obligate integrated DNA provirus, making retroviral vectors attractive vehicles for human gene-therapy. Though most of the host cell genome is available for integration, the process of integration site selection is not random. Retroviruses differ in their choice of chromatin-associated features and also prefer particular nucleotide sequences at the point of insertion. Lentiviruses including HIV-1 preferentially integrate within the bodies of active genes, whereas the prototypical gammaretrovirus Moloney murine leukemia virus (MoMLV) favors strong enhancers and active gene promoter regions. Integration is catalyzed by the viral integrase protein, and recent research has demonstrated that HIV-1 and MoMLV targeting preferences are in large part guided by integrase-interacting host factors (LEDGF/p75 for HIV-1 and BET proteins for MoMLV) that tether viral intasomes to chromatin. In each case, the selectivity of epigenetic marks on histones recognized by the protein tether helps to determine the integration distribution. In contrast, nucleotide preferences at integration sites seem to be governed by the ability for the integrase protein to locally bend the DNA duplex for pairwise insertion of the viral DNA ends. We discuss approaches to alter integration site selection that could potentially improve the safety of retroviral vectors in the clinic. PMID:25147212

Comparative transcriptome analysis reveals key genes potentially related to soluble sugar and organic acid accumulation in watermelon

PubMed Central

Gao, Lei; Zhao, Shengjie; Lu, Xuqiang; He, Nan; Zhu, Hongju; Dou, Junling

2018-01-01

Soluble sugars and organic acids are important components of fruit flavor and have a strong impact on the overall organoleptic quality of watermelon (Citrullus lanatus) fruit. Several studies have analyzed the expression levels of the genes related to soluble sugar accumulation and the dynamic changes in their content during watermelon fruit development and ripening. Nevertheless, to date, there have been no reports on the organic acid content in watermelon or the genes regulating their synthesis. In this study, the soluble sugars and organic acids in watermelon were measured and a comparative transcriptome analysis was performed to identify the key genes involved in the accumulation of these substances during fruit development and ripening. The watermelon cultivar ‘203Z’ and its near-isogenic line (NIL) ‘SW’ (in the ‘203Z’ background) were used as experimental materials. The results suggested that soluble sugar consist of fructose, glucose and sucrose while malic-, citric-, and oxalic acids are the primary organic acids in watermelon fruit. Several differentially expressed genes (DEGs) related to soluble sugar- and organic acid accumulation and metabolism were identified. These include the DEGs encoding raffinose synthase, sucrose synthase (SuSy), sucrose-phosphate synthase (SPSs), insoluble acid invertases (IAI), NAD-dependent malate dehydrogenase (NAD-cyt MDH), aluminum-activated malate transporter (ALMT), and citrate synthase (CS). This is the first report addressing comparative transcriptome analysis via NILs materials in watermelon fruit. These findings provide an important basis for understanding the molecular mechanism that leads to soluble sugar and organic acid accumulation and metabolism during watermelon fruit development and ripening. PMID:29324867

Comparative transcriptome analysis reveals key genes potentially related to soluble sugar and organic acid accumulation in watermelon.

PubMed

Gao, Lei; Zhao, Shengjie; Lu, Xuqiang; He, Nan; Zhu, Hongju; Dou, Junling; Liu, Wenge

2018-01-01

Soluble sugars and organic acids are important components of fruit flavor and have a strong impact on the overall organoleptic quality of watermelon (Citrullus lanatus) fruit. Several studies have analyzed the expression levels of the genes related to soluble sugar accumulation and the dynamic changes in their content during watermelon fruit development and ripening. Nevertheless, to date, there have been no reports on the organic acid content in watermelon or the genes regulating their synthesis. In this study, the soluble sugars and organic acids in watermelon were measured and a comparative transcriptome analysis was performed to identify the key genes involved in the accumulation of these substances during fruit development and ripening. The watermelon cultivar '203Z' and its near-isogenic line (NIL) 'SW' (in the '203Z' background) were used as experimental materials. The results suggested that soluble sugar consist of fructose, glucose and sucrose while malic-, citric-, and oxalic acids are the primary organic acids in watermelon fruit. Several differentially expressed genes (DEGs) related to soluble sugar- and organic acid accumulation and metabolism were identified. These include the DEGs encoding raffinose synthase, sucrose synthase (SuSy), sucrose-phosphate synthase (SPSs), insoluble acid invertases (IAI), NAD-dependent malate dehydrogenase (NAD-cyt MDH), aluminum-activated malate transporter (ALMT), and citrate synthase (CS). This is the first report addressing comparative transcriptome analysis via NILs materials in watermelon fruit. These findings provide an important basis for understanding the molecular mechanism that leads to soluble sugar and organic acid accumulation and metabolism during watermelon fruit development and ripening.

