Sample records for interdomain linker length

  1. Arsenic metalation of seaweed Fucus vesiculosus metallothionein: the importance of the interdomain linker in metallothionein. (United States)

    Ngu, Thanh T; Lee, Janice A; Rushton, Moira K; Stillman, Martin J


    The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protecting agent against toxic metals in the contaminated waters it can grow in. We report the first kinetic pathway data for A3+ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time and temperature dependence of the relative concentrations of apo-rfMT and the five As-containing species have been determined following mixing of As3+ and apo-rfMT using electrospray ionization mass spectrometry (ESI MS). Kinetic analysis of the detailed time-resolved mass spectral data for As3+ metalation allows the simulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consumption of As1- to As4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed here provides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and the Cys9 domain. The rate constants (M(-1) s(-1)) calculated from the fits for the 7-cysteine gamma domain are k1gamma, 19.8, and k2gamma, 1.4, and for the 9-cysteine beta domain are k1beta, 16.3, k2beta, 9.1, and k3beta, 2.2. The activation energies and Arrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which as we show from analysis of the ESI MS data, allows each of its two domains to bind As3+ independently of each other. The analysis provides for the first time an explanation of the differing metal-binding properties of two-domain MTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers) and mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker.

  2. ATPase domain and interdomain linker play a key role in aggregation of mitochondrial Hsp70 chaperone Ssc1. (United States)

    Blamowska, Marta; Sichting, Martin; Mapa, Koyeli; Mokranjac, Dejana; Neupert, Walter; Hell, Kai


    The co-chaperone Hep1 is required to prevent the aggregation of mitochondrial Hsp70 proteins. We have analyzed the interaction of Hep1 with mitochondrial Hsp70 (Ssc1) and the determinants in Ssc1 that make it prone to aggregation. The ATPase and peptide binding domain (PBD) of Hsp70 proteins are connected by a linker segment that mediates interdomain communication between the domains. We show here that the minimal Hep1 binding entity of Ssc1 consists of the ATPase domain and the interdomain linker. In the absence of Hep1, the ATPase domain with the interdomain linker had the tendency to aggregate, in contrast to the ATPase domain with the mutated linker segment or without linker, and in contrast to the PBD. The closest homolog of Ssc1, bacterial DnaK, and a Ssc1 chimera, in which a segment of the ATPase domain of Ssc1 was replaced by the corresponding segment from DnaK, did not aggregate in Delta hep1 mitochondria. The propensity to aggregate appears to be a specific property of the mitochondrial Hsp70 proteins. The ATPase domain in combination with the interdomain linker is crucial for aggregation of Ssc1. In conclusion, our results suggest that interdomain communication makes Ssc1 prone to aggregation. Hep1 counteracts aggregation by binding to this aggregation-prone conformer.

  3. Linker length and flexibility induces new cellobiohydrolase activity of PoCel6A from Penicillium oxalicum. (United States)

    Gao, Le; Wang, Lushan; Jiang, Xukai; Qu, Yinbo


    In a previous study, a novel cellobiohydrolase, PoCel6A, with new enzymatic activity against p-nitrophenyl-β-D-cellobioside (pNPC), was purified from Penicillium oxalicum. The cellulose-binding module and catalytic domain of PoCel6A showed a high degree of sequence similarity with other fungal Cel6As. However, PoCel6A had 11 more amino acids in the linker region than other Cel6As. To evaluate the relationship between the longer linker of PoCel6A and its enzymatic activity, 11 amino acids were deleted from the linker region of PoCel6A. The shortened PoCel6A linker nullified the enzymatic activity against pNPC but dramatically increased the enzyme's capacity for crystalline cellulose degradation. The shortened linker segment appeared to have no effect on the secondary structural conformation of PoCel6A. Another variant (PoCel6A-6pro) with six consecutive proline residues in the interdomain linker had a higher rigid linker, and no enzymatic activity was observed against soluble and insoluble substrate. The flexibility of the linker had an important function in the formation of active cellulase. The length and flexibility of the linker is clearly able to modify the function of PoCel6A and induce new characteristics of Cel6A.

  4. Forced unraveling of chromatin fibers with nonuniform linker DNA lengths (United States)

    Ozer, Gungor; Collepardo-Guevara, Rosana; Schlick, Tamar


    The chromatin fiber undergoes significant structural changes during the cell's life cycle to modulate DNA accessibility. Detailed mechanisms of such structural transformations of chromatin fibers as affected by various internal and external conditions such as the ionic conditions of the medium, the linker DNA length, and the presence of linker histones, constitute an open challenge. Here we utilize Monte Carlo (MC) simulations of a coarse grained model of chromatin with nonuniform linker DNA lengths as found in vivo to help explain some aspects of this challenge. We investigate the unfolding mechanisms of chromatin fibers with alternating linker lengths of 26-62 bp and 44-79 bp using a series of end-to-end stretching trajectories with and without linker histones and compare results to uniform-linker-length fibers. We find that linker histones increase overall resistance of nonuniform fibers and lead to fiber unfolding with superbeads-on-a-string cluster transitions. Chromatin fibers with nonuniform linker DNA lengths display a more complex, multi-step yet smoother process of unfolding compared to their uniform counterparts, likely due to the existence of a more continuous range of nucleosome-nucleosome interactions. This finding echoes the theme that some heterogeneity in fiber component is biologically advantageous.

  5. Hsp70 oligomerization is mediated by an interaction between the interdomain linker and the substrate-binding domain.

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    Francesco A Aprile

    Full Text Available Oligomerization in the heat shock protein (Hsp 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release.

  6. Hsp70 Oligomerization Is Mediated by an Interaction between the Interdomain Linker and the Substrate-Binding Domain (United States)

    Aprile, Francesco A.; Dhulesia, Anne; Stengel, Florian; Roodveldt, Cintia; Benesch, Justin L. P.; Tortora, Paolo; Robinson, Carol V.; Salvatella, Xavier; Dobson, Christopher M.; Cremades, Nunilo


    Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release. PMID:23840795

  7. Preferentially quantized linker DNA lengths in Saccharomyces cerevisiae. (United States)

    Wang, Ji-Ping; Fondufe-Mittendorf, Yvonne; Xi, Liqun; Tsai, Guei-Feng; Segal, Eran; Widom, Jonathan


    The exact lengths of linker DNAs connecting adjacent nucleosomes specify the intrinsic three-dimensional structures of eukaryotic chromatin fibers. Some studies suggest that linker DNA lengths preferentially occur at certain quantized values, differing one from another by integral multiples of the DNA helical repeat, approximately 10 bp; however, studies in the literature are inconsistent. Here, we investigate linker DNA length distributions in the yeast Saccharomyces cerevisiae genome, using two novel methods: a Fourier analysis of genomic dinucleotide periodicities adjacent to experimentally mapped nucleosomes and a duration hidden Markov model applied to experimentally defined dinucleosomes. Both methods reveal that linker DNA lengths in yeast are preferentially periodic at the DNA helical repeat ( approximately 10 bp), obeying the forms 10n+5 bp (integer n). This 10 bp periodicity implies an ordered superhelical intrinsic structure for the average chromatin fiber in yeast.

  8. Preferentially quantized linker DNA lengths in Saccharomyces cerevisiae.

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    Ji-Ping Wang

    Full Text Available The exact lengths of linker DNAs connecting adjacent nucleosomes specify the intrinsic three-dimensional structures of eukaryotic chromatin fibers. Some studies suggest that linker DNA lengths preferentially occur at certain quantized values, differing one from another by integral multiples of the DNA helical repeat, approximately 10 bp; however, studies in the literature are inconsistent. Here, we investigate linker DNA length distributions in the yeast Saccharomyces cerevisiae genome, using two novel methods: a Fourier analysis of genomic dinucleotide periodicities adjacent to experimentally mapped nucleosomes and a duration hidden Markov model applied to experimentally defined dinucleosomes. Both methods reveal that linker DNA lengths in yeast are preferentially periodic at the DNA helical repeat ( approximately 10 bp, obeying the forms 10n+5 bp (integer n. This 10 bp periodicity implies an ordered superhelical intrinsic structure for the average chromatin fiber in yeast.

  9. Intercalating nucleic acids: the influence of linker length and intercalator type on their duplex stabilities. (United States)

    Christensen, Ulf B; Wamberg, Michael; El-Essawy, Farag A G; Ismail, Abd El-Hamid; Nielsen, Christina B; Filichev, Vyacheslav V; Jessen, Carsten H; Petersen, Michael; Pedersen, Erik B


    Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes.

  10. P-Link: A method for generating multicomponent cytochrome P450 fusions with variable linker length

    DEFF Research Database (Denmark)

    Belsare, Ketaki D.; Ruff, Anna Joelle; Martinez, Ronny


    Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P......-LinK),. which was validated by fusing P450(cin) monooxygenase (CinA) to the flavodoxin shuttle protein (CinC). CinC was fused to the C terminus of CinA through a series of 16 amino acid linkers of different lengths in a single experiment employing 3 PCR amplifications. Screening for 2-beta-hydroxy-1,8-cineole...... but also requires only a single cloning and transformation step in order to generate multiple linker variants (1 to 16 amino acids long), making the approach technically simple and robust....

  11. Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication (United States)

    Rustiguel, Joane K.; Soares, Ricardo O. S.; Meisburger, Steve P.; Davis, Katherine M.; Malzbender, Kristina L.; Ando, Nozomi; Dias-Baruffi, Marcelo; Nonato, Maria Cristina


    Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by β-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

  12. Loop-length-dependent SVM prediction of domain linkers for high-throughput structural proteomics. (United States)

    Ebina, Teppei; Toh, Hiroyuki; Kuroda, Yutaka


    The prediction of structural domains in novel protein sequences is becoming of practical importance. One important area of application is the development of computer-aided techniques for identifying, at a low cost, novel protein domain targets for large-scale functional and structural proteomics. Here, we report a loop-length-dependent support vector machine (SVM) prediction of domain linkers, which are loops separating two structural domains. (DLP-SVM is freely available at: approximately domserv/cgi-bin/DLP-SVM.cgi.) We constructed three loop-length-dependent SVM predictors of domain linkers (SVM-All, SVM-Long and SVM-Short), and also built SVM-Joint, which combines the results of SVM-Short and SVM-Long into a single consolidated prediction. The performances of SVM-Joint were, in most aspects, the highest, with a sensitivity of 59.7% and a specificity of 43.6%, which indicated that the specificity and the sensitivity were improved by over 2 and 3% respectively, when loop-length-dependent characteristics were taken into account. Furthermore, the sensitivity and specificity of SVM-Joint were, respectively, 37.6 and 17.4% higher than those of a random guess, and also superior to those of previously reported domain linker predictors. These results indicate that SVMs can be used to predict domain linkers, and that loop-length-dependent characteristics are useful for improving SVM prediction performances.

  13. Tuning cooperativity by controlling the linker length of silica-supported amines in catalysis and CO2 capture. (United States)

    Brunelli, Nicholas A; Didas, Stephanie A; Venkatasubbaiah, Krishnan; Jones, Christopher W


    Cooperative interactions between aminoalkylsilanes and silanols on a silica surface can be controlled by varying the length of the alkyl linker attaching the amine to the silica surface from C1 (methyl) to C5 (pentyl). The linker length strongly affects the catalytic cooperativity of amines and silanols in aldol condensations as well as the adsorptive cooperativity for CO(2) capture. The catalytic cooperativity increases with the linker length up to propyl (C3), with longer, more flexible linkers (up to C5) providing no additional benefit or hindrance. Short linkers (C1 and C2) limit the beneficial amine-silanol cooperativity in aldol condensations, resulting in lower catalytic rates than with the C3+ linkers. For the same materials, the adsorptive cooperativity exhibits similar trends for CO(2) capture efficiency.

  14. Influence of linker length and composition on enzymatic activity and ribosomal binding of neomycin dimers. (United States)

    Watkins, Derrick; Kumar, Sunil; Green, Keith D; Arya, Dev P; Garneau-Tsodikova, Sylvie


    The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA.

  15. Interdomain orientation of cardiac troponin C characterized by paramagnetic relaxation enhancement NMR reveals a compact state. (United States)

    Cordina, Nicole M; Liew, Chu Kong; Gell, David A; Fajer, Piotr G; Mackay, Joel P; Brown, Louise J


    Cardiac troponin C (cTnC) is the calcium binding subunit of the troponin complex that triggers the thin filament response to calcium influx into the sarcomere. cTnC consists of two globular EF-hand domains (termed the N- and C-domains) connected by a flexible linker. While the conformation of each domain of cTnC has been thoroughly characterized through NMR studies involving either the isolated N-domain (N-cTnC) or C-domain (C-cTnC), little attention has been paid to the range of interdomain orientations possible in full-length cTnC that arises as a consequence of the flexibility of the domain linker. Flexibility in the domain linker of cTnC is essential for effective regulatory function of troponin. We have therefore utilized paramagnetic relaxation enhancement (PRE) NMR to assess the interdomain orientation of cTnC. Ensemble fitting of our interdomain PRE measurements reveals that isolated cTnC has considerable interdomain flexibility and preferentially adopts a bent conformation in solution, with a defined range of relative domain orientations.

  16. Effect of linker length between variable domains of single chain variable fragment antibody against daidzin on its reactivity. (United States)

    Yusakul, Gorawit; Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Tanaka, Hiroyuki; Morimoto, Satoshi


    The peptide linker between variable domains of heavy (VH) and light (VL) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of VH-(GGGGS)n-VL (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs.

  17. Tuning the mechanosensitivity of a BK channel by changing the linker length

    Institute of Scientific and Technical Information of China (English)


    Some large-conductance Ca2+ and voltage-activated K+ (BK) channels are activated by membrane stretch. However, the mechanism of mechano-gating of the BK channels is still not well understood. Previous studies have led to the proposal that the tinker-gating ring complex functions as a passive spring, transducing the force generated by intraceilular Ca2+ to the gate to open the channel. This raises the question as to whether membrane stretch is also transmitted to the gate of mechanosensitive (MS) BK channels via the tinker-gating complex. To study this, we changed the linker length in the stretch-activated BK channel (SAKCaC), and examined the effect of membrane stretch on the gating of the resultant mutant channels. Shortening the tinker increased, whereas extending the tinker reduced, the channel mechanosensitivity both in the presence and in the absence of intracellular Ca2+. However, the voltage and Ca2+ sensitivities were not significantly altered by membrane stretch. Furthermore, the SAKCaC became less sensitive to membrane stretch at relatively high intracellular Ca2+ concentrations or membrane depolarization. These observations suggest that once the channel is in the open-state conformation, tension on the spring is partially released and membrane stretch is less effective. Our results are consistent with the idea that membrane stretch is transferred to the gate via the tinker-gating ring complex of the MS BK channels.

  18. S3-S4 linker length modulates the relaxed state of a voltage-gated potassium channel. (United States)

    Priest, Michael F; Lacroix, Jérôme J; Villalba-Galea, Carlos A; Bezanilla, Francisco


    Voltage-sensing domains (VSDs) are membrane protein modules found in ion channels and enzymes that are responsible for a large number of fundamental biological tasks, such as neuronal electrical activity. The VSDs switch from a resting to an active conformation upon membrane depolarization, altering the activity of the protein in response to voltage changes. Interestingly, numerous studies describe the existence of a third distinct state, called the relaxed state, also populated at positive potentials. Although some physiological roles for the relaxed state have been suggested, little is known about the molecular determinants responsible for the development and modulation of VSD relaxation. Several lines of evidence have suggested that the linker (S3-S4 linker) between the third (S3) and fourth (S4) transmembrane segments of the VSD alters the equilibrium between resting and active conformations. By measuring gating currents from the Shaker potassium channel, we demonstrate here that shortening the S3-S4 linker stabilizes the relaxed state, whereas lengthening the linker or splitting it and coinjecting two fragments of the channel have little effect. We propose that natural variations of the length of the S3-S4 linker in various VSD-containing proteins may produce differential VSD relaxation in vivo.

  19. Targeting C-myc G-Quadruplex: Dual Recognition by Aminosugar-Bisbenzimidazoles with Varying Linker Lengths

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    Nihar Ranjan


    Full Text Available G-quadruplexes are therapeutically important biological targets. In this report, we present biophysical studies of neomycin-Hoechst 33258 conjugates binding to a G-quadruplex derived from the C-myc promoter sequence. Our studies indicate that conjugation of neomycin to a G-quadruplex binder, Hoechst 33258, enhances its binding. The enhancement in G-quadruplex binding of these conjugates varies with the length and composition of the linkers joining the neomycin and Hoechst 33258 units.

  20. Structural properties of the linkers connecting the N- and C- terminal domains in the MocR bacterial transcriptional regulators

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    Teresa Milano


    Full Text Available Peptide inter-domain linkers are peptide segments covalently linking two adjacent domains within a protein. Linkers play a variety of structural and functional roles in naturally occurring proteins. In this work we analyze the sequence properties of the predicted linker regions of the bacterial transcriptional regulators belonging to the recently discovered MocR subfamily of the GntR regulators. Analyses were carried out on the MocR sequences taken from the phyla Actinobacteria, Firmicutes, Alpha-, Beta- and Gammaproteobacteria. The results suggest that MocR linkers display phylum-specific characteristics and unique features different from those already described for other classes of inter-domain linkers. They show an average length significantly higher: 31.8 ± 14.3 residues reaching a maximum of about 150 residues. Compositional propensities displayed general and phylum-specific trends. Pro is dominating in all linkers. Dyad propensity analysis indicate Pro–Pro as the most frequent amino acid pair in all linkers. Physicochemical properties of the linker regions were assessed using amino acid indices relative to different features: in general, MocR linkers are flexible, hydrophilic and display propensity for β-turn or coil conformations. Linker sequences are hypervariable: only similarities between MocR linkers from organisms related at the level of species or genus could be found with sequence searches. The results shed light on the properties of the linker regions of the new MocR subfamily of bacterial regulators and may provide knowledge-based rules for designing artificial linkers with desired properties.

  1. Effects of Linker Length and Transient Secondary Structure Elements in the Intrinsically Disordered Notch RAM Region on Notch Signaling. (United States)

    Sherry, Kathryn P; Johnson, Scott E; Hatem, Christine L; Majumdar, Ananya; Barrick, Doug


    Formation of the bivalent interaction between the Notch intracellular domain (NICD) and the transcription factor CBF-1/RBP-j, Su(H), Lag-1 (CSL) is a key event in Notch signaling because it switches Notch-responsive genes from a repressed state to an activated state. Interaction of the intrinsically disordered RBP-j-associated molecule (RAM) region of NICD with CSL is thought to both disrupt binding of corepressor proteins to CSL and anchor NICD to CSL, promoting interaction of the ankyrin domain of NICD with CSL through an effective concentration mechanism. To quantify the role of disorder in the RAM linker region on the effective concentration enhancement of Notch transcriptional activation, we measured the effects of linker length variation on activation. The resulting activation profile has general features of a worm-like chain model for effective concentration. However, deviations from the model for short sequence deletions suggest that RAM contains sequence-specific structural elements that may be important for activation. Structural characterization of the RAM linker with sedimentation velocity analytical ultracentrifugation and NMR spectroscopy reveals that the linker is compact and contains three transient helices and two extended and dynamic regions. To test if these secondary structure elements are important for activation, we made sequence substitutions to change the secondary structure propensities of these elements and measured transcriptional activation of the resulting variants. Substitutions to two of these nonrandom elements (helix 2, extended region 1) have effects on activation, but these effects do not depend on the nature of the substituting residues. Thus, the primary sequences of these elements, but not their secondary structures, are influencing signaling.

  2. Self-organization of Au–CdSe hybrid nanoflowers at different length scales via bi-functional diamine linkers

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    AbouZeid, Khaled Mohamed [Virginia Commonwealth University, Department of Chemistry (United States); Mohamed, Mona Bakr [Cairo University, National Institute of Laser Enhanced Science (NILES) (Egypt); El-Shall, M. Samy, E-mail: [Virginia Commonwealth University, Department of Chemistry (United States)


    This work introduces a series of molecular bridging bi-functional linkers to produce laterally self-assembled nanostructures of the Au–CdSe nanoflowers on different length scales ranging from 10 nm to 100 microns. Assembly of Au nanocrystals within amorphous CdSe rods is found in the early stages of the growth of the Au–CdSe nanoflowers. The Au–CdSe nanoflowers are formed through a one-pot low temperature (150 °C) process where CdSe clusters are adsorbed on the surface of the Au cores, and they then start to form multiple arms and branches resulting in flower-shaped hybrid nanostructures. More complex assembly at a micron length scale can be achieved by means of bi-functional capping agents with appropriate alkyl chain lengths, such as 1,12-diaminododecane.

  3. Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence. (United States)

    Amosova, Olga; Broitman, Steven L; Fresco, Jacques R


    Three identical deoxyoligonucleotide third strands with a 3'-terminal psoralen moiety attached by linkers that differ in length (N = 16, 6 and 4 atoms) and structure were examined for their ability to form triplex-directed psoralen photoproducts with both the mutant T residue of the Sickle Cell beta-globin gene and the comparable wild-type sequence in linear duplex targets. Specificity and yield of UVA (365 nm) and visible (419 nm) light-induced photoadducts were studied. The total photoproduct yield varies with the linker and includes both monoadducts and crosslinks at various available pyrimidine sites. The specificity of photoadduct formation at the desired mutant T residue site was greatly improved by shortening the psoralen linker. In particular, using the N-4 linker, psoralen interaction with the residues of the non-coding duplex strand was essentially eliminated, while modification of the Sickle Cell mutant T residue was maximized. At the same time, the proportion of crosslink formation at the mutant T residue upon UV irradiation was much greater for the N-4 linker. The photoproducts formed with the wild-type target were fully consistent with its single base pair difference. The third strand with the N-4 linker was also shown to bind to a supercoiled plasmid containing the Sickle Cell mutation site, giving photoproduct yields comparable with those observed in the linear mutant target.

  4. An all-atom model of the chromatin fiber containing linker histones reveals a versatile structure tuned by the nucleosomal repeat length.

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    Hua Wong

    Full Text Available In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties.

  5. Accurate distance determination of nucleic acids via Förster resonance energy transfer: implications of dye linker length and rigidity. (United States)

    Sindbert, Simon; Kalinin, Stanislav; Nguyen, Hien; Kienzler, Andrea; Clima, Lilia; Bannwarth, Willi; Appel, Bettina; Müller, Sabine; Seidel, Claus A M


    In Förster resonance energy transfer (FRET) experiments, the donor (D) and acceptor (A) fluorophores are usually attached to the macromolecule of interest via long flexible linkers of up to 15 Å in length. This causes significant uncertainties in quantitative distance measurements and prevents experiments with short distances between the attachment points of the dyes due to possible dye-dye interactions. We present two approaches to overcome the above problems as demonstrated by FRET measurements for a series of dsDNA and dsRNA internally labeled with Alexa488 and Cy5 as D and A dye, respectively. First, we characterize the influence of linker length and flexibility on FRET for different dye linker types (long, intermediate, short) by analyzing fluorescence lifetime and anisotropy decays. For long linkers, we describe a straightforward procedure that allows for very high accuracy of FRET-based structure determination through proper consideration of the position distribution of the dye and of linker dynamics. The position distribution can be quickly calculated with geometric accessible volume (AV) simulations, provided that the local structure of RNA or DNA in the proximity of the dye is known and that the dye diffuses freely in the sterically allowed space. The AV approach provides results similar to molecular dynamics simulations (MD) and is fully consistent with experimental FRET data. In a benchmark study for ds A-RNA, an rmsd value of 1.3 Å is achieved. Considering the case of undefined dye environments or very short DA distances, we introduce short linkers with a propargyl or alkenyl unit for internal labeling of nucleic acids to minimize position uncertainties. Studies by ensemble time correlated single photon counting and single-molecule detection show that the nature of the linker strongly affects the radius of the dye's accessible volume (6-16 Å). For short propargyl linkers, heterogeneous dye environments are observed on the millisecond time scale. A

  6. Investigating the role of chain and linker length on the catalytic activity of an H 2 production catalyst containing a β-hairpin peptide

    Energy Technology Data Exchange (ETDEWEB)

    Reback, Matthew L.; Ginovska, Bojana; Buchko, Garry W.; Dutta, Arnab; Priyadarshani, Nilusha; Kier, Brandon L.; Helm, Monte L.; Raugei, Simone; Shaw, Wendy J.


    Building on our recent report of an active H2 production catalyst [Ni(PPh2NProp-peptide)2]2+ (Prop=para-phenylpropionic acid, peptide (R10)=WIpPRWTGPR-NH2, p=D-proline, and P2N=1-aza-3,6-diphosphacycloheptane) that contains structured -hairpin peptides, here we investigate how H2 production is effected by: (1) the length of the hairpin (eight or ten residues) and (2) limiting the flexibility between the peptide and the core complex by altering the length of the linker: para-phenylpropionic acid (three carbons) or para-benzoic acid (one carbon). Reduction of the peptide chain length from ten to eight residues increases or maintains the catalytic current for H2 production for all complexes, suggesting a non-productive steric interaction at longer peptide lengths. While the structure of the hairpin appears largely intact for the complexes, NMR data are consistent with differences in dynamic behavior which may contribute to the observed differences in catalytic activity. Molecular dynamics simulations demonstrate that complexes with a one-carbon linker have the desired effect of restricting the motion of the hairpin relative to the complex; however, the catalytic currents are significantly reduced compared to complexes containing a three-carbon linker as a result of the electron withdrawing nature of the -COOH group. These results demonstrate the complexity and interrelated nature of the outer coordination sphere on catalysis.

  7. The length of the linker between the epidermal growth factor-like domains in factor VIIa is critical for a productive interaction with tissue factor (United States)

    Persson, Egon; Madsen, Jesper J; Olsen, Ole H


    Formation of the factor VIIa (FVIIa)-tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure-based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)-like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ-carboxyglutamic acid (Gla)-containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly-Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF-mediated stabilization of the active conformation of FVIIa and abrogated TF-induced activity enhancement. In addition, FVIIa variants with short linkers associated 80-fold slower with soluble TF (sTF) as compared with wild-type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca2+-free FVIIa; stepwise truncation resulting in gradually higher activity with des(83–86)-FVIIa reaching the level of Gla-domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo-FVIIa activity. PMID:25234571

  8. DNA Binding and Photocleavage Properties, Cellular Uptake and Localization, and in-Vitro Cytotoxicity of Dinuclear Ruthenium(II) Complexes with Varying Lengths in Bridging Alkyl Linkers. (United States)

    Liu, Ping; Wu, Bao-Yan; Liu, Jin; Dai, Yong-Cheng; Wang, You-Jun; Wang, Ke-Zhi


    Two new dinuclear Ru(II) polypyridyl complexes containing three and ten methylene chains in their bridging linkers are synthesized and characterized. Their calf thymus DNA-binding and plasmid DNA photocleavage behaviors are comparatively studied with a previously reported, six-methylene-containing analog by absorption and luminescence spectroscopy, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, DNA viscosity measurements, DNA thermal denaturation, and agarose gel electrophoresis analyses. Theoretical calculations applying the density functional theory (DFT) method for the three complexes are also performed to understand experimentally observed DNA binding properties. The results show that the two complexes partially intercalate between the base pairs of DNA. Cellular uptake and colocalization studies have demonstrated that the complexes could enter HeLa cells efficiently and localize within lysosomes. The in-vitro antitumor activity against HeLa and MCF-7 tumor cells of the complexes are studied by MTT cytotoxic analysis. A new method, high-content analysis (HCA), is also used to assess cytotoxicity, apoptosis and cell cycle arrest of the three complexes. The results show that the lengths of the alkyl linkers could effectively tune their biological properties and that HCA is suitable for rapidly identifying cytotoxicity and can be substituted for MTT assays to evaluate the cell cytotoxicity of chemotherapeutic agents.

  9. Implications of traffic characteristics on interdomain traffic engineering


    Uhlig, Steve


    This thesis discusses the implications of the traffic characteristics on interdomain traffic engineering with BGP. We first provide an overview of the interdomain traffic control problem. Then, we present results concerning the characteristics of the interdomain traffic, based on the analysis of real traffic traces gathered from non-transit ASes. We discuss the implications of the topological properties of the traffic on interdomain traffic engineering. Based on this knowledge of the...

  10. Implications of traffic characteristics on interdomain traffic engineering


    Uhlig, Steve


    This thesis discusses the implications of the traffic characteristics on interdomain traffic engineering with BGP. We first provide an overview of the interdomain traffic control problem. Then, we present results concerning the characteristics of the interdomain traffic, based on the analysis of real traffic traces gathered from non-transit ASes. We discuss the implications of the topological properties of the traffic on interdomain traffic engineering. Based on this knowledge of the...

  11. Inter-domain synergism is required for efficient feeding of cellulose chain into active site of cellobiohydrolase Cel7A

    DEFF Research Database (Denmark)

    Kont, Riin; Kari, Jeppe; Borch, Kim


    Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme...... of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38...... to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient...

  12. Controlling the dimensionality of metal-metal bonded Rrh(2)(4+) polymers by the length of the linker. A disciplined example of crystal engineering. (United States)

    Cotton, F A; Lin, C; Murillo, C A


    Reaction of the molecular loop [Rh(2)(cis-DAniF)(2)(CH(3)CN)](2)(malonato)(2), DAniF = N,N'-di-p-anisylformamidinate, with 2 equiv of 2,2',3,3',5,5',6,6'-octafluoro-4,4'-biphenyldinitrile gives a linear tubular structure with Rh-Rh distances of 2.435(1) A. A comparison of the structures obtained with this molecular loop but employing other linkers support the idea that longer linkers favor one-dimensional structures while shorter linkers favor two-dimensional structures because of the increased clashing between the large p-anisyl groups. Crystallographic data at 213(2) K for [[Rh(2)(cis-DAniF)(2)](2)(O(2)CCH(2)CO(2))(2)(NCC(6)F(4)C(6)F(4)CN)(2)] x 6.8nCH(2)Cl(2) are: monoclinic, P2(1)/m, a = 11.802(1), b = 28.303(2), c = 18.820(2) A, beta = 95.673(2) degrees, and Z = 2.

  13. A crystal structure of the Dengue virus NS5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication.

    Directory of Open Access Journals (Sweden)

    Yongqian Zhao


    Full Text Available Flavivirus RNA replication occurs within a replication complex (RC that assembles on ER membranes and comprises both non-structural (NS viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase and C-terminal RNA-dependent-RNA polymerase (RdRp domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3 at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV, the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

  14. Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker


    Preissler, Steffen; Chambers, Joseph E.; Crespillo-Casado, Ana; Avezov, Edward; Miranda, Elena; Perez, Juan; Hendershot, Linda M.; Harding, Heather P; Ron, David


    eLife digest Proteins are composed of long chains of amino acids that fold on themselves to form three-dimensional structures. Many proteins are made in a compartment within the cell called the endoplasmic reticulum and ‘chaperone’ proteins help them fold correctly. Cells carefully regulate the levels of chaperone proteins. If there are too few chaperones in the cell, then newly-made proteins may fold incorrectly and interrupt other processes. On the other hand, if too many chaperones are pre...

  15. Verification of Interdomain Routing System Based on Formal Methods

    Institute of Scientific and Technical Information of China (English)

    ZANG Zhiyuan; LUO Guiming; YIN Chongyuan


    In networks,the stable path problem (SPP) usually results in oscillations in interdomain systems and may cause systems to become unstable.With the rapid development of internet technology,the occurrence of SPPs in interdomain systems has quite recently become a significant focus of research.A framework for checking SPPs is presented in this paper with verification of an interdomain routing system using formal methods and the NuSMV software.Sufficient conditions and necessary conditions for determining SPP occurrence are presented with proof of the method's effectiveness.Linear temporal logic was used to model an interdomain routing system and its properties were analyzed.An example is included to demonstrate the method's reliability.

  16. Using Virtualization to Provide Interdomain QoS-enabled Routing

    Directory of Open Access Journals (Sweden)

    Fábio L. Verdi


    Full Text Available Today, the most important aspect related with the Internet architecture is its ossification representing the difficulties to introduce evolutions in the architecture as a way to meet the demands posed by the new requirements as mobility, security, heterogeneity, etc. In this paper we discuss how the network virtualization can be used to support the interdomain QoS-enabled routing. We present the Virtual Topology Service (VTS, a new approach to provide interdomain services taking into account QoS and Traffic Engineering (TE constraints. We advocate in favor of a service layer that offers new mechanisms for interdomain routing without affecting the underlying Internet infrastructure. The VTS abstracts the physical network details of each Autonomous System (AS and is totally integrated with BGP. Two models to obtain VTs were defined, the Push Model and the Pull Model. The latter one uses the Internet hierarchy to get more alternative routes towards a destination. We will show how the VTS and other services such as the end-toend negotiation service work together to provide a complete mechanism for provisioning of interdomain QoS- enabled routes in IP networks. Preliminary evaluation results are also presented.

  17. Novel silicone compatible cross-linkers for controlled functionalization of PDMS networks

    DEFF Research Database (Denmark)

    Madsen, Frederikke Bahrt; Daugaard, Anders Egede; Hvilsted, Søren


    . In order to improve the dielectric properties of PDMS a novel system is developed where push-pull dipoles are grafted to a new silicone compatible cross-linker. The grafted cross-linkers are prepared by reaction of two different push-pull dipole alkynes as well as a fluorescent alkyne with the new azide......-functional cross-linker by click chemistry. The dipole cross-linkers are used to prepare PDMS elastomers of various chains lengths providing different network densities. The functionalized cross-linkers are incorporated successfully into the networks and are well distributed as determined by the fluorescent...

  18. Dipolar cross-linkers for PDMS networks with enhanced dielectric permittivity and low dielectric loss

    DEFF Research Database (Denmark)

    Bahrt, Frederikke; Daugaard, Anders Egede; Hvilsted, Søren


    Dipole grafted cross-linkers were utilized to prepare polydimethylsiloxane (PDMS) elastomers with various chain lengths and with various concentrations of functional cross-linker. The grafted cross-linkers were prepared by reaction of two alkyne-functional dipoles, 1-ethynyl-4-nitrobenzene and 3......-(4-((4-nitrophenyl)diazenyl)phenoxy)-prop-1-yn-1-ylium, with a synthesized silicone compatible azide-functional cross-linker by click chemistry. The thermal, mechanical and electromechanical properties were investigated for PDMS films with 0 to 3.6 wt% of dipole-cross-linker. The relative dielectric permittivity...

  19. Investigation of the redox property of a metalloprotein layer self-assembled on various chemical linkers. (United States)

    Chung, Yong-Ho; Lee, Taek; Min, Junhong; Choi, Jeong-Woo


    Myogloblin, a well-known metalloprotein, was immobilized on a gold surface using various chemical linkers to investigate the length effect of chemical linker on the electron transfer in protein layers, because chemical linkers play roles in the pathway that transfers the electron from the protein to the gold substrate and act as protein immobilization reagents. Chemical linkers with 2, 6, 11, and 16 carbons were utilized to confirm length-effects. The immobilization of protein and chemical linker was validated with surface plasmon resonance (SPR) and atomic force microscopy (AFM). The electrochemical property was evaluated by cyclic voltammetry (CV) and chronocoulometry (CC). In those results, redox peaks of immobilized protein were controlled via the length of chemical linkers, and it could be directly applied to the realization of bioelectronic device.

  20. Privacy-Preserving Interdomain Routing at Internet Scale

    Directory of Open Access Journals (Sweden)

    Asharov Gilad


    Full Text Available The Border Gateway Protocol (BGP computes routes between the organizational networks that make up today’s Internet. Unfortunately, BGP suffers from deficiencies, including slow convergence, security problems, a lack of innovation, and the leakage of sensitive information about domains’ routing preferences. To overcome some of these problems, we revisit the idea of centralizing and using secure multi-party computation (MPC for interdomain routing which was proposed by Gupta et al. (ACM HotNets’12. We implement two algorithms for interdomain routing with state-of-the-art MPC protocols. On an empirically derived dataset that approximates the topology of today’s Internet (55 809 nodes, our protocols take as little as 6 s of topology-independent precomputation and only 3 s of online time. We show, moreover, that when our MPC approach is applied at country/region-level scale, runtimes can be as low as 0.17 s online time and 0.20 s pre-computation time. Our results motivate the MPC approach for interdomain routing and furthermore demonstrate that current MPC techniques are capable of efficiently tackling real-world problems at a large scale.

  1. Provisioning of Inter-Domain QoS-Aware Services

    Institute of Scientific and Technical Information of China (English)

    Fernando Matos; Alexandre Matos; Paulo Simoes; Edmundo Monteiro


    Cooperation among service providers, network providers, and access providers in the Internet allows the creation of new services to offer to customers that are in other domains, thus increasing revenue. However, the Internet heterogeneous environment, where each provider has its own policies, infrastructure and business goals, hinders the deployment of more advanced communication services. This paper presents a Quality of Service (QoS) for Inter-Domain Services (QIDS) model that allows inter-domain QoS-aware services to be defined, configured, and adapted in a dynamic and on-demand fashion, among service providers. This is accomplished by: 1) the use of a common communication channel (business layer) where service providers publish and search for services, and interact with each other to contract and manage these services; 2) the templates to specify the business and technical characteristics of the services; 3) the automatic composition of services using service elements (smaller services) according to performance and service-specific QoS parameters;and 4) the creation and enforcement of configuration rules for the underlying infrastructure. A prototype was implemented to validate QIDS and performance tests were conducted on an inter-domain Border Gateway Protocol (BGP)/Multiprotocol Label Switching (MPLS) Virtual Private Network (VPN) scenario.

  2. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase (United States)

    Biehl, Ralf; Hoffmann, Bernd; Monkenbusch, Michael; Falus, Peter; Préost, Sylvain; Merkel, Rudolf; Richter, Dieter


    Interdomain motions in proteins are essential to enable or promote biochemical function. Neutron spin-echo spectroscopy is used to directly observe the domain dynamics of the protein alcohol dehydrogenase. The collective motion of domains as revealed by their coherent form factor relates to the cleft opening dynamics between the binding and the catalytic domains enabling binding and release of the functional important cofactor. The cleft opening mode hardens as a result of an overall stiffening of the domain complex due to the binding of the cofactor.

  3. Interdomain Contacts Control Native State Switching of RfaH on a Dual-Funneled Landscape.

    Directory of Open Access Journals (Sweden)

    César A Ramírez-Sarmiento


    Full Text Available RfaH is a virulence factor from Escherichia coli whose C-terminal domain (CTD undergoes a dramatic α-to-β conformational transformation. The CTD in its α-helical fold is stabilized by interactions with the N-terminal domain (NTD, masking an RNA polymerase binding site until a specific recruitment site is encountered. Domain dissociation is triggered upon binding to DNA, allowing the NTD to interact with RNA polymerase to facilitate transcription while the CTD refolds into the β-barrel conformation that interacts with the ribosome to activate translation. However, structural details of this transformation process in the context of the full protein remain to be elucidated. Here, we explore the mechanism of the α-to-β conformational transition of RfaH in the full-length protein using a dual-basin structure-based model. Our simulations capture several features described experimentally, such as the requirement of disruption of interdomain contacts to trigger the α-to-β transformation, confirms the roles of previously indicated residues E48 and R138, and suggests a new important role for F130, in the stability of the interdomain interaction. These native basins are connected through an intermediate state that builds up upon binding to the NTD and shares features from both folds, in agreement with previous in silico studies of the isolated CTD. We also examine the effect of RNA polymerase binding on the stabilization of the β fold. Our study shows that native-biased models are appropriate for interrogating the detailed mechanisms of structural rearrangements during the dramatic transformation process of RfaH.

  4. Fusion Protein Linkers: Property, Design and Functionality


    Chen, Xiaoying; Zaro, Jennica; Shen, Wei-Chiang


    As an indispensable component of recombinant fusion proteins, linkers have shown increasing importance in the construction of stable, bioactive fusion proteins. This review covers the current knowledge of fusion protein linkers and summarizes examples for their design and application. The general properties of linkers derived from naturally-occurring multi-domain proteins can be considered as the foundation in linker design. Empirical linkers designed by researchers are generally classified i...

  5. Evolution of Inter-domain Traffic Engineering:Standards, Research and Current Applications

    Institute of Scientific and Technical Information of China (English)


    This paper briefly describes how inter-domain traffic engineering (TE) is evolving and how it can be used for effective commercial networking as well as for high performance networking in Asia and around the globe.

  6. Construction of multiform scFv antibodies using linker peptide

    Institute of Scientific and Technical Information of China (English)

    Shihua Wang; Cengjie Zheng; Ying Liu; Huirong Zheng; Zonghua Wang


    Multiform single chain variable fragments (acFvs) including different length linker scFvs and bispecific seFv were constructed. The linker lengths of 0, 3, 5, 8, 12, and 15 amino acids between VH and VL of antideoxynivalenol (anti-DON) scFv were used to analyze the affinities of scFvs. The affinity constants of these scFvs increased when the linker was lower than 12 amino acids. The affinity constant would not change when the linker was longer than 12 amino acids. Fusion gene of anti-DON seFv and antizearalenone (anti-ZEN) scFv was also constructed through eormection by a short peptide tinker DNA to express a bispecific scFv. The affinity constants assay showed that the two scFvs of fusion bispecific scFv remained their own affinity compared to their parental scFvs. Competitive direct enzyme linked immunosorbent assay was used to detect DON and ZEN in contaminated wheat (Triticum aestivum L.) samples, and the results indicated that this bispecifie acFv was applicable in DON and ZEN detection. This work confirmed that bispecific scFv could be successfully obtained, and might also have an application in diagnosing fungal infection, and breeding transgertic plants.

  7. Peptide linkers for the assembly of semiconductor quantum dot bioconjugates (United States)

    Boeneman, Kelly; Mei, Bing C.; Deschamps, Jeffrey R.; Delehanty, James B.; Mattoussi, Hedi; Medintz, Igor


    The use of semiconductor luminescent quantum dots for the labeling of biomolecules is rapidly expanding, however it still requires facile methods to attach functional globular proteins to biologically optimized quantum dots. Here we discuss the development of controlled variable length peptidyl linkers to attach biomolecules to poly(ethylene) glycol (PEG) coated quantum dots for both in vitro and in vivo applications. The peptides chosen, β-sheets and alpha helices are appended to polyhistidine sequences and this allows for control of the ratio of peptide bioconjugated to QD and the distance from QD to the biomolecule. Recombinant DNA engineering, bacterial peptide expression and Ni-NTA purification of histidine labeled peptides are utilized to create the linkers. Peptide length is confirmed by in vitro fluorescent resonance energy transfer (FRET).

  8. Linker Installation: Engineering Pore Environment with Precisely Placed Functionalities in Zirconium MOFs. (United States)

    Yuan, Shuai; Chen, Ying-Pin; Qin, Jun-Sheng; Lu, Weigang; Zou, Lanfang; Zhang, Qiang; Wang, Xuan; Sun, Xing; Zhou, Hong-Cai


    Precise placement of multiple functional groups in a highly ordered metal-organic framework (MOF) platform allows the tailoring of the pore environment, which is required for advanced applications. To realize this, we present a comprehensive study on the linker installation method, in which a stable MOF with coordinatively unsaturated Zr6 clusters was employed and linkers bearing different functional groups were postsynthetically installed. A Zr-MOF with inherent missing linker sites, namely, PCN-700, was initially constructed under kinetic control. Twelve linkers with different substituents were then designed to study their effect on MOF formation kinetics and therefore resulting MOF structures. Guided by the geometrical analysis, linkers with different lengths were installed into a parent PCN-700, giving rise to 11 new MOFs and each bearing up to three different functional groups in predefined positions. Systematic variation of the pore volume and decoration of pore environment were realized by linker installation, which resulted in synergistic effects including an enhancement of H2 adsorption capacities of up to 57%. In addition, a size-selective catalytic system for aerobic alcohol oxidation reaction is built in PCN-700 through linker installation, which shows high activity and tunable size selectivity. Altogether, these results exemplify the capability of the linker installation method in the pore environment engineering of stable MOFs with multiple functional groups, giving an unparalleled level of control.

  9. Charged Triazole Cross-Linkers for Hyaluronan-Based Hybrid Hydrogels

    Directory of Open Access Journals (Sweden)

    Maike Martini


    Full Text Available Polyelectrolyte hydrogels play an important role in tissue engineering and can be produced from natural polymers, such as the glycosaminoglycan hyaluronan. In order to control charge density and mechanical properties of hyaluronan-based hydrogels, we developed cross-linkers with a neutral or positively charged triazole core with different lengths of spacer arms and two terminal maleimide groups. These cross-linkers react with thiolated hyaluronan in a fast, stoichiometric thio-Michael addition. Introducing a positive charge on the core of the cross-linker enabled us to compare hydrogels with the same interconnectivity, but a different charge density. Positively charged cross-linkers form stiffer hydrogels relatively independent of the size of the cross-linker, whereas neutral cross-linkers only form stable hydrogels at small spacer lengths. These novel cross-linkers provide a platform to tune the hydrogel network charge and thus the mechanical properties of the network. In addition, they might offer a wide range of applications especially in bioprinting for precise design of hydrogels.

  10. Dynamic fuzziness during linker histone action. (United States)

    McBryant, Steven J; Hansen, Jeffrey C


    Linker histones are multi-domain nucleosome binding proteins that stabilize higher order chromatin structures and engage in specific protein-protein interactions. Here we emphasize the structural and functional properties of the linker histone C-terminal domain (CTD), focusing on its intrinsic disorder, interaction-induced secondary structure formation and dynamic fuzziness. We argue that the fuzziness inherent in the CTD is a primary molecular mechanism underlying linker histone function in the nucleus.

  11. Examining Ancient Inter-domain Horizontal Gene Transfer

    Directory of Open Access Journals (Sweden)

    Francisca C. Almeida


    Full Text Available Details of the genomic changes that occurred in the ancestors of Eukarya, Archaea and Bacteria are elusive. Ancient interdomain horizontal gene transfer (IDHGT amongst the ancestors of these three domains has been difficult to detect and analyze because of the extreme degree of divergence of genes in these three domains and because most evidence for such events are poorly supported. In addition, many researchers have suggested that the prevalence of IDHGT events early in the evolution of life would most likely obscure the patterns of divergence of major groups of organisms let alone allow the tracking of horizontal transfer at this level. In order to approach this problem, we mined the E. coli genome for genes with distinct paralogs. Using the 1,268 E. coli K-12 genes with 40% or higher similarity level to a paralog elsewhere in the E. coli genome we detected 95 genes found exclusively in Bacteria and Archaea and 86 genes found in Bacteria and Eukarya. These genes form the basis for our analysis of IDHGT. We also applied a newly developed statistical test (the node height test, to examine the robustness of these inferences and to corroborate the phylogenetically identifi ed cases of ancient IDHGT. Our results suggest that ancient inter domain HGT is restricted to special cases, mostly involving symbiosis in eukaryotes and specific adaptations in prokaryotes. Only three genes in the Bacteria + Eukarya class (Deoxyxylulose-5-phosphate synthase (DXPS, fructose 1,6-phosphate aldolase class II protein and glucosamine-6-phosphate deaminase and three genes–in the Bacteria + Archaea class (ABC-type FE3+ -siderophore transport system, ferrous iron transport protein B, and dipeptide transport protein showed evidence of ancient IDHGT. However, we conclude that robust estimates of IDHGT will be very difficult to obtain due to the methodological limitations and the extreme sequence saturation of the genes suspected of being involved in IDHGT.

  12. Rheology of semiflexible bundle networks with transient linkers. (United States)

    Müller, Kei W; Bruinsma, Robijn F; Lieleg, Oliver; Bausch, Andreas R; Wall, Wolfgang A; Levine, Alex J


    We present a theoretical and computational analysis of the rheology of networks made up of bundles of semiflexible filaments bound by transient cross-linkers. Such systems are ubiquitous in the cytoskeleton and can be formed in vitro using filamentous actin and various cross-linkers. We find that their high-frequency rheology is characterized by a scaling behavior that is quite distinct from that of networks of the well-studied single semiflexible filaments. This regime can be understood theoretically in terms of a length-scale-dependent bending modulus for bundles. Next, we observe new dissipative dynamics associated with the shear-induced disruption of the network at intermediate frequencies. Finally, at low frequencies, we encounter a region of non-Newtonian rheology characterized by power-law scaling. This regime is dominated by bundle dissolution and large-scale rearrangements of the network driven by equilibrium thermal fluctuations.

  13. Solid colloids with surface-mobile linkers. (United States)

    van der Meulen, Stef A J; Helms, Gesa; Dogterom, Marileen


    In this report we review the possibilities of using colloids with surface mobile linkers for the study of colloidal self-assembly processes. A promising route to create systems with mobile linkers is the use of lipid (bi-)layers. These lipid layers can be either used in the form of vesicles or as coatings for hard colloids and emulsion droplets. Inside the lipid bilayers molecules can be inserted via membrane anchors. Due to the fluidity of the lipid bilayer, the anchored molecules remain mobile. The use of different lipid mixtures even allows creating Janus-like particles that exhibit directional bonding if linkers are used which have a preference for a certain lipid phase. In nature mobile linkers can be found e.g. as receptors in cells. Therefore, towards the end of the review, we also briefly address the possibility of using colloids with surface mobile linkers as model systems to mimic cell-cell interactions and cell adhesion processes.

  14. Online charging for IMS-based inter-domain composite services

    NARCIS (Netherlands)

    Le, M.V.; Rumph, F.J.; Huitema, G.B.; Beijnum, B.J.V.; Nieuwenhuis, L.J.M.


    In order to manage financial risks online charging of composite services is becoming increasingly important for service providers to support service delivery in inter-domain environments. The 3rd Generation Partnership Project (3GPP) has developed a framework for off-line and online charging of IMS-

  15. Design of an online charging system to support IMS-based inter-domain composite services

    NARCIS (Netherlands)

    Le, M. van; Huitema, G.B.; Rumph, F.J.; Nieuwenhuis, L.J.M.; Beijnum, B.J.F. van


    For service providers online charging of composite services is necessary in order to manage financial risks of service delivery in multi-domain environments. At service level, inter-domain composite services consist of one or more service components, e.g. access service, IMS communication service or

  16. Design of an online charging system to support IMS-based inter-domain composite services

    NARCIS (Netherlands)

    Le, V.M.; Huitema, G.B.; Rumph, F.J.; Nieuwenhuis, Lambertus Johannes Maria; van Beijnum, Bernhard J.F.; Pfeifer, T.; Bellavista, P.


    For service providers online charging of composite services is necessary in order to manage financial risks of service delivery in multi-domain environments. At service level, inter-domain composite services consist of one or more service components, e.g. access service, IMS communication service or

  17. Monte Carlo analysis of neck linker extension in kinesin molecular motors.

    Directory of Open Access Journals (Sweden)

    Matthew L Kutys


    Full Text Available Kinesin stepping is thought to involve both concerted conformational changes and diffusive movement, but the relative roles played by these two processes are not clear. The neck linker docking model is widely accepted in the field, but the remainder of the step--diffusion of the tethered head to the next binding site--is often assumed to occur rapidly with little mechanical resistance. Here, we investigate the effect of tethering by the neck linker on the diffusive movement of the kinesin head, and focus on the predicted behavior of motors with naturally or artificially extended neck linker domains. The kinesin chemomechanical cycle was modeled using a discrete-state Markov chain to describe chemical transitions. Brownian dynamics were used to model the tethered diffusion of the free head, incorporating resistive forces from the neck linker and a position-dependent microtubule binding rate. The Brownian dynamics and chemomechanical cycle were coupled to model processive runs consisting of many 8 nm steps. Three mechanical models of the neck linker were investigated: Constant Stiffness (a simple spring, Increasing Stiffness (analogous to a Worm-Like Chain, and Reflecting (negligible stiffness up to a limiting contour length. Motor velocities and run lengths from simulated paths were compared to experimental results from Kinesin-1 and a mutant containing an extended neck linker domain. When tethered by an increasingly stiff spring, the head is predicted to spend an unrealistically short amount of time within the binding zone, and extending the neck is predicted to increase both the velocity and processivity, contrary to experiments. These results suggest that the Worm-Like Chain is not an adequate model for the flexible neck linker domain. The model can be reconciled with experimental data if the neck linker is either much more compliant or much stiffer than generally assumed, or if weak kinesin-microtubule interactions stabilize the diffusing

  18. Structure and Functions of Linker Histones. (United States)

    Lyubitelev, A V; Nikitin, D V; Shaytan, A K; Studitsky, V M; Kirpichnikov, M P


    Linker histones such as variants H1, H5, and other similar proteins play an important role in regulation of chromatin structure and dynamics. However, interactions of linker histones with DNA and proteins, as well as specific functions of their different variants, are poorly studied. This is because they acquire tertiary structure only when interacting with a nucleosome, and because of limitations of currently available methods. However, deeper investigation of linker histones and their interactions with other proteins will address a number of important questions - from structure of compacted chromatin to regulation of early embryogenesis. In this review, structures of histone H1 variants and its interaction with chromatin DNA are considered. A possible functional significance of different H1 variants, a role of these proteins in maintaining interphase chromatin structure, and interactions of linker histones with other cellular proteins are also discussed.

  19. Hydroquinone–pyrrole dyads with varied linkers

    Directory of Open Access Journals (Sweden)

    Hao Huang


    Full Text Available A series of pyrroles functionalized in the 3-position with p-dimethoxybenzene via various linkers (CH2, CH2CH2, CH=CH, C≡C has been synthesized. Their electronic properties have been deduced from 1H NMR, 13C NMR, and UV–vis spectra to detect possible interactions between the two aromatic subunits. The extent of conjugation between the subunits is largely controlled by the nature of the linker, with the largest conjugation found with the trans-ethene linker and the weakest with the aliphatic linkers. DFT calculations revealed substantial changes in the HOMO–LUMO gap that correlated with the extent of conjugation found experimentally. The results of this work are expected to open up for use of the investigated compounds as components of redox-active materials in sustainable, organic electrical energy storage devices.

  20. Initial conformation of kinesin's neck linker

    CERN Document Server

    Geng, Yi-Zhao; Liu, Shu-Xia; Yan, Shiwei


    How ATP binding initiates the docking process of kinesin's neck linker is a key question in understanding kinesin mechanism. It is believed that the formation of an extra turn structure by the first three amino acids of neck linker (LYS325, THR326, ILE327 in 2KIN) is crucial for initiating the docking process. But the initial conformation of neck linker (specially the three amino acids of the extra turn) and the neck linker docking initiation mechanism remain unclear. By using molecular dynamics method, we investigate the initial conformation of kinesin's neck linker in the docking process. We find that, in the initial state of NL docking process, NL still has interactions with {\\beta}0 and forms a conformation similar to the "cover-neck bundle" structure proposed by Hwang et al. [Structure 2008, 16(1): 62-71]. From this initial structure, the docking of the "cover-neck bundle" structure can be achieved. The motor head provides a forward force on the initial cover-neck bundle structure through ATP-induced rot...

  1. Geo-Based Inter-Domain Routing (GIDR) Protocol for MANETS (United States)


    routing protocol such as DSDV, AODV is used. The GIDR inter- domain routing protocol chooses the routing path which is marked by red... routing protocols , the scenarios of Figure 7 and 8 have multiple domains, each of which runs different routing protocols , such as AODV , BELLMANFORD...GEO-BASED INTER-DOMAIN ROUTING (GIDR) PROTOCOL FOR MANETS Biao Zhou*, Abhishek Tiwari+, Konglin Zhu*, You Lu$, Mario Gerla*, Anurag Ganguli+,

  2. Crystal Structure of Prp5p Reveals Interdomain Interactions that Impact Spliceosome Assembly

    Directory of Open Access Journals (Sweden)

    Zhi-Min Zhang


    Full Text Available The DEAD-box adenosine triphosphatase (ATPase Prp5p facilitates U2 small nuclear ribonucleoprotein particle (snRNP binding to the intron branch site region during spliceosome assembly. We present crystal structures of S. cerevisiae Prp5p alone and in complex with ADP at 2.12 Å and 1.95 Å resolution. The three-dimensional packing of Prp5p subdomains differs strikingly from that so far observed in other DEAD-box proteins: two RecA-like subdomains adopt an “open state” conformation stabilized by extensive interactions involving sequences that flank the two subdomains. This conformation is distinct from that required for ATP hydrolysis. Consistent with this, Prp5p mutations that destabilize interdomain interactions exhibited increased ATPase activity in vitro and inhibited splicing of suboptimal branch site substrates in vivo, whereas restoration of interdomain interactions reversed these effects. We conclude that the Prp5p open state conformation is biologically relevant and that disruption of the interdomain interaction facilitates a large-scale conformational change of Prp5p during U2 snRNP-branch site recognition.

  3. Butane-1,2,3,4-tetraol-based amphiphilic stereoisomers for membrane protein study: importance of chirality in the linker region

    DEFF Research Database (Denmark)

    Das, Manabendra; Du, Yang; Mortensen, Jonas S.


    of the targeted membrane proteins depending on the chirality of the linker region. These findings indicate an important role for detergent stereochemistry in membrane protein stabilization. In addition, we generally observed enhanced detergent efficacy with increasing alkyl chain length, reinforcing...

  4. Linker histone H1 and protein-protein interactions


    Kalashnikova, Anna A; Rogge, Ryan A.; Hansen, Jeffrey C


    Linker histones H1 are ubiquitous chromatin proteins that play important roles in chromatin compaction, transcription regulation, nucleosome spacing and chromosome spacing. H1 function in DNA and chromatin structure stabilization is well studied and established. The current paradigm of linker histone mode of function considers all other cellular roles of linker histones to be a consequence from H1 chromatin compaction and repression. Here we review the multiple processes regulated by linker h...

  5. Linker histones: History and current perspectives. (United States)

    Crane-Robinson, C


    Although the overall structure of the fifth histone (linker histone, H1) is understood, its location on the nucleosome is only partially defined. Whilst it is clear that H1 helps condense the chromatin fibre, precisely how this is achieved remains to be determined. H1 is not a general gene repressor in that although it must be displaced from transcription start sites for activity to occur, there is only partial loss along the body of genes. How the deposition and removal of H1 occurs in particular need of further study. Linker histones are highly abundant nuclear proteins about which we know too little. Copyright © 2015. Published by Elsevier B.V.

  6. Inter-Domain Roaming Mechanism Transparent to Mobile Nodes among PMIPv6 Networks (United States)

    Park, Soochang; Lee, Euisin; Jin, Min-Sook; Kim, Sang-Ha

    In Proxy Mobile IPv6 (PMIPv6), when a Mobile Node (MN) enters a PMIPv6 domain and attaches to an access link, the router on the access link detects attachment of the MN by the link-layer access. All elements of PMIPv6 including the router then provide network-based mobility management service for the MN. If the MN moves to another router in this PMIPv6 domain, the new router emulates attachment to the previous router by providing same network prefix to the MN. In other words, PMIPv6 provides rapid mobility management based on layer-2 attachment and transparent mobility support to the MN by emulating layer-3 attachment with respect to intra-domain roaming. However, when the MN moves to other PMIPv6 domains, although the domains also provide the network-based mobility management service, the MN should exploit the host-based mobility management protocol, i.e. Mobile IPv6 (MIPv6), for the inter-domain roaming. Hence, this letter proposes the rapid and transparent inter-domain roaming mechanism controlled by the networks adopting PMIPv6.

  7. Combined Intra- and Inter-domain Traffic Engineering using Hot-Potato Aware Link Weights Optimization

    CERN Document Server

    Balon, Simon


    A well-known approach to intradomain traffic engineering consists in finding the set of link weights that minimizes a network-wide objective function for a given intradomain traffic matrix. This approach is inadequate because it ignores a potential impact on interdomain routing. Indeed, the resulting set of link weights may trigger BGP to change the BGP next hop for some destination prefixes, to enforce hot-potato routing policies. In turn, this results in changes in the intradomain traffic matrix that have not been anticipated by the link weights optimizer, possibly leading to degraded network performance. We propose a BGP-aware link weights optimization method that takes these effects into account, and even turns them into an advantage. This method uses the interdomain traffic matrix and other available BGP data, to extend the intradomain topology with external virtual nodes and links, on which all the well-tuned heuristics of a classical link weights optimizer can be applied. A key innovative asset of our ...

  8. Inter-domain interactions of TDP-43 as decoded by NMR. (United States)

    Wei, Yuanyuan; Lim, Liangzhong; Wang, Lu; Song, Jianxing


    TDP-43 inclusions have been found in ∼97% ALS as well as an increasing spectrum of other neurodegenerative diseases including Alzheimer's. TDP-43 contains an ubiquitin-like fold, two RRMs and a prion-like domain, but whether they interact with each other remains unknown due to being intrinsically aggregation-prone. Nevertheless, this knowledge is pivotal to understanding physiological functions and pathological roles of TDP-43. Here as facilitated by our previous discovery which allowed NMR characterization of TDP-43 and its five dissected fragments, we successfully decoded that TDP-43 does have dynamic inter-domain interactions, which are coordinated by the intrinsically-disordered prion-like domain. Thus, TDP-43 appears to undergo conformational exchanges between "closed" and "open" states which are needed for its functions. Our study thus offers a mechanism by which cellular processes might control TDP-43 physiology and proteinopathy by mediating its inter-domain interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Phycobilisomes linker family in cyanobacterial genomes: divergence and evolution

    Directory of Open Access Journals (Sweden)

    Xiangyu Guan, Song Qin, Fangqing Zhao, Xiaowen Zhang, Xuexi Tang


    Full Text Available Cyanobacteria are the oldest life form making important contributions to global CO2 fixation on the Earth. Phycobilisomes (PBSs are the major light harvesting systems of most cyanobacteria species. Recent availability of the whole genome database of cyanobacteria provides us a global and further view on the complex structural PBSs. A PBSs linker family is crucial in structure and function of major light-harvesting PBSs complexes. Linker polypeptides are considered to have the same ancestor with other phycobiliproteins (PBPs, and might have been diverged and evolved under particularly selective forces together. In this paper, a total of 192 putative linkers including 167 putative PBSs-associated linker genes and 25 Ferredoxin-NADP oxidoreductase (FNR genes were detected through whole genome analysis of all 25 cyanobacterial genomes (20 finished and 5 in draft state. We compared the PBSs linker family of cyanobacteria in terms of gene structure, chromosome location, conservation domain, and polymorphic variants, and discussed the features and functions of the PBSs linker family. Most of PBSs-associated linkers in PBSs linker family are assembled into gene clusters with PBPs. A phylogenetic analysis based on protein data demonstrates a possibility of six classes of the linker family in cyanobacteria. Emergence, divergence, and disappearance of PBSs linkers among cyanobacterial species were due to speciation, gene duplication, gene transfer, or gene loss, and acclimation to various environmental selective pressures especially light.

  10. New bis(azobenzocrown)s with dodecylmethylmalonyl linkers as ionophores for sodium selective potentiometric sensors



    Novel biscrowns 1 and 2 were synthesized from 13-membered azobenzocrown ethers containing bromoalkylenoxy chains in para position relative to the azo group. The synthesized diester molecules are dodecylmethylmalonic acid derivatives differing by the linker length. The synthesized compounds have the potential of being used as sodium ionophores in ion-selective electrodes. They were characterized and used as ionophores in classic and miniature, solid contact (screen-printed and glassy carbon) m...

  11. Desmosine-Inspired Cross-Linkers for Hyaluronan Hydrogels (United States)

    Hagel, Valentin; Mateescu, Markus; Southan, Alexander; Wegner, Seraphine V.; Nuss, Isabell; Haraszti, Tamás; Kleinhans, Claudia; Schuh, Christian; Spatz, Joachim P.; Kluger, Petra J.; Bach, Monika; Tussetschläger, Stefan; Tovar, Günter E. M.; Laschat, Sabine; Boehm, Heike


    We designed bioinspired cross-linkers based on desmosine, the cross-linker in natural elastin, to prepare hydrogels with thiolated hyaluronic acid. These short, rigid cross-linkers are based on pyridinium salts (as in desmosine) and can connect two polymer backbones. Generally, the obtained semi-synthetic hydrogels are form-stable, can withstand repeated stress, have a large linear-elastic range, and show strain stiffening behavior typical for biopolymer networks. In addition, it is possible to introduce a positive charge to the core of the cross-linker without affecting the gelation efficiency, or consequently the network connectivity. However, the mechanical properties strongly depend on the charge of the cross-linker. The properties of the presented hydrogels can thus be tuned in a range important for engineering of soft tissues by controlling the cross-linking density and the charge of the cross-linker.

  12. Inter-domain networking innovation on steroids: empowering ixps with SDN capabilities

    KAUST Repository

    Chiesa, Marco


    While innovation in inter-domain routing has remained stagnant for over a decade, Internet exchange points (IXPs) are consolidating their role as economically advantageous interconnection points for reducing path latencies and exchanging ever increasing amounts of traffic. As such, IXPs appear as a natural place to foster network innovation and assess the benefits of SDN, a recent technological trend that has already boosted innovation within data center networks. In this article, we give a comprehensive overview of use cases for SDN at IXPs, which leverage the superior vantage point of an IXP to introduce advanced features like load balancing and DDoS mitigation. We discuss the benefits of SDN solutions by analyzing real-world data from one of the largest IXPs. We also leverage insights into IXP operations to shape benefits not only for members but also for operators.

  13. SLA Establishment with Guaranteed QoS in the Interdomain Network: A Stock Model

    CERN Document Server

    Barth, Dominique; Mautor, Thierry; 10.5121/ijcnc.2011.3413


    The new model that we present in this paper is introduced in the context of guaranteed QoS and resources management in the inter-domain routing framework. This model, called the stock model, is based on a reverse cascade approach and is applied in a distributed context. So transit providers have to learn the right capacities to buy and to stock and, therefore learning theory is applied through an iterative process. We show that transit providers manage to learn how to strategically choose their capacities on each route in order to maximize their benefits, despite the very incomplete information. Finally, we provide and analyse some simulation results given by the application of the model in a simple case where the model quickly converges to a stable state.

  14. Extensive inter-domain lateral gene transfer in the evolution of the human commensal Methanosphaera stadtmanae

    Directory of Open Access Journals (Sweden)

    Mor Nadia Lurie-Weinberger


    Full Text Available Methanosphaera stadtmanae is a commensal methanogenic archaeon found in the human gut. As most of its niche-neighbors are bacteria, it is expected that lateral gene transfer (LGT from bacteria might have contributed to the evolutionary history of this organism. We performed a phylogenomic survey of putative lateral gene transfer events in M. stadtmanae, using a phylogenetic pipeline. Our analysis indicates that a substantial fraction of the proteins of M. stadtmanae are inferred to have been involved in inter-domain LGT. Laterally acquired genes have had a large contribution to surface functions, by providing novel glycosyltransferase functions. In addition, several ABC transporters seem to be of bacterial origin, including the molybdate transporter. Thus, bacterial genes contributed to the adaptation of M. stadtmanae to a host dependent lifestyle by allowing a larger variation in surface structures and increasing transport efficiency in the gut niche which is diverse and competitive

  15. SafeZone: A Hierarchical Inter-Domain Authenticated Source Address Validation Solution

    CERN Document Server

    Li, Jie; Xu, Ke


    Next generation Internet is highly concerned about the issue of reliability. Principally, the foundation of reliability is authentication of the source IP address. With the signature-and-verification based defense mechanisms available today, unfortunately, there is a lack of hierarchical architecture, which makes the structure of the trust alliance excessively flat and single. Moreover, with the increasing scale of the trust alliance, costs of validation grow so quickly that they do not adapt to incremental deployment. Via comparison with traditional solutions, this article proposes a hierarchical, inter-domain authenticated source address validation solution named SafeZone. SafeZone employs two intelligent designs, lightweight tag replacement and a hierarchical partitioning scheme, each of which helps to ensure that SafeZone can construct trustworthy and hierarchical trust alliances without the negative influences and complex operations on de facto networks. Extensive experiments also indicate that SafeZone ...


    Directory of Open Access Journals (Sweden)

    Dominique Barth


    Full Text Available The new model that we present in this paper is introduced in the context of guaranteed QoS andresources management in the inter-domain routing framework. This model, called the stock model, isbased on a reverse cascade approach and is applied in a distributed context. So transit providers have tolearn the right capacities to buy and to stock and, therefore learning theory is applied through aniterative process. We show that transit providers manage to learn how to strategically choose theircapacities on each route in order to maximize their benefits, despite the very incomplete information.Finally, we provide and analyse some simulation results given by the application of the model in a simplecase where the model quickly converges to a stable state.

  17. Estimation of interdomain flexibility of N-terminus of factor H using residual dipolar couplings. (United States)

    Maciejewski, Mateusz; Tjandra, Nico; Barlow, Paul N


    Characterization of segmental flexibility is needed to understand the biological mechanisms of the very large category of functionally diverse proteins, exemplified by the regulators of complement activation, that consist of numerous compact modules or domains linked by short, potentially flexible, sequences of amino acid residues. The use of NMR-derived residual dipolar couplings (RDCs), in magnetically aligned media, to evaluate interdomain motion is established but only for two-domain proteins. We focused on the three N-terminal domains (called CCPs or SCRs) of the important complement regulator, human factor H (i.e., FH1-3). These domains cooperate to facilitate cleavage of the key complement activation-specific protein fragment, C3b, forming iC3b that no longer participates in the complement cascade. We refined a three-dimensional solution structure of recombinant FH1-3 based on nuclear Overhauser effects and RDCs. We then employed a rudimentary series of RDC data sets, collected in media containing magnetically aligned bicelles (disklike particles formed from phospholipids) under three different conditions, to estimate interdomain motions. This circumvents a requirement of previous approaches for technically difficult collection of five independent RDC data sets. More than 80% of conformers of this predominantly extended three-domain molecule exhibit flexions of <40°. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3) could facilitate recognition of C3b via initial anchoring and eventual reorganization of modules to the conformation captured in the previously solved crystal structure of a C3b:FH1-4 complex.

  18. Gold nanoparticles functionalised with fast water exchanging Gd3+ chelates: linker effects on the relaxivity. (United States)

    Ferreira, Miguel F; Gonçalves, Janaina; Mousavi, Bibimaryam; Prata, Maria I M; Rodrigues, Sérgio P J; Calle, Daniel; López-Larrubia, Pilar; Cerdan, Sebastian; Rodrigues, Tiago B; Ferreira, Paula M; Helm, Lothar; Martins, José A; Geraldes, Carlos F G C


    The relaxivity displayed by Gd(3+) chelates immobilized onto gold nanoparticles is the result of the complex interplay between the nanoparticle size, the water exchange rate and the chelate structure. In this work we study the effect of the length of ω-thioalkyl linkers, anchoring fast water exchanging Gd(3+) chelates onto gold nanoparticles, on the relaxivity of the immobilized chelates. Gold nanoparticles functionalized with Gd(3+) chelates of mercaptoundecanoyl and lipoyl amide conjugates of the DO3A-N-(α-amino)propionate chelator were prepared and studied as potential CA for MRI. High relaxivities per chelate, of the order of magnitude 28-38 mM(-1) s(-1) (30 MHz, 25 °C), were attained thanks to simultaneous optimization of the rotational correlation time and of the water exchange rate. Fast local rotational motions of the immobilized chelates around connecting linkers (internal flexibility) still limit the attainable relaxivity. The degree of internal flexibility of the immobilized chelates seems not to be correlated with the length of the connecting linkers. Biodistribution and MRI studies in mice suggest that the in vivo behavior of the gold nanoparticles was determined mainly by size. Small nanoparticles (HD = 3.9 nm) undergo fast renal clearance and avoidance of the RES organs while larger nanoparticles (HD = 4.8 nm) undergo predominantly hepatobiliary excretion. High relaxivities, allied to chelate and nanoparticle stability and fast renal clearance in vivo suggest that functionalized gold nanoparticles hold great potential for further investigation as MRI contrast agents. This study contributes to a better understanding of the effect of linker length on the relaxivity of gold nanoparticles functionalized with Gd(3+) complexes. It is a relevant contribution towards "design rules" for nanostructures functionalized with Gd(3+) chelates as Contrast Agents for MRI and multimodal imaging.

  19. Non-equilibrium fluctuations of a semi-flexible filament driven by active cross-linkers

    CERN Document Server

    Weber, Ines; Schehr, Grégory; Santen, Ludger


    The cytoskeleton is an inhomogeneous network of semi-flexible filaments, which are involved in a wide variety of active biological processes. Although the cytoskeletal filaments can be very stiff and embedded in a dense and cross-linked network, it has been shown that, in cells, they typically exhibit significant bending on all length scales. In this work we propose a model of a semi-flexible filament deformed by different types of cross-linkers for which one can compute and investigate the bending spectrum. Our model allows to couple the evolution of the deformation of the semi-flexible polymer with the stochastic dynamics of linkers which exert transversal forces onto the filament. We observe a $q^{-2}$ dependence of the bending spectrum for some biologically relevant parameters and in a certain range of wavenumbers $q$. However, generically, the spatially localized forcing and the non-thermal dynamics both introduce deviations from the thermal-like $q^{-2}$ spectrum.

  20. A mechanical model for the role of the neck linker during kinesin stepping and gating (United States)

    Wang, HaiYan; He, ChenJuan


    In this paper, considering the different elastic properties in the attached head and the free head, we propose a physical model, in which the free head undergoes a diffusive search in an entropic spring potential formed by undocking the neck linker, and there are asymmetric conformational changes in the attached head formed by docking the neck linker to support the load force and bias the diffusive search to the forward direction. By performing the thermodynamic analysis, we obtain the free energy difference between forward and backward binding sites. And using the Fokker-Planck equation with two absorbing boundaries, we obtain the dependence of the ratio of forward to backward steps on the backward force. Also, within the Michaelis-Menten model, we investigate the dependence of the velocity-load relationship on the effective length of the junction between the two heads. The results show that our model can provide a physical understanding for the processive movement of kinesin.

  1. Structural Mechanisms of Nucleosome Recognition by Linker Histones. (United States)

    Zhou, Bing-Rui; Jiang, Jiansheng; Feng, Hanqiao; Ghirlando, Rodolfo; Xiao, T Sam; Bai, Yawen


    Linker histones bind to the nucleosome and regulate the structure of chromatin and gene expression. Despite more than three decades of effort, the structural basis of nucleosome recognition by linker histones remains elusive. Here, we report the crystal structure of the globular domain of chicken linker histone H5 in complex with the nucleosome at 3.5 Å resolution, which is validated using nuclear magnetic resonance spectroscopy. The globular domain sits on the dyad of the nucleosome and interacts with both DNA linkers. Our structure integrates results from mutation analyses and previous cross-linking and fluorescence recovery after photobleach experiments, and it helps resolve the long debate on structural mechanisms of nucleosome recognition by linker histones. The on-dyad binding mode of the H5 globular domain is different from the recently reported off-dyad binding mode of Drosophila linker histone H1. We demonstrate that linker histones with different binding modes could fold chromatin to form distinct higher-order structures. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Pyrene intercalating nucleic acids with a carbon linker. (United States)

    Østergaard, Michael E; Wamberg, Michael C; Pedersen, Erik B


    We have synthesized a carbon linker analogue of INA (oligonucleotides containing insertions of 1-O-(1-pyrenylmethyl)glycerol). Thermal stability studies showed an increase in melting temperature in favor of the carbon linker analogue. We also synthesized a carbon linker analogue with two pyrenes geminally attached. Fluorescence studies of this intercalating nucleic acid with the pyrene moieties inserted as a bulge showed formation of an excimer band. When a mismatch was introduced at the site of the intercalator, an excimer band was formed for the destabilized duplexes whereas an exciplex band was observed when the stability of the duplex was retained.

  3. MASK: An efficient mechanism to extend inter-domain IP spoofing preventions

    Institute of Scientific and Technical Information of China (English)

    LU XiCheng; L(U) GaoFeng; ZHU PeiDong; CHEN YiJiao


    IP spoofing hinders the efficiency of DDoS defenses. While recent proposals of IP spoofing prevention mechanisms are weak at filtering spoofing packets due to the complexity in maintaining source IP spaces and the Iow incentive of deployments.To address this problem, we propose an efficient mechanism to extend the range of inter-domain IP spoofing prevention called MASK. Source MASK nodes inform destination MASK nodes about the source IP spaces and labels of their neighbor Stub-ASes in order to implement the marking and verification of packets towards the Stub-ASes, and limit the number of MASK peers through the propagation of BGP updates so as to reduce the overheads of computing and storing of labels. By utilizing the method of extending the spoofing prevention to Stub-ASes, MASK can not only enlarge the domain of the spoofing prevention service, but also filter spoofing packets in advance. Through analysis and simulations, we demonstrate MASK's accuracy and effectiveness.

  4. Long-Range Correlations and Memory in the Dynamics of Internet Interdomain Routing.

    Directory of Open Access Journals (Sweden)

    Maksim Kitsak

    Full Text Available Data transfer is one of the main functions of the Internet. The Internet consists of a large number of interconnected subnetworks or domains, known as Autonomous Systems (ASes. Due to privacy and other reasons the information about what route to use to reach devices within other ASes is not readily available to any given AS. The Border Gateway Protocol (BGP is responsible for discovering and distributing this reachability information to all ASes. Since the topology of the Internet is highly dynamic, all ASes constantly exchange and update this reachability information in small chunks, known as routing control packets or BGP updates. In the view of the quick growth of the Internet there are significant concerns with the scalability of the BGP updates and the efficiency of the BGP routing in general. Motivated by these issues we conduct a systematic time series analysis of BGP update rates. We find that BGP update time series are extremely volatile, exhibit long-term correlations and memory effects, similar to seismic time series, or temperature and stock market price fluctuations. The presented statistical characterization of BGP update dynamics could serve as a basis for validation of existing and developing better models of Internet interdomain routing.

  5. Long-Range Correlations and Memory in the Dynamics of Internet Interdomain Routing. (United States)

    Kitsak, Maksim; Elmokashfi, Ahmed; Havlin, Shlomo; Krioukov, Dmitri


    Data transfer is one of the main functions of the Internet. The Internet consists of a large number of interconnected subnetworks or domains, known as Autonomous Systems (ASes). Due to privacy and other reasons the information about what route to use to reach devices within other ASes is not readily available to any given AS. The Border Gateway Protocol (BGP) is responsible for discovering and distributing this reachability information to all ASes. Since the topology of the Internet is highly dynamic, all ASes constantly exchange and update this reachability information in small chunks, known as routing control packets or BGP updates. In the view of the quick growth of the Internet there are significant concerns with the scalability of the BGP updates and the efficiency of the BGP routing in general. Motivated by these issues we conduct a systematic time series analysis of BGP update rates. We find that BGP update time series are extremely volatile, exhibit long-term correlations and memory effects, similar to seismic time series, or temperature and stock market price fluctuations. The presented statistical characterization of BGP update dynamics could serve as a basis for validation of existing and developing better models of Internet interdomain routing.

  6. Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis. (United States)

    Kachaner, David; Pinson, Xavier; El Kadhi, Khaled Ben; Normandin, Karine; Talje, Lama; Lavoie, Hugo; Lépine, Guillaume; Carréno, Sébastien; Kwok, Benjamin H; Hickson, Gilles R; Archambault, Vincent


    Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.

  7. Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein. (United States)

    Kong, Yuelin; Tong, Yue; Gao, Mingming; Chen, Chen; Gao, Xiangdong; Yao, Wenbing


    Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.

  8. Molecular linker-mediated self-assembly of gold nanoparticles: understanding and controlling the dynamics. (United States)

    Abbas, Abdennour; Kattumenu, Ramesh; Tian, Limei; Singamaneni, Srikanth


    This study sheds light on the mechanism and dynamics of self-assembly of gold nanoparticles (AuNPs) using molecular linkers such as aminothiols. An experimental model is established that enables a fine control and prediction of both assembly rate and degree. Furthermore, we have found that under certain conditions, the increase in the molar ratio of linker/AuNPs beyond a certain threshold unexpectedly and dramatically slows down the assembly rate by charge reversal of the surface of nanoparticles. As a result, the assembly rate can be easily tuned to reach a maximum growth within seconds to several days. The decrease of the same molar ratio (linker/AuNPs) below a certain value leads to self-termination of the reaction at different phases of the assembly process, thus providing nanoparticles chains of different length. This work introduces new handles for a rational design of novel self-assembled architectures in a very time-effective manner and contributes to the understanding of the effect of the assembly morphology on the optical properties of gold nanoparticles.

  9. Assembly fabrication of linkers on glass surface and their effect on DNA synthesis and hybridization

    Institute of Scientific and Technical Information of China (English)

    ShenJiayao; XiaoPengfeng; HouPeng; JiMeiju; SunXiao; HeNongyue


    Linkers were assembled on a glass surface based on the hydrolysis and condensation of 3-glycidoxy-propyltrimethoxysilane (GPS). After the assembly of GPS, four approaches were tried to open the ending epoxide group of GPS or to further elongate the linkers. The effect of these approaches on DNA in situ synthesis and hybridization was investigated. For the spacing of the synthesis initiation sites, the wettability of the support and the length of the linking group that attaches the initiation site to the surface have direct influences on the yield of coupling reactions and the subsequent hybridization events. X-ray photoelectron spectroscopy (XPS) and mean contact angles of deionized water of the above slides were measured to assess the linker's characteristics in each procedure. It was proved that the glass slides were successfully modified and became excellent supports for the oligonucleotides synthesis. In addition, it proved best for the in situ oligonueleotides synthesis that a glass slide was in turn treated with ethylenediamine, glutaradehyde, ethanolamine and sodium borohydride solution at ambient temperature after silanized with GPS.

  10. Electronic structure calculations for the study of D-π-A organic sensitizers: Exploring polythiophene linkers

    Energy Technology Data Exchange (ETDEWEB)

    Climent, Clàudia [Departament de Química Física, Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martí i Franquès, 1-11, 08028 Barcelona (Spain); Casanova, David, E-mail: [IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Euskadi, Spain, and Kimika Fakultea, Euskal Herriko Unibertsitatea (UPV/EHU), Donostia, Euskadi, Spain. (Spain)


    Highlights: • We study D-π-A dyes with polythiophene (Tn) or polycyclopentadithiophene (Cn) linkers. • Molecular geometry plays a crucial role in the photophysical properties of organic dyes. • Cn linkers induce lower transition energies and larger oscillator strengths than Tn separators. • We discuss a variety of computational tools to quantify the CT nature of electronic transitions. • We compute ground and excited state oxidation potentials with a long-range corrected functional. - Abstract: In this work we present a detailed study of the atomic and electronic structure of a collection of push–pull organic dyes for high-performance sensitized solar cells (DSSCs). We compare the computed photophysical properties of donor-bridge-acceptor (D-π-A) dyes with polythiophene (Tn) or polycyclopentadithiophene (Cn) conjugated linkers with up to four fused thiophene rings. Excitation energies to lowest excited singlet state have been rationalized by means of fragment and molecular orbitals. Vertical and adiabatic excitation energies are systematically lower for the Cn family and become smaller with the length of the molecular conjugation. We discuss a large variety of computational techniques for the characterization of the charge transfer (CT) nature of the electronic excitation. In addition to standard procedures to quantify CT character, we propose and explain several novel interaction based measures of CT. Finally, we have computed ground and excited state oxidation potentials (GSOP and ESOP) with long-range corrected (LRC) functional.

  11. Crystallization of Galectin-8 Linker Reveals Intricate Relationship between the N-terminal Tail and the Linker

    Directory of Open Access Journals (Sweden)

    Yunlong Si


    Full Text Available Galectin-8 (Gal-8 plays a significant role in normal immunological function as well as in cancer. This lectin contains two carbohydrate recognition domains (CRD connected by a peptide linker. The N-terminal CRD determines ligand binding specificity, whereas the linker has been proposed to regulate overall Gal-8 function, including multimerization and biological activity. Here, we crystallized the Gal-8 N-terminal CRD with the peptide linker using a crystallization condition that contains Ni2+. The Ni2+ ion was found to be complexed between two CRDs via crystal packing contacts. The coordination between Ni2+ and Asp25 plays an indirect role in determining the structure of β-strand F0 and in influencing the linker conformation which could not be defined due to its dynamic nature. The linker was also shortened in situ and crystallized under a different condition, leading to a higher resolution structure refined to 1.08 Å. This crystal structure allowed definition of a short portion of the linker interacting with the Gal-8 N-terminal tail via ionic interactions and hydrogen bonds. Observation of two Gal-8 N-terminal CRD structures implies that the N-terminal tail and the linker may influence each other’s conformation. In addition, under specific crystallization conditions, glycerol could replace lactose and was observed at the carbohydrate binding site. However, glycerol did not show inhibition activity in hemagglutination assay.

  12. Crystallization of Galectin-8 Linker Reveals Intricate Relationship between the N-terminal Tail and the Linker. (United States)

    Si, Yunlong; Wang, Yue; Gao, Jin; Song, Chenyang; Feng, Shiqiong; Zhou, Yifa; Tai, Guihua; Su, Jiyong


    Galectin-8 (Gal-8) plays a significant role in normal immunological function as well as in cancer. This lectin contains two carbohydrate recognition domains (CRD) connected by a peptide linker. The N-terminal CRD determines ligand binding specificity, whereas the linker has been proposed to regulate overall Gal-8 function, including multimerization and biological activity. Here, we crystallized the Gal-8 N-terminal CRD with the peptide linker using a crystallization condition that contains Ni(2+). The Ni(2+) ion was found to be complexed between two CRDs via crystal packing contacts. The coordination between Ni(2+) and Asp25 plays an indirect role in determining the structure of β-strand F0 and in influencing the linker conformation which could not be defined due to its dynamic nature. The linker was also shortened in situ and crystallized under a different condition, leading to a higher resolution structure refined to 1.08 Å. This crystal structure allowed definition of a short portion of the linker interacting with the Gal-8 N-terminal tail via ionic interactions and hydrogen bonds. Observation of two Gal-8 N-terminal CRD structures implies that the N-terminal tail and the linker may influence each other's conformation. In addition, under specific crystallization conditions, glycerol could replace lactose and was observed at the carbohydrate binding site. However, glycerol did not show inhibition activity in hemagglutination assay.

  13. Photocleavable linker for the patterning of bioactive molecules (United States)

    Wegner, Seraphine V.; Sentürk, Oya I.; Spatz, Joachim P.


    Herein, we report the use of a versatile photocleavable nitrobenzyl linker to micropattern a wide variety of bioactive molecules and photorelease them on demand. On one end, the linker has an NHS group that can be coupled with any amine, such as peptides, proteins or amine-linkers, and on the other end an alkyne for convenient attachment to materials with an azide functional group. This linker was conjugated with NTA-amine or the cell adhesion peptide cRGD to enable straightforward patterning of His6-tagged proteins or cells, respectively, on PEGylated glass surfaces. This approach provides a practical way to control the presentation of a wide variety of bioactive molecules with high spatial and temporal resolution. The extent of photocleavage can also be controlled to tune the biomolecule density and degree of cell attachment to the surface.

  14. Linker histones in hormonal gene regulation. (United States)

    Vicent, G P; Wright, R H G; Beato, M


    In the present review, we summarize advances in our knowledge on the role of the histone H1 family of proteins in breast cancer cells, focusing on their response to progestins. Histone H1 plays a dual role in gene regulation by hormones, both as a structural component of chromatin and as a dynamic modulator of transcription. It contributes to hormonal regulation of the MMTV promoter by stabilizing a homogeneous nucleosome positioning, which reduces basal transcription whereas at the same time promoting progesterone receptor binding and nucleosome remodeling. These combined effects enhance hormone dependent gene transcription, which eventually requires H1 phosphorylation and displacement. Various isoforms of histone H1 have specific functions in differentiated breast cancer cells and compact nucleosomal arrays to different extents in vitro. Genome-wide studies show that histone H1 has a key role in chromatin dynamics of hormone regulated genes. A complex sequence of enzymatic events, including phosphorylation by CDK2, PARylation by PARP1 and the ATP-dependent activity of NURF, are required for H1 displacement and gene de-repression, as a prerequisite for further nucleosome remodeling. Similarly, during hormone-dependent gene repression a dedicated enzymatic mechanism controls H1 deposition at promoters by a complex containing HP1γ, LSD1 and BRG1, the ATPase of the BAF complex. Thus, a broader vision of the histone code should include histone H1, as the linker histone variants actively participate in the regulation of the chromatin structure. How modifications of the core histones tails affect H1 modifications and vice versa is one of the many questions that remains to be addressed to provide a more comprehensive view of the histone cross-talk mechanisms.

  15. Linkers Having a Crucial Role in Antibody–Drug Conjugates


    Jun Lu; Feng Jiang; Aiping Lu; Ge Zhang


    Antibody–drug conjugates (ADCs) comprised of a desirable monoclonal antibody, an active cytotoxic drug and an appropriate linker are considered to be an innovative therapeutic approach for targeted treatment of various types of tumors and cancers, enhancing the therapeutic parameter of the cytotoxic drug and reducing the possibility of systemic cytotoxicity. An appropriate linker between the antibody and the cytotoxic drug provides a specific bridge, and thus helps the antibody to selectively...

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

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


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

  17. Inhibition of HBV targeted ribonuclease enhanced by introduction of linker

    Institute of Scientific and Technical Information of China (English)

    Wei-Dong Gong; Jun Liu; Jin Ding; Ya Zhao; Ying-Hui Li; Cai-Fang Xue


    AIM: To construct human eosinophil-derived neurotoxin (hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly4 Ser)3 between them to optimize the molecule folding, which will be used to inhibit HBV replication in vitro.METHODS: Previously constructed pcDNA3. 1(-)/TR was used as a template. Linker sequence was synthesized and annealed to form dslinker, and cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effector molecule and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunofluorescence staining. Radioimmunoassay was used to analyse anti-HBV activity of pcDNA3.1(-)/TNL.Meanwhile, metabolism of cells was evaluated by NTT colorimetry.RESULTS: hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly4Ser)3 between them was successfully constructed. pcDNA3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed compared to pcDNA3. 1(-)/TR (P=0.036, P<0.05).MTT assay suggested that there were no significant differences between groups (P=0.08, P>0.05).CONCLUSION: Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.

  18. Contribution of linker stability to the activities of anticancer immunoconjugates. (United States)

    Alley, Stephen C; Benjamin, Dennis R; Jeffrey, Scott C; Okeley, Nicole M; Meyer, Damon L; Sanderson, Russell J; Senter, Peter D


    The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technological advancement, since the linker half-lives in circulation ( t 1/2 approximately 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepared containing a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 weeks postadministration in mice. In order to assess the impact of improving linker stability beyond mc containing ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac containing ADCs, although the bac linker technology led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide derivative became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new

  19. Manipulation of electrostatic and saccharide linker interactions in the design of efficient glycopolypeptide-based cholera toxin inhibitors. (United States)

    Maheshwari, Ronak; Levenson, Eric A; Kiick, Kristi L


    Multivalent, glycopolymer inhibitors designed for the treatment of disease and pathogen infection have shown improvements in binding correlated with general changes in glycopolymer architecture and composition. We have previously demonstrated that control of glycopolypeptide backbone extension and ligand spacing significantly impacts the inhibition of the cholera toxin B subunit pentamer (CT B(5)) by these polymers. In the studies reported here, we elucidate the role of backbone charge and linker length in modulating the inhibition event. Peptides of the sequence AXPXG (where X is a positive, neutral or negative amino acid), equipped with the alkyne functionality of propargyl glycine, were designed and synthesized via solid-phase peptide synthetic methods and glycosylated via Cu(I)-catalyzed alkyne-azide cycloaddition reactions. The capacity of the glycopeptides to inhibit the binding of the B(5) subunit of cholera toxin was evaluated. These studies indicated that glycopeptides with a negatively charged backbone show improved inhibition of the binding event relative to the other glycopeptides. In addition, variations in the length of the linker between the peptide and the saccharide ligand also affected the inhibition of CT by the glycopeptides. Our findings suggest that, apart from appropriate saccharide spacing and polypeptide chain extension, saccharide linker conformation and the systematic placement of charges on the polypeptide backbone are also significant variables that can be tuned to improve the inhibitory potencies of glycopolypeptide-based multivalent inhibitors.

  20. De novo transcriptome analysis of the red seaweed Gracilaria chilensis and identification of linkers associated with phycobilisomes. (United States)

    Vorphal, María Alejandra; Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Dagnino-Leone, Jorge; Vásquez, José Aleikar; Martínez-Oyanedel, José; Bunster, Marta


    This work reports the results of the Illumina RNA-Seq of a wild population of female haploid plants of Gracilaria chilensis (Bird et al., 1986) (Rhodophyta, Gigartinalis). Most transcripts were de novo assembled in 12,331 contigs with an average length of 1756bp, showing that 96.64% of the sequences were annotated with known proteins. In particular, the identification of linker proteins of phycobilisomes (PBS) is reported. Linker proteins have primary been identified in cyanobacteria but the information available about them in eukaryotic red alga is not complete, and this is the first report in G. chilensis. This resource will also provide the basis for the study of metabolic pathways related to polysaccharide production.

  1. Novel mixing method for cross linker introduction into droplet emulsions

    CSIR Research Space (South Africa)

    Land, KJ


    Full Text Available TAS 2013, Freiburg, Germany, 27-31 October 2013 NOVEL MIXING METHOD FOR CROSS LINKER INTRODUCTION INTO DROPLET EMULSIONS K.J. Land1, 2*, M.M. Mbanjwa1 and J.G. Korvink2 1CSIR, SOUTH AFRICA and 2IMTEK and FRIAS, GERMANY ABSTRACT Microfluidic...

  2. Flame Length (United States)

    Earth Data Analysis Center, University of New Mexico — Flame length was modeled using FlamMap, an interagency fire behavior mapping and analysis program that computes potential fire behavior characteristics. The tool...

  3. Selected C8 two-chain linkers enhance the adenosine A1/A2A receptor affinity and selectivity of caffeine. (United States)

    van der Walt, M M; Terre'Blanche, G


    Recent research exploring C8 substitution on the caffeine core identified 8-(2-phenylethyl)-1,3,7-trimethylxanthine as a non-selective adenosine receptor antagonist. To elaborate further, we included various C8 two-chain-length linkers to enhance adenosine receptor affinity. The results indicated that the unsubstituted benzyloxy linker (1e A1Ki = 1.52 μM) displayed the highest affinity for the A1 adenosine receptor and the para-chloro-substituted phenoxymethyl (1d A2AKi = 1.33 μM) linker the best A2A adenosine receptor affinity. The position of the oxygen revealed that the phenoxymethyl linker favoured A1 adenosine receptor selectivity over the benzyloxy linker and, by introducing a para-chloro substituent, A2A adenosine receptor selectivity was obtained. Selected compounds (1c, 1e) behaved as A1 adenosine receptor antagonists in GTP shift assays and therefore represent selective and non-selective A1 and A2A adenosine receptor antagonists that may have potential for treating neurological disorders.

  4. Open and Closed: The Roles of Linker Histones in Plants and Animals


    Over, Ryan S.; Michaels, Scott D.


    Linker histones play key roles alongside core histones in the regulation and maintenance of chromatin. Here, we illustrate our current understanding of the contributions of linker histones to the cell cycle, development, and chromatin structure in plants and animals.

  5. An acid-stable tert-butyldiarylsilyl (TBDAS) linker for solid-phase organic synthesis. (United States)

    Diblasi, Christine M; Macks, Daniel E; Tan, Derek S


    [reaction: see text] A new, robust tert-butyldiarylsilyl (TBDAS) linker has been developed for solid-phase organic synthesis. This linker is stable to both protic and Lewis acidic reaction conditions, overcoming a significant limitation of previously reported silyl linkers. Solid-phase acetal deprotection, olefination, asymmetric allylation, and silyl protecting group deblocking reactions have been demonstrated with TBDAS-linked substrates.

  6. Micromachined silicon acoustic delay line with 3D-printed micro linkers and tapered input for improved structural stability and acoustic directivity (United States)

    Cho, Y.; Kumar, A.; Xu, S.; Zou, J.


    Recent studies have shown that micromachined silicon acoustic delay lines can provide a promising solution to achieve real-time photoacoustic tomography without the need for complex transducer arrays and data acquisition electronics. To achieve deeper imaging depth and wider field of view, a longer delay time and therefore delay length are required. However, as the length of the delay line increases, it becomes more vulnerable to structural instability due to reduced mechanical stiffness. In this paper, we report the design, fabrication, and testing of a new silicon acoustic delay line enhanced with 3D printed polymer micro linker structures. First, mechanical deformation of the silicon acoustic delay line (with and without linker structures) under gravity was simulated by using finite element method. Second, the acoustic crosstalk and acoustic attenuation caused by the polymer micro linker structures were evaluated with both numerical simulation and ultrasound transmission testing. The result shows that the use of the polymer micro linker structures significantly improves the structural stability of the silicon acoustic delay lines without creating additional acoustic attenuation and crosstalk. In addition, the improvement of the acoustic acceptance angle of the silicon acoustic delay lines was also investigated to better suppress the reception of unwanted ultrasound signals outside of the imaging plane. These two improvements are expected to provide an effective solution to eliminate current limitations on the achievable acoustic delay time and out-of-plane imaging resolution of micromachined silicon acoustic delay line arrays.

  7. A Small Number of Residues Can Determine if Linker Histones Are Bound On or Off Dyad in the Chromatosome. (United States)

    Zhou, Bing-Rui; Feng, Hanqiao; Ghirlando, Rodolfo; Li, Shipeng; Schwieters, Charles D; Bai, Yawen


    Linker histones bind to the nucleosome and regulate the structure and function of chromatin. We have previously shown that the globular domains of chicken H5 and Drosophila H1 linker histones bind to the nucleosome with on- or off-dyad modes, respectively. To explore the determinant for the distinct binding modes, we investigated the binding of a mutant globular domain of H5 to the nucleosome. This mutant, termed GH5_pMut, includes substitutions of five globular domain residues of H5 with the corresponding residues in the globular domain of Drosophila H1. The residues at these five positions play important roles in nucleosome binding by either H5 or Drosophila H1. NMR and spin-labeling experiments showed that GH5_pMut bound to the nucleosome off the dyad. We further found that the nucleosome array condensed by either the GH5_pMut or the globular domain of Drosophila H1 displayed a similar sedimentation coefficient, whereas the same nucleosome array condensed by the wild-type globular domain of H5 showed a much larger sedimentation coefficient. Moreover, NMR and spin-labeling results from the study of the nucleosome in complex with the full-length human linker histone H1.0, whose globular domain shares high sequence conservation with the corresponding globular domain of H5, are consistent with an on-dyad binding mode. Taken together, our results suggest that a small number of residues in the globular domain of a linker histone can control its binding location on the nucleosome and higher-order chromatin structure. Copyright © 2016. Published by Elsevier Ltd.