Proteomic profiling of isogenic primary and metastatic medulloblastoma cell lines reveals differential expression of key metastatic factors.

PubMed

Gu, Shuo; Chen, Kai; Yin, Minzhi; Wu, Zhixiang; Wu, Yeming

2017-05-08

Medulloblastoma is the most common malignant brain tumor in children. Around 30% of medulloblastoma patients are diagnosed with metastasis, which often results in a poor prognosis. Unfortunately, molecular mechanisms of medulloblastoma metastasis remain largely unknown. In this study, we employed the recently developed deep proteome analysis approach to quantitatively profile the expression of >10,000 proteins from CHLA-01-MED and CHLA-01R-MED isogenic cell lines derived from the primary and metastatic tumor of the same patient diagnosed with a group IV medulloblastoma. Using statistical analysis, we identified ~1400 significantly altered proteins between the primary and metastatic cell lines including known factors such as placental growth factor (PLGF), LIM homeobox 1 (LHX1) and prominim 1 (PROM1), as well as the negative regulator secreted protein acidic and cysteine rich (SPARC). Additional transwell experiments and immunohistochemical analysis of clinical medulloblastoma samples implicated yes-associated protein 1 (YAP1) as a potential key factor contributing to metastasis. Taken together, our data broadly defines the metastasis-relevant regulated proteome and provides a precious resource for further investigating potential mechanisms of medulloblastoma metastasis. This study represented the first deep proteome analysis of metastatic medulloblastomas and provided a valuable candidate list of altered proteins in metastatic medulloblastomas. The primary data suggested YAP1 as a potential driver for the metastasis of medulloblastoma. These results open up numerous avenues for further investigating the underlying mechanisms of medulloblastoma metastasis and improving the prognosis of medulloblastoma patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Information verification cryptosystem using one-time keys based on double random phase encoding and public-key cryptography

NASA Astrophysics Data System (ADS)

Zhao, Tieyu; Ran, Qiwen; Yuan, Lin; Chi, Yingying; Ma, Jing

2016-08-01

A novel image encryption system based on double random phase encoding (DRPE) and RSA public-key algorithm is proposed. The main characteristic of the system is that each encryption process produces a new decryption key (even for the same plaintext), thus the encryption system conforms to the feature of the one-time pad (OTP) cryptography. The other characteristic of the system is the use of fingerprint key. Only with the rightful authorization will the true decryption be obtained, otherwise the decryption will result in noisy images. So the proposed system can be used to determine whether the ciphertext is falsified by attackers. In addition, the system conforms to the basic agreement of asymmetric cryptosystem (ACS) due to the combination with the RSA public-key algorithm. The simulation results show that the encryption scheme has high robustness against the existing attacks.

Formal Analysis of Key Integrity in PKCS#11

NASA Astrophysics Data System (ADS)

Falcone, Andrea; Focardi, Riccardo

PKCS#11 is a standard API to cryptographic devices such as smarcards, hardware security modules and usb crypto-tokens. Though widely adopted, this API has been shown to be prone to attacks in which a malicious user gains access to the sensitive keys stored in the devices. In 2008, Delaune, Kremer and Steel proposed a model to formally reason on this kind of attacks. We extend this model to also describe flaws that are based on integrity violations of the stored keys. In particular, we consider scenarios in which a malicious overwriting of keys might fool honest users into using attacker's own keys, while performing sensitive operations. We further enrich the model with a trusted key mechanism ensuring that only controlled, non-tampered keys are used in cryptographic operations, and we show how this modified API prevents the above mentioned key-replacement attacks.

Statistical Inference and Simulation with StatKey

ERIC Educational Resources Information Center

Quinn, Anne

2016-01-01

While looking for an inexpensive technology package to help students in statistics classes, the author found StatKey, a free Web-based app. Not only is StatKey useful for students' year-end projects, but it is also valuable for helping students learn fundamental content such as the central limit theorem. Using StatKey, students can engage in…

33 CFR 117.272 - Boot Key Harbor.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Boot Key Harbor. 117.272 Section 117.272 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.272 Boot Key Harbor. The draw of the Boot Key Harbor drawbridge, mile 0.13, between...

Key Transport and Ammonia Recycling Genes Involved in Aphid Symbiosis Respond to Host-Plant Specialization.

PubMed

Kim, Dohyup; Minhas, Bushra F; Li-Byarlay, Hongmei; Hansen, Allison K

2018-05-25

Microbes are known to influence insect-plant interactions; however, it is unclear if host-plant diet influences the regulation of nutritional insect symbioses. The pea aphid, Acyrthosiphon pisum , requires its nutritional endosymbiont, Buchnera , for the production of essential amino acids. We hypothesize that key aphid genes that regulate the nutritional symbioses respond to host-plant diet when aphids feed on a specialized (alfalfa) compared to a universal host-plant diet (fava), which vary in amino acid profiles. Using RNA-Seq and whole genome bisulfite sequencing, we measured gene expression and DNA methylation profiles for such genes when aphids fed on either their specialized or universal host-plant diets. Our results reveal that when aphids feed on their specialized host-plant they significantly up-regulate and/or hypo-methylate key aphid genes in bacteriocytes related to the amino acid metabolism, including glutamine synthetase in the GOGAT cycle that recycles ammonia into glutamine and the glutamine transporter ApGLNT1 Moreover, regardless of what host-plant aphids feed on we observed significant up-regulation and differential methylation of key genes involved in the amino acid metabolism and the glycine/serine metabolism, a metabolic program observed in proliferating cancer cells potentially to combat oxidative stress. Based on our results, we suggest that this regulatory response of key symbiosis genes in bacteriocytes allows aphids to feed on a suboptimal host-plant that they specialize on. Copyright © 2018, G3: Genes, Genomes, Genetics.

Reconstruction of an Integrated Genome-Scale Co-Expression Network Reveals Key Modules Involved in Lung Adenocarcinoma

PubMed Central

Hosseini Ashtiani, Saman; Moeini, Ali; Nowzari-Dalini, Abbas; Masoudi-Nejad, Ali

2013-01-01

Our goal of this study was to reconstruct a “genome-scale co-expression network” and find important modules in lung adenocarcinoma so that we could identify the genes involved in lung adenocarcinoma. We integrated gene mutation, GWAS, CGH, array-CGH and SNP array data in order to identify important genes and loci in genome-scale. Afterwards, on the basis of the identified genes a co-expression network was reconstructed from the co-expression data. The reconstructed network was named “genome-scale co-expression network”. As the next step, 23 key modules were disclosed through clustering. In this study a number of genes have been identified for the first time to be implicated in lung adenocarcinoma by analyzing the modules. The genes EGFR, PIK3CA, TAF15, XIAP, VAPB, Appl1, Rab5a, ARF4, CLPTM1L, SP4, ZNF124, LPP, FOXP1, SOX18, MSX2, NFE2L2, SMARCC1, TRA2B, CBX3, PRPF6, ATP6V1C1, MYBBP1A, MACF1, GRM2, TBXA2R, PRKAR2A, PTK2, PGF and MYO10 are among the genes that belong to modules 1 and 22. All these genes, being implicated in at least one of the phenomena, namely cell survival, proliferation and metastasis, have an over-expression pattern similar to that of EGFR. In few modules, the genes such as CCNA2 (Cyclin A2), CCNB2 (Cyclin B2), CDK1, CDK5, CDC27, CDCA5, CDCA8, ASPM, BUB1, KIF15, KIF2C, NEK2, NUSAP1, PRC1, SMC4, SYCE2, TFDP1, CDC42 and ARHGEF9 are present that play a crucial role in cell cycle progression. In addition to the mentioned genes, there are some other genes (i.e. DLGAP5, BIRC5, PSMD2, Src, TTK, SENP2, PSMD2, DOK2, FUS and etc.) in the modules. PMID:23874428

Reconstruction of an integrated genome-scale co-expression network reveals key modules involved in lung adenocarcinoma.