  8. Linker polypeptides of the phycobilisome from the cyanobacterium Mastigocladus laminosus. I. Isolation and characterization of phycobiliprotein-linker-polypeptide complexes. (United States)

    Füglistaller, P; Suter, F; Zuber, H


    Phycobilisomes from the cyanobacterium Mastigocladus laminosus cultured in white and red light were isolated and compared with respect to the phycoerythrocyanin (PEC) and linker polypeptide contents. It was verified that the production of PEC is induced by low light intensities. A PEC complex, (alpha PEC beta PEC)6LR34.5,PEC, and a phycocyanin (PC) complex, (alpha PC beta PC)6LR34.5,PC, were isolated from phycobilisomes by Cellex-D anion exchange chromatography and sucrose density gradient centrifugation. The absorption and fluorescence emission maxima of the PEC complex are at 575 and 620 nm and those of the PC complex are at 631 and 647 nm, respectively. The extinction coefficients of the two complexes were determined. From different experiments it was concluded that PEC is present as a hexameric complex, (alpha PEC beta PEC)6LR34.5,PEC, in the phycobilisome. The two linker polypeptides LR34.5,PEC and LR34.5,PC were isolated from their phycobiliprotein complexes by gel filtration on Bio-Gel P-100 in 50% formic acid. A 5-kDa terminal segment of both linker polypeptides was found to influence the hexamer formation of the phycobiliproteins. The same segments have been described to be responsible for the hexamer-hexamer linkage (Yu, M.-H. & Glazer, A.N. (1982) J. Biol. Chem. 257, 3429-3433). A 8.9-kDa linker polypeptide, LR(C)8.9, was isolated from a PEC fraction of the Cellex-D column by Bio-Gel P-100 gel filtration in 50% formic acid. Localisation of this protein within the phycobilisome was attempted. Its most probable function is to terminate the phycobilisomal rods at the end distal to the allophycocyanin core.

  9. Conformational dynamics and the energetics of protein--ligand interactions: role of interdomain loop in human cytochrome P450 reductase. (United States)

    Grunau, Alex; Geraki, Kalotina; Grossmann, J Günter; Gutierrez, Aldo


    A combination of mutagenesis, calorimetry, kinetics, and small-angle X-ray scattering (SAXS) has been used to study the mechanism of ligand binding energy propagation through human cytochrome P450 reductase (CPR). Remarkably, the energetics of 2',5'-ADP binding to R597 at the FAD-binding domain are affected by mutations taking place at an interdomain loop located 60 A away. Either deletion of a 7 amino acid long segment (T236-G237-E238-E239-S240-S241-I242) or its replacement by poly-proline repeats (5 and 10 residues) results in a significant increase in 2',5'-ADP enthalpy of binding (DeltaHB). This is accompanied by a decrease in the number of thermodynamic microstates available for the ligand-CPR complex. Moreover, the estimated heat capacity change (DeltaCp) for this interaction changes from -220 cal mol-1 K-1 in the wild-type enzyme to -580 cal mol-1 K-1 in the deletion mutant. Pre-steady-state kinetics measurements reveal a 50-fold decrease in the microscopic rate for interdomain (FAD --> FMN) electron transfer in the deletion mutant (kobs = 0.4 s-1). Multiple turnover cytochome c reduction assays indicate that these mutations impair the ability of the FMN-binding domain to shuttle electrons from the FAD-binding domain to the cytochrome partner. Binding of 2',5'-ADP to wild-type CPR triggers a large-scale structural rearrangement resulting in the complex having a more compact domain organization, and the maximum molecular dimension (Dmax) decreases from 110 A in ligand-free enzyme to 100 A in the ligand-bound CPR. The SAXS experiments also demonstrate that what is affected by the mutations is indeed the relative diffusional motion of the domains. Furthemore, ab initio shape reconstruction and homology modeling would suggest that-in the deletion mutant-hindering of domain motion occurs concomitantly with dimerization. The results presented here show that the energetics of this highly localized interaction (2',5'-ADP binding) have a global character, and are

  10. 多路径域间路由决策检测协议%Multipath interdomain routing decision verification protocol

    Institute of Scientific and Technical Information of China (English)

    邱修峰; 刘建伟; 伍前红; 刘建华


    由于不同域之间的利益和安全策略存在复杂的竞争与冲突,域间路由安全问题一直具有挑战性。当前相关研究主要集中于路由源可信性与路径信息可信性等方向,但对域间路由策略实施可信性则较少关注。设计了一种多路径域间路由决策检测协议,可验证一个自治域是否遵守从N条目的地相同的候选路径中选择合适的M条路径发送往下游自治域的路由选择决策承诺。分析表明协议正确有效,同时可以抵抗伪造、篡改和重放等攻击,且具有增量部署和尊重参与者隐私的特性。%Because of complicated security policy collisions and twisted interest conflicts between different network do-mains, interdomain routing security has been one challenge of secure routing.At present most of related researches fo-cus on the authentication of routing source and true path, but seldom address another point of whether the routing deci-sion process matches expected policy.A multipath interdomain routing decision verification protocol was designed, which can check whether an AS keeps its promise about choosing appropriate M from N paths that have the same desti-nation and are received from upstream ASs, and sending to a downstream AS.According to the analysis, the protocol is valid and respects the privacy of participants, as well as can resist attacks such as forgery, tampering and replay etc. and deploy on internet incrementally.

  11. Construction of a linker library with widely controllable flexibility for fusion protein design. (United States)

    Li, Gang; Huang, Ziliang; Zhang, Chong; Dong, Bo-Jun; Guo, Ruo-Hai; Yue, Hong-Wei; Yan, Li-Tang; Xing, Xin-Hui


    Flexibility or rigidity of the linker between two fused proteins is an important parameter that affects the function of fusion proteins. In this study, we constructed a linker library with five elementary units based on the combination of the flexible (GGGGS) and the rigid (EAAAK) units. Molecular dynamics (MD) simulation showed that more rigid units in the linkers lead to more helical conformation and hydrogen bonds, and less distance fluctuation between the N- and C-termini of the linker. The diversity of linker flexibility of the linker library was then studied by fluorescence resonance energy transfer (FRET) of cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) fusion proteins, which showed that there is a wide range of distribution of the FRET efficiency. Dissipative particle dynamics (DPD) simulation of CFP-YFP with different linkers also gave identical results with that of FRET efficiency analysis, and we further found that the combination manner of the linker peptide had a remarkable effect on the orientation of CFP and YFP domains. Our studies demonstrated that the construction of the linker library with the widely controllable flexibility could provide appropriate linkers with the desirable characteristics to engineer the fusion proteins with the expected functions.

  12. Nucleosomes, Linker DNA, and Linker Histone form a Unique Structural Motif that Directs the Higher-Order Folding and Compaction of Chromatin (United States)

    Bednar, Jan; Horowitz, Rachel A.; Grigoryev, Sergei A.; Carruthers, Lenny M.; Hansen, Jeffrey C.; Koster, Abraham J.; Woodcock, Christopher L.


    The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes ≈ 1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed ≈ 8 nm from the nucleosome center and remain apposed for 3-5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

  13. Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge. (United States)

    Vishnivetskiy, Sergey A; Hirsch, Joel A; Velez, Maria-Gabriela; Gurevich, Yulia V; Gurevich, Vsevolod V


    Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.

  14. Multifunctionality of the linker histones: an emerging role for protein-protein interactions. (United States)

    McBryant, Steven J; Lu, Xu; Hansen, Jeffrey C


    Linker histones, e.g., H1, are best known for their ability to bind to nucleosomes and stabilize both nucleosome structure and condensed higher-order chromatin structures. However, over the years many investigators have reported specific interactions between linker histones and proteins involved in important cellular processes. The purpose of this review is to highlight evidence indicating an important alternative mode of action for H1, namely protein-protein interactions. We first review key aspects of the traditional view of linker histone action, including the importance of the H1 C-terminal domain. We then discuss the current state of knowledge of linker histone interactions with other proteins, and, where possible, highlight the mechanism of linker histone-mediated protein-protein interactions. Taken together, the data suggest a combinatorial role for the linker histones, functioning both as primary chromatin architectural proteins and simultaneously as recruitment hubs for proteins involved in accessing and modifying the chromatin fiber.

  15. Melting transition in lipid vesicles functionalised by mobile DNA linkers. (United States)

    Bachmann, Stephan Jan; Kotar, Jurij; Parolini, Lucia; Šarić, Anđela; Cicuta, Pietro; Di Michele, Lorenzo; Mognetti, Bortolo Matteo


    We study phase behaviour of lipid-bilayer vesicles functionalised by ligand-receptor complexes made of synthetic DNA by introducing a modelling framework and a dedicated experimental platform. In particular, we perform Monte Carlo simulations that combine a coarse grained description of the lipid bilayer with state of art analytical models for multivalent ligand-receptor interactions. Using density of state calculations, we derive the partition function in pairs of vesicles and compute the number of ligand-receptor bonds as a function of temperature. Numerical results are compared to microscopy and fluorimetry experiments on large unilamellar vesicles decorated by DNA linkers carrying complementary overhangs. We find that vesicle aggregation is suppressed when the total number of linkers falls below a threshold value. Within the model proposed here, this is due to the higher configurational costs required to form inter-vesicle bridges as compared to intra-vesicle loops, which are in turn related to membrane deformability. Our findings and our numerical/experimental methodologies are applicable to the rational design of liposomes used as functional materials and drug delivery applications, as well as to study inter-membrane interactions in living systems, such as cell adhesion.

  16. Construction and Purification of EGF-Linker-TCSKDEL Fusion Proteins%EGF-Linker-TCSKDEL融合蛋白的构建及表达产物的纯化

    Institute of Scientific and Technical Information of China (English)



    目的构建EGF-Linker-TCSKDEL融合蛋白表达载体并纯化目的蛋白。方法以天花粉蛋白基因的改造产物TCSKDEL为毒素,人表皮生长因子EGFR为载体,构建重组免疫毒素EGF-Linker-TCSKDEL。PCR方法扩增EGF-Linker-TCSKDEL基因片段,插入表达载体PET28a中并诱导表达,使用Ni-NTA Agrose亲合层析纯化。结果该融合蛋白以可溶形式表达于大肠杆菌培养上清中,并获得有效纯化。结论成功构建了新型融合蛋白EGF-Linker-TCSKDEL,为其进一步的基础和临床研究奠定基础。%Objective To construct the expression vector of EGF-Linker-TCSKDEL fusion proteins and purify target protein. Methods Recombinant immunotoxin (EGF-Linker-TCSKDEL) was constructed by modified trichosanthin (TCS) with KDEL as toxin and epidermal growth factor (EGF) as a car ier. The DNA fragment of EGF-Linker-TCSKDEL was amplified by using PCR, and the fusion proteins were purified by Ni-NTA Agrose af inity chromatography. Results The fusion protein was expressed in a form of soluble protein and purified ef ectively using af inity chromatography. Conclusion A new EGF-Linker-TCSKDEL fusion proteins was successful y prepared. It laid a foundation of further research in basic and clinical of EGF-Linker-TCSKDEL.

  17. Tunable CO 2 Adsorbents by Mixed-Linker Synthesis and Postsynthetic Modification of Zeolitic Imidazolate Frameworks

    KAUST Repository

    Thompson, Joshua A.


    The incorporation of accessible amine functionality in zeolitic imidazolate frameworks (ZIFs) is used to improve the adsorption selectivity for CO 2/CH4 gas separation applications. Two synthetic approaches are described in this work to introduce functionality into the ZIF: (i) mixed-linker ZIF synthesis with 2-aminobenzimidazole as a substitution linker and (ii) postsynthetic modification of a mixed-linker ZIF with ethylenediamine. Using 2-aminobenzimidazole, a linker with a primary amine functional group, substitution of the ZIF-8 linker during synthesis allows for control over the adsorption properties while maintaining the ZIF-8 structure with up to nearly 50% substitution in the mixed-linker ZIF framework, producing a material with tunable pore size and amine functionality. Alternatively, postsynthetic modification of a mixed-linker ZIF containing an aldehyde functional group produces a ZIF material with a primary amine without detrimental loss of micropore volume by controlling the amount of functional group sites for modification. Both approaches using mixed-linker ZIFs yield new materials that show improvement in adsorption selectivity for the CO 2/CH4 gas pair over ZIF-8 and commercially available adsorbents as well as an increase in the heat of adsorption for CO2 without significant changes to the crystal structure. These results indicate that tuning the surface properties of ZIFs by either mixed-linker synthesis and/or postsynthetic modification may generate new materials with improved gas separation properties, thereby providing a new method for tailoring metal-organic frameworks. © 2013 American Chemical Society.

  18. Desorption of 3,3′-diindolylmethane from imprinted particles: An impact of cross-linker structure on binding capacity and selectivity

    Energy Technology Data Exchange (ETDEWEB)

    Klejn, Dorota; Luliński, Piotr; Maciejewska, Dorota, E-mail:


    Here, seven cross-linkers (six polar diacrylates or dimethacrylates of different lengths between double bonds, and one aromatic-divinylbenzene) were used to examine the impact of the cross-linker on binding capacity and selectivity of 3,3′-diindolylmethane (DIM) imprinted material. DIM participates in the suppression of viability of human ovarian and human breast cancer cell lines, but has low bioavailability. The investigations of novel imprinted polymer matrices for improvement of DIM release could allow to utilize not only a potency of DIM but also similar alkaloids, which are the important compounds with pharmacological activity. The bulk, thermal radical copolymerization of the cross-linkers in the presence of 3,3′-diindolylmethane (the template) and allylamine (the functional monomer) in dimethyl sulfoxide or in carbon tetrachloride (porogens) was carried out. The binding capacities of imprinted and non-imprinted polymers were compared, and two polymers (these were prepared using ethylene glycol dimethacrylate and polyethylene glycol dimethacrylate as the cross-linkers) with the highest selectivity and binding capacity were selected to desorption test. The desorption profile of polymer prepared using polyethylene glycol dimethacrylate as the cross-linker revealed sustained release of 3,3′-diindolylmethane, and this system was selected for further optimization of the cross-linker amounts. The morphology and structure of the selected particles were analyzed using SEM micrographs, {sup 13}C CP/MAS NMR spectroscopy, and BET measurements. The desorption of 3,3′-diindolylmethane from poly(allylamine-co-polyethylene glycol dimethacrylate) particles was in accordance with pseudo-second-order kinetics and the simplified Higuchi model indicated the diffusion controlled release of 3,3′-diindolylmethane. - Graphical abstract: Sustained release of 3,3′-diindolylmethane from cavity in imprinted poly(allylamine-co-polyethylene glycol dimethacrylate

  19. SWI/SNF chromatin remodeling and linker histones in plants. (United States)

    Jerzmanowski, Andrzej


    In yeast and mammals, ATP-dependent chromatin remodeling complexes belonging to the SWI/SNF family play critical roles in the regulation of transcription, cell proliferation, differentiation and development. Homologs of conserved subunits of SWI/SNF-type complexes, including several putative ATPases and other core subunits, have been identified in plants. Here I summarize recent insights in structural organization and functional diversification of putative plant SWI/SNF-type chromatin remodeling complexes and discuss in a broader evolutionary perspective the similarities and differences between plant and yeast/animal SWI/SNF remodeling. I also summarize the current view of localization in nucleosome and dynamic behaviour in chromatin of linker (H1) histones and discuss significance of recent findings indicating that in both plants and mammals histone H1 is involved in determining patterns of DNA methylation at selected loci.

  20. Acylsulfonamide safety-catch linker: promise and limitations for solid–phase oligosaccharide synthesis

    Directory of Open Access Journals (Sweden)

    Jian Yin


    Full Text Available Safety-catch linkers are useful for solid-phase oligosaccharide synthesis as they are orthogonal to many common protective groups. A new acylsulfonamide safety-catch linker was designed, synthesized and employed during glycosylations using an automated carbohydrate synthesizer. The analysis of the cleavage products revealed shortcomings for oligosaccharide synthesis.

  1. Description of a cellulose-binding domain and a linker sequence from Aspergillus fungi

    NARCIS (Netherlands)

    Quentin, M; Ebbelaar, M; Derksen, J; Mariani, C; van der Valk, H

    A family I cellulose-binding domain (CBD) and a serine- and threonine-rich linker peptide were cloned from the fungi Aspergillus japonicus and Aspergillus aculeatus. A glutathione S-transferase (GST) fusion protein comprising GST and a peptide linker with the CBD fused to its C-terminus, was

  2. Linkers, resins, and general procedures for solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Shelton, Anne Pernille Tofteng; Jensen, Knud Jørgen


    and linkers for solid-phase synthesis is a key parameter for successful peptide synthesis. This chapter provides an overview of the most common and useful resins and linkers for the synthesis of peptides with C-terminal amides, carboxylic acids, and more. The chapter finishes with robust protocols for general...

  3. Detection of ligation products of DNA linkers with 5'-OH ends by denaturing PAGE silver stain.

    Directory of Open Access Journals (Sweden)

    Feng Gao

    Full Text Available To explore if DNA linkers with 5'-hydroxyl (OH ends could be joined by commercial T4 and E. coli DNA ligase, these linkers were synthesized by using the solid-phase phosphoramidite method and joined by using commercial T4 and E. coli DNA ligases. The ligation products were detected by using denaturing PAGE silver stain and PCR method. About 0.5-1% of linkers A-B and E-F, and 0.13-0.5% of linkers C-D could be joined by T4 DNA ligases. About 0.25-0.77% of linkers A-B and E-F, and 0.06-0.39% of linkers C-D could be joined by E. coli DNA ligases. A 1-base deletion (-G and a 5-base deletion (-GGAGC could be found at the ligation junctions of the linkers. But about 80% of the ligation products purified with a PCR product purification kit did not contain these base deletions, meaning that some linkers had been correctly joined by T4 and E. coli DNA ligases. In addition, about 0.025-0.1% of oligo 11 could be phosphorylated by commercial T4 DNA ligase. The phosphorylation products could be increased when the phosphorylation reaction was extended from 1 hr to 2 hrs. We speculated that perhaps the linkers with 5'-OH ends could be joined by T4 or E. coli DNA ligase in 2 different manners: (i about 0.025-0.1% of linkers could be phosphorylated by commercial T4 DNA ligase, and then these phosphorylated linkers could be joined to the 3'-OH ends of other linkers; and (ii the linkers could delete one or more nucleotide(s at their 5'-ends and thereby generated some 5'-phosphate ends, and then these 5'-phosphate ends could be joined to the 3'-OH ends of other linkers at a low efficiency. Our findings may probably indicate that some DNA nicks with 5'-OH ends can be joined by commercial T4 or E. coli DNA ligase even in the absence of PNK.

  4. Construction of hierarchically porous metal-organic frameworks through linker labilization (United States)

    Yuan, Shuai; Zou, Lanfang; Qin, Jun-Sheng; Li, Jialuo; Huang, Lan; Feng, Liang; Wang, Xuan; Bosch, Mathieu; Alsalme, Ali; Cagin, Tahir; Zhou, Hong-Cai


    A major goal of metal-organic framework (MOF) research is the expansion of pore size and volume. Although many approaches have been attempted to increase the pore size of MOF materials, it is still a challenge to construct MOFs with precisely customized pore apertures for specific applications. Herein, we present a new method, namely linker labilization, to increase the MOF porosity and pore size, giving rise to hierarchical-pore architectures. Microporous MOFs with robust metal nodes and pro-labile linkers were initially synthesized. The mesopores were subsequently created as crystal defects through the splitting of a pro-labile-linker and the removal of the linker fragments by acid treatment. We demonstrate that linker labilization method can create controllable hierarchical porous structures in stable MOFs, which facilitates the diffusion and adsorption process of guest molecules to improve the performances of MOFs in adsorption and catalysis.

  5. Molecular Cross-Talk between Nonribosomal Peptide Synthetase Carrier Proteins and Unstructured Linker Regions. (United States)

    Harden, Bradley J; Frueh, Dominique P


    Nonribosomal peptide synthetases (NRPSs) employ multiple domains separated by linker regions to incorporate substrates into natural products. During synthesis, substrates are covalently tethered to carrier proteins that translocate between catalytic partner domains. The molecular parameters that govern translocation and associated linker remodeling remain unknown. Here, we used NMR to characterize the structure, dynamics, and invisible states of a peptidyl carrier protein flanked by its linkers. We showed that the N-terminal linker stabilizes and interacts with the protein core while modulating dynamics at specific sites involved in post-translational modifications and/or domain interactions. The results detail the molecular communication between peptidyl carrier proteins and their linkers and could guide efforts in engineering NRPSs to obtain new pharmaceuticals.

  6. On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes (United States)

    Chen, Fan; Gerber, Sabina; Korkhov, Volodymyr M.; Mireku, Samantha; Bucher, Monika; Locher, Kaspar P.; Zenobi, Renato


    We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes. Here, we investigated the capability of N-hydroxysuccinimide (NHS) esters, which react much more specifically, to cross-link membrane protein complexes such as PglK and BtuC2D2. We present clear evidence that NHS esters are capable of stabilizing membrane protein complexes in situ, in the presence of detergents such as DDM, C12E8, and LDAO. The stabilization efficiency strongly depends on the membrane protein structure (i.e, the number of primary amine groups and the distances between primary amines). A minimum number of primary amine groups is required, and the distances between primary amines govern whether a cross-linker with a specific spacer arm length is able to bridge two amine groups.

  7. Enhancing antisense efficacy with multimers and multi-targeting oligonucleotides (MTOs) using cleavable linkers. (United States)

    Subramanian, Romesh R; Wysk, Mark A; Ogilvie, Kathleen M; Bhat, Abhijit; Kuang, Bing; Rockel, Thomas D; Weber, Markus; Uhlmann, Eugen; Krieg, Arthur M


    The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8-15 nucleotides and by the incorporation of high affinity RNA binders such as 2', 4'-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2',4'-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4-5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Effect of the linkers between the zinc fingers in zinc finger protein 809 on gene silencing and nuclear localization

    Energy Technology Data Exchange (ETDEWEB)

    Ichida, Yu, E-mail:; Utsunomiya, Yuko; Onodera, Masafumi


    Zinc finger protein 809 (ZFP809) belongs to the Kruppel-associated box-containing zinc finger protein (KRAB-ZFP) family and functions in repressing the expression of Moloney murine leukemia virus (MoMLV). ZFP809 binds to the primer-binding site (PBS)located downstream of the MoMLV-long terminal repeat (LTR) and induces epigenetic modifications at integration sites, such as repressive histone modifications and de novo DNA methylation. KRAB-ZFPs contain consensus TGEKP linkers between C2H2 zinc fingers. The phosphorylation of threonine residues within linkers leads to the inactivation of zinc finger binding to target sequences. ZFP809 also contains consensus linkers between zinc fingers. However, the function of ZFP809 linkers remains unknown. In the present study, we constructed ZFP809 proteins containing mutated linkers and examined their ability to silence transgene expression driven by MLV, binding ability to MLV PBS, and cellular localization. The results of the present study revealed that the linkers affected the ability of ZFP809 to silence transgene expression. Furthermore, this effect could be partly attributed to changes in the localization of ZFP809 proteins containing mutated linkers. Further characterization of ZFP809 linkers is required for understanding the functions and features of KRAB-ZFP-containing linkers. - Highlights: • ZFP809 has three consensus linkers between the zinc fingers. • Linkers are required for ZFP809 to silence transgene expression driven by MLV-LTR. • Linkers affect the precise nuclear localization of ZFP809.

  9. Calcium binding by the PKD1 domain regulates interdomain flexibility in Vibrio cholerae metalloprotease PrtV. (United States)

    Edwin, Aaron; Rompikuntal, Pramod; Björn, Erik; Stier, Gunter; Wai, Sun N; Sauer-Eriksson, A Elisabeth


    Vibrio cholerae, the causative agent of cholera, releases several virulence factors including secreted proteases when it infects its host. These factors attack host cell proteins and break down tissue barriers and cellular matrix components such as collagen, laminin, fibronectin, keratin, elastin, and they induce necrotic tissue damage. The secreted protease PrtV constitutes one virulence factors of V. cholerae. It is a metalloprotease belonging to the M6 peptidase family. The protein is expressed as an inactive, multidomain, 102 kDa pre-pro-protein that undergoes several N- and C-terminal modifications after which it is secreted as an intermediate variant of 81 kDa. After secretion from the bacteria, additional proteolytic steps occur to produce the 55 kDa active M6 metalloprotease. The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium. However, the molecular mechanism by which calcium controls proteolysis is unknown. In this study, we report the atomic resolution crystal structure of the PKD1 domain from V. cholera PrtV (residues 755-838) determined at 1.1 Å. The structure reveals a previously uncharacterized Ca(2+)-binding site located near linker regions between domains. Conformational changes in the Ca(2+)-free and Ca(2+)-bound forms suggest that Ca(2+)-binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein.

  10. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation. (United States)

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia


    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Gelatin-based biomaterial engineering with anhydride-containing oligomeric cross-linkers. (United States)

    Loth, Tina; Hötzel, Rudi; Kascholke, Christian; Anderegg, Ulf; Schulz-Siegmund, Michaela; Hacker, Michael C


    Chemically cross-linked gelatin hydrogels are versatile cell-adhesive hydrogel materials that have been established for a variety of biomedical applications. The most prominent cross-linker is glutaraldehyde, which, however, has been described to cause compatibility problems and loss of microscopic but relevant structural features. A recently developed oligomeric cross-linker that contains anhydride functionalities was evaluated as cross-linker for the fabrication of gelatin-based hydrogels and microparticles. In a fast curing reaction, hydrogels composed of gelatin and oligomeric cross-linker were fabricated with good conversion over a wide concentration range of constituents and with cross-linkers of different anhydride contents. Hydrogel properties, such as dry weight and mechanics, could be controlled by hydrogel composition and rheological properties correlated to elastic moduli from 1 to 10 kPa. The gels were shown to be cytocompatible and promoted cell adhesion. In soft formulations, cells migrated into the hydrogel bulk. Gelatin microparticles prepared by a standard water-in-oil emulsion technique were also treated with the novel oligomers, and cross-linking degrees matching those obtained with glutaraldehyde were obtained. At the same time, fewer interparticular cross-links were observed. Fluorescein-derivatized cross-linkers yielded labeled microparticles in a concentration-dependent manner. The oligomeric cross-linkers are presented as an efficient and possibly more functional and compatible alternative to glutaraldehyde. The engineered hydrogel materials hold potential for various biomedical applications.

  12. Studies on metal-organic frameworks of Cu(II) with isophthalate linkers for hydrogen storage. (United States)

    Yan, Yong; Yang, Sihai; Blake, Alexander J; Schröder, Martin


    Hydrogen (H2) is a promising alternative energy carrier because of its environmental benefits, high energy density, and abundance. However, development of a practical storage system to enable the "Hydrogen Economy" remains a huge challenge. Metal-organic frameworks (MOFs) are an important class of crystalline coordination polymers constructed by bridging metal centers with organic linkers. MOFs show promise for H2 storage owing to their high surface area and tuneable properties. In this Account, we summarize our research on novel porous materials with enhanced H2 storage properties and describe frameworks derived from 3,5-substituted dicarboxylates (isophthalates) that serve as versatile molecular building blocks for the construction of a range of interesting coordination polymers with Cu(II) ions. We synthesized a series of materials by connecting linear tetracarboxylate linkers to {Cu(II)2} paddlewheel moieties. These materials exhibit high structural stability and permanent porosity. Varying the organic linker modulates the pore size, geometry, and functionality to control the overall H2 adsorption. Our top-performing material in this series has a H2 storage capacity of 77.8 mg g(-1) at 77 K, 60 bar. H2 adsorption at low, medium, and high pressures correlates with the isosteric heat of adsorption, surface area, and pore volume, respectively. Another series, using tribranched C3-symmetric hexacarboxylate ligands with Cu(II), gives highly porous (3,24)-connected frameworks incorporating {Cu(II)2} paddlewheels. Increasing the length of the hexacarboxylate struts directly tunes the porosity of the resultant material from micro- to mesoporosity. These materials show exceptionally high H2 uptakes owing to their high surface area and pore volume. The first member of this family reported adsorbs 111 mg g(-1) of H2, or 55.9 g L(-1), at 77 K, 77 bar, while at 77 K, 1 bar, the material adsorbs 2.3 wt % H2. We and others have since achieved enhanced H2 adsorption in these

  13. Mobile linkers on DNA-coated colloids: valency without patches. (United States)

    Angioletti-Uberti, Stefano; Varilly, Patrick; Mognetti, Bortolo M; Frenkel, Daan


    Colloids coated with single-stranded DNA (ssDNA) can bind selectively to other colloids coated with complementary ssDNA. The fact that DNA-coated colloids (DNACCs) can bind to specific partners opens the prospect of making colloidal "molecules." However, in order to design DNACC-based molecules, we must be able to control the valency of the colloids, i.e., the number of partners to which a given DNACC can bind. One obvious, but not very simple approach is to decorate the colloidal surface with patches of single-stranded DNA that selectively bind those on other colloids. Here we propose a design principle that exploits many-body effects to control the valency of otherwise isotropic colloids. Using a combination of theory and simulation, we show that we can tune the valency of colloids coated with mobile ssDNA, simply by tuning the nonspecific repulsion between the particles. Our simulations show that the resulting effective interactions lead to low-valency colloids self-assembling in peculiar open structures, very different from those observed in DNACCs with immobile DNA linkers.

  14. Distinct binding properties of TIAR RRMs and linker region. (United States)

    Kim, Henry S; Headey, Stephen J; Yoga, Yano M K; Scanlon, Martin J; Gorospe, Myriam; Wilce, Matthew C J; Wilce, Jacqueline A


    The RNA-binding protein TIAR is an mRNA-binding protein that acts as a translational repressor, particularly important under conditions of cellular stress. It binds to target mRNA and DNA via its RNA recognition motif (RRM) domains and is involved in both splicing regulation and translational repression via the formation of "stress granules." TIAR has also been shown to bind ssDNA and play a role in the regulation of transcription. Here we show, using surface plasmon resonance and nuclear magnetic resonance spectroscopy, specific roles of individual TIAR domains for high-affinity binding to RNA and DNA targets. We confirm that RRM2 of TIAR is the major RNA- and DNA-binding domain. However, the strong nanomolar affinity binding to U-rich RNA and T-rich DNA depends on the presence of the six amino acid residues found in the linker region C-terminal to RRM2. On its own, RRM1 shows preferred binding to DNA over RNA. We further characterize the interaction between RRM2 with the C-terminal extension and an AU-rich target RNA sequence using NMR spectroscopy to identify the amino acid residues involved in binding. We demonstrate that TIAR RRM2, together with its C-terminal extension, is the major contributor for the high-affinity (nM) interactions of TIAR with target RNA sequences.

  15. Library of biphenyl privileged substructures using a safety-catch linker approach

    DEFF Research Database (Denmark)

    Severinsen, Rune; Bourne, Gregory T; Tran, Tran T


    A biphenyl privileged structure library containing three attachment points were synthesized using a catechol-based safety-catch linker strategy. The method requires the attachment of a bromo-acid to the linker, followed by a Pd-catalyzed Suzuki cross-coupling reaction. Further derivatization......, activation of the linker with strong acid and aminolysis afforded the respective products in high purity and good overall yield. To show the versatility of the synthesis, a 199-member library was generated. The library samples both conformational and chemical diversity about a well-known privileged...

  16. A Traceless Aryl-Triazene Linker for DNA-Directed Chemistry

    DEFF Research Database (Denmark)

    Hejesen, Christian; Pedersen, Lars Kolster; Gothelf, Kurt Vesterager


    DNA-directed synthesis of encoded combinatorial libraries of small organic compounds most often involves transfer of organic building blocks from one DNA strand to another. This requires cleavable linkers to enable cleavage of the link to the original DNA strand from which the building block...... is transferred. Relatively few cleavable linkers are available for DNA-directed synthesis and most often they leave an amino group at the organic molecule. Here we have extended the application of 10 aryltriazenes as traceless linkers for DNA-directed synthesis. After reaction of one building block...

  17. A Traceless Aryl-Triazene Linker for DNA-Directed Chemistry

    DEFF Research Database (Denmark)

    Hejesen, Christian; Pedersen, Lars Kolster; Gothelf, Kurt Vesterager


    DNA-directed synthesis of encoded combinatorial libraries of small organic compounds most often involves transfer of organic building blocks from one DNA strand to another. This requires cleavable linkers to enable cleavage of the link to the original DNA strand from which the building block...... is transferred. Relatively few cleavable linkers are available for DNA-directed synthesis and most often they leave an amino group at the organic molecule. Here we have extended the application of 10 aryltriazenes as traceless linkers for DNA-directed synthesis. After reaction of one building block...

  18. Design and Synthesis of a Dual Linker for Solid Phase Synthesis of Oleanolic Acid Derivatives

    Directory of Open Access Journals (Sweden)

    Shaorong Wang


    Full Text Available A hydrophilic amino-terminated poly(ethylene glycol-type dual linker for solid phase synthesis of oleanolic acid derivatives using trityl chloride resin was designed and synthesized for the first time. Model reactions in both liquid and solid phase were performed to show the feasibility of its selective cleavage at two different sites. The biological assay results indicated that the long and flexible alkyl ether functionality in the linker is less likely to be critical for the binding event. Following the successful solid-phase synthesis of model compounds, the potential of this dual linker in reaction monitoring and target identification is deemed worthy of further study.

  19. A new highly versatile handle for chemistry on a solid support: the pipecolic linker. (United States)

    Zajdel, Paweł; Nomezine, Gaël; Masurier, Nicolas; Amblard, Muriel; Pawłowski, Maciej; Martinez, Jean; Subra, Gilles


    The design, synthesis, and potential application of the pipecolic linker is presented. This new versatile handle can immobilize primary, secondary, and aromatic amines, as well as alcohols, phenols, and hydrazides, on a solid support. Compared with other linkers, the anchoring step is easy and efficient. The release of final products from the resin proceeds upon acidic treatment with high purities. The pipecolic linker offers the promise of being using in peptide chemistry to produce peptides modified at the N and C terminus, peptidomimetics, as well as small organic molecules.

  20. Fusing two cytochromes b of Rhodobacter capsulatus cytochrome bc1 using various linkers defines a set of protein templates for asymmetric mutagenesis. (United States)

    Czapla, Monika; Borek, Arkadiusz; Sarewicz, Marcin; Osyczka, Artur


    Cytochrome bc(1) (mitochondrial complex III), one of the key enzymes of biological energy conversion, is a functional homodimer in which each monomer contains three catalytic subunits: cytochrome c(1), the iron-sulfur subunit and cytochrome b. The latter is composed of eight transmembrane α-helices which, in duplicate, form a hydrophobic core of a dimer. We show that two cytochromes b can be fused into one 16-helical subunit using a number of different peptide linkers that vary in length but all connect the C-terminus of one cytochrome with the N-terminus of the other. The fusion proteins replace two cytochromes b in the dimer defining a set of available protein templates for introducing mutations that allow breaking symmetry of a dimer. A more detailed comparison of the form with the shortest, 3 amino acid, linker to the form with 12 amino acid linker established that both forms display similar level of structural plasticity to accommodate several, but not all, asymmetric patterns of mutations that knock out individual segments of cofactor chains. While the system based on a fused gene does not allow for the assessments of the functionality of electron-transfer paths in vivo, the family of proteins with fused cytochrome b offers attractive model for detailed investigations of molecular mechanism of catalysis at in vitro/reconstitution level.

  1. Learning a weighted sequence model of the nucleosome core and linker yields more accurate predictions in Saccharomyces cerevisiae and Homo sapiens.

    Directory of Open Access Journals (Sweden)

    Sheila M Reynolds

    Full Text Available DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence-301 base pairs, centered at the position to be scored-with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the

  2. Learning a weighted sequence model of the nucleosome core and linker yields more accurate predictions in Saccharomyces cerevisiae and Homo sapiens. (United States)

    Reynolds, Sheila M; Bilmes, Jeff A; Noble, William Stafford


    DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence-301 base pairs, centered at the position to be scored-with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the bulk of the

  3. Two Principles of Reticular Chemistry Uncovered in a Metal-Organic Framework of Heterotritopic Linkers and Infinite Secondary Building Units. (United States)

    Catarineu, Noelle R; Schoedel, Alexander; Urban, Philipp; Morla, Maureen B; Trickett, Christopher A; Yaghi, Omar M


    Structural diversity of metal-organic frameworks (MOFs) has been largely limited to linkers with at most two different types of coordinating groups. MOFs constructed from linkers with three or more nonidentical coordinating groups have not been explored. Here, we report a robust and porous crystalline MOF, Zn3(PBSP)2 or MOF-910, constructed from a novel linker PBSP (phenylyne-1-benzoate, 3-benzosemiquinonate, 5-oxidopyridine) bearing three distinct types of coordinative functionality. The MOF adopts a complex and previously unreported topology termed tto. Our study suggests that simple, symmetric linkers are not a necessity for formation of crystalline extended structures and that new, more complex topologies are attainable with irregular, heterotopic linkers. This work illustrates two principles of reticular chemistry: first, selectivity for helical over straight rod secondary building units (SBUs) is achievable with polyheterotopic linkers, and second, the pitch of the resulting helical SBUs may be fine-tuned based on the metrics of the polyheterotopic linker.

  4. Structural analysis of the S4-S5 linker of the human KCNQ1 potassium channel. (United States)

    Gayen, Shovanlal; Li, Qingxin; Kang, CongBao


    KCNQ1 plays important roles in the cardiac action potential and consists of an N-terminal domain, a voltage-sensor domain, a pore domain and a C-terminal domain. KCNQ1 is a voltage-gated potassium channel and its channel activity is regulated by membrane potentials. The linker between transmembrane helices 4 and 5 (S4-S5 linker) is important for transferring the conformational changes from the voltage-sensor domain to the pore domain. In this study, the structure of the S4-S5 linker of KCNQ1 was investigated by solution NMR, circular dichroism and fluorescence spectroscopic studies. The S4-S5 linker adopted a helical structure in detergent micelles. The W248 may interact with the cell membrane.

  5. Tethering metal ions to photocatalyst particulate surfaces by bifunctional molecular linkers for efficient hydrogen evolution

    KAUST Repository

    Yu, Weili


    A simple and versatile method for the preparation of photocatalyst particulates modified with effective cocatalysts is presented; the method involves the sequential soaking of photocatalyst particulates in solutions containing bifunctional organic linkers and metal ions. The modification of the particulate surfaces is a universal and reproducible method because the molecular linkers utilize strong covalent bonds, which in turn result in modified monolayer with a small but controlled quantity of metals. The photocatalysis results indicated that the CdS with likely photochemically reduced Pd and Ni, which were initially immobilized via ethanedithiol (EDT) as a linker, were highly efficient for photocatalytic hydrogen evolution from Na2S-Na2SO3-containing aqueous solutions. The method developed in this study opens a new synthesis route for the preparation of effective photocatalysts with various combinations of bifunctional linkers, metals, and photocatalyst particulate materials. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. LRRC45 Is a Centrosome Linker Component Required for Centrosome Cohesion

    Directory of Open Access Journals (Sweden)

    Runsheng He


    Full Text Available During interphase, centrosomes are connected by a proteinaceous linker between the proximal ends of the centrioles, which is important for the centrosomes to function as a single microtubule-organizing center. However, the composition and regulation of centrosomal linker remain largely unknown. Here, we show that LRRC45 is a centrosome linker that localizes at the proximal ends of the centrioles and forms fiber-like structures between them. Depletion of LRRC45 results in centrosome splitting during interphase. Moreover, LRRC45 interacts with both C-Nap1 and rootletin and is phosphorylated by Nek2A at S661 during mitosis. After phosphorylation, both LRRC45 centrosomal localization and fiber-like structures are significantly reduced, which subsequently leads to centrosome separation. Thus, LRRC45 is a critical component of the proteinaceous linker between two centrioles and is required for centrosome cohesion.

  7. The Use of Aryl Hydrazide Linkers for the Solid Phase Synthesis of Chemically Modified Peptides

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Y; Mitchell, A R; Camarero, J A


    Since Merrifield introduced the concept of solid phase synthesis in 1963 for the rapid preparation of peptides, a large variety of different supports and resin-linkers have been developed that improve the efficiency of peptide assembly and expand the myriad of synthetically feasible peptides. The aryl hydrazide is one of the most useful resin-linkers for the synthesis of chemically modified peptides. This linker is completely stable during Boc- and Fmoc-based solid phase synthesis and yet it can be cleaved under very mild oxidative conditions. The present article reviews the use of this valuable linker for the rapid and efficient synthesis of C-terminal modified peptides, head-to-tail cyclic peptides and lipidated peptides.

  8. Identification of novel post-translational modifications in linker histones from chicken erythrocytes. (United States)

    Sarg, Bettina; Lopez, Rita; Lindner, Herbert; Ponte, Inma; Suau, Pedro; Roque, Alicia


    Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC-ESI-MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including N(α)-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics. Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to

  9. Efficient loading of primary alcohols onto a solid phase using a trityl bromide linker

    DEFF Research Database (Denmark)

    Crestey, François; Ottesen, Lars Korsgaard; Jaroszewski, Jerzy Witold


    The Letter describes an improved, rapid and mild strategy for the loading of primary alcohols onto a polystyrene trityl resin via a highly reactive trityl bromide linker. This protocol facilitates an efficient resin loading even of acid-sensitive or heat-labile alcohols, which otherwise require...... of a sensitive alcohol containing an activated aziridine functionality, the use of the trityl bromide linker proved superior to a recently described silver triflate-assisted trityl chloride resin-based procedure....

  10. A dimethacrylate cross-linker cleavable under thermolysis or alkaline hydrolysis conditions: synthesis, polymerization, and degradation. (United States)

    Elladiou, Marios; Patrickios, Costas S


    We develop a new platform based on 2,6-pyridinediethanol diesters for introducing polymer degradability under thermolysis or alkaline hydrolysis conditions, with the latter being rare in polymers. Such labile diesters can be cross-linkers, bifunctional initiators and inimers. We demonstrate the power of this platform through the synthesis of the 2,6-pyridinediethanol dimethacrylate cross-linker, its controlled (co)polymerization, and the thermal and hydrolytic cleavage of its (co)polymers.

  11. Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers. (United States)

    Vroom, Jonathan A; Wang, Clifford L


    We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs. By specifying the sequences of the linkers, desired components can be assembled in any combination and order to generate different plasmid vectors.

  12. Glycosylation efficiencies on different solid supports using a hydrogenolysis-labile linker

    Directory of Open Access Journals (Sweden)

    Mayeul Collot


    Full Text Available Automated oligosaccharide assembly requires suitable linkers to connect the first monosaccharide to a solid support. A new hydrogenolysis-labile linker that is stable under both acidic and basic conditions was designed, synthesized and coupled to different resins. Glycosylation and cleavage efficiencies on these functionalized solid supports were investigated, and restrictions for the choice of solid support for oligosaccharide synthesis were found.

  13. Lecithin-linker formulations for self-emulsifying delivery of nutraceuticals. (United States)

    Chu, Jacquelene; Cheng, Yu-Ling; Rao, A Venketeshwer; Nouraei, Mehdi; Zarate-Muñoz, Silvia; Acosta, Edgar J


    Lecithin-linker microemulsions are formulations produced with soybean lecithin in combination with a highly lipophilic (lipophilic linker) and highly hydrophilic (hydrophilic linkers) surfactant-like additives. In this work, lecithin-linker systems were formulated to produce self-emulsifying delivery systems for β-carotene and β-sitosterol. The concentration of the lipophilic linker, sorbitan monooleate, was adjusted to minimize the formation of liquid crystals. The concentration of hydrophilic linkers, decaglyceryl caprylate/caprate and PEG-6-caprylic/capric glycerides, was gradually increased (scanned) until single phase clear microemulsions were obtained. For these scans, the oil (ethyl caprate) to water ratio was set to 1. The single phase, clear microemulsions were diluted with fed-state simulated intestinal fluid (FeSSIF) and produced stable emulsions, with drop sizes close to 200 nm. Using pseudo-ternary phase diagrams to evaluate the process of dilution of microemulsion preconcentrates (mixtures of oil, lecithin and linkers with little or no water) with FeSSIF, it was determined that self-emulsifying systems are obtained when the early stages of the dilution produce single phase microemulsions. If liquid crystals or multiple phase systems are obtained during those early stages, then the emulsification yields unstable emulsions with large drop sizes. An in vitro permeability study conducted using a Flow-Thru Dialyzer revealed that stable emulsions with drop sizes of 150-300 nm produce large and irreversible permeation of β-carotene to sheep intestine. On the other hand, unstable emulsions produced without the linker combination separated in the dialyzer chamber. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Topological diversity of chromatin fibers: Interplay between nucleosome repeat length, DNA linking number and the level of transcription

    Directory of Open Access Journals (Sweden)

    Davood Norouzi


    Full Text Available The spatial organization of nucleosomes in 30-nm fibers remains unknown in detail. To tackle this problem, we analyzed all stereochemically possible configurations of two-start chromatin fibers with DNA linkers L = 10-70 bp (nucleosome repeat length NRL = 157-217 bp. In our model, the energy of a fiber is a sum of the elastic energy of the linker DNA, steric repulsion, electrostatics, and the H4 tail-acidic patch interaction between two stacked nucleosomes. We found two families of energetically feasible conformations of the fibers—one observed earlier, and the other novel. The fibers from the two families are characterized by different DNA linking numbers—that is, they are topologically different. Remarkably, the optimal geometry of a fiber and its topology depend on the linker length: the fibers with linkers L = 10n and 10n + 5 bp have DNA linking numbers per nucleosome DLk >>-1.5 and -1.0, respectively. In other words, the level of DNA supercoiling is directly related to the length of the inter-nucleosome linker in the chromatin fiber (and therefore, to NRL. We hypothesize that this topological polymorphism of chromatin fibers may play a role in the process of transcription, which is known to generate different levels of DNA supercoiling upstream and downstream from RNA polymerase. A genome-wide analysis of the NRL distribution in active and silent yeast genes yielded results consistent with this assumption.

  15. Estimation of genome length

    Institute of Scientific and Technical Information of China (English)


    The genome length is a fundamental feature of a species. This note outlined the general concept and estimation method of the physical and genetic length. Some formulae for estimating the genetic length were derived in detail. As examples, the genome genetic length of Pinus pinaster Ait. and the genetic length of chromosome Ⅵ of Oryza sativa L. were estimated from partial linkage data.

  16. Role of the S4-S5 linker in CNG channel activation. (United States)

    Kusch, Jana; Zimmer, Thomas; Holschuh, Jascha; Biskup, Christoph; Schulz, Eckhard; Nache, Vasilica; Benndorf, Klaus


    Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus that is located at the intracellular side. The molecular events translating the ligand binding to the pore opening are still unknown. We investigated the role of the S4-S5 linker in the activation process by quantifying its interaction with other intracellular regions. To this end, we constructed chimeric channels in which the N-terminus, the S4-S5 linker, the C-linker, and the CNBD of the retinal CNGA1 subunit were systematically replaced by the respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by thermodynamic double-mutant cycle analysis. We observed that all four intracellular regions, including the relatively short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits, as derived from the Hill coefficient. The interaction energies reveal an interaction of the S4-S5 linker with both the N-terminus and the C-linker, but no interaction with the CNBD.

  17. Inter-domain Embedded Carrying Network Construction in ReFlexNet%面向ReFlexNet的域间嵌入式承载网构建

    Institute of Scientific and Technical Information of China (English)

    齐宁; 汪斌强; 袁博; 张博; 王保进


    To solve the puzzle faced by traditional Internet infrastructure, a new Reconfigurable Flexible Network (ReFlexNet) Infrastructure is proposed. The inter-domain Reconfigurable Embedded Carrying Network (RECN) construction issues in ReFlexNet are discussed. Distributed layered management system and resource management mechanism is proposed. Based on the manner of token passing, a distributed inter-domain RECN construction method is proposed, which can resolve large-scale inter-domain construction problem efficiently. To improve efficiency of token transfer, an improved Token-Ring establishment algorithm is proposed which is called ImprovedSA based on traditional Simulated Annealing (SA) algorithm of solving Hamilton circle problem. It can find a better circle rapidly and effectively via ameliorating the result gained by traditional algorithm.%为解决传统互联网体系结构面临的困境,该文提出了新型的可重构柔性网络(Reconfigurable Flexible Network,ReFlexNet)体系架构.该文针对ReFlexNet中域间嵌入式承载网的构建方法进行了论述,提出了ReFlexNet的分布式分层管理体系和资源管理机制,并基于令牌传递的方式设计了分布式域间嵌入式承载网的构建方法,有效解决了大范围跨域的嵌入式承载网构建问题.为提高令牌传递的效率,在传统求解Hamilton回路的模拟退火算法基础上,提出了改进的令牌环构造算法ImprovedSA,通过对传统算法求得的解进行改良,可快速有效地找到更好的回路.