PubMed

Bidkhori, Gholamreza; Narimani, Zahra; Hosseini Ashtiani, Saman; Moeini, Ali; Nowzari-Dalini, Abbas; Masoudi-Nejad, Ali

2013-01-01

Our goal of this study was to reconstruct a "genome-scale co-expression network" and find important modules in lung adenocarcinoma so that we could identify the genes involved in lung adenocarcinoma. We integrated gene mutation, GWAS, CGH, array-CGH and SNP array data in order to identify important genes and loci in genome-scale. Afterwards, on the basis of the identified genes a co-expression network was reconstructed from the co-expression data. The reconstructed network was named "genome-scale co-expression network". As the next step, 23 key modules were disclosed through clustering. In this study a number of genes have been identified for the first time to be implicated in lung adenocarcinoma by analyzing the modules. The genes EGFR, PIK3CA, TAF15, XIAP, VAPB, Appl1, Rab5a, ARF4, CLPTM1L, SP4, ZNF124, LPP, FOXP1, SOX18, MSX2, NFE2L2, SMARCC1, TRA2B, CBX3, PRPF6, ATP6V1C1, MYBBP1A, MACF1, GRM2, TBXA2R, PRKAR2A, PTK2, PGF and MYO10 are among the genes that belong to modules 1 and 22. All these genes, being implicated in at least one of the phenomena, namely cell survival, proliferation and metastasis, have an over-expression pattern similar to that of EGFR. In few modules, the genes such as CCNA2 (Cyclin A2), CCNB2 (Cyclin B2), CDK1, CDK5, CDC27, CDCA5, CDCA8, ASPM, BUB1, KIF15, KIF2C, NEK2, NUSAP1, PRC1, SMC4, SYCE2, TFDP1, CDC42 and ARHGEF9 are present that play a crucial role in cell cycle progression. In addition to the mentioned genes, there are some other genes (i.e. DLGAP5, BIRC5, PSMD2, Src, TTK, SENP2, PSMD2, DOK2, FUS and etc.) in the modules.

The social context of motorcycle riding and the key determinants influencing rider behavior: a qualitative investigation.

PubMed

Tunnicliff, Deborah; Watson, Barry; White, Katherine M; Lewis, Ioni; Wishart, Darren

2011-08-01

Given the increasing popularity of motorcycle riding and heightened risk of injury or death associated with being a rider, this study explored rider behavior as a determinant of rider safety and, in particular, key beliefs and motivations that influence such behavior. To enhance the effectiveness of future education and training interventions, it is important to understand riders' own views about what influences how they ride. Specifically, this study sought to identify key determinants of riders' behaviors in relation to the social context of riding, including social and identity-related influences relating to the group (group norms and group identity) as well as the self (moral/personal norm and self-identity). Qualitative research was undertaken via group discussions with motorcycle riders (n = 41). The findings revealed that those in the group with which one rides represent an important source of social influence. Also, the motorcyclist (group) identity was associated with a range of beliefs, expectations, and behaviors considered to be normative. Exploration of the construct of personal norm revealed that riders were most cognizant of the "wrong things to do" when riding; among those issues raised was the importance of protective clothing (albeit for the protection of others and, in particular, pillion passengers). Finally, self-identity as a motorcyclist appeared to be important to a rider's self-concept and was likely to influence on-road behavior. Overall, the insight provided by the current study may facilitate the development of interventions including rider training as well as public education and mass media messages. The findings suggest that these interventions should incorporate factors associated with the social nature of riding in order to best align it with some of the key beliefs and motivations underpinning riders' on-road behaviors.

Use of dolutegravir in two INI-experienced patients with multiclass resistance resulted in excellent virological and immunological responses

PubMed Central

Marije Hofstra, Laura; Nijhuis, Monique; Mudrikova, Tania; Fun, Axel; Schipper, Pauline; Schneider, Margriet; Wensing, Annemarie