  18. Different Interfacial Behaviors of Peptides Chemically Immobilized on Surfaces with Different Linker Lengths and via Different Termini (United States)


    sensitivity, selectivity, detection limit, precision, accuracy, reproducibility, working life , and shelf life ) is greatly dependent on the...MA). Potassium phosphate (monobasic and dibasic) solution (PB) (1 M) and tris(2- carboxyethyl) phosphine hydrochloride (TCEP) solution (0.5 M) were...propidium iodide for 20 min in the dark according to the instructions for the LIVE/DEAD BacLight bacterial viability kit (L7012, Invitrogen, Carlsbad, CA

  19. Thermodynamically Guided Synthesis of Mixed-Linker Zr-MOFs with Enhanced Tunability. (United States)

    Yuan, Shuai; Qin, Jun-Sheng; Zou, Lanfang; Chen, Ying-Pin; Wang, Xuan; Zhang, Qiang; Zhou, Hong-Cai


    Guided by thermodynamics, we have synthesized two mixed-linker zirconium-based metal-organic frameworks (Zr-MOFs), namely, PCN-133 and PCN-134. Both of them possess a layer-pillar structure, in which the connection between Zr6 clusters and primary BTB linkers form a (3,6)-connected kdg layer that is further extended into 3D frameworks by auxiliary DCDPS/TCPP linkers (BTB = benzene tribenzoate, DCDPS = 4,4'-dicarboxydiphenyl sulfone, TCPP = tetrakis(4-carboxyphenyl)porphyrin). PCN-134 demonstrates high porosity (N2 uptake of 717 cm(3)·g(-1) and BET surface area of 1946 cm(2)·g(-1)) and excellent chemical stability in aqueous solutions with pH values ranging from 0 to 13. More importantly, PCN-134 tolerates the partial absence of auxiliary linkers leading to structural defects during the assembly process while preserving its framework integrity. Furthermore, the defect density can be systematically controlled by tuning the occupancy of the auxiliary linker, which in turn affects the MOF properties. For instance, the dichromate uptake of PCN-134 is tuned by adjusting the BTB/TCPP ratios, which gives rise to an efficient dichromate absorbent when the TCPP molar ratio in linkers is set as 22%. In addition, the photocatalytic reduction of Cr(VI) in aqueous solution was also performed by PCN-134-22%TCPP which exhibits excellent catalytic activity. This work not only opens up a new synthetic route toward mixed-linker MOFs, but also provides tunable control of MOF defects and, in turn, the properties.

  20. Onset of grain filling is associated with a change in properties of linker histone variants in maize kernels

    DEFF Research Database (Denmark)

    Kalamajka, R.; Finnie, Christine; Grasser, K.D.


    to more basic protein forms suggests a reduction in the level of post-translational modifications of linker histones during kernel development. Analysis of their DNA-binding affinity revealed that the different linker histone gene products bind double-stranded DNA with similar affinity. Interestingly......) initiation of storage synthesis. Six linker histone gene products were identified by MALDI-TOF mass spectrometry. A marked shift of around 4 pH units was observed for the linker histone spot pattern after 2D-gel electrophoresis when comparing the proteins of 11 and 16 dap kernels. The shift from acidic......, the linker histones isolated from 16 dap kernels consistently displayed a lower affinity for DNA than the proteins isolated from 11 dap kernels. These findings suggest that the affinity for DNA of the linker histones may be regulated by post-translational modification and that the reduction in DNA affinity...

  1. Spectroscopic study of the light-harvesting protein C-phycocyanin associated with colorless linker peptides

    Energy Technology Data Exchange (ETDEWEB)

    Pizarro, Shelly A.


    The phycobilisome (PBS) light-harvesting antenna is composed of chromophore-containing biliproteins and 'colorless' linker peptides and is structurally designed to support unidirectional transfer of excitation energy from the periphery of the PBS to its core. The linker peptides have a unique role in this transfer process by modulating the spectral properties of the associated biliprotein. There is only one three-dimensional structure of a biliprotein/linker complex available to date (APC/LC7.8) and the mechanism of interaction between these two proteins remains unknown. This study brings together a detailed spectroscopic characterization of C-Phycocyanin (PC)-linker complexes (isolated from Synechococcus sp. PCC 7002) with proteomic analysis of the linker amino acid sequences to produce a model for biliprotein/linker interaction. The amino acid sequences of the rod linkers [LR8.9, LR32.3 and LRC28.5] were examined to identify evolutionarily conserved regions important to either the structure or function of this protein family. Although there is not one common homologous site among all the linkers, there are strong trends across each separate subset (LC, LR and LRC) and the N-terminal segments of both LR32.3 and LRC28.5 display multiple regions of similarity with other linkers. Predictions of the secondary structure of LR32.3 and LRC28.5, and comparison to the crystal structure of LC7.8, further narrowed the candidates for interaction sites with the PC chromophores. Measurements of the absorption, fluorescence, CD and excitation anisotropy of PC trimer, PC/LR32.3, and PC/LRC28.5, document the spectroscopic effect of each linker peptide on the PC chromophores at a series of temperatures (298 to 77 K). Because LR32.3 and LRC28.5 modulate the PC trimer spectral properties in distinct manners, it suggests different chromophore-interaction mechanisms for each linker. The low temperature absorbance spectrum of the PC trimer is consistent with an excitonic

  2. Spectroscopic study of the light-harvesting protein C-phycocyanin associated with colorless linker peptides

    Energy Technology Data Exchange (ETDEWEB)

    Pizarro, Shelly Ann [Univ. of California, Berkeley, CA (United States)


    The phycobilisome (PBS) light-harvesting antenna is composed of chromophore-containing biliproteins and 'colorless' linker peptides and is structurally designed to support unidirectional transfer of excitation energy from the periphery of the PBS to its core. The linker peptides have a unique role in this transfer process by modulating the spectral properties of the associated biliprotein. There is only one three-dimensional structure of a biliprotein/linker complex available to date (APC/LC7.8) and the mechanism of interaction between these two proteins remains unknown. This study brings together a detailed spectroscopic characterization of C-Phycocyanin (PC)-linker complexes (isolated from Synechococcus sp. PCC 7002) with proteomic analysis of the linker amino acid sequences to produce a model for biliprotein/linker interaction. The amino acid sequences of the rod linkers [LR8.9, LR32.3 and LRC28.5] were examined to identify evolutionarily conserved regions important to either the structure or function of this protein family. Although there is not one common homologous site among all the linkers, there are strong trends across each separate subset (LC, LR and LRC) and the N-terminal segments of both LR32.3 and LRC28.5 display multiple regions of similarity with other linkers. Predictions of the secondary structure of LR32.3 and LRC28.5, and comparison to the crystal structure of LC7.8, further narrowed the candidates for interaction sites with the PC chromophores. Measurements of the absorption, fluorescence, CD and excitation anisotropy of PC trimer, PC/LR32.3, and PC/LRC28.5, document the spectroscopic effect of each linker peptide on the PC chromophores at a series of temperatures (298 to 77 K

  3. A High-Throughput Small Molecule Screen for C. elegans Linker Cell Death Inhibitors (United States)

    Schwendeman, Andrew R.; Shaham, Shai


    Programmed cell death is a ubiquitous process in metazoan development. Apoptosis, one cell death form, has been studied extensively. However, mutations inactivating key mammalian apoptosis regulators do not block most developmental cell culling, suggesting that other cell death pathways are likely important. Recent work in the nematode Caenorhabditis elegans identified a non-apoptotic cell death form mediating the demise of the male-specific linker cell. This cell death process (LCD, linker cell-type death) is morphologically conserved, and its molecular effectors also mediate axon degeneration in mammals and Drosophila. To develop reagents to manipulate LCD, we established a simple high-throughput screening protocol for interrogating the effects of small molecules on C. elegans linker cell death in vivo. From 23,797 compounds assayed, 11 reproducibly block linker cell death onset. Of these, five induce animal lethality, and six promote a reversible developmental delay. These results provide proof-of principle validation of our screening protocol, demonstrate that developmental progression is required for linker cell death, and suggest that larger scale screens may identify LCD-specific small-molecule regulators that target the LCD execution machinery. PMID:27716809

  4. The measles virus phosphoprotein interacts with the linker domain of STAT1

    Energy Technology Data Exchange (ETDEWEB)

    Devaux, Patricia, E-mail:; Priniski, Lauren; Cattaneo, Roberto


    The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainly to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway.

  5. A Partial Calcium-Free Linker Confers Flexibility to Inner-Ear Protocadherin-15. (United States)

    Powers, Robert E; Gaudet, Rachelle; Sotomayor, Marcos


    Tip links of the inner ear are protein filaments essential for hearing and balance. Two atypical cadherins, cadherin-23 and protocadherin-15, interact in a Ca(2+)-dependent manner to form tip links. The largely unknown structure and mechanics of these proteins are integral to understanding how tip links pull on ion channels to initiate sensory perception. Protocadherin-15 has 11 extracellular cadherin (EC) repeats. Its EC3-4 linker lacks several of the canonical Ca(2+)-binding residues, and contains an aspartate-to-alanine polymorphism (D414A) under positive selection in East Asian populations. We present structures of protocadherin-15 EC3-5 featuring two Ca(2+)-binding linker regions: canonical EC4-5 linker binding three Ca(2+) ions, and non-canonical EC3-4 linker binding only two Ca(2+) ions. Our structures and biochemical assays reveal little difference between the D414 and D414A variants. Simulations predict that the partial Ca(2+)-free EC3-4 linker exhibits increased flexural flexibility without compromised mechanical strength, providing insight into the dynamics of tip links and other atypical cadherins.

  6. Synthesis of tetravalent LacNAc-glycoclusters as high-affinity cross-linker against Erythrina cristagalli agglutinin. (United States)

    Ogata, Makoto; Chuma, Yasushi; Yasumoto, Yoshinori; Onoda, Takashi; Umemura, Myco; Usui, Taichi; Park, Enoch Y


    Four kinds of tetravalent double-headed glycoclusters [(LacNAc)4-DHGs] were designed with linkers of varying lengths consisting of alkanedioic carboxyamido groups (C6, C12, C18 and C24) between two bi-antennary LacNAc-glycosides. These glycoclusters served as high-affinity cross-linking ligands for the LacNAc-binding lectin Erythrina cristagalli agglutinin (ECA). The binding activity and cross-linking between each ligand and ECA were characterized by a hemagglutination inhibition (HI) assay, isothermal titration calorimetry (ITC), a quantitative precipitation assay and dynamic light scattering (DLS). For the precipitation assay and DLS measurement, the synthesized (LacNAc)4-DHGs were found to be capable of binding and precipitating the ECA as multivalent ligands. ITC analysis indicated the binding of (LacNAc)4-DHGs was driven by a favorable enthalpy change. Furthermore, the entropy penalty from binding (LacNAc)4-DHGs clearly decreased in a spacer length-dependent manner. The binding affinities of flexible (LacNAc)4-DHGs (C18 and C24) with long spacers were found to be more favorable than those of the clusters having short spacers (C6 and C12). These results were supported by molecular dynamics simulations with explicit water molecules for the tetravalent glycoclusters with ECA. We concluded that the subtle modification in the epitope-presenting scaffolds exerts the significant effect in the recognition efficiency involved in the LacNAc moieties by ECA.

  7. Chaperone-like effect of the linker on the isolated C-terminal domain of rabbit muscle creatine kinase. (United States)

    Chen, Zhe; Chen, Xiang-Jun; Xia, Mengdie; He, Hua-Wei; Wang, Sha; Liu, Huihui; Gong, Haipeng; Yan, Yong-Bin


    Intramolecular chaperones (IMCs), which are specific domains/segments encoded in the primary structure of proteins, exhibit chaperone-like activity against the aggregation of the other domains in the same molecule. In this research, we found that the truncation of the linker greatly promoted the thermal aggregation of the isolated C-terminal domain (CTD) of rabbit muscle creatine kinase (RMCK). Either the existence of the linker covalently linked to CTD or the supply of the synthetic linker peptide additionally could successfully protect the CTD of RMCK against aggregation in a concentration-dependent manner. Truncated fragments of the linker also behaved as a chaperone-like effect with lower efficiency, revealing the importance of its C-terminal half in the IMC function of the linker. The aggregation sites in the CTD of RMCK were identified by molecular dynamics simulations. Mutational analysis of the three key hydrophobic residues resulted in opposing effects on the thermal aggregation between the CTD with intact or partial linker, confirming the role of linker as a lid to protect the hydrophobic residues against exposure to solvent. These observations suggested that the linkers in multidomain proteins could act as IMCs to facilitate the correct folding of the aggregation-prone domains. Furthermore, the intactness of the IMC linker after proteolysis modulates the production of off-pathway aggregates, which may be important to the onset of some diseases caused by the toxic effects of aggregated proteolytic fragments.

  8. Modeling neck linker of kinesin motor movement with MRSR stochastic differential equation (United States)

    Razali, Wan Qashishah Akmal Wan; Ramli, Siti Norafidah Mohd; Radiman, Shahidan


    Stochastic differential equation has a significant role in a range of biological areas including molecular motor like kinesin motor. Mean-reverting square root (MRSR) stochastic differential equation is commonly used in economics and finance areas. In this study, we use the MRSR stochastic differential equation to model neck linker motion of kinesin motor by considering the possibilities of rightward direction and occasionally in the leftward direction of kinesin movements. This neck linker docking model of kinesin motor incorporates the conformational change in the chemical kinetics and the tethered diffusion of the free head of kinesin motor. Here, we demonstrate this model by using Hookean spring method which referred to the stiffness model of neck linker. The motion of kinesin motor seems to be well described to move in unidirectional way with volatile behavior based on MRSR rather than common stochastic differential equation [DOI 10.1007/s11538-011-9697-6].

  9. Smart linkers in polymer-drug conjugates for tumor-targeted delivery. (United States)

    Chang, Minglu; Zhang, Fang; Wei, Ting; Zuo, Tiantian; Guan, Yuanyuan; Lin, Guimei; Shao, Wei


    To achieve effective chemotherapy, many types of drug delivery systems have been developed for the specific environments in tumor tissues. Polymer-drug conjugates are increasingly used in tumor therapy due to several significant advantages over traditional delivery systems. In the fabrication of polymer-drug conjugates, a smart linker is an important component that joins two fragments or molecules together and can be cleared by a specific stimulus, which results in targeted drug delivery and controlled release. By regulating the conjugation between the drug and the nanocarriers, stimulus-sensitive systems based on smart linkers can offer high payloads, certified stability, controlled release and targeted delivery. In this review, we summarize the current state of smart linkers (e.g. disulfide, hydrazone, peptide, azo) used recently in various polymer-drug conjugate-based delivery systems with a primary focus on their sophisticated design principles and drug delivery mechanisms as well as in vivo processes.

  10. A linker peptide with high affinity towards silica-containing materials. (United States)

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L


    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.

  11. Wheat Gluten Blends with Maleic Anhydride-Functionalized Polyacrylate Cross-Linkers for Improved Properties. (United States)

    Diao, Cheng; Xia, Hongwei; Parnas, Richard S


    A family of polyacrylate-based cross-linkers was synthesized to maximize the toughness of high Tg, high modulus wheat gluten blends in the glassy state. Mechanical testing and damping measurements were conducted to provide an example where the work of fracture and strength of the blend substantially exceeds polystyrene while maintaining flexure stiffness in excess of 3 GPa. The new rubbery cross-linkers, polymethyl acrylate-co-maleic anhydride and polyethyl acrylate-co-maleic anhydride, improve WG mechanical properties and reduce water absorption simultaneously. MDSC, FTIR, HPLC, and NMR data confirmed the cross-linking reaction with wheat gluten. Flexural, DMA, and water absorption testing were carried out to characterize the property improvements. DMA was conducted to investigate the relationship between energy damping and mechanical property improvement. If the cross-linker damping temperature is close to the testing temperature, the entire sample exhibits high damping, toughness, and strength.

  12. Replacement of the human topoisomerase linker domain with the plasmodial counterpart renders the enzyme camptothecin resistant

    DEFF Research Database (Denmark)

    Arnò, Barbara; D'Annessa, Ilda; Tesauro, Cinzia;


    , but it is characterized by a much faster religation rate. The hybrid enzyme is also camptothecin resistant. A 3D structure of the hybrid enzyme has been built and its structural-dynamical properties have been analyzed by molecular dynamics simulation. The analysis indicates that the swapped plasmodial linker samples......A human/plasmodial hybrid enzyme, generated by swapping the human topoisomerase IB linker domain with the corresponding domain of the Plasmodium falciparum enzyme, has been produced and characterized. The hybrid enzyme displays a relaxation activity comparable to the human enzyme...... in the modulation of the topoisomerase IB activity....

  13. A photolabile linker for the solid-phase synthesis of peptide hydrazides and heterocycles. (United States)

    Qvortrup, Katrine; Komnatnyy, Vitaly V; Nielsen, Thomas E


    A photolabile hydrazine linker for the solid-phase synthesis of peptide hydrazides and hydrazine-derived heterocycles is presented. The developed protocols enable the efficient synthesis of structurally diverse peptide hydrazides derived from the standard amino acids, including those with side-chain protected residues at the C-terminal of the resulting peptide hydrazide, and are useful for the synthesis of dihydropyrano[2,3-c]pyrazoles. The linker is compatible with most commonly used coupling reagents and protecting groups for solid-phase peptide synthesis.

  14. Impact of metal-alkoxide functionalized linkers on H2 binding: A density functional study (United States)

    Banu, Tahamida; Ghosh, Avik; Das, Abhijit K.


    The effect of metal-alkoxide functionalization of different organic linkers on the H2 binding is investigated employing DFT approach. While analyzing the H2 binding interaction of magnesium-alkoxide modified benzene, naphthalene, anthracene and pyrene linkers, we find their comparable affinity toward H2 molecules. Six-member alkoxide ring containing naphthalene and pyrene systems interact with the H2 molecules in a comparatively better way than their five-member analogues. AIM, NBO and LMO-EDA analyses have been performed to comprehend the bonding nature between Mg center and the H2 molecules. Polarization along with the charge transfer interactions play significant role in stabilizing the systems.

  15. A Photolabile Linker for the Solid-Phase Synthesis of Peptide Hydrazides and Heterocycles

    DEFF Research Database (Denmark)

    Qvortrup, Katrine; Komnatnyy, Vitaly V.; Nielsen, Thomas Eiland


    A photolabile hydrazine linker for the solid-phase synthesis of peptide hydrazides and hydrazine-derived heterocycles is presented. The developed protocols enable the efficient synthesis of structurally diverse peptide hydrazides derived from the standard amino adds, including those with side-cha......-chain protected residues at the C-terminal of the resulting peptide hydrazide, and are useful for the synthesis of dihydropyrano[2,3-c]pyrazoles. The linker is compatible with most commonly used coupling reagents and protecting groups for solid-phase peptide synthesis....

  16. Measuring Thermodynamic Length

    Energy Technology Data Exchange (ETDEWEB)

    Crooks, Gavin E


    Thermodynamic length is a metric distance between equilibrium thermodynamic states. Among other interesting properties, this metric asymptotically bounds the dissipation induced by a finite time transformation of a thermodynamic system. It is also connected to the Jensen-Shannon divergence, Fisher information, and Rao's entropy differential metric. Therefore, thermodynamic length is of central interestin understanding matter out of equilibrium. In this Letter, we will consider how to denethermodynamic length for a small system described by equilibrium statistical mechanics and how to measure thermodynamic length within a computer simulation. Surprisingly, Bennett's classic acceptance ratio method for measuring free energy differences also measures thermodynamic length.

  17. Photosensitizer (PS)-cyanine dye (CD) conjugates: Impact of the linkers joining the PS and CD moieties and their orientation in tumor-uptake and photodynamic therapy (PDT). (United States)

    James, Nadine S; Joshi, Penny; Ohulchanskyy, Tymish Y; Chen, Yihui; Tabaczynski, Walter; Durrani, Farukh; Shibata, Masayuki; Pandey, Ravindra K


    To investigate the impact of linker(s) joining the photosensitizer HPPH [3-(1'-hexyloxy) ethyl-3-devinylpyropheophorbide-a] and the cyanine dye (CD) in tumor-imaging and photodynamic therapy (dual-function agents), a series of HPPH-CD conjugates were synthesized. The modifications were done in an attempt to minimize Forster Resonance Energy Transfer (FRET) between the two chromophores and maximize singlet oxygen production. Among the conjugates containing variable length of linkers, the HPPH-CD conjugate, in which the photosensitizer (PS) and the CD was joined by four Carbon [(CH2)4] units showed higher tumor uptake, improved tumor contrast and limited skin uptake in mice bearing Colon-26 (BALB/c) or U87 tumors in Nude mice. The bi-functional agents in which the HPPH was linked at the meta-position of phenyl-substituted CD 5, 6 and 7 showed longer tumor response (cure) than the corresponding para-substituted analogs 2, 3, and 4, which suggests that the orientation of the PS and CD moieties within the conjugate also makes a substantial difference in tumor-specificity. Compared to HPPH, the singlet oxygen yields of all the HPPH-CD conjugates were significantly low, and required a higher therapeutic dose to achieve the same in vivo response obtained by HPPH-PDT alone. However, conjugate 6 produced a higher singlet oxygen yield with reduced FRET and exhibited enhanced long-term PDT efficacy in mice bearing Colon-26 (BALB/c) and U87 tumors (nude) than its counterparts, including our lead compound (HPPH-CD), making it the most efficacious of the series. Thus, these conjugates bearing cyanine dye moiety (CD) provide an opportunity of imaging deeply seated tumors for fluorescence-guided surgery with an option of PDT.

  18. General Approach for Introduction of Various Chemical Labels in Specific RNA Locations Based on Insertion of Amino Linkers

    Directory of Open Access Journals (Sweden)

    Dmitri Graifer


    Full Text Available Introduction of reporter groups at designed RNA sites is a widely accepted approach to gain information about the molecular environment of RNAs in their complexes with other biopolymers formed during various cellular processes. A general approach to obtain RNAs bearing diverse reporter groups at designed locations is based on site-specific insertion of groups containing primary aliphatic amine functions (amino linkers with their subsequent selective derivatization by appropriate chemicals. This article is a brief review on methods for site-specific introduction of amino linkers in different RNAs. These methods comprise: (i incorporation of a nucleoside carrying an amino-linker or a function that can be substituted with it into oligoribonucleotides in the course of their chemical synthesis; (ii assembly of amino linker-containing RNAs from short synthetic fragments via their ligation; (iii synthesis of amino linker-modified RNAs using T7 RNA polymerase; (iv insertion of amino linkers into unmodified RNAs at functional groups of a certain type such as the 5'-phosphates and N7 of guanosine residues and (v introduction of an amino linker into long highly structured RNAs exploiting an approach based on sequence-specific modification of nucleic acids. Particular reporter groups used for derivatization of amino linker-containing RNAs together with types of RNA derivatives obtained and fields of their application are presented.

  19. A photolabile linker for the solid-phase synthesis of 4-substituted NH-1,2,3-triazoles

    DEFF Research Database (Denmark)

    Qvortrup, Katrine; Nielsen, Thomas Eiland


    A novel photolabile linker for solid-phase synthesis is presented. The linker displays an azido handle for copper-catalyzed azide–alkyne cycloaddition reactions with a variety of alkynes, remains intact under typical solid-phase reaction conditions, and enables a mild photolytic release of 4-subs...

  20. Backbone amide linker strategy for the synthesis of 1,4-triazole-containing cyclic tetra- and pentapeptides

    NARCIS (Netherlands)

    Springer, J.; de Cuba, K.R.; Calvet-Vitale, S.; Geenevasen, J.A.J.; Hermkens, P.H.H.; Hiemstra, H.; van Maarseveen, J.H.


    A backbone amide linker strategy was chosen for the solid-phase synthesis of triazole-containing Cyclic tetra- and pentapeptides. An alkyne-substituted linker derived from 4-hydroxy-2-methoxybenzaldehyde was elongated by using standard "Fmoc-based" solid phase chemistry and terminated by coupling of

  1. Novel cross-linkers for PDMS networks for controlled and well distributed grafting of functionalities by click chemistry

    DEFF Research Database (Denmark)

    Bahrt, Frederikke; Dimitrov, Ivaylo; Daugaard, Anders Egede


    An azide-containing, trifunctional vinyl cross-linker for silicone networks has been synthesized. The cross-linker has through Cu(i) catalyzed 1,3-cycloaddition been reacted with six different alkyne-containing chemical groups which each possess a particular functionality. The functional cross-li...... The Royal Society of Chemistry....

  2. Characterization, structure and function of linker polypeptides in phycobilisomes of cyanobacteria and red algae: an overview. (United States)

    Liu, Lu-Ning; Chen, Xiu-Lan; Zhang, Yu-Zhong; Zhou, Bai-Cheng


    Cyanobacteria and red algae have intricate light-harvesting systems comprised of phycobilisomes that are attached to the outer side of the thylakoid membrane. The phycobilisomes absorb light in the wavelength range of 500-650 nm and transfer energy to the chlorophyll for photosynthesis. Phycobilisomes, which biochemically consist of phycobiliproteins and linker polypeptides, are particularly wonderful subjects for the detailed analysis of structure and function due to their spectral properties and their various components affected by growth conditions. The linker polypeptides are believed to mediate both the assembly of phycobiliproteins into the highly ordered arrays in the phycobilisomes and the interactions between the phycobilisomes and the thylakoid membrane. Functionally, they have been reported to improve energy migration by regulating the spectral characteristics of colored phycobiliproteins. In this review, the progress regarding linker polypeptides research, including separation approaches, structures and interactions with phycobiliproteins, as well as their functions in the phycobilisomes, is presented. In addition, some problems with previous work on linkers are also discussed.

  3. Assembly of bipolar microtubule structures by passive cross-linkers and molecular motors (United States)

    Johann, D.; Goswami, D.; Kruse, K.


    During cell division, sister chromatids are segregated by the mitotic spindle, a bipolar assembly of interdigitating antiparallel polar filaments called microtubules. The spindle contains the midzone, a stable region of overlapping antiparallel microtubules, that is essential for maintaining bipolarity. Although a lot is known about the molecular players involved, the mechanism underlying midzone formation and maintenance is still poorly understood. We study the interaction of polar filaments that are cross-linked by molecular motors moving directionally and by passive cross-linkers diffusing along microtubules. Using a particle-based stochastic model, we find that the interplay of motors and passive cross-linkers can generate a stable finite overlap between a pair of antiparallel polar filaments. We develop a mean-field theory to study this mechanism in detail and investigate the influence of steric interactions between motors and passive cross-linkers on the overlap dynamics. In the presence of interspecies steric interactions, passive cross-linkers mimic the behavior of molecular motors and stable finite overlaps are generated even for non-cross-linking motors. Finally, we develop a mean-field theory for a bundle of aligned polar filaments and show that they can self-organize into a spindlelike pattern. Our work suggests possible ways as to how cells can generate spindle midzones and control their extensions.

  4. Cellular delivery and antisense effects of peptide nucleic acid conjugated to polyethyleneimine via disulfide linkers

    DEFF Research Database (Denmark)

    Berthold, Peter R; Shiraishi, Takehiko; Nielsen, Peter E


    for PNA due to the (inherent) charge neutrality of PNA. However, PEI could function as an efficient scaffold for PNA via chemical conjugation. Accordingly, we modified PEI with the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (with and without a PEG...

  5. A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study

    DEFF Research Database (Denmark)

    Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano;


    with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively...

  6. Novel nucleosomal particles containing core histones and linker DNA but no histone H1. (United States)

    Cole, Hope A; Cui, Feng; Ocampo, Josefina; Burke, Tara L; Nikitina, Tatiana; Nagarajavel, V; Kotomura, Naoe; Zhurkin, Victor B; Clark, David J


    Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these 'proto-chromatosomes' are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.


    Institute of Scientific and Technical Information of China (English)

    SUN Weimin; LUO Juntao; HUANG Wenqiang; ZHU Xiaoxia


    A use of Sulfonate ester as a linker in synthesis of ω-aminoalkanols was reported. Diols were tethered onto polystyryl sulfonyl chloride resin, yielding sulfonate resins (2). After cleaved by diethyl amine, diisopropylamine and propylamine respectively, three ω-aminoalkanols were obtained.


    Institute of Scientific and Technical Information of China (English)

    SUNWeimin; ZHUXiaoxia; 等


    A use of sulfonate ester as a linker in synthesis of w-aminoalkanols was reporte.Diols were tethered onto polystyryl sulfonyl chloride resin,yielding sulfonate resins(2).After cleaved by diethyl amine,diisopropylamine and propylamine respectively,three w-aminoalkanlos were obtained.

  9. Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker. (United States)

    Mo, Xuejun; Li, Qiushi; Yi Lui, Lena Wai; Zheng, Baixue; Kang, Chiang Huen; Nugraha, Bramasta; Yue, Zhilian; Jia, Rui Rui; Fu, Hong Xia; Choudhury, Deepak; Arooz, Talha; Yan, Jie; Lim, Chwee Teck; Shen, Shali; Hong Tan, Choon; Yu, Hanry


    Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications.

  10. Solid-phase synthesis of polyfunctional polylysine dendrons using aldehyde linkers

    DEFF Research Database (Denmark)

    Svenssen, Daniel K.; Mirsharghi, Sahar; Boas, Ulrik


    A straightforward method for the solid-phase synthesis of C-terminally modified polylysine dendrons has been developed by applying bisalkoxybenzaldehyde and trisalkoxybenzaldehyde linkers. The method has been used for the synthesis of polylysine dendrons with a variety of C-terminal ‘tail groups’...

  11. Identification of a minimal functional linker in human topoisomerase I by domain swapping with Cre recombinase

    DEFF Research Database (Denmark)

    Hougaard, Rikke Frøhlich; Juul, Sissel; Vinther, Maria


    . In this study we replace 86 amino acids including the linker domain of the cellular type IB topoisomerase, human topoisomerase I, with four, six, or eight amino acids from the corresponding short loop region in Cre recombinase. In vitro characterization of the resulting chimeras, denoted Cropos, reveals...

  12. A two-photon activatable amino acid linker for the induction of fluorescence. (United States)

    Friedrich, Felix; Klehs, Kathrin; Fichte, Manuela A H; Junek, Stephan; Heilemann, Mike; Heckel, Alexander


    A new one- and two-photon activatable fluorophore based on ATTO565 was developed using a photolabile linker that simultaneously acts as a quencher. It is especially interesting for protein and peptide applications because it can be incorporated by standard peptide chemistry. The application of the new fluorogenic construct in super-resolution microscopy of antibody conjugates is shown.

  13. Novel pH-Sensitive Cationic Lipids with Linear Ortho Ester Linkers for Gene Delivery (United States)

    Chen, Haigang; Zhang, Huizhen; Thor, Der; Rahimian, Roshanak; Guo, Xin


    In an effort to develop pH-sensitive lipoplexes for efficient gene delivery, we report three novel cationic lipids containing a linear ortho ester linker that conjugates either the headgroup (Type I) or one hydrocarbon chain (Type II) with the rest of the lipid molecule. The cationic lipids carry either an iodide or a chloride counterion. Compared to our previously reported cyclic ortho ester linker, the linear ortho ester linker facilitated the construction of cationic liposomes and lipoplexes with different helper lipids. The chloride counterion not only facilitated the hydration of the lipid films during liposome construction, but also enhanced the hydrolysis of the ortho ester linker in the lipoplexes. After incubation at endosomal pH 5.5, the Type I lipoplexes aggregated and destabilized the endosome-mimicking model liposomes, but not the Type II lipoplexes. The helper lipids (DOPE or cholesterol) of the lipoplexes enhanced the pH-sensitivity of the Type I lipoplexes. In CV-1 cells (monkey kidney fibroblast), the Type I ortho ester-based lipoplexes, especially those with the chloride counterion, significantly improved the gene transfection efficiency, in some cases by more than 100 fold, compared to their pH-insensitive counterparts consisting of DOTAP. The gene transfection efficiency of the ortho ester-based lipoplexes was well correlated with their rate of aggregation and membrane destabilization in response to the endosomal pH 5.5. PMID:22480493

  14. Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases. (United States)

    Naumann, Todd A; Wicklow, Donald T; Price, Neil P J


    Cmps (chitinase-modifying proteins) are fungal proteases that truncate plant class IV chitinases by cleaving near their N-termini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine-cysteine bond within the hevein domain. In the present paper we describe a new type of cmp, polyglycine hydrolases, as proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine linker of plant class IV chitinases. Polyglycine hydrolases were purified from Cochliobolus carbonum (syn. Bipolaris zeicola; Bz-cmp) and Epicoccum sorghi (syn. Phoma sorghina; Es-cmp) and were shown to cleave three different maize class IV chitinase substrates. The proteolytic cleavage sites were assessed by SDS/PAGE and MALDI-TOF-MS and indicated the cleavage of multiple peptide bonds within the polyglycine linker regions. Site-directed mutagenesis was used to produce mutants of maize ChitB chitinase in which two serine residues in its linker were systematically modified to glycine. Serine to glycine changes in the ChitB linker resulted in higher susceptibility to truncation by Bz-cmp and altered substrate specificity for Bz-cmp and Es-cmp, such that different glycine-glycine peptide bonds were cleaved. Removal of the hevein domain led to loss of Es-cmp activity, indicating that interactions outside of the active site are important for recognition. Our findings demonstrate that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. This novel proteolysis of polyglycine motifs is previously unreported, but the specificity is similar to that of bacterial lysostaphin proteases, which cleave pentaglycine cross-links from peptidoglycan.

  15. An intelligent inter-domain routing scheme under the consideration of diffserv QoS and energy saving in multi-domain software-defined flexible optical networks (United States)

    Zhao, Jijun; Li, Fengyun; Ren, Danping; Hu, Jinhua; Yao, Qiuyan; Li, Wei


    Large scale multi-domain software-defined optical networks (SDON) provisioning has become a key area with increased scalable bandwidth services across wider network regions. Although distributed schemes could achieve lightpath routing by the ergodic process of domain boundary nodes, it increases the complexity of the signaling procedure and deployment of the interface. Moreover, the physical impairments are always the main factor of the infrastructure layer in SDON, which affect the transmission quality of the signal. Meanwhile, with increasing energy consumption of the Internet, it is imperative to design energy-efficient networks. To address the above issues, in this paper, an intelligent inter-domain routing scheme, which is supported by hierarchical control plane architecture, is presented based on sub-topology graph under the consideration of diffserv quality-of-service (QoS) and energy saving. The proposed scheme could achieve multi-domain quality of transmission (QoT), energy aware routing and spectrum assignment (RSA). We explore the scenarios where the multi-domain SDON achieve energy efficiency on the basis of meeting the QoT requirement. The blocking, energy consumption and average set up delay performances of the proposed schemes are estimated. The results indicate that the introduction of sub-topology in multi-domain RSA scheme has the better performance comparing with the distributed scheme.

  16. Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism (United States)

    Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.


    Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention. PMID:28291250

  17. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3. (United States)

    Floor, Stephen N; Condon, Kendall J; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A


    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.

  18. Construction of a novel coarse grain model for simulations of HIV capsid assembly to capture the backbone structure and inter-domain motions in solution

    Directory of Open Access Journals (Sweden)

    Xin Qiao


    Full Text Available We show the construction of a novel coarse grain model for simulations of HIV capsid assembly based on four structural models of HIV capsid proteins: isolated hexamer 3H47.pdb, tubular assembly 3J34.pdb, isolated pentamer 3P05.pdb and C-terminus dimer 2KOD.pdb. The data demonstrates the derivation of inter-domain motions from all atom Molecular Dynamics simulations and comparison with the motions derived from the analysis of solution NMR results defined in 2M8L.pdb. Snapshots from a representative Monte Carlo simulation with 128 dimeric subunit proteins based on 3J34.pdb are shown in addition to the quantitative analysis of its assembly pathway. Movies of the assembly process are compiled with snapshots of representative simulations of four structural models. The methods and data in this article were utilized in Qiao et al. (in press [1] to probe the mechanism of polymorphism and curvature control of HIV capsid assembly.

  19. Toughening of a Carbon-Fibre Composite Using Electrospun Poly(Hydroxyether of Bisphenol A Nanofibrous Membranes Through Inverse Phase Separation and Inter-Domain Etherification

    Directory of Open Access Journals (Sweden)

    Bronwyn Fox


    Full Text Available The interlaminar toughening of a carbon fibre reinforced composite by interleaving a thin layer (~20 microns of poly(hydroxyether of bisphenol A (phenoxy nanofibres was explored in this work. Nanofibres, free of defect and averaging several hundred nanometres, were produced by electrospinning directly onto a pre-impregnated carbon fibre material (Toray G83C at various concentrations between 0.5 wt % and 2 wt %. During curing at 150 °C, phenoxy diffuses through the epoxy resin to form a semi interpenetrating network with an inverse phase type of morphology where the epoxy became the co-continuous phase with a nodular morphology. This type of morphology improved the fracture toughness in mode I (opening failure and mode II (in-plane shear failure by up to 150% and 30%, respectively. Interlaminar shear stress test results showed that the interleaving did not negatively affect the effective in-plane strength of the composites. Furthermore, there was some evidence from DMTA and FT-IR analysis to suggest that inter-domain etherification between the residual epoxide groups with the pendant hydroxyl groups of the phenoxy occurred, also leading to an increase in glass transition temperature (~7.5 °C.

  20. Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry. (United States)

    Song, Hongjian; Olsen, Ole H; Persson, Egon; Rand, Kasper D


    Factor VIIa (FVIIa) is a trypsin-like protease that plays an important role in initiating blood coagulation. Very limited structural information is available for the free, inactive form of FVIIa that circulates in the blood prior to vascular injury and the molecular details of its activity enhancement remain elusive. Here we have applied hydrogen/deuterium exchange mass spectrometry coupled to electron transfer dissociation to pinpoint individual residues in the heavy chain of FVIIa whose conformation and/or local interaction pattern changes when the enzyme transitions to the active form, as induced either by its cofactor tissue factor or a covalent active site inhibitor. Identified regulatory residues are situated at key sites across one continuous surface of the protease domain spanning the TF-binding helix across the activation pocket to the calcium binding site and are embedded in elements of secondary structure and at the base of flexible loops. Thus these residues are optimally positioned to mediate crosstalk between functional sites in FVIIa, particularly the cofactor binding site and the active site. Our results unambiguously show that the conformational allosteric activation signal extends to the EGF1 domain in the light chain of FVIIa, underscoring a remarkable intra- and interdomain allosteric regulation of this trypsin-like protease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Biodistribution of the chimeric monoclonal antibody U36 radioiodinated with a closo-dodecaborate-containing linker. Comparison with other radioiodination methods.

    NARCIS (Netherlands)

    Nestor, M; Persson, M; Cheng, J.; Tolmachev, V; Dongen, van G.A.M.S.; Anniko, M; Kairemo, K


    We have evaluated the applicability of the [(4-isothiocyanatobenzylammonio)undecahydro-closo-dodecaborate (1-)] (DABI) linker molecule for antibody radiohalogenation and compared it to radiohalogenation using the linker N-succinimidyl 4-iodobenzoate (PIB) and to direct radiohalogenation using Chlora

  2. Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts (United States)

    Guaitoli, Giambattista; Raimondi, Francesco; Gilsbach, Bernd K.; Gómez-Llorente, Yacob; Deyaert, Egon; Renzi, Fabiana; Li, Xianting; Schaffner, Adam; Jagtap, Pravin Kumar Ankush; Boldt, Karsten; von Zweydorf, Felix; Gotthardt, Katja; Lorimer, Donald D.; Yue, Zhenyu; Burgin, Alex; Janjic, Nebojsa; Sattler, Michael; Versées, Wim; Ueffing, Marius; Ubarretxena-Belandia, Iban; Kortholt, Arjan; Gloeckner, Christian Johannes


    Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson’s disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity—together with the LRR domain—to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD. PMID:27357661

  3. Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1. (United States)

    Bednar, Jan; Garcia-Saez, Isabel; Boopathi, Ramachandran; Cutter, Amber R; Papai, Gabor; Reymer, Anna; Syed, Sajad H; Lone, Imtiaz Nisar; Tonchev, Ognyan; Crucifix, Corinne; Menoni, Hervé; Papin, Christophe; Skoufias, Dimitrios A; Kurumizaka, Hitoshi; Lavery, Richard; Hamiche, Ali; Hayes, Jeffrey J; Schultz, Patrick; Angelov, Dimitar; Petosa, Carlo; Dimitrov, Stefan


    Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. CyTargetLinker: a cytoscape app to integrate regulatory interactions in network analysis.

    Directory of Open Access Journals (Sweden)

    Martina Kutmon

    Full Text Available INTRODUCTION: The high complexity and dynamic nature of the regulation of gene expression, protein synthesis, and protein activity pose a challenge to fully understand the cellular machinery. By deciphering the role of important players, including transcription factors, microRNAs, or small molecules, a better understanding of key regulatory processes can be obtained. Various databases contain information on the interactions of regulators with their targets for different organisms, data recently being extended with the results of the ENCODE (Encyclopedia of DNA Elements project. A systems biology approach integrating our understanding on different regulators is essential in interpreting the regulation of molecular biological processes. IMPLEMENTATION: We developed CyTargetLinker (, a Cytoscape app, for integrating regulatory interactions in network analysis. Recently we released CyTargetLinker as one of the first apps for Cytoscape 3. It provides a user-friendly and flexible interface to extend biological networks with regulatory interactions, such as microRNA-target, transcription factor-target and/or drug-target. Importantly, CyTargetLinker employs identifier mapping to combine various interaction data resources that use different types of identifiers. RESULTS: Three case studies demonstrate the strength and broad applicability of CyTargetLinker, (i extending a mouse molecular interaction network, containing genes linked to diabetes mellitus, with validated and predicted microRNAs, (ii enriching a molecular interaction network, containing DNA repair genes, with ENCODE transcription factor and (iii building a regulatory meta-network in which a biological process is extended with information on transcription factor, microRNA and drug regulation. CONCLUSIONS: CyTargetLinker provides a simple and extensible framework for biologists and bioinformaticians to integrate different regulatory interactions

  5. Development of Bioorthogonally Degradable Linkers and Polymers Using alpha-Azidoethers (United States)

    Rajagopalan, Chandrasekhar Ramasubramanian

    Degradable polymers have gained a lot of attention in recent years for applications in biotechnology and medicine. External control over polymer degradation can be obtained by incorporating functional groups that cleave in the presence of triggers that would normally be absent in biological environments, i.e. are bioorthogonal. This thesis explores the use of chemically cleavable alpha-azidoethers as a new method to obtain external control over the degradation behavior of polymers. My first goal is to illustrate the potential of alpha-azidoethers toward developing cleavable linkers. We have studied the relationship between alpha-azidoether structure and hydrolytic stability, to prepare linkers that withstand background hydrolytic cleavage until they are exposed to the cleaving trigger. The cleavage kinetics of the alpha-azidoether functional group was quantified. In addition to the conventionally used tris(2-carboxyethyl)phosphine (TCEP), dihydrolipoic acid (DHLA), a previously unexplored, biocompatible reducing agent, was also evaluated as a cleaving trigger. Based on these results, we have proposed design rules for utilizing alpha-azidoethers as cleavable linkers in applications that require bioorthogonal control over linker cleavage. Secondly, the alpha-azidoether cleavable linker chemistry was implemented into the development of polymeric materials. Two different types of polymers were developed. Polyamides incorporating alpha-azidoethers along the backbone were synthesized, and their physical properties and chemically triggered degradation behavior were characterized. The degradation timescale of these polymers can be tuned simply by manipulating the concentration of the externally applied chemical trigger. The alpha-azidoether functional group was then utilized to develop a unique triggered-release polymeric adhesive for potential applications in dental adhesive formulations. A methacrylamide-phosphonate adhesive monomer incorporating an alpha

  6. Aromatic fumaronitrile core-based donor-linker-acceptor-linker-donor (D-pi-A-pi-D) compounds: synthesis and photophysical properties. (United States)

    Panthi, Krishna; Adhikari, Ravi M; Kinstle, Thomas H


    A new class of aromatic fumaronitrile core-based compounds with different donors and linkers has been synthesized and well characterized. Compounds 1 and 2 have indole and 2-phenylindole groups as electron donors, respectively. Compounds 3 and 4 have a diphenylamino group as the electron donor, and compound 5 has a 3,6-di-tert-butylcarbazole group as an electron donor. These compounds absorb in the blue-to-green region and emit in the blue-to-red region depending on the electron donor, linker, and solvents. The quantum yields of fluorescence of these compounds in solution are measured and found to be moderate, but in solid states, they are high. These compounds display strong emission solvatochromism that is reflected by a large shift in their fluorescence emission maxima on changing the solvents. This change is accompanied by a successive decrease in fluorescence intensity. The fluorescence lifetimes of these compounds are measured in different solvent and found to vary from compounds with solvents, concentration, and excitation energy have been studied. The correlation between the functional group and optical properties has been established to some extent. The ability of these compounds to function as colorimetric and luminescence pH sensors is demonstrated with color changes and luminescence switching upon the addition of trifluoroacetic acid. The potentiality of these compounds for application in optoelectronics has been optically assessed.

  7. Minimum Length - Maximum Velocity

    CERN Document Server

    Panes, Boris


    We study a framework where the hypothesis of a minimum length in space-time is complemented with the notion of reference frame invariance. It turns out natural to interpret the action of the obtained reference frame transformations in the context of doubly special relativity. As a consequence of this formalism we find interesting connections between the minimum length properties and the modified velocity-energy relation for ultra-relativistic particles. For example we can predict the ratio between the minimum lengths in space and time using the results from OPERA about superluminal neutrinos.

  8. cis-Apa: a practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage. (United States)

    Baron, Alice; Verdié, Pascal; Martinez, Jean; Lamaty, Frédéric


    A new linker cis-5-aminopent-3-enoic acid (cis-Apa) was prepared for the synthesis of cyclic pseudopeptides by cyclization-cleavage by using ring-closing methatesis (RCM). We developed a new synthetic pathway for the preparation of the cis-Apa linker that was tested in the cyclization-cleavage process of different RGD peptide sequences. Different macrocyclic peptidomimetics were prepared by using this integrated microwave-assisted method, showing that the readily available cis-Apa amino acid is well adapted as a linker in the cyclization-cleavage process.

  9. Synthesis of Biotin Linkers with the Activated Triple Bond Donor [p-(N-propynoylaminotoluic Acid] (PATA for Efficient Biotinylation of Peptides and Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Martina Jezowska


    Full Text Available Biotin is an important molecule for modern biological studies including, e.g., cellular transport. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows ready and specific detection. As a consequence methods for the attachment of biotin to various biological targets are of high importance, especially when they are very selective and can also proceed in water. One useful method is Hüisgen dipolar [3+2]-cycloaddition, commonly referred to as “click chemistry”. As we reported recently, the activated triple bond donor p-(N-propynoylaminotoluic acid (PATA gives excellent results when used for conjugations at submicromolar concentrations. Thus, we have designed and synthesized two biotin linkers, with different lengths equipped with this activated triple bond donor and we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution and on solid support with excellent yields of conversion.

  10. Effects of branched O-glycosylation on a semiflexible peptide linker. (United States)

    Johnson, Quentin R; Lindsay, Richard J; Raval, Sherin R; Dobbs, Jeremy S; Nellas, Ricky B; Shen, Tongye


    Glycosylation is an essential modification of proteins and lipids by the addition of carbohydrate residues. These attached carbohydrates range from single monomers to elaborate branched glycans. Here, we examine how the level of glycosylation affects the conformation of a semiflexible peptide linker using the example of the hinge peptide from immunoglobulin A. Three sets of atomistic models of this hinge peptide with varying degrees of glycosylation are constructed to probe how glycosylation affects the physical properties of the linker. We found that glycosylation greatly altered the predominant conformations of the peptide, causing it to become elongated in reference to the unglycosylated form. Furthermore, glycosylation restricts the conformational exploration of the peptide. At the residue level, glycans are found to introduce a bias for the formation of more extended secondary structural elements for glycosylated serines. Additionally, the flexibility of this semiflexible proline-rich peptide is significantly reduced by glycosylation.

  11. Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

    DEFF Research Database (Denmark)

    Pedersen, Nis Borbye; Wang, Shengjun; Narimatsu, Yoshiki;


    , which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide GalNAc...... transferases. Probing into which isoform(s) contributed to glycosylation of the linker regions of the LDLR class A repeats by in vitro enzyme assays suggested a major role of GalNAc-T11. This was supported by expression of LDLR in HEK293 cells, where knock-out of the GalNAc-T11 isoform resulted in the loss...

  12. Structural and dynamic properties of linker histone H1 binding to DNA

    CERN Document Server

    Dootz, Rolf; Pfohl, Thomas


    Found in all eukaryotic cells, linker histones H1 are known to bind to and rearrange nucleosomal linker DNA. In vitro, the fundamental nature of H1/DNA interactions has attracted wide interest among research communities - for biologists from a chromatin organization deciphering point of view, and for physicists from the study of polyelectrolyte interactions point of view. Hence, H1/DNA binding processes, structural and dynamical information about these self-assemblies is of broad importance. Targeting a quantitative understanding of H1 induced DNA compaction mechanisms our strategy is based on using small angle X-ray microdiffraction in combination with microfluidics. The usage of microfluidic hydrodynamic focusing devices facilitate a microscale control of these self-assembly processes. In addition, the method enables time-resolved access to structure formation in situ, in particular to transient intermediate states. The observed time dependent structure evolution shows that the interaction of H1 with DNA ca...

  13. Quantum-dot-sensitized solar cells: understanding linker molecules through theory and experiment. (United States)

    Margraf, Johannes T; Ruland, Andrés; Sgobba, Vito; Guldi, Dirk M; Clark, Timothy


    We have investigated the role of linker molecules in quantum-dot-sensitized solar cells (QDSSCs) using density-functional theory (DFT) and experiments. Linkers not only govern the number of attached QDs but also influence charge separation, recombination, and transport. Understanding their behavior is therefore not straightforward. DFT calculations show that mercaptopropionic acid (MPA) and cysteine (Cys) exhibit characteristic binding configurations on TiO(2) surfaces. This information is used to optimize the cell assembly process, yielding Cys-based cells that significantly outperform MPA cells, and reach power conversion efficiencies (PCE) as high as 2.7% under AM 1.5 illumination. Importantly, the structural information from theory also helps understand the cause for this improved performance.

  14. The H1 linker histones: multifunctional proteins beyond the nucleosomal core particle. (United States)

    Hergeth, Sonja P; Schneider, Robert


    The linker histone H1 family members are a key component of chromatin and bind to the nucleosomal core particle around the DNA entry and exit sites. H1 can stabilize both nucleosome structure and higher-order chromatin architecture. In general, H1 molecules consist of a central globular domain with more flexible tail regions at both their N- and C-terminal ends. The existence of multiple H1 subtypes and a large variety of posttranslational modifications brings about a considerable degree of complexity and makes studying this protein family challenging. Here, we review recent progress in understanding the function of linker histones and their subtypes beyond their role as merely structural chromatin components. We summarize current findings on the role of H1 in heterochromatin formation, transcriptional regulation and embryogenesis with a focus on H1 subtypes and their specific modifications. © 2015 The Authors.

  15. Linker histones: novel insights into structure-specific recognition of the nucleosome. (United States)

    Cutter, Amber R; Hayes, Jeffrey J


    Linker histones (H1s) are a primary component of metazoan chromatin, fulfilling numerous functions, both in vitro and in vivo, including stabilizing the wrapping of DNA around the nucleosome, promoting folding and assembly of higher order chromatin structures, influencing nucleosome spacing on DNA, and regulating specific gene expression. However, many molecular details of how H1 binds to nucleosomes and recognizes unique structural features on the nucleosome surface remain undefined. Numerous, confounding studies are complicated not only by experimental limitations, but the use of different linker histone isoforms and nucleosome constructions. This review summarizes the decades of research that has resulted in several models of H1 association with nucleosomes, with a focus on recent advances that suggest multiple modes of H1 interaction in chromatin, while highlighting the remaining questions.

  16. Randomized Terminal Linker-dependent PCR: A Versatile and Sensitive Method for Detection of DNA Damage

    Institute of Scientific and Technical Information of China (English)


    Objective To design and develop a novel, sensitive and versatile method for in vivo foot printing and studies of DNA damage, such as DNA adducts and strand breaks. Methods Starting with mammalian genomic DNA, single-stranded products were made by repeated primer extension, these products were ligated to a double-stranded linker having a randomized 3′ overhang, and used for PCR.DNA breaks in p53 gene produced by restriction endonuclease AfaI were detected by using this new method followed by Southern hybridization with DIG-labeled probe. Results This randomized terminal linker-dependent PCR (RDPCR) method could generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR), and it was more rapid, convenient and accurate than the terminal transferase-dependent PCR (TDPCR). Conclusion DNA strand breakage can be detected sensitively in the gene level by RDPCR. Any lesion that blocks primer extension should be detectable.

  17. DNA-duplex linker for AFM-SELEX of DNA aptamer against human serum albumin. (United States)

    Takenaka, Musashi; Okumura, Yuzo; Amino, Tomokazu; Miyachi, Yusuke; Ogino, Chiaki; Kondo, Akihiko


    DNA-duplex interactions in thymines and adenins are used as a linker for the novel methodology of Atomic Force Microscope-Systematic Evolution of Ligands by EXpotential enrichment (AFM-SELEX). This study used the hydrogen bonds in 10 mer of both thymines (T10) and adenines (A10). Initially, the interactive force in T10-A10 was measured by AFM, which returned an average interactive force of approximately 350pN. Based on this result, DNA aptamers against human serum albumin could be selected in the 4th round, and 15 different clones could be sequenced. The lowest dissociation constant of the selected aptamer was identified via surface plasmon resonance, and it proved to be identical to that of the commercial aptamer. Therefore, specific hydrogen bonds in DNA can be useful linkers for AFM-SELEX. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Telomere length and depression

    DEFF Research Database (Denmark)

    Wium-Andersen, Marie Kim; Ørsted, David Dynnes; Rode, Line


    BACKGROUND: Depression has been cross-sectionally associated with short telomeres as a measure of biological age. However, the direction and nature of the association is currently unclear. AIMS: We examined whether short telomere length is associated with depression cross-sectionally as well...... as prospectively and genetically. METHOD: Telomere length and three polymorphisms, TERT, TERC and OBFC1, were measured in 67 306 individuals aged 20-100 years from the Danish general population and associated with register-based attendance at hospital for depression and purchase of antidepressant medication....... RESULTS: Attendance at hospital for depression was associated with short telomere length cross-sectionally, but not prospectively. Further, purchase of antidepressant medication was not associated with short telomere length cross-sectionally or prospectively. Mean follow-up was 7.6 years (range 0...

  19. Myofilament length dependent activation

    Energy Technology Data Exchange (ETDEWEB)

    de Tombe, Pieter P.; Mateja, Ryan D.; Tachampa, Kittipong; Mou, Younss Ait; Farman, Gerrie P.; Irving, Thomas C. (IIT); (Loyola)


    The Frank-Starling law of the heart describes the interrelationship between end-diastolic volume and cardiac ejection volume, a regulatory system that operates on a beat-to-beat basis. The main cellular mechanism that underlies this phenomenon is an increase in the responsiveness of cardiac myofilaments to activating Ca{sup 2+} ions at a longer sarcomere length, commonly referred to as myofilament length-dependent activation. This review focuses on what molecular mechanisms may underlie myofilament length dependency. Specifically, the roles of inter-filament spacing, thick and thin filament based regulation, as well as sarcomeric regulatory proteins are discussed. Although the 'Frank-Starling law of the heart' constitutes a fundamental cardiac property that has been appreciated for well over a century, it is still not known in muscle how the contractile apparatus transduces the information concerning sarcomere length to modulate ventricular pressure development.

  20. A Characteristic Particle Length

    CERN Document Server

    Roberts, Mark D


    It is argued that there are characteristic intervals associated with any particle that can be derived without reference to the speed of light $c$. Such intervals are inferred from zeros of wavefunctions which are solutions to the Schr\\"odinger equation. The characteristic length is $\\ell=\\beta^2\\hbar^2/(8Gm^3)$, where $\\beta=3.8\\dots$; this length might lead to observational effects on objects the size of a virus.

  1. Moiety and linker site heterologies for highly sensitive immunoanalysis of cyprodinil in fermented alcoholic drinks


    Esteve Turrillas, Francesc Albert; Mercader Badia, Josep Vicent; Agulló, Consuelo; Abad Somovilla, Antonio; Abad Fuentes, Antonio


    Cyprodinil is a new-generation anilinopyrimidine fungicide widely used in crop protection and frequently found in fruits. In this study, novel derivatives of cyprodinil with linker site heterologies were synthesized and employed in order to produce antibodies with enhanced affinity. Moreover, moiety-heterologous haptens were designed and prepared for assay sensitivity improvement. Two competitive enzyme-linked immunosorbent assays for the analysis of this active substance were developed using...

  2. A New Achiral Linker Reagent for the Incorporation of Multiple Amino Groups Into Oligonucleotides

    DEFF Research Database (Denmark)


    The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide wit......, to a method for preparing a labelled oligonucleotide, and to the use of the labelled oligonucleotide as hybridisation probe, in polymerase chain reactions (PCR), in nucleic acid sequencing, in cloning recombinant DNA and $i(in vitro) mutagenesis....

  3. The S4-S5 linker couples voltage sensing and activation of pacemaker channels. (United States)

    Chen, J; Mitcheson, J S; Tristani-Firouzi, M; Lin, M; Sanguinetti, M C


    Voltage-gated channels are normally opened by depolarization and closed by repolarization of the membrane. Despite sharing significant sequence homology with voltage-gated K(+) channels, the gating of hyperpolarization-activated, cyclic-nucleotide-gated (HCN) pacemaker channels has the opposite dependence on membrane potential: hyperpolarization opens, whereas depolarization closes, these channels. The mechanism and structural basis of the process that couples voltage sensor movement to HCN channel opening and closing is not understood. On the basis of our previous studies of a mutant HERG (human ether-a-go-go-related gene) channel, we hypothesized that the intracellular linker that connects the fourth and fifth transmembrane domains (S4-S5 linker) of HCN channels might be important for channel gating. Here, we used alanine-scanning mutagenesis of the HCN2 S4-S5 linker to identify three residues, E324, Y331, and R339, that when mutated disrupted normal channel closing. Mutation of a basic residue in the S4 domain (R318Q) prevented channel opening, presumably by disrupting S4 movement. However, channels with R318Q and Y331S mutations were constitutively open, suggesting that these channels can open without a functioning S4 domain. We conclude that the S4-S5 linker mediates coupling between voltage sensing and HCN channel activation. Our findings also suggest that opening of HCN and related channels corresponds to activation of a gate located near the inner pore, rather than recovery of channels from a C-type inactivated state.

  4. Roles of the linker region of RNA helicase A in HIV-1 RNA metabolism.

    Directory of Open Access Journals (Sweden)

    Li Xing

    Full Text Available RNA helicase A (RHA promotes multiple steps in HIV-1 production including transcription and translation of viral RNA, annealing of primer tRNA(Lys3 to viral RNA, and elevating the ratio of unspliced to spliced viral RNA. At its amino terminus are two double-stranded RNA binding domains (dsRBDs that are essential for RHA-viral RNA interaction. Linking the dsRBDs to the core helicase domain is a linker region containing 6 predicted helices. Working in vitro with purified mutant RHAs containing deletions of individual helices reveals that this region may regulate the enzyme's helicase activity, since deletion of helix 2 or 3 reduces the rate of unwinding RNA by RHA. The biological significance of this finding was then examined during HIV-1 production. Deletions in the linker region do not significantly affect either RHA-HIV-1 RNA interaction in vivo or the incorporation of mutant RHAs into progeny virions. While the partial reduction in helicase activity of mutant RHA containing a deletion of helices 2 or 3 does not reduce the ability of RHA to stimulate viral RNA synthesis, the promotion of tRNA(Lys3 annealing to viral RNA is blocked. In contrast, deletion of helices 4 or 5 does not affect the ability of RHA to promote tRNA(Lys3 annealing, but reduces its ability to stimulate viral RNA synthesis. Additionally, RHA stimulation of viral RNA synthesis results in an increased ratio of unspliced to spliced viral RNA, and this increase is not inhibited by deletions in the linker region, nor is the pattern of splicing changed within the ∼ 4.0 kb or ∼ 1.8 kb HIV-1 RNA classes, suggesting that RHA's effect on suppressing splicing is confined mainly to the first 5'-splice donor site. Overall, the differential responses to the mutations in the linker region of RHA reveal that RHA participates in HIV-1 RNA metabolism by multiple distinct mechanisms.

  5. Equilibrium CO bond lengths (United States)

    Demaison, Jean; Császár, Attila G.


    Based on a sample of 38 molecules, 47 accurate equilibrium CO bond lengths have been collected and analyzed. These ultimate experimental (reEX), semiexperimental (reSE), and Born-Oppenheimer (reBO) equilibrium structures are compared to reBO estimates from two lower-level techniques of electronic structure theory, MP2(FC)/cc-pVQZ and B3LYP/6-311+G(3df,2pd). A linear relationship is found between the best equilibrium bond lengths and their MP2 or B3LYP estimates. These (and similar) linear relationships permit to estimate the CO bond length with an accuracy of 0.002 Å within the full range of 1.10-1.43 Å, corresponding to single, double, and triple CO bonds, for a large number of molecules. The variation of the CO bond length is qualitatively explained using the Atoms in Molecules method. In particular, a nice correlation is found between the CO bond length and the bond critical point density and it appears that the CO bond is at the same time covalent and ionic. Conditions which permit the computation of an accurate ab initio Born-Oppenheimer equilibrium structure are discussed. In particular, the core-core and core-valence correlation is investigated and it is shown to roughly increase with the bond length.

  6. Observations on oesophageal length. (United States)

    Kalloor, G J; Deshpande, A H; Collis, J L


    The subject of oesophageal length is discussed. The great variations in the length of the oesophagus in individual patients is noted, and the practical use of its recognition in oesophageal surgery is stressed. An apprasial of the various methods available for this measurement is made; this includes the use of external chest measurement, endoscopic measurement, and the measurement of the level of the electrical mucosal potential change. Correlative studies of these various methods are made, and these show a very high degree of significance. These studies involved simultaneous measurement of external and internal oesophageal length in 26 patients without a hiatal hernia or gastro-oesophageal length in 26 patients without a hiatal hernia or gastro-oesophageal reflux symptoms, 42 patients with sliding type hiatal hernia, and 17 patients with a peptic stricture in association with hiatal hernia. The method of measuring oesophageal length by the use of the external chest measurement, that is, the distance between the lower incisor teeth and the xiphisternum, measured with the neck fully extended and the patient lying supine, is described in detail, its practical application in oesophageal surgery is illustrated, and its validity tested by internal measurements. The findings of this study demonstrate that the external chest measurement provides a mean of assessing the true static length of the oesophagus, corrected for the size of the individual. Images PMID:941114

  7. Dissecting interdomain communication within cAPK regulatory subunit type IIβ using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS) (United States)

    Zawadzki, Kerri M.; Hamuro, Yoshitomo; Kim, Jack S.; Garrod, Siv; Stranz, David D.; Taylor, Susan S.; Woods, Virgil L.


    cAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RIIβ subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain. PMID:12930997

  8. Efficient Naphthalenediimide-Based Hole Semiconducting Polymer with Vinylene Linkers between Donor and Acceptor Units

    KAUST Repository

    Zhang, Lei


    We demonstrate a new method to reverse the polarity and charge transport behavior of naphthalenediimide (NDI)-based copolymers by inserting a vinylene linker between the donor and acceptor units. The vinylene linkers minimize the intrinsic steric congestion between the NDI and thiophene moieties to prompt backbone planarity. The polymers with vinylene linkers exhibit electron n-channel transport characteristics under vacuum, similar to the benchmark polymer, P(NDI2OD-T2). To our surprise, when the polymers are measured in air, the dominant carrier type switches from n- to p-type and yield hole mobilities up to 0.45 cm(2) s(-1) with hole to electron mobility ratio of three (mu(h)/mu(e), similar to 3), which indicates that the hole density in the active layer can be significantly increased by exposure to air. This increase is consistent with the intrinsic more delocalized nature of the highest occupied molecular orbital of the charged vinylene polymer, as estimated by density functional theory (DFT) calculations, which facilitates hole transport within the polymer chains. This is the first demonstration of an efficient NDI-based hole semiconducting polymer, which will enable new developments in all-polymer solar cells, complementary circuits, and dopable polymers for use in thermoelectrics.

  9. The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms. (United States)

    Wang, Huanyu; Ge, Zhongqi; Walsh, Scott T R; Parthun, Mark R


    Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.

  10. Fivefold increase of hydrogen uptake in MOF74 through linker decorations (United States)

    Arter, C. A.; Zuluaga, S.; Harrison, D.; Welchman, E.; Thonhauser, T.


    We present ab initio results for linker decorations in Mg-MOF74, i.e., attaching various metals M =Li, Na, K, Sc, Cr, Mn, Fe, Ni, Cu, Zn, Rb, Pd, Ag, and Pt near the ring of the linker, creating new strong adsorption sites and thus maximizing small-molecule uptake. We find that in most cases these decorations influence the overall form and structure of Mg-MOF74 only marginally. After the initial screening, we chose metals that bind favorably to the linker and further investigated adsorption of H2,CO2, and H2O for M =Li , Na, K, and Sc. For the case of H2 we show that up to 24 additional guest molecules can be adsorbed in the metal-organic framework (MOF) unit cell, with binding energies comparable to the original open-metal sites at the six corners of the channel. This leads to a fivefold increase of the molecule uptake in Mg-MOF74, with tremendous impact on many applications in general and hydrogen storage in particular, where the gravimetric hydrogen density increases from 1.63 to 7.28 mass % and the volumetric density increases from 15.10 to 75.50 g H2L-1 .

  11. PDP-CON: prediction of domain/linker residues in protein sequences using a consensus approach. (United States)

    Chatterjee, Piyali; Basu, Subhadip; Zubek, Julian; Kundu, Mahantapas; Nasipuri, Mita; Plewczynski, Dariusz


    The prediction of domain/linker residues in protein sequences is a crucial task in the functional classification of proteins, homology-based protein structure prediction, and high-throughput structural genomics. In this work, a novel consensus-based machine-learning technique was applied for residue-level prediction of the domain/linker annotations in protein sequences using ordered/disordered regions along protein chains and a set of physicochemical properties. Six different classifiers-decision tree, Gaussian naïve Bayes, linear discriminant analysis, support vector machine, random forest, and multilayer perceptron-were exhaustively explored for the residue-level prediction of domain/linker regions. The protein sequences from the curated CATH database were used for training and cross-validation experiments. Test results obtained by applying the developed PDP-CON tool to the mutually exclusive, independent proteins of the CASP-8, CASP-9, and CASP-10 databases are reported. An n-star quality consensus approach was used to combine the results yielded by different classifiers. The average PDP-CON accuracy and F-measure values for the CASP targets were found to be 0.86 and 0.91, respectively. The dataset, source code, and all supplementary materials for this work are available at for noncommercial use.

  12. Synthesis of hyaluronan haloacetates and biology of novel cross-linker-free synthetic extracellular matrix hydrogels. (United States)

    Serban, Monica A; Prestwich, Glenn D


    Hyaluronan (HA) derivatives containing thiol-reactive electrophilic esters were prepared to react with thiol-modified macromolecules to give cross-linker-free hydrogels. Specifically, HA was converted to two haloacetate derivatives, HA bromoacetate (HABA) and HA iodoacetate (HAIA). In cytotoxicity assays, these reactive macromolecules predictably induced cell death in a dose-dependent manner. Cross-linker-free synthetic extracellular matrix (sECM) hydrogels were prepared by thiol alkylation using HAIA and HABA as polyvalent electrophiles and thiol-modified HA (CMHA-S) with or without thiol-modified gelatin (Gtn-DTPH) as polyvalent nucleophiles. When primary human fibroblasts were seeded on the surface of the sECMs containing only the electrophilic HA haloacetate and nucleophilic CMHA-S components, no significant cytoadherence was observed. Cell attachment and viability was 17% (HABA) to 30% (HAIA) lower on HA haloacetate cross-linked hydrogels than on CMHA-S that had been oxidatively cross-linked via disulfide-bonds. In contrast, sECMs that included Gtn-DTPH allowed fibroblasts to attach, spread, and proliferate. Taken together, the HA haloacetates are attractive candidates for producing cross-linker-free sECM biomaterials that can function either as anti-adhesive barriers or as cytoadhesive sECMs for cell culture in pseudo-3-D.

  13. Bifunctional bridging linker-assisted synthesis and characterization of TiO2/Au nanocomposites (United States)

    Žunič, Vojka; Kurtjak, Mario; Suvorov, Danilo


    Using a simple organic bifunctional bridging linker, titanium dioxide (TiO2) nanoparticles were coupled with the Au nanoparticles to form TiO2/Au nanocomposites with a variety of Au loadings. This organic bifunctional linker, meso-2,3-dimercaptosuccinic acid, contains two types of functional groups: (i) the carboxyl group, which enables binding to the TiO2, and (ii) the thiol group, which enables binding to the Au. In addition, the organic bifunctional linker acts as a stabilizing agent to prevent the agglomeration and growth of the Au particles, resulting in the formation of highly dispersed Au nanoparticles. To form the TiO2/Au nanocomposites in a simple way, we deliberately applied a synthetic method that simultaneously ensures: (i) the capping of the Au nanoparticles and (ii) the binding of different amounts of Au to the TiO2. The TiO2/Au nanocomposites formed with this method show enhanced UV and Vis photocatalytic activities when compared to the pure TiO2 nanopowders.

  14. Linker proteins enable ultrafast excitation energy transfer in the phycobilisome antenna system of Thermosynechococcus vulcanus. (United States)

    Nganou, C; David, L; Adir, N; Mkandawire, M


    We applied a femtosecond flash method, using induced transient absorption changes, to obtain a time-resolved view of excitation energy transfer in intact phycobilisomes of Thermosynechococcus vulcanus at room temperature. Our measurement of an excitation energy transfer rate of 888 fs in phycobilisomes shows the existence of ultrafast kinetics along the phycocyanin rod subcomplex to the allophycocyanin core that is faster than expected for previous excitation energy transfer based on Förster theory in phycobilisomes. Allophycocyanin in the core further transfers energy to the terminal emitter(s) in 17 ps. In the phycobilisome, rod doublets composed of hexameric phycocyanin discs and internal linker proteins are arranged in a parallel fashion, facilitating direct rod-rod interactions. Excitonic splitting likely drives rod absorption at 635 nm as a result of strong coupling between β84 chromophores (20 ± 1 Å) in adjacent hexamers. In comparison to the absorbance of the phycobilisome antenna system of the cyanobacterium Acaryochloris marina, which possesses a single rod structure, the linkers in T. vulcanus rods induce a 17 nm red shift in the absorbance spectrum. Furthermore, the kinetics of 888 fs indicates that the presence of the linker protein induces ultrafast excitation energy transfer between phycocyanin and allophycocyanin inside the phycobilisome, which is faster than all previous excitation energy transfer in phycobilisome subunits or sub-complexes reported to date.

  15. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K; Illemann, Martin;


    MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full...

  16. A photolabile linker for the solid-phase synthesis of 4-substituted NH-1,2,3-triazoles. (United States)

    Qvortrup, Katrine; Nielsen, Thomas E


    This communication presents the synthesis of a novel photolabile azidolinker based on the o-nitroveratryl group. The application of this linker for the synthesis and photolytic release of NH-1,2,3-triazoles is described.

  17. Sensitive Photoacoustic IR Spectroscopy for the Characterization of Amino/Azido Mixed-Linker Metal-Organic Frameworks. (United States)

    Canivet, Jerome; Lysenko, Vladimir; Lehtinen, Jaakko; Legrand, Alexandre; Wisser, Florian M; Quadrelli, Elsje Alessandra; Farrusseng, David


    Photoacoustic Fourier-transform infrared spectroscopy makes it possible to determine the organic composition of mixed-linker metal-organic frameworks. The sound produced upon IR irradiation enables the discrimination of azido and amino linkers in three different MOF platforms with a sensitivity that is two orders of magnitude higher than that achieved using classic IR analysis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Mixed-linker zeolitic imidazolate framework mixed-matrix membranes for aggressive CO2 separation from natural gas

    KAUST Repository

    Thompson, Joshua A.


    Zeolitic imidazolate framework (ZIF) materials are a promising subclass of metal-organic frameworks (MOF) for gas separations. However, due to the deleterious effects of gate-opening phenomena associated with organic linker rotation near the limiting pore apertures of ZIFs, there have been few demonstrations of improved gas separation properties over pure polymer membranes when utilizing ZIF materials in composite membranes for CO2-based gas separations. Here, we report a study of composite ZIF/polymer membranes, containing mixed-linker ZIF materials with ZIF-8 crystal topologies but composed of different organic linker compositions. Characterization of the mixed-linker ZIFs shows that the mixed linker approach offers control over the porosity and pore size distribution of the materials, as determined from nitrogen physisorption and Horváth-Kawazoe analysis. Single gas permeation measurements on mixed-matrix membranes reveal that inclusion of mixed-linker ZIFs yields membranes with better ideal CO2/CH4 selectivity than membranes containing ZIF-8. This improvement is shown to likely occur from enhancement in the diffusion selectivity of the membranes associated with controlling the pore size distribution of the ZIF filler. Mixed-gas permeation experiments show that membranes with mixed-linker ZIFs display an effective plasticization resistance that is not typical of the pure polymeric matrix. Overall, we demonstrate that mixed-linker ZIFs can improve the gas separation properties in composite membranes and may be applicable to aggressive CO2 concentrations in natural gas feeds. © 2013 Elsevier Inc. All rights reserved.

  19. Synthesis and characterization of a linker for primary amines used in the solid phase organic synthesis of spermidine

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Emerson T. da; San Gil, Rosane A.S.; Lima, Edson L.S. [Universidade Federal do Rio de Janeiro (IQ/UFRJ), RJ (Brazil). Inst. de Quimica; Caldarelli, Stefano [Aix-Marseille Univ., Marseille (France). Campus de Saint Jerome; Ziarelli, Fabio [Aix-Marseille Universite Spectropole - Federation de Sciences Chimiques de Marseille, Campus de Saint Jerome (France)


    A linker resin for the synthesis of functionalized spermidine in good yield is described, along with its characterization by infrared (IR), {sup 13}C solid-state nuclear magnetic resonance with cross polarization and magic angle spinning ({sup 13}C CPMAS NMR) and {sup 1}H high resolution magic angle spinning nuclear magnetic resonance ({sup 1}H HRMAS NMR). This linker has been regenerated after cleavage of spermidine and re-used without loss of efficiency. (author)

  20. Hydrogen physisorption on metal-organic framework linkers and metalated linkers: a computational study of the factors that control binding strength. (United States)

    Tsivion, Ehud; Long, Jeffrey R; Head-Gordon, Martin


    In order for hydrogen gas to be used as a fuel, it must be stored in sufficient quantity on board the vehicle. Efforts are being made to increase the hydrogen storage capabilities of metal-organic frameworks (MOFs) by introducing unsaturated metal sites into their linking element(s), as hydrogen adsorption centers. In order to devise successful hydrogen storage strategies there is a need for a fundamental understanding of the weak and elusive hydrogen physisorption interaction. Here we report our findings from the investigation of the weak intermolecular interactions of adsorbed hydrogen molecules on MOF-linkers by using cluster models. Since physical interactions such as dispersion and polarization have a major contribution to attraction energy, our approach is to analyze the adsorption interaction using energy decomposition analysis (EDA) that distinguishes the contribution of the physical interactions from the charge-transfer (CT) "chemical" interaction. Surprisingly, it is found that CT from the adsorbent to the σ*(H2) orbital is present in all studied complexes and can contribute up to approximately -2 kJ/mol to the interaction. When metal ions are present, donation from the σ(H2) → metal Rydberg-like orbital, along with the adsorbent → σ*(H2) contribution, can contribute from -2 to -10 kJ/mol, depending on the coordination mode. To reach a sufficient adsorption enthalpy for practical usage, the hydrogen molecule must be substantially polarized. Ultimately, the ability of the metalated linker to polarize the hydrogen molecule is highly dependent on the geometry of the metal ion coordination site where a strong electrostatic dipole or quadrupole moment is required.

  1. Complete gene sequence of spider attachment silk protein (PySp1) reveals novel linker regions and extreme repeat homogenization. (United States)

    Chaw, Ro Crystal; Saski, Christopher A; Hayashi, Cheryl Y


    Spiders use a myriad of silk types for daily survival, and each silk type has a unique suite of task-specific mechanical properties. Of all spider silk types, pyriform silk is distinct because it is a combination of a dry protein fiber and wet glue. Pyriform silk fibers are coated with wet cement and extruded into "attachment discs" that adhere silks to each other and to substrates. The mechanical properties of spider silk types are linked to the primary and higher-level structures of spider silk proteins (spidroins). Spidroins are often enormous molecules (>250 kDa) and have a lengthy repetitive region that is flanked by relatively short (∼100 amino acids), non-repetitive amino- and carboxyl-terminal regions. The amino acid sequence motifs in the repetitive region vary greatly between spidroin type, while motif length and number underlie the remarkable mechanical properties of spider silk fibers. Existing knowledge of pyriform spidroins is fragmented, making it difficult to define links between the structure and function of pyriform spidroins. Here, we present the full-length sequence of the gene encoding pyriform spidroin 1 (PySp1) from the silver garden spider Argiope argentata. The predicted protein is similar to previously reported PySp1 sequences but the A. argentata PySp1 has a uniquely long and repetitive "linker", which bridges the amino-terminal and repetitive regions. Predictions of the hydrophobicity and secondary structure of A. argentata PySp1 identify regions important to protein self-assembly. Analysis of the full complement of A. argentata PySp1 repeats reveals extreme intragenic homogenization, and comparison of A. argentata PySp1 repeats with other PySp1 sequences identifies variability in two sub-repetitive expansion regions. Overall, the full-length A. argentata PySp1 sequence provides new evidence for understanding how pyriform spidroins contribute to the properties of pyriform silk fibers. Copyright © 2017 The Authors. Published by

  2. Mappability and Read Length

    Directory of Open Access Journals (Sweden)

    Wentian eLi


    Full Text Available Power-law distributions are the main functional form forthe distribution of repeat size and repeat copy number in the human genome. When the genome is broken into fragments for sequencing, the limited size offragments and reads may prevent an unique alignment of repeatsequences to the reference sequence. Repeats in the human genome canbe as long as $10^4$ bases, or $10^5-10^6$ bases when allowing for mismatches between repeat units. Sequence reads from these regions are therefore unmappable when the read length is in the range of $10^3$ bases.With the read length of exactly 1000 bases, slightly more than 1% of theassembled genome, and slightly less than 1% of the 1kbreads, are unmappable, excluding the unassembled portion of the humangenome (8% in GRCh37. The slow decay (long tail ofthe power-law function implies a diminishing return in convertingunmappable regions/reads to become mappable with the increase of theread length, with the understanding that increasing read length willalways move towards the direction of 100% mappability.

  3. Telomere Length and Mortality

    DEFF Research Database (Denmark)

    Kimura, Masayuki; Hjelmborg, Jacob V B; Gardner, Jeffrey P


    telomeres predicted the death of the first co-twin better than the mTRFL did (mTRFL: 0.56, 95% confidence interval (CI): 0.49, 0.63; mTRFL(50): 0.59, 95% CI: 0.52, 0.66; mTRFL(25): 0.59, 95% CI: 0.52, 0.66; MTRFL: 0.60, 95% CI: 0.53, 0.67). The telomere-mortality association was stronger in years 3-4 than......Leukocyte telomere length, representing the mean length of all telomeres in leukocytes, is ostensibly a bioindicator of human aging. The authors hypothesized that shorter telomeres might forecast imminent mortality in elderly people better than leukocyte telomere length. They performed mortality...... analysis in 548 same-sex Danish twins (274 pairs) aged 73-94 years, of whom 204 pairs experienced the death of one or both co-twins during 9-10 years of follow-up (1997-2007). From the terminal restriction fragment length (TRFL) distribution, the authors obtained the mean TRFL (mTRFL) and the mean values...

  4. Engineering of a novel Ca{sup 2+}-regulated kinesin molecular motor using a calmodulin dimer linker

    Energy Technology Data Exchange (ETDEWEB)

    Shishido, Hideki [Department of Bioinformatics, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577 (Japan); Maruta, Shinsaku, E-mail: [Department of Bioinformatics, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577 (Japan)


    Highlights: Black-Right-Pointing-Pointer Engineered kinesin-M13 and calmodulin involving single cysteine were prepared. Black-Right-Pointing-Pointer CaM mutant was cross-linked to dimer by bifunctional thiol reactive reagent. Black-Right-Pointing-Pointer Kinesin-M13 was dimerized via CaM dimer in the presence of calcium. Black-Right-Pointing-Pointer Function of the engineered kinesin was regulated by a Ca{sup 2+}-calmodulin dimer linker. -- Abstract: The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have 'on-off' control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesin monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca{sup 2+}-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca{sup 2+}-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.

  5. A novel technique for rat liver transplantation using Quick Linker system: a preliminary result. (United States)

    Oldani, Graziano; Maestri, Marcello; Gaspari, Annalisa; Lillo, Ettore; Angelastri, Giacomo; Lenti, Luca Matteo; Rademacher, Johannes; Alessiani, Mario; Dionigi, Paolo


    The clinical success of liver transplantation is founded upon years of experimental research. Since Kamada and colleagues developed the "two-cuff" technique, the rat has become the best model for extensive investigations. Although the Kamada technique is technically complex and not easy to master, it is still the mainstay of orthotopic liver transplantation in rodents. We have developed a modified three-cuff version of this technique that facilitates anastomosis and markedly reduces warm ischemia time. The new technique involves a set of five microinstruments (the Quick-Linker system) designed and manufactured by our group. It was tested in male Lewis rats (group 1, donors n = 10, recipients n = 10). The graft was explanted as usual and standard cuffs were attached to the portal vein and the supra- and infrahepatic vena cavae. Corresponding vessels in the recipient were isolated, and Quicker-Linker holding rings were attached to each. The vessels were then clamped and the native organ removed. Once the graft was positioned in the recipient's abdomen, the holding rings attached to the recipient vessels and the cuffs applied to graft vessels were automatically aligned and joined with the aid of a special alignment tool. Warm ischemia times were always inferior to 6 minutes. Survival at postoperative day 10 was 80%. Liver function was well preserved in all of the surviving rats. The Quick-Linker technique significantly shortens warm ischemia time and allows rapid anastomosis that is relatively independent of operator skill. It can be considered a reliable option for microsurgeons looking for quick results and high success rates.

  6. Regional flexibility in the S4-S5 linker regulates hERG channel closed-state stabilization. (United States)

    Hull, Christina M; Sokolov, Stanislav; Van Slyke, Aaron C; Claydon, Tom W


    hERG K(+) channel function is vital for normal cardiac rhythm, yet the mechanisms underlying the unique biophysical characteristics of the channel, such as slow activation and deactivation gating, are incompletely understood. The S4-S5 linker is thought to transduce voltage sensor movement to opening of the pore gate, but may also integrate signals from cytoplasmic domains. Previously, we showed that substitutions of G546 within the S4-S5 linker destabilize the closed state of the channel. Here, we present results of a glycine-scan in the background of 546L. We demonstrate site-specific restoration of WT-like activation which suggests that flexibility in the N-terminal portion of the S4-S5 linker is critical for the voltage dependence of hERG channel activation. In addition, we show that the voltage dependence of deactivation, which was recently shown to be left-shifted from that of activation due to voltage sensor mode-shift, is also modulated by the S4-S5 linker. The G546L mutation greatly attenuated the coupling of voltage sensor mode-shift to the pore gate without altering the mode-shift itself. Indeed, all of the S4-S5 linker mutations tested similarly reduced coupling of the mode-shift to the pore gate. These data demonstrate a key role for S4-S5 linker in the unique activation and deactivation gating of hERG channels. Furthermore, uncoupling of the mode-shift to the pore by S4-S5 linker mutations parallels the effects of mutations in the N-terminus suggestive of functional interactions between the two regions.

  7. The DEK1 Calpain Linker Functions in Three-Dimensional Body Patterning in Physcomitrella patens1[OPEN (United States)

    Demko, Viktor; Mekhlif, Ahmed Khaleel


    The DEFECTIVE KERNEL1 (DEK1) calpain is a conserved 240-kD key regulator of three-dimensional body patterning in land plants acting via mitotic cell plane positioning. The activity of the cytosolic C-terminal calpain protease is regulated by the membrane-anchored DEK1 MEM, which is connected to the calpain via the 600-amino acid residue Linker. Similar to the calpain and MEM domains, the Linker is highly conserved in the land plant lineage, the similarity dropping sharply compared with orthologous charophyte sequences. Using site-directed mutagenesis, we studied the effect on Physcomitrella patens development by deleting the Linker and two conserved Linker motifs. The results show that removal of the Linker has nearly the same effect as removal of the entire DEK1 gene. In contrast, deletion of the conserved Laminin_G3 (LG3) domain had a milder effect, perturbing leafy gametophore patterning and archegonia development. The LG3 domain from Marchantia polymorpha is fully functional in P. patens, whereas angiosperm sequences are not functional. Deletion of a C-terminal Linker subsegment containing a potential calpain autolytic site severely disturbs gametophore development. Finally, changing one of the three calpain active-site amino acid residues results in the same phenotype as deleting the entire DEK1 gene. Based on the conserved nature of animal and DEK1 calpains, we propose that the DEK1 MEM-Linker complex inactivates the calpain by forcing apart the two calpain subunits carrying the three amino acids of the active site. PMID:27506240

  8. SEVA Linkers: A Versatile and Automatable DNA Backbone Exchange Standard for Synthetic Biology

    DEFF Research Database (Denmark)

    Kim, Se Hyeuk; Cavaleiro, Mafalda; Rennig, Maja


    DNA vectors serve to maintain and select recombinant DNA in cell factories, and as design complexity increases, there is a greater need for well-characterized parts and methods for their assembly. Standards in synthetic biology are top priority, but standardizing molecular cloning contrasts...... flexibility, and different researchers prefer and master different molecular technologies. Here, we describe a new, highly versatile and automatable standard “SEVA linkers” for vector exchange. SEVA linkers enable backbone swapping with 20 combinations of classical enzymatic restriction/ligation, Gibson...... to the synthetic biology community....

  9. Atomic Structure of Clathrin: A β Propeller Terminal Domain Joins an α Zigzag Linker


    ter Haar, Ernst; Musacchio, Andrea; Harrison, Stephen C.; Kirchhausen, Tomas


    Clathrin triskelions form the lattice that organizes recruitment of proteins to coated pits and helps drive vesiculation of the lipid bilayer. We report the crystal structure at 2.6 Å resolution of a 55 kDa N-terminal fragment from the 190 kDa clathrin heavy chain. The structure comprises the globular “terminal domain” and the linker that joins it to the end of a triskelion leg. The terminal domain is a seven-blade β propeller, a structure well adapted to interaction with multiple partners, s...

  10. Photosensitized cleavage of some olefins as potential linkers to be used in drug delivery (United States)

    Dinache, Andra; Smarandache, Adriana; Simon, Agota; Nastasa, Viorel; Tozar, Tatiana; Pascu, Alexandru; Enescu, Mironel; Khatyr, Abderrahim; Sima, Felix; Pascu, Mihail-Lucian; Staicu, Angela


    A study of photosensitized cleavage of different olefins as potential linkers for drug carrier complexes is reported. The role of singlet oxygen and the kinetic rates for light induced reactions were estimated by time-resolved measurements of singlet oxygen phosphorescence (at 1270 nm) obtained via 532 nm pulse laser excitation of a photosensitizer. The mixture of each studied olefin with verteporfin (used as photosensitizer) were exposed to low energy visible radiation. The rate constants for singlet oxygen quenching by studied olefins were determined. The irradiated solutions were investigated by FTIR spectroscopy and potential photoproducts were suggested. The experimental results were compared with simulations made by DFT method.

  11. Synthesis and molecular modeling of antimicrobial active fluoroquinolone-pyrazine conjugates with amino acid linkers. (United States)

    Panda, Siva S; Detistov, Oleksandr S; Girgis, Adel S; Mohapatra, Prabhu P; Samir, Ahmed; Katritzky, Alan R


    Novel fluoroquinolone-pyrazine conjugates 7a-h with amino acid linkers were synthesized in good yields utilizing benzotriazole chemistry. Antimicrobial bioassay showed that the synthesized bis-conjugates have antimicrobial properties comparable to the parent drugs. Compound 7h showed superior antibacterial activity against Staphylococcus aureus and Streptococcus pyogenes (MIC=74.6 μM and 149.3 μM, respectively). This matched well with the estimated values obtained from 3D-pharmacophore and 2D-QSAR studies (MIC=67 μM and 92.9 μM, respectively). Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Relativistic length agony continued

    Directory of Open Access Journals (Sweden)

    Redžić D.V.


    Full Text Available We made an attempt to remedy recent confusing treatments of some basic relativistic concepts and results. Following the argument presented in an earlier paper (Redžić 2008b, we discussed the misconceptions that are recurrent points in the literature devoted to teaching relativity such as: there is no change in the object in Special Relativity, illusory character of relativistic length contraction, stresses and strains induced by Lorentz contraction, and related issues. We gave several examples of the traps of everyday language that lurk in Special Relativity. To remove a possible conceptual and terminological muddle, we made a distinction between the relativistic length reduction and relativistic FitzGerald-Lorentz contraction, corresponding to a passive and an active aspect of length contraction, respectively; we pointed out that both aspects have fundamental dynamical contents. As an illustration of our considerations, we discussed briefly the Dewan-Beran-Bell spaceship paradox and the ‘pole in a barn’ paradox. [Projekat Ministarstva nauke Republike Srbije, br. 171028

  13. Ground Wood Fiber Length Distributions


    Lauri Ilmari Salminen; Sari Liukkonen; Alava, Mikko J.


    This study considers ground wood fiber length distributions arising from pilot grindings. The empirical fiber length distributions appear to be independent of wood fiber length as well as feeding velocity. In terms of mathematics the fiber fragment distributions of ground wood pulp combine an exponential distribution for high-length fragments and a power-law distribution for smaller lengths. This implies that the fiber length distribution is influenced by the stone surface. A fragmentation-ba...

  14. Modeling signal propagation mechanisms and ligand-based conformational dynamics of the Hsp90 molecular chaperone full-length dimer.

    Directory of Open Access Journals (Sweden)

    Giulia Morra


    Full Text Available Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine "hot spots" involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a "conformational selection model" of the Hsp90 mechanism, whereby the protein may

  15. Mutations of the S4-S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes. (United States)

    Sanguinetti, M C; Xu, Q P


    1. The structural basis for the activation gate of voltage-dependent K+ channels is not known, but indirect evidence has implicated the S4-S5 linker, the cytoplasmic region between the fourth and fifth transmembrane domains of the channel subunit. We have studied the effects of mutations in the S4-S5 linker of HERG (human ether-á-go-go-related gene), a human delayed rectifier K+ channel, in Xenopus oocytes. 2. Mutation of acidic residues (D540, E544) in the S4-S5 linker of HERG channels to neutral (Ala) or basic (Lys) residues accelerated the rate of channel deactivation. Most mutations greatly accelerated the rate of activation. However, E544K HERG channels activated more slowly than wild-type HERG channels. 3. Mutation of residues in the S4-S5 linker had little or no effect on fast inactivation, consistent with independence of HERG channel activation and inactivation 4. In response to large hyperpolarizations, D540K HERG channels can reopen into a state that is distinct from the normal depolarization-induced open state. It is proposed that substitution of a negatively charged Asp with the positively charged Lys disrupts a subunit interaction that normally stabilizes the channel in a closed state at negative transmembrane potentials. 5. The results indicate that the S4-S5 linker is a crucial component of the activation gate of HERG channels.

  16. Facile Synthesis and Characterizations of MOF-5 Coordination Polymer with Various Metal Linker Ratios for Ammonia Gas Storage (United States)

    Misran, Halina; Aminuddin, A. M.; Zini, F. A. M.; Ghazali, M. J.; Ramesh, S.


    Metal-organic framework coordination polymer materials of MOF-5 with Fm3m cubic structure and tetrahedral structure were successfully synthesized at room temperature and pressure using liquid-crystal (i.e. surfactant) templating method and direct mixing method. The crystallinity increased as metal (Zn)/linker (benzenedicarboxylic acid, BDC) ratios were varied from 0.3 to 0.5. However, the cubic structure of evacuated framework was relatively disrupted at metal/linker ratio of 0.7. Direct-mixing synthesis approach resulted in MOF-5 with layered tetragonal structure due to the increased mobility of the organic-linker as the metal/linker ratios were increased. On the other hand, liquid-crystal templating method resulted in MOF-5 with cubic structure with sizes of ca. 3-5 micron. Changing the solvent from dimethylformamide to chloroform during the synthesis affected the size of the single cubic to decrease from 5 micron to 3 micron, respectively. Furthermore, MOF-5 prepared with metal/linker ratio 0.5 and aged in solvents with higher crystallinity exhibited higher ammonia adsorption uptake at ca. 7.2%.

  17. A minimal phycobilisome: fusion and chromophorylation of the truncated core-membrane linker and phycocyanin. (United States)

    Tang, Kun; Zeng, Xiao-Li; Yang, Yi; Wang, Zhi-Bin; Wu, Xian-Jun; Zhou, Ming; Noy, Dror; Scheer, Hugo; Zhao, Kai-Hong


    Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli, the fused phycobiliproteins absorbed light in the range of 610-660nm, and fluoresced at ~670nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540-575nm, and fluorescing at ~585nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly.

  18. Hybrid Zeolitic Imidazolate Frameworks: Controlling Framework Porosity and Functionality by Mixed-Linker Synthesis

    KAUST Repository

    Thompson, Joshua A.


    Zeolitic imidazolate frameworks (ZIFs) are a subclass of nanoporous metal-organic frameworks (MOFs) that exhibit zeolite-like structural topologies and have interesting molecular recognition properties, such as molecular sieving and gate-opening effects associated with their pore apertures. The synthesis and characterization of hybrid ZIFs with mixed linkers in the framework are described in this work, producing materials with properties distinctly different from the parent frameworks (ZIF-8, ZIF-90, and ZIF-7). NMR spectroscopy is used to assess the relative amounts of the different linkers included in the frameworks, whereas nitrogen physisorption shows the evolution of the effective pore size distribution in materials resulting from the framework hybridization. X-ray diffraction shows these hybrid materials to be crystalline. In the case of ZIF-8-90 hybrids, the cubic space group of the parent frameworks is continuously maintained, whereas in the case of the ZIF-7-8 hybrids there is a transition from a cubic to a rhombohedral space group. Nitrogen physisorption data reveal that the hybrid materials exhibit substantial changes in gate-opening phenomena, either occurring at continuously tunable partial pressures of nitrogen (ZIF-8-90 hybrids) or loss of gate-opening effects to yield more rigid frameworks (ZIF-7-8 hybrids). With this synthetic approach, significant alterations in MOF properties may be realized to suit a desired separation or catalytic process. © 2012 American Chemical Society.

  19. Fine mapping of posttranslational modifications of the linker histone H1 from Drosophila melanogaster.

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    Ana Villar-Garea

    Full Text Available The linker histone H1 binds to the DNA in between adjacent nucleosomes and contributes to chromatin organization and transcriptional control. It is known that H1 carries diverse posttranslational modifications (PTMs, including phosphorylation, lysine methylation and ADP-ribosylation. Their biological functions, however, remain largely unclear. This is in part due to the fact that most of the studies have been performed in organisms that have several H1 variants, which complicates the analyses. We have chosen Drosophila melanogaster, a model organism, which has a single H1 variant, to approach the study of the role of H1 PTMs during embryonic development. Mass spectrometry mapping of the entire sequence of the protein showed phosphorylation only in the ten N-terminal amino acids, mostly at S10. For the first time, changes in the PTMs of a linker H1 during the development of a multicellular organism are reported. The abundance of H1 monophosphorylated at S10 decreases as the embryos age, which suggests that this PTM is related to cell cycle progression and/or cell differentiation. Additionally, we have found a polymorphism in the protein sequence that can be mistaken with lysine methylation if the analysis is not rigorous.

  20. Nucleosome linker proteins HMGB1 and histone H1 differentially enhance DNA ligation reactions. (United States)

    Yamanaka, Shiho; Katayama, Eisaku; Yoshioka, Ken-ichi; Nagaki, Sumiko; Yoshida, Michiteru; Teraoka, Hirobumi


    We previously reported that HMGB1, which originally binds to chromatin in a manner competitive with linker histone H1 to modulate chromatin structure, enhances both intra-molecular and inter-molecular ligations. In this paper, we found that histone H1 differentially enhances ligation reaction of DNA double-strand breaks (DSB). Histone H1 stimulated exclusively inter-molecular ligation reaction of DSB with DNA ligase IIIbeta and IV, whereas HMGB1 enhanced mainly intra-molecular ligation reaction. Electron microscopy of direct DNA-protein interaction without chemical cross-linking visualized that HMGB1 bends and loops linear DNA to form compact DNA structure and that histone H1 is capable of assembling DNA in tandem arrangement with occasional branches. These results suggest that differences in the enhancement of DNA ligation reaction are due to those in alteration of DNA configuration induced by these two linker proteins. HMGB1 and histone H1 may function in non-homologous end-joining of DSB repair and V(D)J recombination in different manners.

  1. Linker-Induced Anomalous Emission of Organic-Molecule Conjugated Metal-Oxide Nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Turkowski, Volodymyr; Babu, Suresh; Le, Duy; Kumar, Amit; Haldar, Manas K.; Wagh, Anil V.; Hu, Zhongjian; Karakoti, Ajay S.; Gesquiere, Andre J.; Law, Benedict; Mallik, Sanku; Rahman, Talat S.; Leuenberger, Michael N.; Seal, Sudipta


    Semiconductor nanoparticles conjugated with organic- and dye-molecules to yield high efficiency visible photoluminescence (PL) hold great potential for many future technological applications. We show that folic acid (FA)-conjugated to nanosize TiO2 and CeO2 particles demonstrates a dramatic increase of photoemission intensity at wavelengths between 500 and 700 nm when derivatized using aminopropyl trimethoxysilane (APTMS) as spacer-linker molecules between the metal oxide and FA. Using density-functional theory (DFT) and time-dependent DFT calculations we demonstrate that the strong increase of the PL can be explained by electronic transitions between the titania surface oxygen vacancy (OV) states and the low-energy excited states of the FA/APTMS molecule anchored onto the surface oxygen bridge sites in close proximity to the OVs. We suggest this scenario to be a universal feature for a wide class of metal oxide nanoparticles, including nanoceria, possessing a similar band gap (3 eV) and with a large surface-vacancy-related density of electronic states. We demonstrate that the molecule-nanoparticle linker can play a crucial role in tuning the electronic and optical properties of nanosystems by bringing optically active parts of the molecule and of the surface close to each other.