2014-01-01

Introduction Dolutegravir is a second generation integrase inhibitor with a proposed high genetic barrier to resistance. However, in clinical trials, decreased virological response was seen in a subset of patients with prior exposure to raltegravir and multiple integrase resistance mutations. Methods We describe two cases of HIV subtype B-infected patients starting dolutegravir after previous failure on a raltegravir-containing regimen with extensive resistance. Genotypic analysis was performed using population sequencing and 454 ultradeep sequencing of integrase at time of raltegravir exposure. Results Both patients were diagnosed in early 1990s and received mono- and dual therapy, followed by several cART-regimens. Due to presence of extensive resistance, the genotypic susceptibility score of these regimens never reached a score >2 and never resulted in sustained virological suppression despite good adherence. Early 2012, the clinical condition of patient 1 worsened during persistent failure of a mega-cART regimen despite excellent drug levels. Six major PI, six minor PI, seven NRTI, six NNRTI and two INI mutations plus DM-virus were detected (Table 1). Ultra-deep sequencing of integrase showed the selection of Q148R, E138K+Q148K, and N155H variants and phenotypic raltegravir resistance was demonstrated. After addition of dolutegravir and enfuvirtide to the failing regimen (zidovudine, lamivudine, tenofovir, etravirine, darunavir/ritonavir, maraviroc), viral load (VL) decreased from 244,000 to <20 cps/mL within five months, CD4-count increased (33 to 272 mm3) and the clinical condition improved substantially. In patient 2, similar worsening of the clinical condition was observed late 2012 during persistent failure on mega-cART. Five major PI, six minor PI, nine NRTI, seven NNRTI and one INI mutation plus DM-virus were detected. Ultra-deep sequencing showed selection of N155H, followed by Q95K and V151I variants and phenotypic raltegravir resistance was

Practical issues in quantum-key-distribution postprocessing

NASA Astrophysics Data System (ADS)

Fung, Chi-Hang Fred; Ma, Xiongfeng; Chau, H. F.

2010-01-01

Quantum key distribution (QKD) is a secure key generation method between two distant parties by wisely exploiting properties of quantum mechanics. In QKD, experimental measurement outcomes on quantum states are transformed by the two parties to a secret key. This transformation is composed of many logical steps (as guided by security proofs), which together will ultimately determine the length of the final secret key and its security. We detail the procedure for performing such classical postprocessing taking into account practical concerns (including the finite-size effect and authentication and encryption for classical communications). This procedure is directly applicable to realistic QKD experiments and thus serves as a recipe that specifies what postprocessing operations are needed and what the security level is for certain lengths of the keys. Our result is applicable to the BB84 protocol with a single or entangled photon source.

Enhanced diffie-hellman algorithm for reliable key exchange

NASA Astrophysics Data System (ADS)

Aryan; Kumar, Chaithanya; Vincent, P. M. Durai Raj

2017-11-01

The Diffie -Hellman is one of the first public-key procedure and is a certain way of exchanging the cryptographic keys securely. This concept was introduced by Ralph Markel and it is named after Whitfield Diffie and Martin Hellman. Sender and Receiver make a common secret key in Diffie-Hellman algorithm and then they start communicating with each other over the public channel which is known to everyone. A number of internet services are secured by Diffie -Hellman. In Public key cryptosystem, the sender has to trust while receiving the public key of the receiver and vice-versa and this is the challenge of public key cryptosystem. Man-in-the-Middle attack is very much possible on the existing Diffie-Hellman algorithm. In man-in-the-middle attack, the attacker exists in the public channel, the attacker receives the public key of both sender and receiver and sends public keys to sender and receiver which is generated by his own. This is how man-in-the-middle attack is possible on Diffie-Hellman algorithm. Denial of service attack is another attack which is found common on Diffie-Hellman. In this attack, the attacker tries to stop the communication happening between sender and receiver and attacker can do this by deleting messages or by confusing the parties with miscommunication. Some more attacks like Insider attack, Outsider attack, etc are possible on Diffie-Hellman. To reduce the possibility of attacks on Diffie-Hellman algorithm, we have enhanced the Diffie-Hellman algorithm to a next level. In this paper, we are extending the Diffie -Hellman algorithm by using the concept of the Diffie -Hellman algorithm to get a stronger secret key and that secret key is further exchanged between the sender and the receiver so that for each message, a new secret shared key would be generated. The second secret key will be generated by taking primitive root of the first secret key.

10 CFR 95.18 - Key personnel.

Code of Federal Regulations, 2011 CFR

2011-01-01

... INFORMATION AND RESTRICTED DATA Physical Security § 95.18 Key personnel. The senior management official and... Clearance. Other key management officials, as determined by the CSA, must be granted an access authorization... organization's policies or practices in the performance of activities involving classified information. This...

An Inexpensive Device for Teaching Public Key Encryption

ERIC Educational Resources Information Center

Pendegraft, Norman

2009-01-01

An inexpensive device to assist in teaching the main ideas of Public Key encryption and its use in class to illustrate the operation of public key encryption is described. It illustrates that there are two keys, and is particularly useful for illustrating that privacy is achieved by using the public key. Initial data from in class use seem to…

Setting objectives for managing Key deer

USGS Publications Warehouse

Diefenbach, Duane R.; Wagner, Tyler; Stauffer, Glenn E.