  2. Bimane fluorescence scanning suggests secondary structure near the S3-S4 linker of BK channels. (United States)

    Semenova, Nina P; Abarca-Heidemann, Karin; Loranc, Eva; Rothberg, Brad S


    Gating of large conductance Ca(2+)-activated K(+) channels (BK or maxi-K channels) is controlled by a Ca(2+)-sensor, formed by the channel cytoplasmic C-terminal domain, and a voltage sensor, formed by its S0-S4 transmembrane helices. Here we analyze structural properties of a portion of the BK channel voltage sensing domain, the S3-S4 linker, using fluorescence lifetime spectroscopy. Single residues in the S3-S4 linker region were substituted with cysteine, and the cysteine-substituted mutants were expressed in CHO cells and covalently labeled with the sulfhydryl-reactive fluorophore monobromo-trimethylammonio-bimane (qBBr). qBBr fluorescence is quenched by tryptophan and, to a lesser extent, tyrosine side chains. We found that qBBr fluorescence in several of the labeled cysteine-substituted channels shows position-specific quenching, as indicated by increase of the brief lifetime component of the qBBr fluorescence decay. Quenching was reduced with the mutation W203F (in the S4 segment), suggesting that Trp-203 acts as a quenching group. Our results suggest a working hypothesis for the secondary structure of the BK channel S3-S4 region, and places residues Leu-204, Gly-205, and Leu-206 within the extracellular end of the S4 helix.

  3. DNA methylation affects nuclear organization, histone modifications, and linker histone binding but not chromatin compaction. (United States)

    Gilbert, Nick; Thomson, Inga; Boyle, Shelagh; Allan, James; Ramsahoye, Bernard; Bickmore, Wendy A


    DNA methylation has been implicated in chromatin condensation and nuclear organization, especially at sites of constitutive heterochromatin. How this is mediated has not been clear. In this study, using mutant mouse embryonic stem cells completely lacking in DNA methylation, we show that DNA methylation affects nuclear organization and nucleosome structure but not chromatin compaction. In the absence of DNA methylation, there is increased nuclear clustering of pericentric heterochromatin and extensive changes in primary chromatin structure. Global levels of histone H3 methylation and acetylation are altered, and there is a decrease in the mobility of linker histones. However, the compaction of both bulk chromatin and heterochromatin, as assayed by nuclease digestion and sucrose gradient sedimentation, is unaltered by the loss of DNA methylation. This study shows how the complete loss of a major epigenetic mark can have an impact on unexpected levels of chromatin structure and nuclear organization and provides evidence for a novel link between DNA methylation and linker histones in the regulation of chromatin structure.

  4. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

    Directory of Open Access Journals (Sweden)

    Shih Ping Yao


    Full Text Available Abstract Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal antibody (mAb C, is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57 of transgenic pigs (F0 generation. Conclusions Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

  5. discouraged by queue length

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    P. R. Parthasarathy


    Full Text Available The transient solution is obtained analytically using continued fractions for a state-dependent birth-death queue in which potential customers are discouraged by the queue length. This queueing system is then compared with the well-known infinite server queueing system which has the same steady state solution as the model under consideration, whereas their transient solutions are different. A natural measure of speed of convergence of the mean number in the system to its stationarity is also computed.

  6. Tyrosine Residues from the S4-S5 Linker of Kv11.1 Channels Are Critical for Slow Deactivation. (United States)

    Ng, Chai-Ann; Gravel, Andrée E; Perry, Matthew D; Arnold, Alexandre A; Marcotte, Isabelle; Vandenberg, Jamie I


    Slow deactivation of Kv11.1 channels is critical for its function in the heart. The S4-S5 linker, which joins the voltage sensor and pore domains, plays a critical role in this slow deactivation gating. Here, we use NMR spectroscopy to identify the membrane-bound surface of the S4S5 linker, and we show that two highly conserved tyrosine residues within the KCNH subfamily of channels are membrane-associated. Site-directed mutagenesis and electrophysiological analysis indicates that Tyr-542 interacts with both the pore domain and voltage sensor residues to stabilize activated conformations of the channel, whereas Tyr-545 contributes to the slow kinetics of deactivation by primarily stabilizing the transition state between the activated and closed states. Thus, the two tyrosine residues in the Kv11.1 S4S5 linker play critical but distinct roles in the slow deactivation phenotype, which is a hallmark of Kv11.1 channels.

  7. A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations. (United States)

    Zmolek, Wesley; Bañas, Stefanie; Barfield, Robyn M; Rabuka, David; Drake, Penelope M


    Antibody-drug conjugates represent a growing class of biologic drugs that use the targeted specificity of an antibody to direct the localization of a small molecule drug, often a cytotoxic payload. After conjugation, antibody-drug conjugate preparations typically retain a residual amount of free (unconjugated) linker-payload. Monitoring this free small molecule drug component is important due to the potential for free payload to mediate unintended (off-target) toxicity. We developed a simple RP-HPLC/MRM-MS-based assay that can be rapidly employed to quantify free linker-payload. The method uses low sample volumes and offers an LLOQ of 10nM with 370pg on column. This analytical approach was used to monitor free linker-payload removal during optimization of the tangential flow filtration manufacturing step.

  8. Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias

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    Shuji Mizumoto


    Full Text Available Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

  9. The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating. (United States)

    Ng, Chai Ann; Perry, Matthew D; Tan, Peter S; Hill, Adam P; Kuchel, Philip W; Vandenberg, Jamie I


    Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.

  10. A Linker for the Solid-Phase Synthesis of Hydroxamic Acids and Identification of HDAC6 Inhibitors. (United States)

    Bang, Claus G; Jensen, Jakob F; O'Hanlon Cohrt, Emil; Olsen, Lasse B; Siyum, Saba G; Mortensen, Kim T; Skovgaard, Tine; Berthelsen, Jens; Yang, Liang; Givskov, Michael; Qvortrup, Katrine; Nielsen, Thomas E


    We herein present broadly useful, readily available and nonintegral hydroxylamine linkers for the routine solid-phase synthesis of hydroxamic acids. The developed protocols enable the efficient synthesis and release of a wide range of hydroxamic acids from various resins, relying on high control and flexibility with respect to reagents and synthetic processes. A trityl-based hydroxylamine linker was used to synthesize a library of peptide hydroxamic acids. The inhibitory effects of the compounds were examined for seven HDAC enzyme subtypes using a chemiluminescence-based assay.

  11. Photoresponsive porous materials: the design and synthesis of photochromic diarylethene-based linkers and a metal-organic framework. (United States)

    Patel, Dinesh G Dan; Walton, Ian M; Cox, Jordan M; Gleason, Cody J; Butzer, David R; Benedict, Jason B


    The synthesis and characterization of novel photochromic diarylethene-based linkers for use in metal–organic frameworks is described including crystal structure analysis of nearly all reaction intermediates. The bis-carboxylated dithien-3-ylphenanthrenes can be prepared under relatively mild conditions in high yield and were subsequently used to create a photoresponsive metal–organic framework, UBMOF-1. While the photochromism of the ligand TPDC in solution is fully reversible, the cycloreversion reaction is suppressed when this linker is incorporated into the crystalline framework of UBMOF-1.

  12. Silyl-based alkyne-modifying linker for the preparation of C-terminal acetylene-derivatized protected peptides. (United States)

    Strack, Martin; Langklotz, Sina; Bandow, Julia E; Metzler-Nolte, Nils; Albada, H Bauke


    A novel linker for the synthesis of C-terminal acetylene-functionalized protected peptides is described. This SAM1 linker is applied in the manual Fmoc-based solid-phase peptide synthesis of Leu-enkephalin and in microwave-assisted automated synthesis of Maculatin 2.1, an antibacterial peptide that contains 18 amino acid residues. For the cleavage, treatment with tetramethylammonium fluoride results in protected acetylene-derivatized peptides. Alternatively, a one-pot cleavage-click procedure affords the protected 1,2,3-triazole conjugate in high yields after purification.

  13. Ground Wood Fiber Length Distributions

    Directory of Open Access Journals (Sweden)

    Lauri Ilmari Salminen


    Full Text Available This study considers ground wood fiber length distributions arising from pilot grindings. The empirical fiber length distributions appear to be independent of wood fiber length as well as feeding velocity. In terms of mathematics the fiber fragment distributions of ground wood pulp combine an exponential distribution for high-length fragments and a power-law distribution for smaller lengths. This implies that the fiber length distribution is influenced by the stone surface. A fragmentation-based model is presented that allows reproduction of the empirical results.

  14. Selective histone deacetylase 6 inhibitors bearing substituted urea linkers inhibit melanoma cell growth. (United States)

    Bergman, Joel A; Woan, Karrune; Perez-Villarroel, Patricio; Villagra, Alejandro; Sotomayor, Eduardo M; Kozikowski, Alan P


    The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet nonselective inhibitor. Structure-activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5 g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of ∼600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6-selective inhibitors that possess antiproliferative effects against melanoma cells.

  15. Speaking tasks for the assessment of English: the use of linkers

    Directory of Open Access Journals (Sweden)

    Marcos Peñate Cabrera


    Full Text Available Although assessment experts have been researching the relationship between the different types of tasks and their level of difficulty, this process of analysis should be pursued further in order to acquire a more accurate vision of the variables involved. In this article we study the three types of tasks most frequently used in the different models of oral exams (one-to-one interview based on a photo, one-to-one interview based on a comic strip and a dialogue in pairs. In order to carry out this study we assessed 244 pupils from the second year of Bachillerato belonging to nine different schools and having an A2 level of English. When contrasting the 3 types of tasks, our aim was to analyse the amount and complexity of the oral production, namely the use of linkers, by means of statistical tests of homogeneity and specificity.

  16. Solid-phase synthesis of lidocaine and procainamide analogues using backbone amide linker (BAL) anchoring. (United States)

    Shannon, Simon K; Peacock, Mandy J; Kates, Steven A; Barany, George


    New solid-phase strategies have been developed for the synthesis of lidocaine (1) and procainamide (2) analogues, using backbone amide linker (BAL) anchoring. Both sets were prepared starting from a common resin-bound intermediate, followed by four general steps: (i) attachment of a primary aliphatic or aromatic amine to the solid support via reductive amination (as monitored by a novel test involving reaction of 2,4-dinitrophenylhydrazine with residual aldehyde groups); (ii) acylation of the resultant secondary amine; (iii) displacement of halide with an amine; and (iv) trifluoroacetic acid-mediated release from the support. A manual parallel strategy was followed to provide 60 novel compounds, of which two dozen have not been previously described. In most cases, initial crude purities were >80%, and overall isolated yields were in the 40-88% range.

  17. Cytoskeletal Linker Protein Dystonin Is Not Critical to Terminal Oligodendrocyte Differentiation or CNS Myelination.

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    Samantha F Kornfeld

    Full Text Available Oligodendrocyte differentiation and central nervous system myelination require massive reorganization of the oligodendrocyte cytoskeleton. Loss of specific actin- and tubulin-organizing factors can lead to impaired morphological and/or molecular differentiation of oligodendrocytes, resulting in a subsequent loss of myelination. Dystonin is a cytoskeletal linker protein with both actin- and tubulin-binding domains. Loss of function of this protein results in a sensory neuropathy called Hereditary Sensory Autonomic Neuropathy VI in humans and dystonia musculorum in mice. This disease presents with severe ataxia, dystonic muscle and is ultimately fatal early in life. While loss of the neuronal isoforms of dystonin primarily leads to sensory neuron degeneration, it has also been shown that peripheral myelination is compromised due to intrinsic Schwann cell differentiation abnormalities. The role of this cytoskeletal linker in oligodendrocytes, however, remains unclear. We sought to determine the effects of the loss of neuronal dystonin on oligodendrocyte differentiation and central myelination. To address this, primary oligodendrocytes were isolated from a severe model of dystonia musculorum, Dstdt-27J, and assessed for morphological and molecular differentiation capacity. No defects could be discerned in the differentiation of Dstdt-27J oligodendrocytes relative to oligodendrocytes from wild-type littermates. Survival was also compared between Dstdt-27J and wild-type oligodendrocytes, revealing no significant difference. Using a recently developed migration assay, we further analysed the ability of primary oligodendrocyte progenitor cell motility, and found that Dstdt-27J oligodendrocyte progenitor cells were able to migrate normally. Finally, in vivo analysis of oligodendrocyte myelination was done in phenotype-stage optic nerve, cerebral cortex and spinal cord. The density of myelinated axons and g-ratios of Dstdt-27J optic nerves was normal, as

  18. Hooked on Cryogels: A Carbamate Linker Based Depot for Slow Drug Release. (United States)

    Aydin, Duygu; Arslan, Mehmet; Sanyal, Amitav; Sanyal, Rana


    Poly(ethylene glycol) (PEG) based bulk hydrogels and cryogels containing activated carbonate groups as amine reactive handles to facilitate drug conjugations through carbamate linkages were fabricated and evaluated as slow releasing drug reservoirs. As an initial approach, photopolymerization of N-hydroxysuccinimide (NHS)-activated carbonate functional group containing monomer and PEG-methacrylate in the presence of a cross-linker was utilized to obtain bulk hydrogels with high gel conversions. The resultant hydrogels possessed moderate water uptake (170-340%) which was dependent on the monomer ratios. These hydrogels were functionalized with an anticancer drug, namely, doxorubicin. Surprisingly, while negligible drug release was observed from the bulk hydrogels under normal pH, only about 6% drug release was observed under acidic condition. Limited swelling of these hydrogels as well as lack of porous structure as deduced from scanning electron microscopy analysis might explain the poor drug release. To enhance the drug releasing capacity of these hydrogels that might stem from the increased porosity, reactive carbonate group bearing cryogels were synthesized. Compared to the bulk hydrogels, cryogels were highly porous in structure and also possessed much higher swelling capacity (1150-1500%). As a result of these distinctions, a 7-fold enhancement in drug release was observed for the cryogel system compared to the relating hydrogel. In vitro studies demonstrated that the anticancer drug doxorubicin conjugated through carbamate linkers to the cryogels was released and proved effective against MDA-MB-231 human breast cancer cells. Overall, a novel class of slow releasing nontoxic hydrogel and cryogel scaffolds with potential applications as anticancer drug reservoirs was realized.

  19. Nile tilapia skin collagen sponge modified with chemical cross-linkers as a biomedical hemostatic material. (United States)

    Sun, Leilei; Li, Bafang; Jiang, Dandan; Hou, Hu


    Nile tilapia skin collagen sponges were fabricated by freeze-drying technology and modified with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the presence of N-hydroxysuccinimide (EDC/NHS), genipin+PBS, genipin+ethanol, tea polyphenol (TP), nordihydroguaiaretic acid (NDGA) and diphenyl phosphoryl azide (DPPA). Physicochemical and biological properties, micromorphology and compatibility before and after modification were investigated to evaluate collagen sponge as a hemostatic biomedical material. The mechanical property of collagen sponges strengthened after cross-linking. The elongation at break of cross-linked collagen sponges decreased except for EDC/NHS, which was close to that of non-crosslinked. The collagen sponge cross-linked with EDC/NHS exhibited the highest hygroscopicity in comparison with other cross-linkers. The resistance to collagenase biodegradation of collagen sponges after cross-linking strengthened significantly except for NDGA. Collagen sponges cross-linked with EDC/NHS, TP and NDGA maintained high porosity (97-98%), similar to non-crosslinked (98.42%). Collagen sponges could shorten the blood coagulation time. From the variations of the FTIR spectrum pattern and SEM, DPPA could change the secondary structure of collagen and destroy the spongy structure of collagen sponge, which was not suitable for the cross-linking of collagen sponge. Whereas, EDC/NHS was recognized as a perfect cross-linker owing to its excellent properties and porous microstructure. All fabricated collagen sponges were recognized to be biocompatible by the hemolysis assay in vitro. Therefore, collagen sponge modified with EDC/NHS could be used as a perfect biomedical hemostatic material. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Temperature-triggered release of a liquid cross-linker micro-encapsulated in a glassy polymer for low temperature curing

    NARCIS (Netherlands)

    Senatore, D.; Cate, A.T. ten; Laven, J.; Benthem, R.A.T.M. van; With, G. de


    In order to prevent a liquid epoxy cross-linker from premature, Arrhenius-law predicted, reaction with an acid-functional polyester resin, the liquid cross-linker has been physically separated from the resin by encapsulation while release is only possible by a temperature-controlled trigger. The gla

  1. Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails. (United States)

    Stützer, Alexandra; Liokatis, Stamatios; Kiesel, Anja; Schwarzer, Dirk; Sprangers, Remco; Söding, Johannes; Selenko, Philipp; Fischle, Wolfgang


    Post-translational histone modifications and linker histone incorporation regulate chromatin structure and genome activity. How these systems interface on a molecular level is unclear. Using biochemistry and NMR spectroscopy, we deduced mechanistic insights into the modification behavior of N-terminal histone H3 tails in different nucleosomal contexts. We find that linker histones generally inhibit modifications of different H3 sites and reduce H3 tail dynamics in nucleosomes. These effects are caused by modulations of electrostatic interactions of H3 tails with linker DNA and largely depend on the C-terminal domains of linker histones. In agreement, linker histone occupancy and H3 tail modifications segregate on a genome-wide level. Charge-modulating modifications such as phosphorylation and acetylation weaken transient H3 tail-linker DNA interactions, increase H3 tail dynamics, and, concomitantly, enhance general modifiability. We propose that alterations of H3 tail-linker DNA interactions by linker histones and charge-modulating modifications execute basal control mechanisms of chromatin function. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Synthesis of Selective Butyrylcholinesterase Inhibitors Coupled between α-Lipoic Acid and Polyphenols by Using 2-(Piperazin-1-yl)ethanol Linker

    Energy Technology Data Exchange (ETDEWEB)

    Yeun, Go Heun; Lee, Seung Hwan; LIm, Yong Bae; Lee, Hye Sook; Lee, Bong Ho; Park, Jeong Ho [Hanbat National Univ., Daejeon (Korea, Republic of); Won, Mooho [Kangwon National Univ., Chuncheon (Korea, Republic of)


    In the previous paper (Bull. Korean Chem. Soc., 2011, 32, 2997), the hybrid molecules between α-lipoic acid (ALA) and polyphenols (PPs) connected with neutral 2-(2-aminoethoxy)ethanol linker (linker-1) showed new biological activity such as butyrylcholinesterase (BuChE) inhibition. In order to increase the binding affinity of the hybrid compounds to cholinesterase (ChE), the neutral 2-(2-aminoethoxy)ethanol (linker 1) was switched to the cationic 2-(piperazin-1-yl)ethanol linker (linker 2). The IC{sub 50} values of the linker-2 hybrid molecules for BuChE inhibition were lower than those of linker-1 hybrid molecules (except 9-2) and they also had the same great selectivity for BuChE over AChE (> 800 fold) as linker-1 hybrid molecules. ALA-acetyl caffeic acid (10-2, ALA-AcCA) was shown as an effective inhibitor of BuChE (IC{sub 50} = 0.44 ± 0.24 μM). A kinetic study using 7-2 showed that it is the same mixed type inhibition as 7-1. Its inhibition constant (Ki) to BuChE is 4.3 ± 0.09 μM.

  3. Preparation of value-added metal-organic frameworks (MOFs) using waste PET bottles as source of acid linker

    CSIR Research Space (South Africa)

    Dyosiba, Xoliswa


    Full Text Available Metal-organic frameworks (MOFs) have been considered with various novel applications and the development of cost-effective processes is essential for further practices. In this work, waste PET bottles were demonstrated as the source of acid linker...

  4. Phenyl amide linker improves the pharmacokinetics and pharmacodynamics of N-terminally mono-PEGylated human growth hormone. (United States)

    Wu, Ling; Ji, Shaoyang; Shen, Lijuan; Hu, Tao


    Human growth hormone (hGH) suffers from a short plasma half-life of ∼15 min, necessitating frequent injections to maintain its physiological effect. PEGylation, conjugation of polyethylene glycol (PEG), is an effective strategy to prolong the plasma half-life of hGH. However, PEGylation can significantly decrease the bioactivity of hGH. Thus, a new PEGylation approach is desired to improve the pharmacokinetics (PK) and pharmacodynamics (PD) of the PEGylated hGH. In the present study, two N-terminally mono-PEGylated hGHs were prepared using aldehyde chemistry. Phenyl amide and ethyl moieties were used as the linkers between PEG and hGH, respectively. The hydrodynamic volume, proteolytic sensitivity, and immunogenicity of the PEGylated hGH with phenyl amide linker (hGH-phenyl-PEG) were lower than those of the one with propyl linker (hGH-prop-PEG). In addition, hGH-phenyl-PEG showed a higher in vitro bioactivity and better PK and PD than hGH-prop-PEG. The better PK of hGH-phenyl-PEG was mainly due to its lower proteolytic sensitivity and low immunogenicity. The better PD of hGH-phenyl-PEG was mainly due to its higher in vitro bioactivity. Thus, the phenyl amide linker can improve the overall pharmacological profiles of the PEGylated hGH. Our study is expected to advance the development of long-acting protein biotherapeutics with high therapeutic efficacy.

  5. Mechanistic Evaluation of Motion in Redox-Driven Rotaxanes Reveals Longer Linkers Hasten Forward Escape's and Hinder Backward Translations

    DEFF Research Database (Denmark)

    Andersen, S. S.; Share, A. I.; Poulsen, B. L.;


    of the glycol linker connecting the two stations (n = 5, 8, 11, and 23 atoms). We undertook the first mechanistic study of the full cycle of motion in this class of molecular switch using cyclic voltammetry. The kinetics parameters (k, Delta G(double dagger) of switching were determined at different...

  6. Photochemical Reactions of the LOV and LOV-Linker Domains of the Blue Light Sensor Protein YtvA

    NARCIS (Netherlands)

    Choi, S.; Nakasone, Y.; Hellingwerf, K.J.; Terazima, M.


    YtvA is a blue light sensor protein composed of an N-terminal LOV (light-oxygen-voltage) domain, a linker helix, and the C-terminal sulfate transporter and anti-sigma factor antagonist domain. YtvA is believed to act as a positive regulator for light and salt stress responses by regulating the

  7. Efficient synthesis of diverse heterobifunctionalized clickable oligo(ethylene glycol) linkers: potential applications in bioconjugation and targeted drug delivery. (United States)

    Goswami, Lalit N; Houston, Zachary H; Sarma, Saurav J; Jalisatgi, Satish S; Hawthorne, M Frederick


    Herein we describe the sequential synthesis of a variety of azide-alkyne click chemistry-compatible heterobifunctional oligo(ethylene glycol) (OEG) linkers for bioconjugation chemistry applications. Synthesis of these bioorthogonal linkers was accomplished through desymmetrization of OEGs by conversion of one of the hydroxyl groups to either an alkyne or azido functionality. The remaining distal hydroxyl group on the OEGs was activated by either a 4-nitrophenyl carbonate or a mesylate (-OMs) group. The -OMs functional group served as a useful precursor to form a variety of heterobifunctionalized OEG linkers containing different highly reactive end groups, e.g., iodo, -NH(2), -SH and maleimido, that were orthogonal to the alkyne or azido functional group. Also, the alkyne- and azide-terminated OEGs are useful for generating larger discrete poly(ethylene glycol) (PEG) linkers (e.g., PEG(16) and PEG(24)) by employing a Cu(I)-catalyzed 1,3-dipolar cycloaddition click reaction. The utility of these clickable heterobifunctional OEGs in bioconjugation chemistry was demonstrated by attachment of the integrin (α(v)β(3)) receptor targeting peptide, cyclo-(Arg-Gly-Asp-D-Phe-Lys) (cRGfKD) and to the fluorescent probe sulfo-rhodamine B. The synthetic methodology presented herein is suitable for the large scale production of several novel heterobifunctionalized OEGs from readily available and inexpensive starting materials.

  8. One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA. (United States)

    Mochizuki, Yuki; Biyani, Manish; Tsuji-Ueno, Sachika; Suzuki, Miho; Nishigaki, Koichi; Husimi, Yuzuru; Nemoto, Naoto


    A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.

  9. Binary functionalization of H:Si(111) surfaces by alkyl monolayers with different linker atoms enhances monolayer stability and packing. (United States)

    Arefi, Hadi H; Nolan, Michael; Fagas, Giorgos


    Alkyl monolayer modified Si forms a class of inorganic-organic hybrid materials with applications across many technologies such as thin-films, fuel/solar-cells and biosensors. Previous studies have shown that the linker atom, through which the monolayer binds to the Si substrate, and any tail group in the alkyl chain, can tune the monolayer stability and electronic properties. In this paper we study the H:Si(111) surface functionalized with binary SAMs: these are composed of alkyl chains that are linked to the surface by two different linker groups. Aiming to enhance SAM stability and increase coverage over singly functionalized Si, we examine with density functional theory simulations that incorporate vdW interactions, a range of linker groups which we denote as -X-(alkyl) with X = CH2, O(H), S(H) or NH(2) (alkyl = C6 and C12 chains). We show how the stability of the SAM can be enhanced by adsorbing alkyl chains with two different linkers, e.g. Si-[C, NH]-alkyl, through which the adsorption energy is increased compared to functionalization with the individual -X-alkyl chains. Our results show that it is possible to improve stability and optimum coverage of alkyl functionalized SAMs linked through a direct Si-C bond by incorporating alkyl chains linked to Si through a different linker group, while preserving the interface electronic structure that determines key electronic properties. This is important since any enhancement in stability and coverage to give more densely packed monolayers will result in fewer defects. We also show that the work function can be tuned within the interval of 3.65-4.94 eV (4.55 eV for bare H:Si(111)).

  10. Photochemical Reactions of the LOV and LOV-Linker Domains of the Blue Light Sensor Protein YtvA. (United States)

    Choi, Seokwoo; Nakasone, Yusuke; Hellingwerf, Klaas J; Terazima, Masahide


    YtvA is a blue light sensor protein composed of an N-terminal LOV (light-oxygen-voltage) domain, a linker helix, and the C-terminal sulfate transporter and anti-σ factor antagonist domain. YtvA is believed to act as a positive regulator for light and salt stress responses by regulating the σB transcription factor. Although its biological function has been studied, the reaction dynamics and molecular mechanism underlying the function are not well understood. To improve our understanding of the signaling mechanism, we studied the reaction of the LOV domain (YLOV, amino acids 26-127), the LOV domain with its N-terminal extension (N-YLOV, amino acids 1-127), the LOV domain with its C-terminal linker helix (YLOV-linker, amino acids 26-147), and the YLOV domain with the N-terminal extension and the C-terminal linker helix (N-YLOV-linker, amino acids 1-147) using the transient grating method. The signals of all constructs showed adduct formation, thermal diffusion, and molecular diffusion. YLOV showed no change in the diffusion coefficient (D), while the other three constructs showed a significant decrease in D within ∼70 μs of photoexcitation. This indicates that conformational changes in both the N- and C-terminal helices of the YLOV domain indeed do occur. The time constant in the YtvA derivatives was much faster than the corresponding dynamics of phototropins. Interestingly, an additional reaction was observed as a volume expansion as well as a slight increase in D only when both helices were included. These findings suggest that although the rearrangement of the N- and C-terminal helices occurs independently on the fast time scale, this change induces an additional conformational change only when both helices are present.

  11. Self-assembled micelles composed of doxorubicin conjugated Y-shaped PEG-poly(glutamic acid)2 copolymers via hydrazone linkers. (United States)

    Sui, Bowen; Xu, Hui; Jin, Jian; Gou, Jingxin; Liu, Jingshuo; Tang, Xing; Zhang, Yu; Xu, Jinghua; Zhang, Hongfeng; Jin, Xiangqun


    In this work, micelles composed of doxorubicin-conjugated Y-shaped copolymers (YMs) linked via an acid-labile linker were constructed. Y-shaped copolymers of mPEG-b-poly(glutamate-hydrazone-doxorubicin)2 and linear copolymers of mPEG-b-poly(glutamate-hydrazone-doxorubicin) were synthesized and characterized. Particle size, size distribution, morphology, drug loading content (DLC) and drug release of the micelles were determined. Alterations in size and DLC of the micelles could be achieved by varying the hydrophobic block lengths. Moreover, at fixed DLCs, YMs showed a smaller diameter than micelles composed of linear copolymers (LMs). Also, all prepared micelles showed sustained release behaviors under physiological conditions over 72 h. DOX loaded in YMs was released more completely, with 30% more drug released in acid. The anti-tumor efficacy of the micelles against HeLa cells was evaluated by MTT assays, and YMs exhibited stronger cytotoxic effects than LMs in a dose- and time-dependent manner. Cellular uptake studied by CLSM indicated that YMs and LMs were readily taken up by HeLa cells. According to the results of this study, doxorubicin-conjugated Y-shaped PEG-(polypeptide)2 copolymers showed advantages over linear copolymers, like assembling into smaller nanoparticles, faster drug release in acid, which may correspond to higher cellular uptake and enhanced extracellular/intracellular drug release, indicating their potential in constructing nano-sized drug delivery systems.


    Directory of Open Access Journals (Sweden)



    Full Text Available BACKGROUND Estimation of gestational age of fetus is of great medicolegal importance. Multiple parameters of the fetal anatomical measurements are in use. However, gestational age assessment may be difficult in fetus with anencephaly, hydrocephalus, short limb dysplasia, post mortem destruction or in mutilated case. Study of literature suggests that fetal foot has a characteristic pattern of normal growth and the fetal foot shows gradual increase in length relative to the length of the embryo and could be used to estimate gestational age. The purpose of the present study is to determine the accuracy in estimating gestational age using fetal foot and hand length by studying its relation with crown rump length in the foetuses of Manipuri origin. AIMS AND OBJECTIVES 1 To study the relationship between fetal crown rump length and fetal hand and foot length, thereby determining the accuracy in estimating gestational age by a cross-sectional study. MATERIALS AND METHODS A total of 100 formalin fixed fetuses of Manipuri origin, obtained from the Department of Obstetrics and Gynaecology, Regional Institute of Medical Sciences, Imphal, were included in the study, carried out in the Department of Anatomy, from February 2015 to July 2015. The parameters studied were crown rump length, foot length and hand length of fetuses. The data was analysed using SPSS software by regression analysis. Graphs were also plotted to determine pattern of growth and their correlation with crown rump length if any. RESULTS A total of 100 fetuses were studied, of which 43 were females and 57 were males. The mean foot length and hand length progressively increased with increase in crown rump length. Measurements were not significantly different in right or left side or among male and female fetuses. A statistically significant linear relationship was seen between foot length and crown rump length of the fetus (r=0.980, p<0.0001 and hand length and crown rump length of the fetus


    Energy Technology Data Exchange (ETDEWEB)



    We determine the impedance of a cylindrical metal tube (resistor) of radius a, length g, and conductivity {sigma}, attached at each end to perfect conductors of semi-infinite length. Our main interest is in the asymptotic behavior of the impedance at high frequency, k >> 1/a. In the equilibrium regime, , the impedance per unit length is accurately described by the well-known result for an infinite length tube with conductivity {sigma}. In the transient regime, ka{sup 2} >> g, we derive analytic expressions for the impedance and wakefield.

  14. Polyketide chain length control by chain length factor. (United States)

    Tang, Yi; Tsai, Shiou-Chuan; Khosla, Chaitan


    Bacterial aromatic polyketides are pharmacologically important natural products. A critical parameter that dictates product structure is the carbon chain length of the polyketide backbone. Systematic manipulation of polyketide chain length represents a major unmet challenge in natural product biosynthesis. Polyketide chain elongation is catalyzed by a heterodimeric ketosynthase. In contrast to homodimeric ketosynthases found in fatty acid synthases, the active site cysteine is absent from the one subunit of this heterodimer. The precise role of this catalytically silent subunit has been debated over the past decade. We demonstrate here that this subunit is the primary determinant of polyketide chain length, thereby validating its designation as chain length factor. Using structure-based mutagenesis, we identified key residues in the chain length factor that could be manipulated to convert an octaketide synthase into a decaketide synthase and vice versa. These results should lead to novel strategies for the engineered biosynthesis of hitherto unidentified polyketide scaffolds.

  15. Curve Length Estimation using Vertix Chain Code Curve Length Estimation

    Directory of Open Access Journals (Sweden)

    Habibollah Haron


    Full Text Available Most of the applications in image analysis are based on Freeman chain code. In this paper, for the first time, vertex chain code (VCC proposed by Bribiesca is applied to improve length estimation of the 2D digitized curve. The chain code has some preferences such as stable in shifting, turning, mirroring movement of image and has normalized starting point. Due to the variety of length estimator methods, we focused on the three specific techniques. First, the way Bribiesca proposed which is based on counting links between vertices; second, based on maximum length digital straight segments (DSSs and lastly local metrics. The results of these length estimators with the real perimeter are compared. Results thus obtained exhibits thatlength estimation using VCC is nearest to the actual length.

  16. The S1-S2 linker determines the distinct pH sensitivity between ZmK2.1 and KAT1. (United States)

    Wang, Li; Yang, Shun-Ying; Guo, Man-Yuan; Huang, Ya-Nan; Sentenac, Hervé; Véry, Anne-Aliénor; Su, Yan-Hua


    Efficient stomatal opening requires activation of KAT-type K(+) channels, which mediate K(+) influx into guard cells. Most KAT-type channels are functionally facilitated by extracellular acidification. However, despite sequence and structural homologies, the maize counterpart of Arabidopsis KAT1 (ZmK2.1) is resistant to pH activation. To understand the structural determinant that results in the differential pH activation of these counterparts, we analysed chimeric channels and channels with point mutations for ZmK2.1 and its closest Arabidopsis homologue KAT1. Exchange of the S1-S2 linkers altered the pH sensitivity between the two channels, suggesting that the S1-S2 linker is essentially involved in the pH sensitivity. The effects of D92 mutation within the linker motif together with substitution of the first half of the linker largely resemble the effects of substitution of the complete linker. Topological modelling predicts that one of the two cysteines located on the outer face section of the S5 domain may serve as a potential titratable group that interacts with the S1-S2 linker. The difference between ZmK2.1 and KAT1 is predicted to be the result of the distance of the stabilized linkers from the titratable group. In KAT1, residue K85 within the linker forms a hydrogen bond with C211 that enables the pH activation; conversely, the linker of ZmK2.1 is distantly located and thus does not interact with the equivalent titration group (C208). Thus, in addition to the known structural contributors to the proton activation of KAT channels, we have uncovered a previously unidentified component that is strongly involved in this complex proton activation network.

  17. The concerted contribution of the S4-S5 linker and the S6 segment to the modulation of a Kv channel by 1-alkanols. (United States)

    Bhattacharji, Aditya; Kaplan, Benjamin; Harris, Thanawath; Qu, Xiaoguang; Germann, Markus W; Covarrubias, Manuel


    Gating of voltage-gated K(+) channels (K(v) channels) depends on the electromechanical coupling between the voltage sensor and activation gate. The main activation gate of K(v) channels involves the COOH-terminal section of the S6 segment (S6-b) and the S4-S5 linker at the intracellular mouth of the pore. In this study, we have expanded our earlier work to probe the concerted contribution of these regions to the putative amphipathic 1-alkanol site in the Shaw2 K(+) channel. In the S4-S5 linker, we found a direct energetic correlation between alpha-helical propensity and the inhibition of the Shaw2 channel by 1-butanol. Spectroscopic structural analyses of the S4-S5 linker supported this correlation. Furthermore, the analysis of chimeric Shaw2 and K(v)3.4 channels that exchanged their corresponding S4-S5 linkers showed that the potentiation induced by 1-butanol depends on the combination of a single mutation in the S6 PVPV motif (PVAV) and the presence of the Shaw2 S4-S5 linker. Then, using tandem-heterodimer subunits, we determined that this potentiation also depends on the number of S4-S5 linkers and PVAV mutations in the K(v) channel tetramer. Consistent with the critical contribution of the Shaw2 S4-S5 linker, the equivalent PVAV mutation in certain mammalian K(v) channels with divergent S4-S5 linkers conferred weak potentiation by 1-butanol. Overall, these results suggest that 1-alkanol action in Shaw2 channels depends on interactions involving the S4-S5 linker and the S6-b segment. Therefore, we propose that amphiphilic general anesthetic agents such as 1-alkanols may modulate gating of the Shaw2 K(+) channel by an interaction with its activation gate.

  18. Molecular motor-induced instabilities and cross linkers determine biopolymer organization.

    Energy Technology Data Exchange (ETDEWEB)

    Smith, D.; Ziebert, F.; Humphrey, D.; Duggan, C.; Steinbeck, M.; Zimmermann, W.; Kas, J.; Materials Science Division; Univ. of Leipzig; Univ. of Texas at Austin; Univ. Bayreuth


    All eukaryotic cells rely on the active self-organization of protein filaments to form a responsive intracellular cytoskeleton. The necessity of motility and reaction to stimuli additionally requires pathways that quickly and reversibly change cytoskeletal organization. While thermally driven order-disorder transitions are, from the viewpoint of physics, the most obvious method for controlling states of organization, the timescales necessary for effective cellular dynamics would require temperatures exceeding the physiologically viable temperature range. We report a mechanism whereby the molecular motor myosin II can cause near-instantaneous order-disorder transitions in reconstituted cytoskeletal actin solutions. When motor-induced filament sliding diminishes, the actin network structure rapidly and reversibly self-organizes into various assemblies. Addition of stable cross linkers was found to alter the architectures of ordered assemblies. These isothermal transitions between dynamic disorder and self-assembled ordered states illustrate that the interplay between passive crosslinking and molecular motor activity plays a substantial role in dynamic cellular organization.

  19. Gold nanoparticles deposited on linker-free silicon substrate and embedded in aluminum Schottky contact. (United States)

    Gorji, Mohammad Saleh; Razak, Khairunisak Abdul; Cheong, Kuan Yew


    Given the enormous importance of Au nanoparticles (NPs) deposition on Si substrates as the precursor for various applications, we present an alternative approach to deposit Au NPs on linker-free n- and p-type Si substrates. It is demonstrated that, all conditions being similar, there is a significant difference between densities of the deposited NPs on both substrates. The Zeta-potential and polarity of charges surrounding the hydroxylamine reduced seeded growth Au NPs, are determined by a Zetasizer. To investigate the surface properties of Si substrates, contact angle measurement is performed. Field-emission scanning electron microscope is then utilized to distinguish the NPs density on the substrates. Finally, Al/Si Schottky barrier diodes with embedded Au NPs are fabricated, and their structural and electrical characteristics are further evaluated using an energy-filtered transmission electron microscope and current-voltage measurements, respectively. The results reveal that the density of NPs is significantly higher on n-type Si substrate and consequently has more pronounced effects on the electrical characteristics of the diode. It is concluded that protonation of Si-OH group on Si surface in low pH is responsible for the immobilization of Au NPs, which eventually contributes to the lowering of barrier height and enhances the electrical characteristics.

  20. Graphitic carbon nanofiber (GCNF)/polymer materials. I. GCNF/epoxy monoliths using hexanediamine linker molecules. (United States)

    Zhong, Wei-Hong; Li, Jiang; Xu, Luoyu R; Michel, Jason A; Sullivan, Lisa M; Lukehart, Charles M


    Processing methods have been optimized for the formation of graphitic carbon nanofiber (GCNF)/epoxy nanocomposites containing GCNFs highly dispersed throughout a thermoset epoxy matrix. GCNFs having a herringbone atomic structure are surface-derivatized with bifunctional hexanediamine linker molecules (GCNF-HDA) capable of covalent binding to an epoxy matrix during thermal curing and are cut to smaller dimension using high-power ultrasonication. GCNF-HDA nanofibers are dispersed in epoxy resin at 0.3 wt.% loading using variable levels of ultrasonication processing prior to thermal curing. Effects of sonication power on the quality of the GCNF-HDA/epoxy material obtained after curing have been determined from flexural property measurements, thermomechanical analysis and SEM/TEM imaging. GCNF-HDA/epoxy material of the highest quality is obtained using low-power sonication, although high-power sonication for short periods gives improved flexural properties without lowering the glass transition temperature. Good dispersion and polymer wetting of the GCNF component is evident on the nanoscale.

  1. Role of H1 linker histones in mammalian development and stem cell differentiation. (United States)

    Pan, Chenyi; Fan, Yuhong


    H1 linker histones are key chromatin architectural proteins facilitating the formation of higher order chromatin structures. The H1 family constitutes the most heterogeneous group of histone proteins, with eleven non-allelic H1 variants in mammals. H1 variants differ in their biochemical properties and exhibit significant sequence divergence from one another, yet most of them are highly conserved during evolution from mouse to human. H1 variants are differentially regulated during development and their cellular compositions undergo dramatic changes in embryogenesis, gametogenesis, tissue maturation and cellular differentiation. As a group, H1 histones are essential for mouse development and proper stem cell differentiation. Here we summarize our current knowledge on the expression and functions of H1 variants in mammalian development and stem cell differentiation. Their diversity, sequence conservation, complex expression and distinct functions suggest that H1s mediate chromatin reprogramming and contribute to the large variations and complexity of chromatin structure and gene expression in the mammalian genome. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Linker histone variant H1T targets rDNA repeats. (United States)

    Tani, Ruiko; Hayakawa, Koji; Tanaka, Satoshi; Shiota, Kunio


    H1T is a linker histone H1 variant that is highly expressed at the primary spermatocyte stage through to the early spermatid stage of spermatogenesis. While the functions of the somatic types of H1 have been extensively investigated, the intracellular role of H1T is unclear. H1 variants specifically expressed in germ cells show low amino acid sequence homology to somatic H1s, which suggests that the functions or target loci of germ cell-specific H1T differ from those of somatic H1s. Here, we describe the target loci and function of H1T. H1T was expressed not only in the testis but also in tumor cell lines, mouse embryonic stem cells (mESCs), and some normal somatic cells. To elucidate the intracellular localization and target loci of H1T, fluorescent immunostaining and ChIP-seq were performed in tumor cells and mESCs. We found that H1T accumulated in nucleoli and predominantly targeted rDNA repeats, which differ from somatic H1 targets. Furthermore, by nuclease sensitivity assay and RT-qPCR, we showed that H1T repressed rDNA transcription by condensing chromatin structure. Imaging analysis indicated that H1T expression affected nucleolar formation. We concluded that H1T plays a role in rDNA transcription, by distinctively targeting rDNA repeats.

  3. Bis-isatin hydrazones with novel linkers: Synthesis and biological evaluation as cytotoxic agents. (United States)

    Ibrahim, Hany S; Abou-Seri, Sahar M; Ismail, Nasser S M; Elaasser, Mahmoud M; Aly, Mohamed H; Abdel-Aziz, Hatem A


    Many bis-isatins and isatins with hydrazide extension were reported to have a potential anti-proliferative effects against different cancer cell lines and cancer targets. In this study, four series of bis-isatins with hydrazide linkers were synthesized. These compounds were investigated for their antitumor activity by assessing their cytotoxic potency against HepG2, MCF-7 and HCT-116 cancer cell lines. Compound 21c possessed significant cytotoxic activity against MCF-7 (IC50 = 1.84 μM) and HCT-116 (IC50 = 3.31 μM) that surpasses the activity of doxorubicin against both cell lines (MCF-7; IC50 = 2.57 μM and HCT-116; IC50 = 3.70 μM). Cell cycle analysis and annexin V-FITC staining of MCF-7 cells treated with 21c suggested that the cytotoxic effect of the compound could be attributed to its pro-apoptotic activity.

  4. Linker-free conjugation and specific cell targeting of antibody functionalized iron-oxide nanoparticles (United States)

    Xu, Yaolin; Baiu, Dana C.; Sherwood, Jennifer A.; McElreath, Meghan R.; Qin, Ying; Lackey, Kimberly H.; Otto, Mario; Bao, Yuping


    Specific targeting is a key step to realize the full potential of iron oxide nanoparticles in biomedical applications, especially tumor-associated diagnosis and therapy. Here, we developed anti-GD2 antibody conjugated iron oxide nanoparticles for highly efficient neuroblastoma cell targeting. The antibody conjugation was achieved through an easy, linker-free method based on catechol reactions. The targeting efficiency and specificity of the antibody-conjugated nanoparticles to GD2-positive neuroblastoma cells were confirmed by flow cytometry, fluorescence microscopy, Prussian blue staining and transmission electron microscopy. These detailed studies indicated that the receptor-recognition capability of the antibody was fully retained after conjugation and the conjugated nanoparticles quickly attached to GD2-positive cells within four hours. Interestingly, longer treatment (12 h) led the cell membrane-bound nanoparticles to be internalized into cytosol, either by directly penetrating the cell membrane or escaping from the endosomes. Last but importantly, the uniquely designed functional surfaces of the nanoparticles allow easy conjugation of other bioactive molecules. PMID:26660881

  5. Chitosan hydrogel formation using laccase activated phenolics as cross-linkers. (United States)

    Huber, Daniela; Tegl, Gregor; Baumann, Martina; Sommer, Eva; Gorji, Elham Ghorbani; Borth, Nicole; Schleining, Gerhard; Nyanhongo, Gibson S; Guebitz, Georg M


    Chitosan hydrogels are gaining increasing interest for biomedical applications due to attractive properties such as biocompatibility. In order to replace toxic chemical cross-linkers for hydrogel formation, we investigated the cross-linking potential of laccase oxidized phenolics. HPLC-TOF-MS and ATR-FTIR demonstrated that phenolics were bond to glucosamine as chitosan model substrate. Phenolics concentrations required for hydrogel formation varied from 500μM for catechol to 5000μM for sinapic acid. The hydrogels showed different swelling and release properties assessed using methylene blue release as a model. Laccase oxidized caffeic acid and pyrogallol-chitosan hydrogels showed excellent behavior in up-taking water with a swelling of 208.7% for caffeic acid. Biocompatibility results did not show any significant inhibition of growth of HEK293 cell line when phenolics like catechol or eugenol were used. Therefore, this study demonstrates that laccase oxidized phenolics are potential cross-linking agents of chitosan as a novel green approach to synthesizing chitosan hydrogels.

  6. Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome. (United States)

    Yue, Hongjun; Fang, He; Wei, Sijie; Hayes, Jeffrey J; Lee, Tae-Hee


    Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.

  7. Linker histone H1 and H3K56 acetylation are antagonistic regulators of nucleosome dynamics. (United States)

    Bernier, Morgan; Luo, Yi; Nwokelo, Kingsley C; Goodwin, Michelle; Dreher, Sarah J; Zhang, Pei; Parthun, Mark R; Fondufe-Mittendorf, Yvonne; Ottesen, Jennifer J; Poirier, Michael G


    H1 linker histones are highly abundant proteins that compact nucleosomes and chromatin to regulate DNA accessibility and transcription. However, the mechanisms that target H1 regulation to specific regions of eukaryotic genomes are unknown. Here we report fluorescence measurements of human H1 regulation of nucleosome dynamics and transcription factor (TF) binding within nucleosomes. H1 does not block TF binding, instead it suppresses nucleosome unwrapping to reduce DNA accessibility within H1-bound nucleosomes. We then investigated H1 regulation by H3K56 and H3K122 acetylation, two transcriptional activating histone post translational modifications (PTMs). Only H3K56 acetylation, which increases nucleosome unwrapping, abolishes H1.0 reduction of TF binding. These findings show that nucleosomes remain dynamic, while H1 is bound and H1 dissociation is not required for TF binding within the nucleosome. Furthermore, our H3K56 acetylation measurements suggest that a single-histone PTM can define regions of the genome that are not regulated by H1.

  8. Interaction between the sodium channel inactivation linker and domain III S4-S5. (United States)

    Smith, M R; Goldin, A L


    The III-IV linker (L(III-IV)) of the rat brain sodium channel is critical for fast inactivation, possibly forming a fast inactivation particle. Inactivation can be disrupted by mutation of a conserved alanine at position 1329 in the S4-S5 loop of domain III. Combination of a charged mutation at 1329 with a compensatory (opposite) charge mutation at position 1489 in L(III-IV) partially restores inactivation of the channel. The compensatory charge mutant channel has a single-channel mean open time that is similar to that of the wild-type channel and is approximately 50 times shorter than that of the L(III-IV) mutant channel. The results of thermodynamic cycle analysis indicate that the mutations in domain III S4-S5 and L(III-IV) have a coupling energy of 2.8 kcal/mol, indicating that the two mutations act interdependently. These data suggest that L(III-IV) interacts directly with A1329, which may form part of the docking site if L(III-IV) is a fast inactivation particle.

  9. Graft linker immobilization for spatial control of protein immobilization inside fused microchips. (United States)

    Shirai, Kentaro; Renberg, Björn; Sato, Kae; Mawatari, Kazuma; Konno, Tomohiro; Ishihara, Kazuhiko; Kitamori, Takehiko


    Fused silica glass microchips have several attractive features for lab-on-a-chip applications; they can be machined with excellent precision down to nanospace; are stable; transparent and can be modified with a range of silanization agents to change channel surface properties. For immobilization, however, ligands must be added after bonding, since the harsh bonding conditions using heat or hydrofluoric acid would remove all prior immobilized ligands. For spatial control over immobilization, UV-mediated immobilization offers several advantages; spots can be created in parallel, the feature size can be made small, and spatial control over patterns and positions is excellent. However, UV sensitive groups are often based on hydrophobic chemical moieties, which unfortunately result in greater non-specific binding of biomolecules, especially proteins. Here, we present techniques in which any -CH(x) (x=1,2,3) containing surface coating can be used as foundation for grafting a hydrophilic linker with a chemical anchor, a carboxyl group, to which proteins and amine containing molecules can be covalently coupled. Hence, the attractive features of many well-known protein and biomolecule repelling polymer coatings can be utilized while achieving site-specific immobilization only to pre-determined areas within the bonded microchips.

  10. Impedance of Finite Length Resistor

    CERN Document Server

    Krinsky, Samuel; Podobedov, Boris


    We determine the impedance of a cylindrical metal tube (resistor) of radius a and length g, attached at each end to perfect conductors of semi-infinite length. Our main interest is in the behavior of the impedance at high frequency (k>>1/a). In the equilibrium regime, ka2

  11. Line Lengths and Starch Scores. (United States)

    Moriarty, Sandra E.


    Investigates readability of different line lengths in advertising body copy, hypothesizing a normal curve with lower scores for shorter and longer lines, and scores above the mean for lines in the middle of the distribution. Finds support for lower scores for short lines and some evidence of two optimum line lengths rather than one. (SKC)

  12. The solution structure of the S4-S5 linker of the hERG potassium channel. (United States)

    Gayen, Shovanlal; Li, Qingxin; Kang, CongBao


    The human ether-à-go-go related gene (hERG) encodes a protein that forms a voltage-gated potassium channel and plays an important role in the heart by controlling the rapid delayed rectifier K(+) current (I(Kr)). The S4-S5 linker was shown to be important for the gating of the hERG channel. Nuclear magnetic resonance study showed that a peptide derived from the S4-S5 linker had no well-ordered structure in aqueous solution and adopted a 3(10) -helix (E544-Y545-G546) structure in detergent micelles. The existence of an amphipathic helix was confirmed, which may be important for interaction with cell membrane. Close contact between side chains of residues R541 and E544 was observed, which may be important for its regulation of channel gating.

  13. Development of a Novel, Oxidatively Activated, Safety-Catch Linker for Solid-Phase Asymmetric Organic Synthesis (SPOS)

    Institute of Scientific and Technical Information of China (English)

    LIN,Jun; Hjalmar Skarphedinsson; Stepehen G.Davies


    @@ Solid-phase asymmetric organic synthesis has become a very important synthetic strategy within the organic chemistry community.[1] Critical to success in SPOS is a linking strategy which allows both the substrate to be loaded and the product released efficiently from the polymeric support. A safety catch linker[2] (SCL) is in principle a linking molecule orthogonal to the reaction conditions of the library synthesis, which can be easily activated by a simple chemical transformation to allow efficient cleavage of the products from the polymer under mild conditions. In order to introduce the SuperQuat chiral auxiliaries[3] for SOPS, we report herein design and synthesis of a novel safety catch linker for asymmetric conjugate addition reactions.

  14. Postassembly Transformation of a Catalytically Active Composite Material, Pt@ZIF-8, via Solvent-Assisted Linker Exchange. (United States)

    Stephenson, Casey J; Hupp, Joseph T; Farha, Omar K


    2-Methylimidazolate linkers of Pt@ZIF-8 are exchanged with imidazolate using solvent-assisted linker exchange (SALE) to expand the apertures of the parent material and create Pt@SALEM-2. Characterization of the material before and after SALE was performed. Both materials are active as catalysts for the hydrogenation of 1-octene, whereas the hydrogenation of cis-cyclohexene occurred only with Pt@SALEM-2, consistent with larger apertures for the daughter material. The largest substrate, β-pinene, proved to be unreactive with H2 when either material was employed as a candidate catalyst, supporting the contention that substrate molecules, for both composites, must traverse the metal-organic framework component in order to reach the catalytic nanoparticles.

  15. The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5. (United States)

    Muretta, Joseph M; Jun, Yonggun; Gross, Steven P; Major, Jennifer; Thomas, David D; Rosenfeld, Steven S


    Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction--including switch-1 and the neck linker--how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as "structural kinetics," to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.

  16. Gestation length in farmed reindeer. (United States)

    Shipka, M P; Rowell, J E


    Reindeer (Rangifer tarandus tarundus) are the only cervids indigenous to the arctic environment. In Alaska, reindeer are a recognized agricultural species and an economic mainstay for many native populations. Traditionally raised in extensive free-ranging systems, a recent trend toward intensive farming requires a more in-depth knowledge of reproductive management. Reported gestation length in reindeer varies, ranging from 198 to 229 d in studies performed at the University of Alaska Fairbanks. A switchback study that manipulated only breeding date demonstrated a mean increase in gestation length of 8.5 d among females bred early in the season. The negative correlation between conception date and gestation length is consistent with reindeer research at other locations and reports of variable gestation length in a growing number of domestic and non-domestic species. This paper reviews the phenomenon in reindeer and discusses some of the factors known to affect gestation length as well as possible areas for future research.

  17. Reserved-Length Prefix Coding

    CERN Document Server

    Baer, Michael B


    Huffman coding finds an optimal prefix code for a given probability mass function. Consider situations in which one wishes to find an optimal code with the restriction that all codewords have lengths that lie in a user-specified set of lengths (or, equivalently, no codewords have lengths that lie in a complementary set). This paper introduces a polynomial-time dynamic programming algorithm that finds optimal codes for this reserved-length prefix coding problem. This has applications to quickly encoding and decoding lossless codes. In addition, one modification of the approach solves any quasiarithmetic prefix coding problem, while another finds optimal codes restricted to the set of codes with g codeword lengths for user-specified g (e.g., g=2).

  18. Xe Adsorption and Separation Properties of a Series of Microporous Metal–Organic Frameworks (MOFs) with V-Shaped Linkers

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Debasis; Elsaidi, Sameh K.; Thallapally, Praveen K.


    A series of microporous Metal-Organic Frameworks (MOFs) constructed by a V-shaped linker, 4,4’-sul-fonyldibenzoic acid were evaluated for their Xe gas adsorption properties. In particular, a cadmium based MOF exhibit noteworthy Xe adsorption and separation properties in presence of other gases under nuclear reprocessing conditions. The difference in Xe adsorption and separation properties of the materials were attributed towards their topology and pore size.

  19. Facile synthesis of one-dimensional organometallic-organic hybrid polymers based on a diphosphorus complex and flexible bipyridyl linkers. (United States)

    Elsayed Moussa, M; Attenberger, B; Peresypkina, E V; Fleischmann, M; Balázs, G; Scheer, M


    The selective synthesis of a series of new "ladderlike" one-dimensional organometallic-organic hybrid polymers is shown. The polymers are obtained from the reaction of the diphosphorus ligand complex [Cp2Mo2(CO)4(η(2)-P2)] with the copper salt [Cu(CH3CN)4]BF4 in the presence of flexible organic bipyridyl linkers in high selectivity. This unique behaviour is supported by DFT calculations.

  20. Interaction of local anesthetics with a peptide encompassing the IV/S4-S5 linker of the Na+ channel. (United States)

    Fraceto, Leonardo F; Oyama, Sérgio; Nakaie, Clóvis R; Spisni, Alberto; de Paula, Eneida; Pertinhez, Thelma A


    The peptide pIV/S4-S5 encompasses the cytoplasmic linker between helices S4-S5 in domain IV of the voltage-gated Na+ channel, residues 1644-1664. The interaction of two local anesthetics (LA), lidocaine and benzocaine, with pIV/S4-S5 has been studied by DOSY, heteronuclear NMR 1H-15N-HSQC spectroscopy and computational methods. DOSY indicates that benzocaine, a neutral ester, exhibits stronger interaction with pIV/S4-S5 than lidocaine, a charged amine-amide. Weighted average chemical shifts, Deltadelta(1H-15N), show that benzocaine affects residues L1653, M1655 and S1656 while lidocaine slightly perturbs residues I1646, L1649 and A1659, L1660, near the N- and C-terminus, respectively. Computational methods confirmed the stability of the benzocaine binding and the existence of two binding sites for lidocaine. Even considering that the approach of studying the peptide in the presence of a co-solvent (TFE/H2O, 30%/70% v/v) has an inherently limited implication, our data strongly support the existence of multiple LA binding sites in the IV/S4-S5 linker, as suggested in the literature. In addition, we consider that LA can bind to the S4-S5 linker with diverse binding modes and strength since this linker is part of the receptor for the "inactivation gate particle". Conditions for devising new functional studies, aiming to better understand Na+ channel functionality as well as the various facets of LA pharmacological activity are proposed in this work.

  1. Design, synthesis and biological evaluation of benzamide and phenyltetrazole derivatives with amide and urea linkers as BCRP inhibitors. (United States)

    Gujarati, Nehaben A; Zeng, Leli; Gupta, Pranav; Chen, Zhe-Sheng; Korlipara, Vijaya L


    Breast cancer resistant protein (BCRP/ABCG2), a 72kDa plasma membrane transporter protein is a member of ABC transporter superfamily. Increased expression of BCRP causes increased efflux and therefore, reduced intracellular accumulation of many unrelated chemotherapeutic agents leading to multidrug resistance (MDR). A series of 31 benzamide and phenyltetrazole derivatives with amide and urea linkers has been synthesized to serve as potential BCRP inhibitors in order to overcome BCRP-mediated MDR. The target derivatives were tested for their cytotoxicity and reversal effects in human non-small cell lung cancer cell line H460 and mitoxantrone resistant cell line H460/MX20 using the MTT assay. In the benzamide series, compounds 6 and 7 exhibited a fold resistance of 1.51 and 1.62, respectively at 10µM concentration which is similar to that of FTC, a known BCRP inhibitor. Compounds 27 and 31 were the most potent analogues in the phenyltetrazole series with amide linker with a fold resistance of 1.39 and 1.32, respectively at 10µM concentration. For the phenyltetrazole series with urea linker, 38 exhibited a fold resistance of 1.51 which is similar than that of FTC and is the most potent compound in this series. The target compounds did not exhibit reversal effect in P-gp overexpressing resistant cell line SW620/Ad300 suggesting that they are selective BCRP inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Linker-extended native cyanovirin-N facilitates PEGylation and potently inhibits HIV-1 by targeting the glycan ligand.

    Directory of Open Access Journals (Sweden)

    Jia Chen

    Full Text Available Cyanovirin-N (CVN potently inhibits human immunodeficiency virus type 1 (HIV-1 infection, but both cytotoxicity and immunogenicity have hindered the translation of this protein into a viable therapeutic. A molecular docking analysis suggested that up to 12 residues were involved in the interaction of the reverse parallel CVN dimer with the oligosaccharide targets, among which Leu-1 was the most prominent hot spot residue. This finding provided a possible explanation for the lack of anti-HIV-1 activity observed with N-terminal PEGylated CVN. Therefore, linker-CVN (LCVN was designed as a CVN derivative with a flexible and hydrophilic linker (Gly4Ser3 at the N-terminus. The N-terminal α-amine of LCVN was PEGylated to create 10 K PEG-aldehyde (ALD-LCVN. LCVN and 10 K PEG-ALD-LCVN retained the specificity and affinity of CVN for high mannose N-glycans. Moreover, LCVN exhibited significant anti-HIV-1 activity with attenuated cytotoxicity in the HaCaT keratinocyte cell line and MT-4 T lymphocyte cell lines. 10 K PEG-ALD-LCVN also efficiently inactivated HIV-1 with remarkably decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. The linker-extended CVN and the mono-PEGylated derivative were determined to be promising candidates for the development of an anti-HIV-1 agent. This derivatization approach provided a model for the PEGylation of biologic candidates without introducing point mutations.

  3. Linker-extended native cyanovirin-N facilitates PEGylation and potently inhibits HIV-1 by targeting the glycan ligand. (United States)

    Chen, Jia; Huang, Dane; Chen, Wei; Guo, Chaowan; Wei, Bo; Wu, Chongchao; Peng, Zhou; Fan, Jun; Hou, Zhibo; Fang, Yongsheng; Wang, Yifei; Kitazato, Kaio; Yu, Guoying; Zou, Chunbin; Qian, Chuiwen; Xiong, Sheng


    Cyanovirin-N (CVN) potently inhibits human immunodeficiency virus type 1 (HIV-1) infection, but both cytotoxicity and immunogenicity have hindered the translation of this protein into a viable therapeutic. A molecular docking analysis suggested that up to 12 residues were involved in the interaction of the reverse parallel CVN dimer with the oligosaccharide targets, among which Leu-1 was the most prominent hot spot residue. This finding provided a possible explanation for the lack of anti-HIV-1 activity observed with N-terminal PEGylated CVN. Therefore, linker-CVN (LCVN) was designed as a CVN derivative with a flexible and hydrophilic linker (Gly4Ser)3 at the N-terminus. The N-terminal α-amine of LCVN was PEGylated to create 10 K PEG-aldehyde (ALD)-LCVN. LCVN and 10 K PEG-ALD-LCVN retained the specificity and affinity of CVN for high mannose N-glycans. Moreover, LCVN exhibited significant anti-HIV-1 activity with attenuated cytotoxicity in the HaCaT keratinocyte cell line and MT-4 T lymphocyte cell lines. 10 K PEG-ALD-LCVN also efficiently inactivated HIV-1 with remarkably decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. The linker-extended CVN and the mono-PEGylated derivative were determined to be promising candidates for the development of an anti-HIV-1 agent. This derivatization approach provided a model for the PEGylation of biologic candidates without introducing point mutations.

  4. PIP2 controls voltage-sensor movement and pore opening of Kv channels through the S4-S5 linker. (United States)

    Rodriguez-Menchaca, Aldo A; Adney, Scott K; Tang, Qiong-Yao; Meng, Xuan-Yu; Rosenhouse-Dantsker, Avia; Cui, Meng; Logothetis, Diomedes E


    Voltage-gated K(+) (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4-S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP(2) depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP(2). Gating current measurements revealed that PIP(2) constrains the movement of the sensor through interactions with the S4-S5 linker. Thus, PIP(2) controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them.

  5. A model study of artificial linker system using self-assembled calix[4]arene derivative monolayers for protein immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Minsu; An, Won Gun; Kim, Jae-Ho; Choi, Heung-Jin; Kim, Sung-Hoon; Han, Moon-Hi; Koh, Kwangnak


    The attachment of biomolecules, in particular proteins, onto solid supports is fundamental in the development of advanced biosensors, biochips, bioreactors, and many diagnostic techniques. In addition, the effective investigation of biomolecular structure and function with chip-based modern instruments often requires effective attachment of the biomolecule to a substrate. For this reason, it is very important to construct well-characterized linker system that can immobilize protein efficiently. Here, we investigate the formation of self-assembled monolayers (SAMs) with calix[4]arene ethylester and carboxylic acid derivatives that can serve as a model system for protein immobilization at solid surfaces. The calix[4]arene derivative monolayers were formed on Au surface and carefully characterized by atomic force microscopy (AFM), Fourier transform infrared reflection absorption spectroscopy (FTIR-RAS) and surface plasmon resonance (SPR). Immobilization process of protein using bovine serum albumin (BSA) on the artificial linker layer was measured by SPR. The surface concentration of BSA was calculated by simulation of experimental SPR data. The surface concentration of BSA on the carboxylic acid form was higher than that of the ethylester. These results can help in modeling and understanding of protein immobilization on the linker layer.

  6. Reactivation of VX-inhibited AChE by novel oximes having two oxygen atoms in the linker. (United States)

    Kuca, Kamil; Cabal, Jiri; Jun, Daniel; Musilek, Kamil; Soukup, Ondrej; Pohanka, Miroslav; Pejchal, Jaroslav; Oh, Kyung-Ae; Yang, Garp Yeol; Jung, Young-Sik


    Two newly developed AChE reactivators possessing two oxime groups in 4-position of the pyridinium rings with linkers CH(2)O(CH(2))(2)OCH(2) and CH(2)O(CH(2))(4)OCH(2) were tested for their potency to reactivate VX-inhibited AChE. Their reactivation potency was compared with currently available oximes such as pralidoxime, obidoxime and HI-6. Appropriate constants (affinity towards the intact and inhibited enzyme, reactivation rate) characterizing the reactivation process were determined. According to the data obtained, a new oxime with CH(2)O(CH(2))(2)OCH(2) linker reached as high reactivation potency as HI-6. The percentage of reactivation of the oxime with CH(2)O(CH(2))(2)OCH(2) linker was comparable to that of obidoxime at a concentration 10(-3)M. Hence, these oximes may be worthy of future development for the treatment of nerve agent intoxications, especially, with lipophilic agents such as soman and cyclosarin.

  7. Diketopyrrolopyrrole Polymers with Thienyl and Thiazolyl Linkers for Application in Field-Effect Transistors and Polymer Solar Cells. (United States)

    Yu, Yaping; Wu, Yang; Zhang, Andong; Li, Cheng; Tang, Zheng; Ma, Wei; Wu, Yonggang; Li, Weiwei


    Conjugated polymers consisting of diketopyrrolopyrrole (DPP) units have been successfully applied in field-effect transistors (FETs) and polymer solar cells (PSCs), while most of the DPP polymers were designed as symmetric structures containing identical aromatic linkers. In this manuscript, we design a new asymmetric DPP polymer with varied aromatic linkers in the backbone for application in FETs and PSCs. The designation provides the chance to finely adjust the energy levels of conjugated polymers so as to influence the device performance. The asymmetric polymer exhibits highly crystalline properties, high hole mobilities of 3.05 cm(2) V(-1) s(-1) in FETs, and a high efficiency of 5.9% in PSCs with spectra response from 300 to 850 nm. Morphology investigation demonstrates that the asymmetric polymer has a large crystal domain in blended thin films, indicating that the solar cell performance can be further enhanced by optimizing the microphase separation. The study reveals that the asymmetric design via adjusting the aromatic linkers in DPP polymers is a useful route toward flexible electronic devices.

  8. The PathLinker app: Connect the dots in protein interaction networks [version 1; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Daniel P. Gil


    Full Text Available PathLinker is a graph-theoretic algorithm for reconstructing the interactions in a signaling pathway of interest. It efficiently computes multiple short paths within a background protein interaction network from the receptors to transcription factors (TFs in a pathway. We originally developed PathLinker to complement manual curation of signaling pathways, which is slow and painstaking. The method can be used in general to connect any set of sources to any set of targets in an interaction network. The app presented here makes the PathLinker functionality available to Cytoscape users. We present an example where we used PathLinker to compute and analyze the network of interactions connecting proteins that are perturbed by the drug lovastatin.

  9. Minimum length-maximum velocity (United States)

    Panes, Boris


    We study a framework where the hypothesis of a minimum length in space-time is complemented with the notion of reference frame invariance. It turns out natural to interpret the action of the obtained reference frame transformations in the context of doubly special relativity. As a consequence of this formalism we find interesting connections between the minimum length properties and the modified velocity-energy relation for ultra-relativistic particles. For example, we can predict the ratio between the minimum lengths in space and time using the results from OPERA on superluminal neutrinos.

  10. Linker-assisted immunoassay and liquid chromatography/mass spectrometry for the analysis of glyphosate (United States)

    Lee, E.A.; Zimmerman, L.R.; Bhullar, B.S.; Thurman, E.M.


    A novel, sensitive, linker-assisted enzyme-linked immunosorbent assay (L'ELISA) was compared to on-line solidphase extraction (SPE) with high-performance liquid chromatography/mass spectrometry (HPLC/MS) for the analysis of glyphosate in surface water and groundwater samples. The L'ELISA used succinic anhydride to derivatize glyphosate, which mimics the epitotic attachment of glyphosate to horseradish peroxidase hapten. Thus, L'ELISA recognized the derivatized glyphosate more effectively (detection limit of 0.1 ??g/L) and with increased sensitivity (10-100 times) over conventional ELISA and showed the potential for other applications. The precision and accuracy of L'ELISA then was compared with on-line SPE/HPLC/MS, which detected glyphosate and its degradate derivatized with 9-fluorenylmethyl chloroformate using negative-ion electrospray (detection limit 0.1 ??g/L, relative standard deviation ??15%). Derivatization efficiency and matrix effects were minimized by adding an isotope-labeled glyphosate (2-13C15N). The accuracy of L'ELISA gave a false positive rate of 18% between 0.1 and 1.0 ??g/L and a false positive rate of only 1% above 1.0 ??g/L. The relative standard deviation was ??20%. The correlation of L'ELISA and HPLC/MS for 66 surface water and groundwater samples was 0.97 with a slope of 1.28, with many detections of glyphosate and its degradate in surface water but not in groundwater.

  11. Post-translational modifications of linker histone H1 variants in mammals (United States)

    Starkova, T. Yu; Polyanichko, A. M.; Artamonova, T. O.; Khodorkovskii, M. A.; Kostyleva, E. I.; Chikhirzhina, E. V.; Tomilin, A. N.


    The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2–H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

  12. Preparation and in vivo evaluation of novel linkers for {sup 211}At labeling of proteins

    Energy Technology Data Exchange (ETDEWEB)

    Talanov, Vladimir S. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Yordanov, Alexander T. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Garmestani, Kayhan [Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Milenic, Diane E. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Arora, Hans C. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Plascjak, Paul S. [Department of Nuclear Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Eckelman, William C. [Department of Nuclear Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Waldmann, Thomas A. [Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Brechbiel, Martin W. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)]. E-mail:


    The syntheses, radiolabeling, antibody conjugation and in vivo evaluation of new linkers for {sup 211}At labeling of monoclonal antibodies are described. Syntheses of the N-succinimidyl esters and labeling with {sup 211}At to form succinimidyl 4-methoxymethyl-3-[{sup 211}At]astatobenzoate (9) and succinimidyl 4-methylthiomethyl-3-[{sup 211}At]astatobenzoate (11) from the corresponding bromo-aryl esters is reported. Previously reported succinimidyl N-{l_brace}4-[{sup 211}At]astatophenethyl{r_brace}succinamate (SAPS) is employed as a standard of in vivo stability. Each agent is conjugated with Herceptin in parallel with their respective {sup 125}I analogue, succinimidyl 4-methoxymethyl-3-[{sup 125}I]iodobenzoate (10), succinimidyl 4-methylthiomethyl-3-[{sup 125}I]iodobenzoate (12) and succinimidyl N-{l_brace}4-[{sup 125}I]iodophenethyl{r_brace}succinamate (SIPS), respectively, for comparative assessment in LS-174T xenograft-bearing mice. With 9 and 11, inclusion of an electron pair donor in the ortho position does not appear to provide in vivo stability comparable to SAPS. Variables in radiolabeling chemistry of these three agents with {sup 211}At are notable. Sequential elimination of acetic acid and oxidizing agent, N-chlorosuccinimide (NCS), from the {sup 211}At radiolabeling protocol for forming SAPS improves yield, product purity and consistency. NCS appears to be critical for the radiolabeling of 6 with {sup 211}At. Formation of 11, however, is found to require the absence of NCS. Elimination of acetic acid is found to have no effect on radiolabeling efficiency or yield for either of these reactions.

  13. Tetrathiafulvalene-fused porphyrins via quinoxaline linkers: symmetric and asymmetric donor-acceptor systems. (United States)

    Jia, Hongpeng; Schmid, Belinda; Liu, Shi-Xia; Jaggi, Michael; Monbaron, Philippe; Bhosale, Sheshanath V; Rivadehi, Shadi; Langford, Steven J; Sanguinet, Lionel; Levillain, Eric; El-Khouly, Mohamed E; Morita, Ysushi; Fukuzumi, Shunichi; Decurtins, Silvio


    A tetrathiafulvalene (TTF) donor is annulated to porphyrins (P) via quinoxaline linkers to form novel symmetric P-TTF-P triads 1 a-c and asymmetric P-TTF dyads 2 a,b in good yields. These planar and extended π-conjugated molecules absorb light over a wide region of the UV/Vis spectrum as a result of additional charge-transfer excitations within the donor-acceptor assemblies. Quantum-chemical calculations elucidate the nature of the electronically excited states. The compounds are electrochemically amphoteric and primarily exhibit low oxidation potentials. Cyclic voltammetric and spectroelectrochemical studies allow differentiation between the TTF and porphyrin sites with respect to the multiple redox processes occurring within these molecular assemblies. Transient absorption measurements give insight into the excited-state events and deliver corresponding kinetic data. Femtosecond transient absorption spectra in benzonitrile may suggest the occurrence of fast charge separation from TTF to porphyrin in dyads 2 a,b but not in triads 1 a-c. Clear evidence for a photoinduced and relatively long lived charge-separated state (385 ps lifetime) is obtained for a supramolecular coordination compound built from the ZnP-TTF dyad and a pyridine-functionalized C(60) acceptor unit. This specific excited state results in a (ZnP-TTF)(⋅+) ⋅⋅⋅(C(60) py)(⋅-) state. The binding constant of Zn(II) ⋅⋅⋅py is evaluated by constructing a Benesi-Hildebrand plot based on fluorescence data. This plot yields a binding constant K of 7.20×10(4)  M(-1), which is remarkably high for bonding of pyridine to ZnP. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Fabrication of electrospun HPGL scaffolds via glycidyl methacrylate cross-linker: Morphology, mechanical and biological properties. (United States)

    Baratéla, Fernando José Costa; Higa, Olga Zazuco; Dos Passos, Esdras Duarte; de Queiroz, Alvaro Antonio Alencar


    Electrospinning is a suitable method to produce scaffolds composed of nanoscale to microscale fibers, which are comparable to the extracellular matrix (ECM). Hyperbranched polyglycerol (HPGL) is a highly biocompatible polyether polyol potentially useful for the design of fibrous scaffolds mimicking the ECM architecture. However, scaffolds developed from HPGL have poor mechanical properties and morphological stability in the aqueous environments required for tissue engineering applications. This work reports the production of stable electrospun HPGL scaffolds (EHPGLS) using glycidyl methacrylate (GMA) as cross-linker to enhance the water stability and mechanical property of electrospun HPGL. The diameter and morphology of the produced EHPGLS were analyzed by scanning electron microscopy (SEM). It was observed that electrical fields in the range of 0.2kV·cm(-1) to 1.0kV·cm(-1) decrease the average fiber diameter of EHPGLS. The increase in porosity of EHPGLS with GMA concentration indicates the in situ formation of a heterogeneous structure resultant from the phase separation during crosslinking of HPGL by GMA. EHPGLS containing 20% (w/w) GMA concentration possessed highest tensile strength (295.4±11.32kPa), which is approximately 58 times higher than that of non-crosslinked EHPGLS (5.1±2.12kPa). The MTS cell viability results showed that the EHPGLS have no significant cytotoxicity effect on Chinese hamster ovary (CHO-K1) cells. Scanning electron microscopy (SEM) indicates that the cultured BALB/3T3 fibroblasts cells were able to keep contact each other's, thus forming a homogeneous monolayer on the internal surface of the EHPGLS. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Linker design for the modular assembly of multifunctional and targeted platinum(ii)-containing anticancer agents. (United States)

    Ding, S; Bierbach, U


    A versatile and efficient modular synthetic platform was developed for assembling multifunctional conjugates and targeted forms of platinum-(benz)acridines, a class of highly cytotoxic DNA-targeted hybrid agents. The synthetic strategy involved amide coupling between succinyl ester-modified platinum compounds (P1, P2) and a set of 11 biologically relevant primary and secondary amines (N1-N11). To demonstrate the feasibility and versatility of the approach, a structurally and functionally diverse range of amines was introduced. These include biologically active molecules, such as rucaparib (a PARP inhibitor), E/Z-endoxifen (an estrogen receptor antagonist), and a quinazoline-based tyrosine kinase inhibitor. Micro-scale reactions in Eppendorf tubes or on 96-well plates were used to screen for optimal coupling conditions in DMF solution with carbodiimide-, uronium-, and phosphonium-based compounds, as well as other common coupling reagents. Reactions with the phosphonium-based coupling reagent PyBOP produced the highest yields and gave the cleanest conversions. Furthermore, it was demonstrated that the chemistry can also be performed in aqueous media and is amenable to parallel synthesis based on multiple consecutive reactions in DMF in a "one-tube" format. In-line LC-MS was used to assess the stability of the conjugates in physiologically relevant buffers. Hydrolysis of the conjugates occurs at the ester moiety and is facilitated by the aquated metal moiety under low-chloride ion conditions. The rate of ester cleavage greatly depends on the nature of the amine component. Potential applications of the linker technology are discussed.

  16. Extracellular Linkers Completely Transplant the Voltage Dependence from Kv1.2 Ion Channels to Kv2.1. (United States)

    Elinder, Fredrik; Madeja, Michael; Zeberg, Hugo; Århem, Peter


    The transmembrane voltage needed to open different voltage-gated K (Kv) channels differs by up to 50 mV from each other. In this study we test the hypothesis that the channels' voltage dependences to a large extent are set by charged amino-acid residues of the extracellular linkers of the Kv channels, which electrostatically affect the charged amino-acid residues of the voltage sensor S4. Extracellular cations shift the conductance-versus-voltage curve, G(V), by interfering with these extracellular charges. We have explored these issues by analyzing the effects of the divalent strontium ion (Sr(2+)) on the voltage dependence of the G(V) curves of wild-type and chimeric Kv channels expressed in Xenopus oocytes, using the voltage-clamp technique. Out of seven Kv channels, Kv1.2 was found to be most sensitive to Sr(2+) (50 mM shifted G(V) by +21.7 mV), and Kv2.1 to be the least sensitive (+7.8 mV). Experiments on 25 chimeras, constructed from Kv1.2 and Kv2.1, showed that the large Sr(2+)-induced G(V) shift of Kv1.2 can be transferred to Kv2.1 by exchanging the extracellular linker between S3 and S4 (L3/4) in combination with either the extracellular linker between S5 and the pore (L5/P) or that between the pore and S6 (LP/6). The effects of the linker substitutions were nonadditive, suggesting specific structural interactions. The free energy of these interactions was ∼20 kJ/mol, suggesting involvement of hydrophobic interactions and/or hydrogen bonds. Using principles from double-layer theory we derived an approximate linear equation (relating the voltage shifts to altered ionic strength), which proved to well match experimental data, suggesting that Sr(2+) acts on these channels mainly by screening surface charges. Taken together, these results highlight the extracellular surface potential at the voltage sensor as an important determinant of the channels' voltage dependence, making the extracellular linkers essential targets for evolutionary selection.

  17. Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: structural and functional significance of the intermodular linker

    Directory of Open Access Journals (Sweden)

    Svetlana Petkun


    Full Text Available Non-cellulosomal processive endoglucanase 9I (Cel9I from Clostridium thermocellum is a modular protein, consisting of a family-9 glycoside hydrolase (GH9 catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b, separated by linker regions. GH9 does not show cellulase activity when expressed without CBM3c and CBM3b and the presence of the CBM3c was previously shown to be essential for endoglucanase activity. Physical reassociation of independently expressed GH9 and CBM3c modules (containing linker sequences restored 60–70% of the intact Cel9I endocellulase activity. However, the mechanism responsible for recovery of activity remained unclear. In this work we independently expressed recombinant GH9 and CBM3c with and without their interconnecting linker in Escherichia coli. We crystallized and determined the molecular structure of the GH9/linker-CBM3c heterodimer at a resolution of 1.68 Å to understand the functional and structural importance of the mutual spatial orientation of the modules and the role of the interconnecting linker during their re-association. Enzyme activity assays and isothermal titration calorimetry were performed to study and compare the effect of the linker on the re-association. The results indicated that reassembly of the modules could also occur without the linker, albeit with only very low recovery of endoglucanase activity. We propose that the linker regions in the GH9/CBM3c endoglucanases are important for spatial organization and fixation of the modules into functional enzymes.

  18. Evaluation of the effects of chemically different linkers on hepatic accumulations, cell tropism and gene silencing ability of cholesterol-conjugated antisense oligonucleotides. (United States)

    Wada, Shunsuke; Yasuhara, Hidenori; Wada, Fumito; Sawamura, Motoki; Waki, Reiko; Yamamoto, Tsuyoshi; Harada-Shiba, Mariko; Obika, Satoshi


    Cholesterol conjugation of oligonucleotides is an attractive way to deliver the oligonucleotides specifically to the liver. However cholesterol-conjugated antisense oligonucleotides (ASOs) mainly accumulate in non-parenchymal cells (NPCs) such as Kupffer cells. In this study, to increase the hepatic accumulation of cholesterol-conjugated ASOs, we prepared a variety of linkers for cholesterol conjugation to anti-Pcsk9 ASOs and examined their effects on pharmacological parameters. Hepatic accumulation of ASO was dramatically increased with cholesterol conjugation. The increase in hepatic accumulation depended largely on the linker chemistry of each cholesterol-conjugated ASO. In addition to hepatic accumulation, the cell tropism of each cholesterol-conjugated ASO tended to depend on their linker. Although a linker bearing a disulfide bond accumulated mainly in NPCs, hexamethylene succinimide linker accumulated mainly in hepatocytes. To estimate the benefits of releasing ASO from the conjugated cholesterol in hepatocyte, we designed another linker based on hexamethylene succinimide, which has a phosphodiester bond between the linker and the ASO. The cholesterol-conjugated ASO bearing such a phosphodiester bond showed a significantly improved Pcsk9 mRNA inhibitory effect compared to its counterpart, cholesterol-conjugated ASO with a phosphorothioate bond, while the hepatic accumulation of both cholesterol-conjugated ASOs was comparable, indicating the effectiveness of removing the conjugated cholesterol for ASO activity. In toxicity analysis, some of the linkers induced lethal toxicities when they were injected at high concentrations (>600μM). These toxicities were attributed to decreased platelet levels in the blood, suggesting an interaction between cholesterol-conjugated ASO and platelets. Our findings may provide a guideline for the design of molecule-conjugated ASOs. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Spectroscopic investigation of photoinduced charge-transfer processes in FTO/TiO2/N719 photoanodes with and without covalent attachment through silane-based linkers. (United States)

    Pandit, Bill; Luitel, Tulashi; Cummins, Dustin R; Thapa, Arjun K; Druffel, Thad; Zamborini, Frank; Liu, Jinjun


    Understanding electron-transfer (ET) processes in dye-sensitized solar cells (DSSCs) is crucial to improving their device performance. Recently, covalent attachment of dye molecules to mesoporous semiconductor nanoparticle films via molecular linkers has been employed to increase the stability of DSSC photoanodes. The power conversion efficiency (PCE) of these DSSCs, however, is lower than DSSCs with conventional unmodified photoanodes in this study. Ultrafast transient absorption pump-probe spectroscopy (TAPPS) has been used to study the electron injection process from N719 dye molecules to TiO2 nanoparticles (NPs) in DSSC photoanodes with and without the presence of two silane-based linker molecules: 3-aminopropyltriethoxysilane (APTES) and p-aminophenyltrimethoxysilane (APhS). Ultrafast biphasic electron injection kinetics were observed in all three photoanodes using a 530 nm pump wavelength and 860 nm probe wavelength. Both the slow and fast decay components, attributed to electron injection from singlet and triplet excited states, respectively, of the N719 dye to the TiO2 conduction band, are hindered by the molecular linkers. The hindering effect is less significant with the APhS linker than the APTES linker and is more significant for the singlet-state channel than the triplet-state one. Electron injection from the vibrationally excited states is less affected by the linkers. The spectroscopic results are interpreted on the basis of the standard ET theory and can be used to guide selection of molecular linkers for DSSCs with better device performance. Other factors that affect the efficiency and stability of the DSSCs are also discussed. The relatively lower PCE of the covalently attached photoanodes is attributed to the multilayer and aggregation of the dye molecules as well as the linkers.

  20. Pairing versus quarteting coherence length

    CERN Document Server

    Delion, Doru S


    We systematically analyse the coherence length in even-even nuclei. The pairing coherence length in the spin-singlet channel for the effective density dependent delta (DDD) and Gaussian interaction is estimated. We consider in our calculations bound states as well as narrow resonances. It turns out that the pairing gaps given by the DDD interaction are similar to those of the Gaussian potential if one renormalizes the radial width to the nuclear radius. The correlations induced by the pairing interaction have in all considered cases a long range character inside the nucleus and decrease towards the surface. The mean coherence length is larger than the geometrical radius for light nuclei and approaches this value for heavy nuclei. The effect of the temperature and states in continuum is investigated. Strong shell effects are evidenced, especially for protons. We generalize this concept to quartets by considering similar relations, but between proton and neutron pairs. The quartet coherence length has a similar...

  1. Role of the external NH2 linker on the conformation of surface immobilized single strand DNA probes and their SERS detection (United States)

    He, Lijie; Langlet, Michel; Stambouli, Valerie


    The conformation and topological properties of DNA single strand probe molecules attached on solid surfaces are important, notably for the performances of devices such as biosensors. Commonly, the DNA probes are tethered to the surface using external linkers such as NH2. In this study, the role and influence of this amino-linker on the immobilization way and conformation of DNA probes on Ag nanoparticle surface is emphasized using Surface Enhanced Raman Spectroscopy (SERS). We compare the SERS spectra and their reproducibility in the case of two groups of DNA polybase probes which are polyA, polyC, polyT, and polyG. In the first group, the polybases exhibit an external NH2 functional linker while in the second group the polybases are NH2-free. The results show that the reproducibility of SERS spectra is enhanced in the case of the first group. It leads us to propose two models of polybase conformation on Ag surface according to the presence or the absence of the external NH2 linker. In the presence of the NH2 external linker, the latter would act as a major anchoring point. As a result, the polybases are much ordered with a less random orientation than in the case of NH2-free polybases. Consequently, in view of further in situ hybridization for biosensing applications, it is strongly recommended to use NH2 linker functionalized DNA probes.

  2. Length Invisibilization of Tachyonic Neutrinos (United States)

    Estakhr, Ahmad Reza


    Faster than the speed of light particle such as tachyonic neutrino due to its superluminal nature disapper and is undetectable. L = iΩ-1Lo where, i =√{ - 1 } is imaginary Number, Ω = 1 /√{βs2 - 1 } is Estakhr's Omega factor, L is the Superluminal Length, Lo is the proper length, βs =Vs / c > 1 is superluminal speed parameter, Vs is Superluminal velocity and c is speed of light.

  3. A specific interdomain interaction preserves the structural and binding properties of the ModA protein from the phytopathogen Xanthomonas citri domain interaction and transport in ModA. (United States)

    Santacruz-Perez, Carolina; Pegos, Vanessa Rodrigues; Honorato, Rodrigo V; Verli, Hugo; Lindahl, Erik; Barbosa, João Alexandre Ribeiro Gonçalves; Balan, Andrea


    The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. Substitution of the Lys linker with the β-Ala linker dramatically decreased the renal uptake of 99mTc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone peptides. (United States)

    Flook, Adam M; Yang, Jianquan; Miao, Yubin


    The purpose of this study was to examine whether the substitution of the Lys linker with the β-Ala could reduce the renal uptake of (99m)Tc-labeled Arg-X-Asp-conjugated and X-Ala-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-β-Ala-(Arg(11))CCMSH (1) {c[Arg-Ser-Asp-dTyr-Asp]-β-Ala-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RTD-β-Ala-(Arg(11))CCMSH (2), RVD-β-Ala-(Arg(11))CCMSH (3), RAD-β-Ala-(Arg(11))CCMSH (4), NAD-β-Ala-(Arg(11))CCMSH (5), and EAD-β-Ala-(Arg(11))CCMSH (6) peptides were synthesized and evaluated for their melanocortin 1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of their (99m)Tc-conjugates were determined in B16/F1 melanoma-bearing C57 mice. The substitution of the Lys linker with β-Ala linker dramatically reduced the renal uptake of all six (99m)Tc-peptides. (99m)Tc-4 exhibited the highest melanoma uptake (15.66 ± 6.19% ID/g) and the lowest kidney uptake (20.18 ± 3.86% ID/g) among these (99m)Tc-peptides at 2 h postinjection. The B16/F1 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using (99m)Tc-4 as an imaging probe.

  5. D-A conjugated polymers containing substituted thiophene, 1,3,4-oxadiazole and non-conjugation linkers: Synthesis and study of optical and electrochemical properties

    Indian Academy of Sciences (India)



    In this communication, we report synthesis and characterization of new D-A conjugated polymers (P1-P3) consisting of electron-donating (D) 3,4-didodecyloxythiophene, electron-accepting (A) 1,3,4- oxadiazole unit and non-conjugation linkers. The conjugated segment in P1-P3 contains only five aromatic rings resulting in short conjugation length, but has an alternate D-A arrangement which significantly enhances the intramolecular charge transfer (ICT) interaction within the segment. As a result, these polymers exhibited lowoptical band gap in the range 2.51–2.76 eV. Fluorescence emission studies revealed that the polymer thin films emit intense blue light with emission maxima in the wavelength rage 430–480 nm. All three polymers undergo both oxidation and reduction processes under electrochemical conditions. Further, these polymers (P1–P3) exhibit low-lying HOMO and LUMO levels as a result of D-A structure of the conjugated segment. Polymer light-emitting devices were fabricated using these polymers as emissive layer with a device configuration of ITO/MoO₃/polymer/LiF/Al. The test device based on P2 emitted blue light with a low threshold voltage of 5 V. Z-scan studies reveal that the polymers exhibit a strong optical limiting behavior. The value of the nonlinear absorption coefficient (β) of polymers is of the order 10⁻¹¹m/W which indicates that these materials may be accomplished for fabricating optical limiters.

  6. Persistence Length of Stable Microtubules (United States)

    Hawkins, Taviare; Mirigian, Matthew; Yasar, M. Selcuk; Ross, Jennifer


    Microtubules are a vital component of the cytoskeleton. As the most rigid of the cytoskeleton filaments, they give shape and support to the cell. They are also essential for intracellular traffic by providing the roadways onto which organelles are transported, and they are required to reorganize during cellular division. To perform its function in the cell, the microtubule must be rigid yet dynamic. We are interested in how the mechanical properties of stable microtubules change over time. Some ``stable'' microtubules of the cell are recycled after days, such as in the axons of neurons or the cilia and flagella. We measured the persistence length of freely fluctuating taxol-stabilized microtubules over the span of a week and analyzed them via Fourier decomposition. As measured on a daily basis, the persistence length is independent of the contour length. Although measured over the span of the week, the accuracy of the measurement and the persistence length varies. We also studied how fluorescently-labeling the microtubule affects the persistence length and observed that a higher labeling ratio corresponded to greater flexibility. National Science Foundation Grant No: 0928540 to JLR.

  7. Linker for activation of T cells contributes to airway inflammation in an asthmatic mouse model

    Institute of Scientific and Technical Information of China (English)

    GUO Xue-jun; REN Lian-ping; SUN Yi-ping; ZHOU Min; XU Wei-guo


    Background Allergic asthma is associated with airway inflammation and hyperresponsiveness caused by dysregulated production of cytokines secreted by allergen-specific helper T-type 2 (Th2) cells. The linker for activation of T cells (LAT)is a membrane-associated adaptor protein, which has been shown to take part in regulating T cell receptor (TCR)signaling and T cell homeostasis. In this study, we established an asthmatic mouse model to examine the changes in LAT levels during allergic airway disease and the effects of LAT transgenic expression on airway inflammation.Methods T ceils from mouse lung tissues were isolated from allergen challenged (ovalbumin (OVA)) and control mice,and the purity of these isolated T cells was examined by fluorescence-activated cell sorter (FACS). Semi-quantitative RT-PCR and Western blotting were used to detect the expression of the LAT gene and LAT protein, respectively. After an intranasally administered mixture of pCMV-HA-LAT plasmid and Lipofectamine 2000, 24 hours before and 72 hours after allergen challenge, the BALF cell count and the differential cytologies were studied. In addition, IL-4 and IFN-γ levels in the BALF were determined by ELISA, and pathological changes in lung tissues were observed.Results LAT protein and mRNA expression were decreased in lung T cells in a mouse model of allergen-induced airway disease. After intranasal administration of pCMV-HA-LAT, histopathological examination of the lungs showed that intervention with LAT overexpression prevented mice from developing airway inflammation, and the number of total cells,eosinophils, neutrophils, and lymphocytes in the BALF was reduced significantly compared with the OVA sensitized and challenged group. In addition, the Th2 cytokine IL-4 decreased, while the Th1 cytokine IFN-Y increased compared to the OVA sensitized and challenged group or the OVA sensitized group plus pCMV-HA treatment.Conclusion This study demonstrates that LAT might effectively diminish Th2

  8. 1D coordination polymers with polychalcogenides as linkers between metal atoms

    Energy Technology Data Exchange (ETDEWEB)

    Kysliak, Oleksandr; Beck, Johannes, E-mail:


    The reactions of zinc metal with elemental selenium and selenium/sulfur mixtures in liquid ammonia or methylamine under solvothermal conditions in closed glass ampoules at 50 °C lead within some days specifically to [Zn(NH{sub 3}){sub 2}Se{sub 4}]{sub n} (1), [Zn(MeNH{sub 2}){sub 2}Se{sub 4}]{sub n} (2), [Zn(NH{sub 3}){sub 2}Se{sub 2.23}S{sub 1.77}]{sub n} (3). From MnCl{sub 2}, Rb{sub 2}Se and excess Se in n-butylamine [Mn({sup n}BuNH{sub 2}){sub 4}Se{sub 6}]{sub n} (4) is obtained after prolonged reaction time at ambient temperature. The compounds are sensitive towards air and loss of NH{sub 3} or the amine ligands. The crystal structures were determined by single crystal diffraction at low temperatures. As a common structural feature, all compounds represent 1D coordination polymers with polychalcogenide chains as linkers between the metal atoms and consist of infinite [M–Ch{sub m}–]{sub n} chains (M=Zn, Mn; Ch{sub m}=Se{sub 4}, (S/Se){sub 4}, Se{sub 6}). The Zn central atoms in 1–3 have tetrahedral coordination with two amine ligands, the Mn atoms in 4 have octahedral coordination with four amine ligands and cis position of the two Se atoms. Raman spectra of 1–3 show the stretching mode vibrations of the Ch{sub 4} groups. The observation of S–S, S–Se, and Se–Se vibration bands in the spectrum of 3 indicates the presence of mixed S/Se polyanions. An optical band gap of 1.86(5) eV was determined for 2 by diffuse reflectance spectroscopy. - Graphical abstract: The reaction of Zn and Se in liquid methylamine yields dark red [Zn(NH{sub 2}CH{sub 3})Se{sub 4}], a 1D coordination polymer consisting of helical Zn–Se{sub 4}–Zn– chains. - Highlights: • A series of 1D coordination polymers consisting of metal amine complexes concatenated by polychalcogenide ions is presented. • Syntheses were performed as solvothermal reactions in liquid ammonia, liquid methylamine and n-butylamine. • Crystal structures are dominated by helices [M–Ch{sub m

  9. IMF Length Scales and Predictability: The Two Length Scale Medium (United States)

    Collier, Michael R.; Szabo, Adam; Slavin, James A.; Lepping, R. P.; Kokubun, S.


    We present preliminary results from a systematic study using simultaneous data from three spacecraft, Wind, IMP 8 (Interplanetary Monitoring Platform) and Geotail to examine interplanetary length scales and their implications on predictability for magnetic field parcels in the typical solar wind. Time periods were selected when the plane formed by the three spacecraft included the GSE (Ground Support Equipment) x-direction so that if the parcel fronts were strictly planar, the two adjacent spacecraft pairs would determine the same phase front angles. After correcting for the motion of the Earth relative to the interplanetary medium and deviations in the solar wind flow from radial, we used differences in the measured front angle between the two spacecraft pairs to determine structure radius of curvature. Results indicate that the typical radius of curvature for these IMF parcels is of the order of 100 R (Sub E). This implies that there are two important IMF (Interplanetary Magnetic Field) scale lengths relevant to predictability: (1) the well-established scale length over which correlations observed by two spacecraft decay along a given IMF parcel, of the order of a few tens of Earth radii and (2) the scale length over which two spacecraft are unlikely to even observe the same parcel because of its curvature, of the order of a hundred Earth radii.

  10. Protein structure prediction guided by cross-linking restraints - A systematic evaluation of the impact of the cross-linking spacer length

    CERN Document Server

    Hofmann, Tommy; Meiler, Jens; Kalkhof, Stefan


    Recent development of high-resolution mass spectrometry (MS) instruments enables chemical cross-linking (XL) to become a high-throughput method for obtaining structural information about proteins. Restraints derived from XL-MS experiments have been used successfully for structure refinement and protein-protein docking. However, one formidable question is under which circumstances XL-MS data might be sufficient to determine a protein's tertiary structure de novo? Answering this question will not only include understanding the impact of XL-MS data on sampling and scoring within a de novo protein structure prediction algorithm, it must also determine an optimal cross-linker type and length for protein structure determination. While a longer cross-linker will yield more restraints, the value of each restraint for protein structure prediction decreases as the restraint is consistent with a larger conformational space. In this study, the number of cross-links and their discriminative power was systematically analyz...

  11. CEBAF Upgrade Bunch Length Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Ahmad, Mahmoud [Old Dominion Univ., Norfolk, VA (United States)


    Many accelerators use short electron bunches and measuring the bunch length is important for efficient operations. CEBAF needs a suitable bunch length because bunches that are too long will result in beam interruption to the halls due to excessive energy spread and beam loss. In this work, bunch length is measured by invasive and non-invasive techniques at different beam energies. Two new measurement techniques have been commissioned; a harmonic cavity showed good results compared to expectations from simulation, and a real time interferometer is commissioned and first checkouts were performed. Three other techniques were used for measurements and comparison purposes without modifying the old procedures. Two of them can be used when the beam is not compressed longitudinally while the other one, the synchrotron light monitor, can be used with compressed or uncompressed beam.