2014-01-01

The U.S. Fish and Wildlife Service (FWS) is responsible for the protection and management of Key deer (Odocoileus virginianus clavium) because the species is listed as Endangered under the Endangered Species Act (ESA). The purpose of the ESA is to protect and recover imperiled species and the ecosystems upon which they depend. There are a host of actions that could possibly be undertaken to recover the Key deer population, but without a clearly defined problem and stated objectives it can be difficult to compare and evaluate alternative actions. In addition, management goals and the acceptability of alternative management actions are inherently linked to stakeholders, who should be engaged throughout the process of developing a decision framework. The purpose of this project was to engage a representative group of stakeholders to develop a problem statement that captured the management problem the FWS must address with Key deer and identify objectives that, if met, would help solve the problem. In addition, the objectives were organized in a hierarchical manner (i.e., an objectives network) to show how they are linked, and measurable attributes were identified for each objective. We organized a group of people who represented stakeholders interested in and potentially affected by the management of Key deer. These stakeholders included individuals who represented local, state, and federal governments, non-governmental organizations, the general public, and local businesses. This stakeholder group met five full days over the course of an eight-week period to identify objectives that would address the following problem:“As recovery and removal from the Endangered Species list is the purpose of the Endangered Species Act, the U.S. Fish and Wildlife Service needs a management approach that will ensure a sustainable, viable, and healthy Key deer population. Urbanization has affected the behavior and population dynamics of the Key deer and the amount and characteristics

Key stages of material expansion in dielectrics upon femtosecond laser ablation revealed by double-color illumination time-resolved microscopy

NASA Astrophysics Data System (ADS)

Garcia-Lechuga, Mario; Solis, Javier; Siegel, Jan

2018-03-01

The physical origin of material removal in dielectrics upon femtosecond laser pulse irradiation (800 nm, 120 fs pulse duration) has been investigated at fluences slightly above ablation threshold. Making use of a versatile pump-probe microscopy setup, the dynamics and different key stages of the ablation process in lithium niobate have been monitored. The use of two different illumination wavelengths, 400 and 800 nm, and a rigorous image analysis combined with theoretical modelling, enables drawing a clear picture of the material excitation and expansion stages. Immediately after excitation, a dense electron plasma is generated. Few picoseconds later, direct evidence of a rarefaction wave propagating into the bulk is obtained, with an estimated speed of 3650 m/s. This process marks the onset of material expansion, which is confirmed by the appearance of transient Newton rings, which dynamically change during the expansion up to approximately 1 ns. Exploring delays up to 15 ns, a second dynamic Newton ring pattern is observed, consistent with the formation of a second ablation front propagating five times slower than the first one.

Quantum Public Key Cryptosystem Based on Bell States

NASA Astrophysics Data System (ADS)

Wu, WanQing; Cai, QingYu; Zhang, HuanGuo; Liang, XiaoYan

2017-11-01

Classical public key cryptosystems ( P K C), such as R S A, E I G a m a l, E C C, are no longer secure in quantum algorithms, and quantum cryptography has become a novel research topic. In this paper we present a quantum asymmetrical cryptosystem i.e. quantum public key cryptosystem ( Q P K C) based on the Bell states. In particular, in the proposed QPKC the public key are given by the first n particles of Bell states and generalized Pauli operations. The corresponding secret key are the last n particles of Bell states and the inverse of generalized Pauli operations. The proposed QPKC encrypts the message using a public key and decrypts the ciphertext using a private key. By H o l e v o ' s theorem, we proved the security of the secret key and messages during the QPKC.

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

PubMed

Yang, Yuting; Gourinath, S; Kovács, Mihály; Nyitray, László; Reutzel, Robbie; Himmel, Daniel M; O'Neall-Hennessey, Elizabeth; Reshetnikova, Ludmilla; Szent-Györgyi, Andrew G; Brown, Jerry H; Cohen, Carolyn

2007-05-01

Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.

A computational analysis of the three isoforms of glutamate dehydrogenase reveals structural features of the isoform EC 1.4.1.4 supporting a key role in ammonium assimilation by plants

PubMed Central

Jaspard, Emmanuel

2006-01-01

Background There are three isoforms of glutamate dehydrogenase. The isoform EC 1.4.1.4 (GDH4) catalyses glutamate synthesis from 2-oxoglutarate and ammonium, using NAD(P)H. Ammonium assimilation is critical for plant growth. Although GDH4 from animals and prokaryotes are well characterized, there are few data concerning plant GDH4, even from those whose genomes are well annotated. Results A large set of the three GDH isoforms was built resulting in 116 non-redundant full polypeptide sequences. A computational analysis was made to gain more information concerning the structure – function relationship of GDH4 from plants (Eukaryota, Viridiplantae). The tested plant GDH4 sequences were the two ones known to date, those of Chlorella sorokiniana. This analysis revealed several structural features specific of plant GDH4: (i) the lack of a structure called "antenna"; (ii) the NAD(P)-binding motif GAGNVA; and (iii) a second putative coenzyme-binding motif GVLTGKG together with four residues involved in the binding of the reduced form of NADP. Conclusion A number of structural features specific of plant GDH4 have been found. The results reinforce the probable key role of GDH4 in ammonium assimilation by plants. Reviewers This article was reviewed by Tina Bakolitsa (nominated by Eugene Koonin), Martin Jambon (nominated by Laura Landweber), Sandor Pangor and Franck Eisenhaber. PMID:17173671

Key lessons for designing health literacy professional development courses.

PubMed

Naccarella, Lucio; Murphy, Bernice

2018-02-01

Health literacy courses for health professionals have emerged in response to health professionals' perceived lack of understanding of health literacy issues, and their failure to routinely adopt health literacy practices. Since 2013 in Victoria, Australia, the Centre for Culture, Ethnicity and Health has delivered an annual health literacy demonstration training course that it developed. Course development and delivery partners included HealthWest Partnership and cohealth. The courses are designed to develop the health literacy knowledge, skills and organisational capacity of the health and community services sector in the western metropolitan region of Melbourne. This study presents key learnings from evaluation data from three health literacy courses using Wenger's professional educational learning design framework. The framework has three educational learning architecture components (engagement, imagination and alignment) and four educational learning architecture dimensions (participation, emergent, local/global, identification). Participatory realist evaluation approaches and qualitative methods were used. The evaluations revealed that the health literacy courses are developing leadership in health literacy, building partnerships among course participants, developing health literacy workforce knowledge and skills, developing ways to use and apply health literacy resources and are serving as a catalyst for building organisational infrastructure. Although the courses were not explicitly developed or implemented using Wenger's educational learning design pedagogic features, the course structure (i.e. facilitation role of course coordinators, providing safe learning environments, encouraging small group work amongst participants, requiring participants to conduct mini-projects and sponsor organisation buy-in) provided opportunities for engagement, imagination and alignment. Wenger's educational learning design framework can inform the design of future key

Coherent one-way quantum key distribution

NASA Astrophysics Data System (ADS)

Stucki, Damien; Fasel, Sylvain; Gisin, Nicolas; Thoma, Yann; Zbinden, Hugo

2007-05-01

Quantum Key Distribution (QKD) consists in the exchange of a secrete key between two distant points [1]. Even if quantum key distribution systems exist and commercial systems are reaching the market [2], there are still improvements to be made: simplify the construction of the system; increase the secret key rate. To this end, we present a new protocol for QKD tailored to work with weak coherent pulses and at high bit rates [3]. The advantages of this system are that the setup is experimentally simple and it is tolerant to reduced interference visibility and to photon number splitting attacks, thus resulting in a high efficiency in terms of distilled secret bits per qubit. After having successfully tested the feasibility of the system [3], we are currently developing a fully integrated and automated prototype within the SECOQC project [4]. We present the latest results using the prototype. We also discuss the issue of the photon detection, which still remains the bottleneck for QKD.

Cryptic Diversity of African Tigerfish (Genus Hydrocynus) Reveals Palaeogeographic Signatures of Linked Neogene Geotectonic Events

PubMed Central

Goodier, Sarah A. M.; Cotterill, Fenton P. D.; O'Ryan, Colleen; Skelton, Paul H.; de Wit, Maarten J.

2011-01-01

The geobiotic history of landscapes can exhibit controls by tectonics over biotic evolution. This causal relationship positions ecologically specialized species as biotic indicators to decipher details of landscape evolution. Phylogeographic statistics that reconstruct spatio-temporal details of evolutionary histories of aquatic species, including fishes, can reveal key events of drainage evolution, notably where geochronological resolution is insufficient. Where geochronological resolution is insufficient, phylogeographic statistics that reconstruct spatio-temporal details of evolutionary histories of aquatic species, notably fishes, can reveal key events of drainage evolution. This study evaluates paleo-environmental causes of mitochondrial DNA (mtDNA) based phylogeographic records of tigerfishes, genus Hydrocynus, in order to reconstruct their evolutionary history in relation to landscape evolution across Africa. Strong geographical structuring in a cytochrome b (cyt-b) gene phylogeny confirms the established morphological diversity of Hydrocynus and reveals the existence of five previously unknown lineages, with Hydrocynus tanzaniae sister to a clade comprising three previously unknown lineages (Groups B, C and D) and H. vittatus. The dated phylogeny constrains the principal cladogenic events that have structured Hydrocynus diversity from the late Miocene to the Plio-Pleistocene (ca. 0–16 Ma). Phylogeographic tests reveal that the diversity and distribution of Hydrocynus reflects a complex history of vicariance and dispersals, whereby range expansions in particular species testify to changes to drainage basins. Principal divergence events in Hydrocynus have interfaced closely with evolving drainage systems across tropical Africa. Tigerfish evolution is attributed to dominant control by pulses of geotectonism across the African plate. Phylogenetic relationships and divergence estimates among the ten mtDNA lineages illustrates where and when local tectonic

33 CFR 334.610 - Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted areas and danger zone.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 33 Navigation and Navigable Waters 3 2014-07-01 2014-07-01 false Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted areas and danger zone. 334.610 Section 334.610 Navigation and... RESTRICTED AREA REGULATIONS § 334.610 Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted...

33 CFR 334.610 - Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted areas and danger zone.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 33 Navigation and Navigable Waters 3 2011-07-01 2011-07-01 false Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted areas and danger zone. 334.610 Section 334.610 Navigation and... RESTRICTED AREA REGULATIONS § 334.610 Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted...

33 CFR 334.610 - Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted areas and danger zone.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 false Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted areas and danger zone. 334.610 Section 334.610 Navigation and... RESTRICTED AREA REGULATIONS § 334.610 Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted...

33 CFR 334.610 - Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted areas and danger zone.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 33 Navigation and Navigable Waters 3 2012-07-01 2012-07-01 false Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted areas and danger zone. 334.610 Section 334.610 Navigation and... RESTRICTED AREA REGULATIONS § 334.610 Key West Harbor, at U.S. Naval Base, Key West, Fla.; naval restricted...

Multicomponent reactions provide key molecules for secret communication.

PubMed

Boukis, Andreas C; Reiter, Kevin; Frölich, Maximiliane; Hofheinz, Dennis; Meier, Michael A R

2018-04-12

A convenient and inherently more secure communication channel for encoding messages via specifically designed molecular keys is introduced by combining advanced encryption standard cryptography with molecular steganography. The necessary molecular keys require large structural diversity, thus suggesting the application of multicomponent reactions. Herein, the Ugi four-component reaction of perfluorinated acids is utilized to establish an exemplary database consisting of 130 commercially available components. Considering all permutations, this combinatorial approach can unambiguously provide 500,000 molecular keys in only one synthetic procedure per key. The molecular keys are transferred nondigitally and concealed by either adsorption onto paper, coffee, tea or sugar as well as by dissolution in a perfume or in blood. Re-isolation and purification from these disguises is simplified by the perfluorinated sidechains of the molecular keys. High resolution tandem mass spectrometry can unequivocally determine the molecular structure and thus the identity of the key for a subsequent decryption of an encoded message.

Secret key distillation from shielded two-qubit states

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

Bae, Joonwoo

The quantum states corresponding to a secret key are characterized using the so-called private states, where the key part consisting of a secret key is shielded by the additional systems. Based on the construction, it was shown that a secret key can be distilled from bound entangled states. In this work, I consider the shielded two-qubit states in a key-distillation scenario and derive the conditions under which a secret key can be distilled using the recurrence protocol or the two-way classical distillation, advantage distillation together with one-way postprocessing. From the security conditions, it is shown that a secret key canmore » be distilled from bound entangled states in a much wider range. In addition, I consider the case that in which white noise is added to quantum states and show that the classical distillation protocol still works despite a certain amount of noise although the recurrence protocol does not.« less