  12. Keeping disease at arm's length

    DEFF Research Database (Denmark)

    Lassen, Aske Juul


    active ageing change everyday life with chronic disease, and how do older people combine an active life with a range of chronic diseases? The participants in the study use activities to keep their diseases at arm’s length, and this distancing of disease at the same time enables them to engage in social...... and physical activities at the activity centre. In this way, keeping disease at arm’s length is analysed as an ambiguous health strategy. The article shows the importance of looking into how active ageing is practised, as active ageing seems to work well in the everyday life of the older people by not giving...... emphasis to disease. The article is based on ethnographic fieldwork and uses vignettes of four participants to show how they each keep diseases at arm’s length....

  13. Spreading lengths of Hermite polynomials

    CERN Document Server

    Sánchez-Moreno, P; Manzano, D; Yáñez, R; 10.1016/


    The Renyi, Shannon and Fisher spreading lengths of the classical or hypergeometric orthogonal polynomials, which are quantifiers of their distribution all over the orthogonality interval, are defined and investigated. These information-theoretic measures of the associated Rakhmanov probability density, which are direct measures of the polynomial spreading in the sense of having the same units as the variable, share interesting properties: invariance under translations and reflections, linear scaling and vanishing in the limit that the variable tends towards a given definite value. The expressions of the Renyi and Fisher lengths for the Hermite polynomials are computed in terms of the polynomial degree. The combinatorial multivariable Bell polynomials, which are shown to characterize the finite power of an arbitrary polynomial, play a relevant role for the computation of these information-theoretic lengths. Indeed these polynomials allow us to design an error-free computing approach for the entropic moments (w...

  14. Continuous lengths of oxide superconductors (United States)

    Kroeger, Donald M.; List, III, Frederick A.


    A layered oxide superconductor prepared by depositing a superconductor precursor powder on a continuous length of a first substrate ribbon. A continuous length of a second substrate ribbon is overlaid on the first substrate ribbon. Sufficient pressure is applied to form a bound layered superconductor precursor powder between the first substrate ribbon and the second substrate ribbon. The layered superconductor precursor is then heat treated to establish the oxide superconducting phase. The layered oxide superconductor has a smooth interface between the substrate and the oxide superconductor.

  15. Overview of bunch length measurements.

    Energy Technology Data Exchange (ETDEWEB)

    Lumpkin, A. H.


    An overview of particle and photon beam bunch length measurements is presented in the context of free-electron laser (FEL) challenges. Particle-beam peak current is a critical factor in obtaining adequate FEL gain for both oscillators and self-amplified spontaneous emission (SASE) devices. Since measurement of charge is a standard measurement, the bunch length becomes the key issue for ultrashort bunches. Both time-domain and frequency-domain techniques are presented in the context of using electromagnetic radiation over eight orders of magnitude in wavelength. In addition, the measurement of microbunching in a micropulse is addressed.

  16. Improving the antitumor activity of squalenoyl-paclitaxel conjugate nanoassemblies by manipulating the linker between paclitaxel and squalene. (United States)

    Caron, Joachim; Maksimenko, Andrei; Wack, Séverine; Lepeltier, Elise; Bourgaux, Claudie; Morvan, Estelle; Leblanc, Karine; Couvreur, Patrick; Desmaële, Didier


    A series of new lipid prodrugs of paclitaxel, which can be formulated as nanoassemblies, are described. These prodrugs which are designed to overcome the limitations due to the systemic toxicity and low water solubility of paclitaxel consist of a squalene chain bound to the 2'-OH of paclitaxel through a 1,4-cis,cis-dienic linker. This design allows the squalene-conjugates to self-assemble as nanoparticular systems while preserving an efficient release of the free drug, thanks to the dienic spacer. The size, steric hindrance, and functional groups of the spacer have been modulated. All these prodrugs self-assemble into nanosized aggregates in aqueous solution as characterized by dynamic light scattering and transmission electron microscopy and appear stable in water for several days as determined by particle size measurement. In vitro biological assessment shows that these squalenoyl-paclitaxel nanoparticles display notable cytotoxicity on several tumor cell lines including A549 lung cell line, colon cell line HT-29, or KB 3.1 nasopharyngeal epidermoid cell line. The cis,cis-squalenyl-deca-5,8-dienoate prodrug show improved activity over simple 2'-squalenoyl-paclitaxel prodrug highlighting the favourable effect of the dienic linker. The antitumor efficacy of the nanoassemblies constructed with the more active prodrugs has been investigated on human lung (A549) carcinoma xenograft model in mice. The prodrug bearing the cis,cis-deca-5,8-dienoyl linker shows comparable antitumor efficacy to the parent drug, but reveals a much lower subacute toxicity as seen in body weight loss. Thus, nanoparticles with the incorporated squalenoyl paclitaxel prodrug may prove useful for replacement of the toxic Cremophor EL.

  17. Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation. (United States)

    Feng, Jing; Yang, Weishan; Xie, Zili; Xiang, Fang; Cao, Zhijian; Li, Wenxin; Hu, Hongzhen; Chen, Zongyun; Wu, Yingliang


    Among the three extracellular domains of the tetrameric voltage-gated K(+) (Kv) channels consisting of six membrane-spanning helical segments named S1-S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2-4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.

  18. Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers. (United States)

    Decher, Niels; Chen, Jun; Sanguinetti, Michael C


    Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have a transmembrane topology that is highly similar to voltage-gated K(+) channels, yet HCN channels open in response to membrane hyperpolarization instead of depolarization. The structural basis for the "inverted" voltage dependence of HCN gating and how voltage sensing by the S1-S4 domains is coupled to the opening of the intracellular gate formed by the S6 domain are unknown. Coupling could arise from interaction between specific residues or entire transmembrane domains. We previously reported that the mutation of specific residues in the S4-S5 linker of HCN2 (i.e. Tyr-331 and Arg-339) prevented normal channel closure presumably by disruption of a crucial interaction with the activation gate. Here we hypothesized that the C-linker, a carboxyl terminus segment that connects S6 to the cyclic nucleotide binding domain, interacts with specific residues of the S4-S5 linker to mediate coupling. The recently solved structure of the C-linker of HCN2 indicates that an alpha-helix (the A'-helix) is located near the end of each S6 domain, the presumed location of the activation gate. Ala-scanning mutagenesis of the end of S6 and the A'-helix identified five residues that were important for normal gating as mutations disrupted channel closure. However, partial deletion of the C-linker indicated that the presence of only two of these residues was required for normal coupling. Further mutation analyses suggested that a specific electrostatic interaction between Arg-339 of the S4-S5 linker and Asp-443 of the C-linker stabilizes the closed state and thus participates in the coupling of voltage sensing and activation gating in HCN channels.

  19. A pull-down method with a biotinylated bait protein prepared by cell-free translation using a puromycin linker. (United States)

    Mochizuki, Yuki; Kohno, Fumiaki; Nishigaki, Koichi; Nemoto, Naoto


    In this paper, we demonstrate a novel pull-down method that dramatically reduces the cost and preparation time of a bait protein by cell-free translation with a puromycin linker. With the C-terminus of the bait protein linked to biotin through a puromycin molecule after the translation reaction and subsequent mRNA degradation by RNase, the prey protein was easily pulled down by streptavidin-coated magnetic beads in a test tube. Three fluorescent prey protein types were tested and confirmed by gel electrophoresis to be pulled down easily and rapidly, depending on their affinity. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Adapting photosynthesis to the near-infrared: non-covalent binding of phycocyanobilin provides an extreme spectral red-shift to phycobilisome core-membrane linker from Synechococcus sp. PCC7335. (United States)

    Miao, Dan; Ding, Wen-Long; Zhao, Bao-Qing; Lu, Lu; Xu, Qian-Zhao; Scheer, Hugo; Zhao, Kai-Hong


    Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to reaction centers via two energy traps, allophycocyanin B and the core-membrane linker, ApcE1 (conventional ApcE). The covalently bound phycocyanobilin (PCB) of ApcE1 absorbs near 660 nm and fluoresces near 675 nm. In cyanobacteria capable of near infrared photoacclimation, such as Synechococcus sp. PCC7335, there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm. We expressed the chromophore domain of the extra core-membrane linker from Synechococcus sp. PCC7335, ApcE2, in E. coli together with enzymes generating the chromophore, PCB. The resulting chromoproteins, PCB-ApcE2(1-273) and the more truncated PCB-ApcE2(24-245), absorb at 700 nm and fluoresce at 714 nm. The red shift of ~40 nm compared with canonical ApcE1 results from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,3(1) double bond is preserved. The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273) and monomeric-ApcE2(24-245) absorbed and fluoresced similarly. Chromophorylation of ApcE2 with phycoerythrobilin- or phytochromobilin resulted in similar red shifts (absorption at 615 and 711 nm, fluorescence at 628 or 726 nm, respectively), compared to the covalently bound chromophores. The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores. Brightly fluorescent biomarking was exemplified in E. coli by single-plasmid transformation.

  1. 3.5A cryoEM structure of hepatitis B virus core assembled from full-length core protein.

    Directory of Open Access Journals (Sweden)

    Xuekui Yu

    Full Text Available The capsid shell of infectious hepatitis B virus (HBV is composed of 240 copies of a single protein called HBV core antigen (HBc. An atomic model of a core assembled from truncated HBc was determined previously by X-ray crystallography. In an attempt to obtain atomic structural information of HBV core in a near native, non-crystalline environment, we reconstructed a 3.5Å-resolution structure of a recombinant core assembled from full-length HBc by cryo electron microscopy (cryoEM and derived an atomic model. The structure shows that the 240 molecules of full-length HBc form a core with two layers. The outer layer, composed of the N-terminal assembly domain, is similar to the crystal structure of the truncated HBc, but has three differences. First, unlike the crystal structure, our cryoEM structure shows no disulfide bond between the Cys61 residues of the two subunits within the dimer building block, indicating such bond is not required for core formation. Second, our cryoEM structure reveals up to four more residues in the linker region (amino acids 140-149. Third, the loops in the cryoEM structures containing this linker region in subunits B and C are oriented differently (~30° and ~90° from their counterparts in the crystal structure. The inner layer, composed of the C-terminal arginine-rich domain (ARD and the ARD-bound RNAs, is partially-ordered and connected with the outer layer through linkers positioned around the two-fold axes. Weak densities emanate from the rims of positively charged channels through the icosahedral three-fold and local three-fold axes. We attribute these densities to the exposed portions of some ARDs, thus explaining ARD's accessibility by proteases and antibodies. Our data supports a role of ARD in mediating communication between inside and outside of the core during HBV maturation and envelopment.

  2. Length Normalization in XML Retrieval

    NARCIS (Netherlands)

    Kamps, Jaap; Rijke, Maarten de; Sigurbjörnsson, Börkur


    The full paper appeared as: J. Kamps, M. de Rijke, and B. Sigurbj¨ornsson, “Length Normalization in XML Retrieval,” In: Proceedings 27th Annual International ACM SIGIR Conference (SIGIR 2004), pages 80-87, 2004.

  3. Finite length Taylor Couette flow (United States)

    Streett, C. L.; Hussaini, M. Y.


    Axisymmetric numerical solutions of the unsteady Navier-Stokes equations for flow between concentric rotating cylinders of finite length are obtained by a spectral collocation method. These representative results pertain to two-cell/one-cell exchange process, and are compared with recent experiments.

  4. 7 Length-weight relationship

    African Journals Online (AJOL)


    from which stomachs were extracted for the analysis of the food contents, using frequency of occurrence, numerical and ... both fish species showed strong correlation between the weight and length with correlation coefficient (r) and ..... Basic data on the assessment of Sphyraena ... review of methods and their application.

  5. Persistence length of dendronized polymers

    NARCIS (Netherlands)

    Mikhailov, I.V.; Darinskii, A.A.; Zhulina, E.B.; Borisov, O.V.; Leermakers, F.A.M.


    We present numerical results for the thermodynamic rigidity and induced persistence length of dendronized polymers with systematically varied topology of their grafts obtained by the Scheutjens-Fleer self-consistent field method. The results were compared to predictions of an analytical mean-fiel

  6. Fractional baud-length coding

    Directory of Open Access Journals (Sweden)

    J. Vierinen


    Full Text Available We present a novel approach for modulating radar transmissions in order to improve target range and Doppler estimation accuracy. This is achieved by using non-uniform baud lengths. With this method it is possible to increase sub-baud range-resolution of phase coded radar measurements while maintaining a narrow transmission bandwidth. We first derive target backscatter amplitude estimation error covariance matrix for arbitrary targets when estimating backscatter in amplitude domain. We define target optimality and discuss different search strategies that can be used to find well performing transmission envelopes. We give several simulated examples of the method showing that fractional baud-length coding results in smaller estimation errors than conventional uniform baud length transmission codes when estimating the target backscatter amplitude at sub-baud range resolution. We also demonstrate the method in practice by analyzing the range resolved power of a low-altitude meteor trail echo that was measured using a fractional baud-length experiment with the EISCAT UHF system.

  7. Cavity length below chute aerators

    Institute of Scientific and Technical Information of China (English)


    It is proved that air entrainment is one of the efficient measures dealing with cavi-tation control for the release works of hydropower projects. There are many factors to be considered in designing a chute aerator. One of the most important factors concerns the cavity length below the aerator,which has outstanding effects on air entrainment against cavitation damage. It is crucial to determine reasonable emergence angle for the calculation of the cavity length. In the present paper the overall effects of structural and hydraulic parameters on the emergence angle of the flow from the aerator were analyzed. Four improved expressions of the emer-gence angle with weight coefficient were investigated through experimental data of 68 points observed from 12 aerators of 6 hydropower projects,of both model and prototype,on the basis of error theory. A method to calculate the cavity length be-low aerators was suggested,which considers overall effects of the above men-tioned parameters. Comparison between the method in this paper and the other five methods of calculating the cavity length showed that the present method is much more reliable than the existing methods while the mean error of the method is less than others.

  8. Cavity length below chute aerators

    Institute of Scientific and Technical Information of China (English)

    WU JianHua; RUAN ShiPing


    It is proved that air entrainment is one of the efficient measures dealing with cavitation control for the release works of hydropower projects. There are many factors to be considered in designing a chute aerator. One of the most important factors concerns the cavity length below the aerator, which has outstanding effects on air entrainment against cavitation damage. It is crucial to determine reasonable emergence angle for the calculation of the cavity length. In the present paper the overall effects of structural and hydraulic parameters on the emergence angle of the flow from the aerator were analyzed. Four improved expressions of the emergence angle with weight coefficient were investigated through experimental data of 68 points observed from 12 aerators of 6 hydropower projects, of both model and prototype, on the basis of error theory. A method to calculate the cavity length below aerators was suggested, which considers overall effects of the above mentioned parameters. Comparison between the method in this paper and the other five methods of calculating the cavity length showed that the present method is much more reliable than the existing methods while the mean error of the method is less than others.

  9. Cyclic Codes of Length 2

    Indian Academy of Sciences (India)

    Manju Pruthi


    In this paper explicit expressions of + 1 idempotents in the ring $R = F_q[X]/\\langle X^{2^m}-1\\rangle$ are given. Cyclic codes of length 2 over the finite field , of odd characteristic, are defined in terms of their generator polynomials. The exact minimum distance and the dimension of the codes are obtained.

  10. Chromatin condensing functions of the linker histone C-terminal domain are mediated by specific amino acid composition and intrinsic protein disorder. (United States)

    Lu, Xu; Hamkalo, Barbara; Parseghian, Missag H; Hansen, Jeffrey C


    Linker histones bind to the nucleosomes and linker DNA of chromatin fibers, causing changes in linker DNA structure and stabilization of higher order folded and oligomeric chromatin structures. Linker histones affect chromatin structure acting primarily through their approximately 100-residue C-terminal domain (CTD). We have previously shown that the ability of the linker histone H1 degrees to alter chromatin structure was localized to two discontinuous 24-/25-residue CTD regions (Lu, X., and Hansen, J. C. (2004) J. Biol. Chem. 279, 8701-8707). To determine the biochemical basis for these results, we have characterized chromatin model systems assembled with endogenous mouse somatic H1 isoforms or recombinant H1 degrees CTD mutants in which the primary sequence has been scrambled, the amino acid composition mutated, or the location of various CTD regions swapped. Our results indicate that specific amino acid composition plays a fundamental role in molecular recognition and function by the H1 CTD. Additionally, these experiments support a new molecular model for CTD function and provide a biochemical basis for the redundancy observed in H1 isoform knockout experiments in vivo.

  11. Wall to membrane linkers, stretch activated channels, and the detection of tension, voltage, temperature, auxin, and pH (United States)

    Pickard, B. G.


    Introduction. The higher plant is a heterogeneous, mechanically prestressed structure continually subject to shifting forces. When a cell grows in a plant at gravitropic equilibrium, it must create localized maxima of shear in walls of neighboring cells. Such mechanical stress and strain are likely detected in a variety of ways. However, tension-sensitive ion channels are of particular interest because it appears that they are elaborately evolved for sensory function. We hypothesize that 1) the patchy patterns of high shear are focused via wall-to-membrane linkers onto the plasma membrane, where 2) they are translated by mechanosensory cation channels into corresponding patterns of high cytosolic Ca2+, which 3) initiate local enhancement of wall expansion. Further, we hypothesize that the local promotion of enhancement is achieved at least in part by local intensification of auxin transport across the plasma membrane. By implication, when an organ is asymmetrically pressed, rubbed, or bent or when it is displaced in the gravitational field, the net asymmetry of shear stress occurring across the organ would lead to asymmetric redistribution of auxin and corrective asymmetric growth. We shall describe a representative mechanosensitive Ca(2+) -selective cation channel (MCaC) with susceptibilities to xenobiotics implicating it as a force transducer in thigmo- and gravitropism. Then, we shall consider whether a putative wall-to-membrane linker (WML) could be a key feature of the molecular architecture permitting the stress distributed in the wall system to be focused on the channels.

  12. Mutations within the S4-S5 linker alter voltage sensor constraints in hERG K+ channels. (United States)

    Van Slyke, Aaron C; Rezazadeh, Saman; Snopkowski, Mischa; Shi, Patrick; Allard, Charlene R; Claydon, Tom W


    Human ether-a-go-go related gene (hERG) channel gating is associated with slow activation, yet the mechanistic basis for this is unclear. Here, we examine the effects of mutation of a unique glycine residue (G546) in the S4-S5 linker on voltage sensor movement and its coupling to pore gating. Substitution of G546 with residues possessing different physicochemical properties shifted activation gating by ∼-50 mV (with the exception of G546C). With the activation shift taken into account, the time constant of activation was also accelerated, suggesting a stabilization of the closed state by ∼1.6-4.3 kcal/mol (the energy equivalent of one to two hydrogen bonds). Predictions of the α-helical content of the S4-S5 linker suggest that the presence of G546 in wild-type hERG provides flexibility to the helix. Deactivation gating was affected differentially by the G546 substitutions. G546V induced a pronounced slow component of closing that was voltage-independent. Fluorescence measurements of voltage sensor movement in G546V revealed a slow component of voltage sensor return that was uncoupled from charge movement, suggesting a direct effect of the mutation on voltage sensor movement. These data suggest that G546 plays a critical role in channel gating and that hERG channel closing involves at least two independently modifiable reconfigurations of the voltage sensor.

  13. An A-T linker adapter polymerase chain reaction method for chromosome walking without restriction site cloning bias. (United States)

    Trinh, Quoclinh; Xu, Wentao; Shi, Hui; Luo, Yunbo; Huang, Kunlun


    A-T linker adapter polymerase chain reaction (PCR) was modified and employed for the isolation of genomic fragments adjacent to a known DNA sequence. The improvements in the method focus on two points. The first is the modification of the PO(4) and NH(2) groups in the adapter to inhibit the self-ligation of the adapter or the generation of nonspecific products. The second improvement is the use of the capacity of rTaq DNA polymerase to add an adenosine overhang at the 3' ends of digested DNA to suppress self-ligation in the digested DNA and simultaneously resolve restriction site clone bias. The combination of modifications in the adapter and in the digested DNA leads to T/A-specific ligation, which enhances the flexibility of this method and makes it feasible to use many different restriction enzymes with a single adapter. This novel A-T linker adapter PCR overcomes the inherent limitations of the original ligation-mediated PCR method such as low specificity and a lack of restriction enzyme choice. Moreover, this method also offers higher amplification efficiency, greater flexibility, and easier manipulation compared with other PCR methods for chromosome walking. Experimental results from 143 Arabidopsis mutants illustrate that this method is reliable and efficient in high-throughput experiments.

  14. One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy (United States)

    Casini, Arturo; MacDonald, James T.; Jonghe, Joachim De; Christodoulou, Georgia; Freemont, Paul S.; Baldwin, Geoff S.; Ellis, Tom


    Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications. PMID:24153110

  15. One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy. (United States)

    Casini, Arturo; MacDonald, James T; De Jonghe, Joachim; Christodoulou, Georgia; Freemont, Paul S; Baldwin, Geoff S; Ellis, Tom


    Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications.

  16. Reversal of HCN channel voltage dependence via bridging of the S4-S5 linker and Post-S6. (United States)

    Prole, David L; Yellen, Gary


    Voltage-gated ion channels possess charged domains that move in response to changes in transmembrane voltage. How this movement is transduced into gating of the channel pore is largely unknown. Here we show directly that two functionally important regions of the spHCN1 pacemaker channel, the S4-S5 linker and the C-linker, come into close proximity during gating. Cross-linking these regions with high-affinity metal bridges or disulfide bridges dramatically alters channel gating in the absence of cAMP; after modification the polarity of voltage dependence is reversed. Instead of being closed at positive voltage and activating with hyperpolarization, modified channels are closed at negative voltage and activate with depolarization. Mechanistically, this reversal of voltage dependence occurs as a result of selectively eliminating channel deactivation, while retaining an existing inactivation process. Bridging also alters channel activation by cAMP, showing that interaction of these two regions can also affect the efficacy of physiological ligands.

  17. The HCN4 channel mutation D553N associated with bradycardia has a C-linker mediated gating defect. (United States)

    Netter, Michael F; Zuzarte, Marylou; Schlichthörl, Günter; Klöcker, Nikolaj; Decher, Niels


    The D553N mutation located in the C-linker of the cardiac pacemaker channel HCN4 is thought to cause sino-atrial dysfunction via a pronounced dominant-negative trafficking defect. Since HCN4 mutations usually have a minor defect in channel gating, it was our aim to further characterize the disease causing mechanism of D553N. Fluorescence microscopy, FACS, TEVC and patch-clamp recordings were performed to characterize D553N. Surprisingly, we found that D553N channels reach the plasma membrane and have no apparent trafficking defect. Co-expression of D553N with HCN4 also revealed no dominant-negative effect on wild-type channels. Consistent with the normal cell surface expression of D553N, it was possible to extensively characterize D553N mutants in Xenopus oocytes and mammalian cells. D553N channels generate currents with reduced amplitude, while the kinetics of activation and deactivation are not altered. While the regulation of D553N by tyrosine kinases is normal, we observed a change in the cAMP regulation which however cannot account for the strong loss-of-function of the mutant. The pronounced current reduction and the regular surface expression indicate a major gating defect of the C-linker gate. We hypothesize that the D553N mutation stabilizes a previously reported salt bridge important for the gating of the channel. Copyright © 2012 S. Karger AG, Basel.

  18. Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence. (United States)

    Gromek, Katarzyna A; Meddaugh, Hannah R; Wrobel, Russell L; Suchy, Fabian P; Bingman, Craig A; Primm, John G; Fox, Brian G


    Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Role of autoantibodies against the linker subdomains of envoplakin and periplakin in the pathogenesis of paraneoplastic pemphigus

    Institute of Scientific and Technical Information of China (English)

    LI Jing; BU Ding-fang; HUANG Yong-chu; ZHU Xue-jun


    Background The presence of autoantibodies against multiple epidermal proteins is an important feature in paraneoplastic pemphigus (PNP). Circulating anti-desmoglein 3 autoantibody, the major pathogenic autoantibody in pemphigus vulgaris (PV), has been proved pathogenic in PNP. Because of many clinical differences between PNP and PV, we speculate about the involvement of other autoantibodies in the pathogenesis of PNP. Envoplakin (EPL) and periplakin (PPL) are recognized by most PNP sere. Their linker subdomains are highly homologous and necessary for the association of intermediate filaments.Methods We characterized the autoantibodies against the linker subdomains of EPL and PPL in PNP patients' sera and their associated tumors by enzyme-linked immunosorbent assay (ELISA) and immunofluorence. We also applied the purified autoantibodies against EPL and PPL from PNP sera to cultured human epidermal keratinocytes (HEK), to evaluate the changes of cell-cell adhesion.Results Autoantibodies against EPL and PPL were detected in most PNP patients by ELISA, and the decrease of these autoantibodies after removal of the tumors was roughly comparable to the improvement of clinical symptoms. Cultured tumor cells from PNP patients secreted these autoantibodies. Specific immunoglobulin receptors for EPL and PPL were found on B lymphocytes in tumors from PNP. Furthermore, purified anti-EPL and anti-PPL autoantibodies from PNP sere were capable of dissociating cultured human epidermal keratinocytes.Conclusion Autoantibodies against EPL and PPL may also be pathogenic in PNP.

  20. Thioredoxin as a fusion tag for carrier-driven crystallization. (United States)

    Corsini, Lorenzo; Hothorn, Michael; Scheffzek, Klaus; Sattler, Michael; Stier, Gunter


    Structural investigations are frequently hindered by difficulties in obtaining diffracting crystals of the target protein. Here, we report the crystallization and structure solution of the U2AF homology motif (UHM) domain of splicing factor Puf60 fused to Escherichia coli thioredoxin A. Both modules make extensive crystallographic contacts, contributing to a well-defined crystal lattice with clear electron density for both the thioredoxin and the Puf60-UHM module. We compare two short linker sequences between the two fusion domains, GSAM and GSPPM, for which only the GSAM-linked fusion protein yielded diffracting crystals. While specific interdomain contacts are not observed for both fusion proteins, NMR relaxation data in solution indicate reduced interdomain mobility between the Trx and Puf60-UHM modules. The GSPPM-linked fusion protein is significantly more flexible, albeit both linker sequences have the same number of degrees of torsional freedom. Our analysis provides a rationale for the crystallization of the GSAM-linked fusion protein and indicates that in this case, a four-residue linker between thioredoxin A and the fused target may represent the maximal length for crystallization purposes. Our data provide an experimental basis for the rational design of linker sequences in carrier-driven crystallization and identify thioredoxin A as a powerful fusion partner that can aid crystallization of difficult targets.

  1. Length of a Hanging Cable

    Directory of Open Access Journals (Sweden)

    Eric Costello


    Full Text Available The shape of a cable hanging under its own weight and uniform horizontal tension between two power poles is a catenary. The catenary is a curve which has an equation defined by a hyperbolic cosine function and a scaling factor. The scaling factor for power cables hanging under their own weight is equal to the horizontal tension on the cable divided by the weight of the cable. Both of these values are unknown for this problem. Newton's method was used to approximate the scaling factor and the arc length function to determine the length of the cable. A script was written using the Python programming language in order to quickly perform several iterations of Newton's method to get a good approximation for the scaling factor.


    Institute of Scientific and Technical Information of China (English)

    Petar Petrov


    For given data (ti,yi), I= 0,1,…,n,0 = t0 <t1 <…<tn = 1we study constrained interpolation problem of Favard type inf{‖f"‖∞|f∈W2∞[0,1],f(ti)=yi,i=0,…,n,l(f;[0,1])≤l0}, wherel(f";[0,1])=∫1 0 / 1+f'2(x)dx is the arc length off in [0,1]. We prove the existence of a solution f* of the above problem, that is a quadratic spline with a second derivative f"* , which coincides with one of the constants - ‖f"*‖∞,0,‖f"*‖∞ between every two consecutive knots. Thus, we extend a result ofKarlin concerning Favard problem, to the case of restricted length interpolation.

  3. Minimal Length, Measurability and Gravity

    Directory of Open Access Journals (Sweden)

    Alexander Shalyt-Margolin


    Full Text Available The present work is a continuation of the previous papers written by the author on the subject. In terms of the measurability (or measurable quantities notion introduced in a minimal length theory, first the consideration is given to a quantum theory in the momentum representation. The same terms are used to consider the Markov gravity model that here illustrates the general approach to studies of gravity in terms of measurable quantities.

  4. Structure of a double-domain phosphagen kinase reveals an asymmetric arrangement of the tandem domains. (United States)

    Wang, Zhiming; Qiao, Zhu; Ye, Sheng; Zhang, Rongguang


    Tandem duplications and fusions of single genes have led to magnificent expansions in the divergence of protein structures and functions over evolutionary timescales. One of the possible results is polydomain enzymes with interdomain cooperativities, few examples of which have been structurally characterized at the full-length level to explore their innate synergistic mechanisms. This work reports the crystal structures of a double-domain phosphagen kinase in both apo and ligand-bound states, revealing a novel asymmetric L-shaped arrangement of the two domains. Unexpectedly, the interdomain connections are not based on a flexible hinge linker but on a rigid secondary-structure element: a long α-helix that tethers the tandem domains in relatively fixed positions. Besides the connective helix, the two domains also contact each other directly and form an interdomain interface in which hydrogen bonds and hydrophobic interactions further stabilize the L-shaped domain arrangement. Molecular-dynamics simulations show that the interface is generally stable, suggesting that the asymmetric domain arrangement crystallographically observed in the present study is not a conformational state simply restrained by crystal-packing forces. It is possible that the asymmetrically arranged tandem domains could provide a structural basis for further studies of the interdomain synergy.

  5. A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function. (United States)

    Kaur, Gurjot; Pinggera, Alexandra; Ortner, Nadine J; Lieb, Andreas; Sinnegger-Brauns, Martina J; Yarov-Yarovoy, Vladimir; Obermair, Gerald J; Flucher, Bernhard E; Striessnig, Jörg


    L-type voltage-gated Ca(2+) channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca(2+) levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation

    DEFF Research Database (Denmark)

    Wang, Zhenxing; D'Annessa, Ilda; Tesauro, Cinzia;


    –DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in Phe. The mutation gives rise to drug resistance in vivo as observed through a viability assay of yeast cells. In vitro activity...... assays show that the mutant is characterized by a fast religation rate, only partially reduced by the presence of the drug. Comparative molecular dynamics simulations of the native and mutant proteins indicate that the mutation of Gly717 affects the motion orientation of the linker domain, changing its...

  7. Toward the Rational Design of Galactosylated Glycoclusters That Target Pseudomonas aeruginosa Lectin A (LecA): Influence of Linker Arms That Lead to Low-Nanomolar Multivalent Ligands. (United States)

    Wang, Shuai; Dupin, Lucie; Noël, Mathieu; Carroux, Cindy J; Renaud, Louis; Géhin, Thomas; Meyer, Albert; Souteyrand, Eliane; Vasseur, Jean-Jacques; Vergoten, Gérard; Chevolot, Yann; Morvan, François; Vidal, Sébastien


    Anti-infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA-targeting glycoclusters have been synthesized. Nine aromatic galactose aglycons were investigated with three different linker arms that connect the central mannopyranoside core. A low-nanomolar (Kd =19 nm, microarray) ligand with a tyrosine-based linker arm could be identified in a structure-activity relationship study. Molecular modeling of the glycoclusters bound to the lectin tetramer was also used to rationalize the binding properties observed.

  8. Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker. (United States)

    Thouta, Samrat; Hull, Christina M; Shi, Yu Patrick; Sergeev, Valentine; Young, James; Cheng, Yen M; Claydon, Thomas W


    Slow deactivation of hERG channels is critical for preventing cardiac arrhythmia yet the mechanistic basis for the slow gating transition is unclear. Here, we characterized the temporal sequence of events leading to voltage sensor stabilization upon membrane depolarization. Progressive increase in step depolarization duration slowed voltage-sensor return in a biphasic manner (τfast = 34 ms, τslow = 2.5 s). The faster phase of voltage-sensor return slowing correlated with the kinetics of pore opening. The slower component occurred over durations that exceeded channel activation and was consistent with voltage sensor relaxation. The S4-S5 linker mutation, G546L, impeded the faster phase of voltage sensor stabilization without attenuating the slower phase, suggesting that the S4-S5 linker is important for communications between the pore gate and the voltage sensor during deactivation. These data also demonstrate that the mechanisms of pore gate-opening-induced and relaxation-induced voltage-sensor stabilization are separable. Deletion of the distal N-terminus (Δ2-135) accelerated off-gating current, but did not influence the relative contribution of either mechanism of stabilization of the voltage sensor. Lastly, we characterized mode-shift behavior in hERG channels, which results from stabilization of activated channel states. The apparent mode-shift depended greatly on recording conditions. By measuring slow activation and deactivation at steady state we found the "true" mode-shift to be ∼15 mV. Interestingly, the "true" mode-shift of gating currents was ∼40 mV, much greater than that of the pore gate. This demonstrates that voltage sensor return is less energetically favorable upon repolarization than pore gate closure. We interpret this to indicate that stabilization of the activated voltage sensor limits the return of hERG channels to rest. The data suggest that this stabilization occurs as a result of reconfiguration of the pore gate upon opening by

  9. Pharmacologically induced erect penile length and stretched penile length are both good predictors of post-inflatable prosthesis penile length. (United States)

    Osterberg, E C; Maganty, A; Ramasamy, R; Eid, J F


    Inflatable penile prosthesis (IPP) remains the gold standard for the surgical treatment of refractory erectile dysfunction; however, current literature to aid surgeons on how best to counsel patients on their postoperative inflated penile length is lacking. The aim of this study was to identify preoperative parameters that could better predict postoperative penile length following insertion of an IPP. Twenty men were enrolled in a prospective study examining penile lengths before and after IPP surgery. Patients with Peyronie's disease were excluded from this analysis. Baseline preoperative characteristics, including body mass index, history of hypertension, diabetes, Sexual Health Inventory for Men scores and/or prior radical prostatectomy were recorded. All patients underwent implantation with a three-piece inflatable Coloplast penile prosthesis. We compared stretched penile length to pharmacologically induced erect lengths. Postoperatively, we measured inflated penile lengths at 6 weeks and assessed patients' perception of penile size at 12 weeks. The median (±interquartile range) stretched penile length and pharmacologically induced erect penile length was 15 (±3) and 14.25 (±2) cm, respectively (P=0.5). Median post-prosthesis penile length (13.5±2.13 cm) was smaller than preoperative pharmacologically induced length (P=0.02) and preoperative stretched penile length (P=0.01). The majority of patients (70%) had a decrease in penile length (median loss 0.5±1.5 cm); however, this loss was perceptible by 43% of men. Stretched penile length and pharmacologically induced erect penile length were equally good predictors of postoperative inflated length (Spearman's correlation 0.8 and 0.9, respectively). Pharmacologically induced erect penile length and stretched penile lengths are equal predictors of post-prosthesis penile length. The majority of men will experience some decrease in penile length following prosthesis implantation; however <50% report a

  10. Testing of Cotton Fiber Length

    Institute of Scientific and Technical Information of China (English)

    刘若华; 李汝勤


    To understand the influences of actual sampling conditions on cotton fiber length testing, this article presents a theoretic study on the distributions and fibrogram of the sample taken out by sampler from ideal sliver at a certain angle. From the distributions expression it can be found that the size of the sampler and the sampling angle are important factors which affect sampling, but if the sampling width is narrow enough, the influence of the sampling angle on the distributions and fibrogram is small enough to be omitted. This is an important conclusion for sampling, in light of this, some suggestions for designing new type sampler are put forward.

  11. Generalizations of Brandl's theorem on Engel length (United States)

    Quek, S. G.; Wong, K. B.; Wong, P. C.


    Let n Engel cycle generated by g and h. The length of the Engel cycle is m-n. A group G is said to have Engel length r, if all the length of the Engel cycles in G divides r. In this paper we discuss the Brandl's theorem on Engel length and give some of its generalizations.

  12. Hydrophobicity, topography in membranes and photosensitization of silicon phthalocyanines with axial ligands of varying lengths. (United States)

    Sholto, Alan; Ehrenberg, Benjamin


    Six amphiphilic silicon phthalocyanines (SiPc's) axially linked to a dimethylated amino alkyl group of varying length have been examined for their potential suitability as photosensitizers for photodynamic therapy (PDT). This group of molecules was chosen because the length of the axial ligand might place the chromophoric part of the molecule at different vertical depths in the membrane and possibly affect the extent of membrane localized damage caused by singlet oxygen. We tested the relative penetration depth of the SiPc groups in the membrane by the extent to which their fluorescence was quenched by external iodide ions. We also measured singlet oxygen quantum yields of the SiPc's in a liposome membrane, using the fluorescent target for singlet oxygen, 9,10-dimethylanthracene. The hydrophobicity parameters, LogP, were calculated and were also measured. Some correlation was found between them and Kb's, the binding constants for liposomes. The effect of the axial ligand's length is less striking than in similar cases with hematoporphyrins and protoporphyrins. We link this smaller effect with a bending of the linker chain that enables, sterically, a better positioning of the sensitizer molecules within the ordered lipid layer structure.

  13. Expression, purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker. (United States)

    Bradley, Luke H; Bricken, Michael L; Randle, Charlotte


    Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: all library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.

  14. BA-BGP:一种基于备份AS通告的域间路由协议%BA-BGP:A New Inter-Domain Routing Protocol Based on Backup AS Advertisement

    Institute of Scientific and Technical Information of China (English)

    胡乔林; 苏金树


    BGP协议通过触发全局、反应式收敛应对网络拓扑和策略变化,然而由于其收敛时间过长、收敛过程中大量AS经历不可达、环路,造成大量转发中断,难以支持VoIP、远程医疗等关键业务的应用.本文提出了一种基于备份AS通告的新型域间路由协议BA-BGP,在不影响BGP协议动态性的基础上,通过在更新报文中增加备份AS属性,使AS节点可以获取到达目标的备份AS地址,当节点面临瞬时失效时可将报文封装转发到与备份AS关联的路径,从而降低了转发中断.大量模拟实验证明,通过采用具有互联网特征的拓扑以及广泛采用的BGP策略,BA-BGP有效降低了链路失效引起的瞬时失效率与转发中断时间,增强了网络可靠性.%The BGP routing protocol triggers global and reactive convergence to dynamically adapt to network topology and policy changes, Nevertheless, the long convergence time, transient disconnectivity and forwarding loop during the routing convergence process lead to forwarding disruption, which makes BGP hard to support the critical business applications such as VolP, interactive tele-surgery.In this paper, We present BA-BGP, a novel inter-domain routing protocol based on backup AS advertisement.BA-BGP does not affect the BGP dynamic behavior, AS node learns the backup AS address associated to destination by adding the Backup AS attribute in the update packets.BA-BGP provides a feasible encapsulation forwarding path associated backup AS when the AS node experience failure, which reduces the forwarding disruption time.Through a detailed evaluation on the Internet characteristic topology and the widely-used BGP policy, we show that BA-BGP improves network survivability effectively,and reduces the transient failure and forwarding disruption time in case of link failure.

  15. MFT2-BGP:基于多转发树的无中断域间路由协议%MFT2-BGP: Achieving Disruption-Free Inter-Domain Routing Protocol Using Multiple Forwarding Trees

    Institute of Scientific and Technical Information of China (English)

    胡乔林; 彭伟; 陈新; 苏金树


    BGP通过触发全局、反应式收敛应对链路或节点失效引起的拓扑变化,然而BGP协议收敛时间长、收敛过程中的瞬时失效严重降低了数据平面转发性能,难以支持关键业务流量.该文提出了容忍失效的MFT2 -BGP,通过利用路径标识符以较低的消息开销构造符合BGP策略的多转发树,使得每个AS获得多样性路径,当出现瞬时失效时,在不改变协议动态性的情况下,允许节点动态切换报文转发路径以实现无中断报文转发,通过嵌入“失效根源信息”以降低收敛时间,抑制瞬时失效以降低路由系统的扰动.通过在Internet-like拓扑上的大量实验表明,在链路失效场景中与其它协议相比,MFT2-BGP能有效改善收敛时间,降低转发中断时间,改善路由系统稳定性.%BGP triggers global and reactive routing convergence to dynamically adapt to network topology and policy changes such as link or node failures. Nevertheless, BGP faces the challenge of long convergence time, transient failure during the routing convergence, which affects the forwarding performance so as to hard to support mission critical traffic. This paper presents the protocol for achieving failure tolerant inter-domain routing protocol named MFT2-BGP, it constructs policy-compliant multiple forwarding trees using path identifier with low message and process overhead, which make every AS discovery diversity path, Every AS can switch traffic to backup path dynamically to achieve disruption-free forwarding without comprising the routing behavior in the presence of transient failure scenario. MFT2-BGP reduces the convergence time by carrying "root cause notification" in update messages and reduces the churn of routing system by suppressing non-necessary routing updates. Detailed experiments on internet-like topology suggest that MFT2-BGP reduces convergence time and forwarding disruption time in link failure scenarios, enhances the routing stability than other

  16. The linker domain of the Ha-Ras hypervariable region regulates interactions with exchange factors, Raf-1 and phosphoinositide 3-kinase. (United States)

    Jaumot, Montserrat; Yan, Jun; Clyde-Smith, Jodi; Sluimer, Judith; Hancock, John F


    Ha-Ras and Ki-Ras have different distributions across plasma membrane microdomains. The Ras C-terminal anchors are primarily responsible for membrane micro-localization, but recent work has shown that the interaction of Ha-Ras with lipid rafts is modulated by GTP loading via a mechanism that requires the hypervariable region (HVR). We have now identified two regions in the HVR linker domain that regulate Ha-Ras raft association. Release of activated Ha-Ras from lipid rafts is blocked by deleting amino acids 173-179 or 166-172. Alanine replacement of amino acids 173-179 but not 166-172 restores wild type micro-localization, indicating that specific N-terminal sequences of the linker domain operate in concert with a more C-terminal spacer domain to regulate Ha-Ras raft association. Mutations in the linker domain that confine activated Ha-RasG12V to lipid rafts abrogate Raf-1, phosphoinositide 3-kinase, and Akt activation and inhibit PC12 cell differentiation. N-Myristoylation also prevents the release of activated Ha-Ras from lipid rafts and inhibits Raf-1 activation. These results demonstrate that the correct modulation of Ha-Ras lateral segregation is critical for downstream signaling. Mutations in the linker domain also suppress the dominant negative phenotype of Ha-RasS17N, indicating that HVR sequences are essential for efficient interaction of Ha-Ras with exchange factors in intact cells.

  17. N- and C-terminal domains determine differential nucleosomal binding geometry and affinity of linker histone isotypes H1(0) and H1c. (United States)

    Vyas, Payal; Brown, David T


    Eukaryotic linker or H1 histones modulate DNA compaction and gene expression in vivo. In mammals, these proteins exist as multiple isotypes with distinct properties, suggesting a functional significance to the heterogeneity. Linker histones typically have a tripartite structure composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain. We hypothesized that the variable terminal domains of individual subtypes contribute to their functional heterogeneity by influencing chromatin binding interactions. We developed a novel dual color fluorescence recovery after photobleaching assay system in which two H1 proteins fused to spectrally separable fluorescent proteins can be co-expressed and their independent binding kinetics simultaneously monitored in a single cell. This approach was combined with domain swap and point mutagenesis to determine the roles of the terminal domains in the differential binding characteristics of the linker histone isotypes, mouse H1(0) and H1c. Exchanging the N-terminal domains between H1(0) and H1c changed their overall binding affinity to that of the other variant. In contrast, switching the C-terminal domains altered the chromatin interaction surface of the globular domain. These results indicate that linker histone subtypes bind to chromatin in an intrinsically specific manner and that the highly variable terminal domains contribute to differences between subtypes. The methods developed in this study will have broad applications in studying dynamic properties of additional histone subtypes and other mobile proteins.

  18. The ortho backbone amide linker (o-BAL) is an easily prepared and highly acid-labile handle for solid-phase synthesis

    DEFF Research Database (Denmark)

    Boas, Ulrik; Brask, Jesper; Christensen, J.B.;


    The tris(alkoxy)benzyl backbone amide linker (BAL) has found widespread application in solid-phase synthesis. The key intermediate for preparation of para BAL (p-BAL) is 2,6-dimethoxy-4-hydroxybenzaldehyde; several reports on its synthesis have appeared. However, the ortho analogue of the handle (o...

  19. Synthesis of highly fluorescent and thio-linkers stabilize gold quantum dots and nano clusters in DMF for bio-labeling

    Energy Technology Data Exchange (ETDEWEB)

    Rastogi, Shiva K., E-mail: [University of Idaho, Department of Chemistry (United States); Denn, Benjamin D.; Branen, A. Larry [University of Idaho, Coeur D' Alene, Biosensors and Nanotechnology Application Laboratory (BNAL) (United States)


    This study demonstrates a one versus two-step synthesis of fluorescent gold quantum dots (F-AuQDs) and nano clusters (F-AuNCs) functionalized with thiolated organic linkers using reduction of gold precursor in N,N Prime -dimethylformamide in 1 h of reaction. The F-AuQDs and F-AuNCs show fluorescence emission at 425 {+-} 5 nm upon excitation at 345 {+-} 5 nm of wavelength, with good water solubility and stability. Five different thiolated organic binary linkers consisting of various functional groups including: carboxylic acid, hydroxyl, and aromatic amine, were conjugated with the F-AuQDs and F-AuNCs. The formation mechanism and functionalization of the F-AuQDs and F-AuNCs was characterized using UV-vis absorption spectra, UV-vis light, fluorescent emission spectra, pH, TEM, and FTIR. The fluorescence emission of the F-AuQDs and F-AuNCs is greatly dependent on the thio-linker. This novel one-step approach provides facile and fast synthesis of F-AuQDs and F-AuNCs over the two-step method, with less than 5 h of reaction and workup compared to more than 28 h of reaction for the two-step approach. These thio-linker functionalized F-AuQDs and F-AuNCs have a wide application in fluorescent labeling of biomolecules, optical devices, imaging, energy transfer, and biosensing.

  20. Synthetic incorporation of Nile Blue into DNA using 2′-deoxyriboside substitutes: Representative comparison of (R- and (S-aminopropanediol as an acyclic linker

    Directory of Open Access Journals (Sweden)

    Daniel Lachmann


    Full Text Available The Nile Blue chromophore was incorporated into oligonucleotides using “click” chemistry for the postsynthetic modification of oligonucleotides. These were synthesized using DNA building block 3 bearing an alkyne group and reacted with the azide 4. (R-3-amino-1,2-propanediol was applied as the linker between the phosphodiester bridges. Two sets of DNA duplexes were prepared. One set carried the chromophore in an A-T environment, the second set in a G-C environment. Both were characterized by optical spectroscopy. Sequence-dependent fluorescence quenching was applied as a sensitive tool to compare the stacking interactions with respect to the chirality of the acyclic linker attachment. The results were compared to recent results from duplexes that carried the Nile Blue label in a sequentially and structurally identical context, except for the opposite chirality of the linker ((S-3-amino-1,2-propandiol. Only minor, negligible differences were observed. Melting temperatures, UV–vis absorption spectra together with fluorescence quenching data indicate that Nile Blue stacks perfectly between the adjacent base pairs regardless of whether it has been attached via an S- or R-configured linker. This result was supported by geometrically optimized DNA models.

  1. Traceless Azido Linker for the Solid-Phase Synthesis of NH-1,2,3-Triazoles via Cu-Catalyzed Azide-Alkyne Cycloaddition Reactions

    DEFF Research Database (Denmark)

    Cohrt, Anders Emil; Jensen, Jakob Feldthusen; Nielsen, Thomas Eiland


    A broadly useful acid-labile traceless azido linker for the solid-phase synthesis of NH-1,2,3-triazoles is presented. A variety of alkynes were efficiently immobilized on a range of polymeric supports by Cu(I)-mediated azide-alkyne cycloadditions. Supported triazoles showed excellent compatibility...

  2. Mutational analysis of dimeric linkers in peri- and cytoplasmic domains of histidine kinase DctB reveals their functional roles in signal transduction. (United States)

    Liu, Jiwei; Yang, Jianguo; Wen, Jin; Yang, Yun; Wei, Xiaolu; Zhang, Xiaodong; Wang, Yi-Ping


    Membrane-associated histidine kinases (HKs) in two-component systems respond to environmental stimuli by autophosphorylation and phospho-transfer. HK typically contains a periplasmic sensor domain that regulates the cytoplasmic kinase domain through a conserved cytoplasmic linker. How signal is transduced from the ligand-binding site across the membrane barrier remains unclear. Here, we analyse two linker regions of a typical HK, DctB. One region connects the first transmembrane helix with the periplasmic Per-ARNT-Sim domains, while the other one connects the second transmembrane helix with the cytoplasmic kinase domains. We identify a leucine residue in the first linker region to be essential for the signal transduction and for maintaining the delicate balance of the dimeric interface, which is key to its activities. We also show that the other linker, belonging to the S-helix coiled-coil family, plays essential roles in signal transduction inside the cell. Furthermore, by combining mutations with opposing activities in the two regions, we show that these two signalling transduction elements are integrated to produce a combined effect on the final activity of DctB.

  3. A Series of Robust Copper-Based Triazolyl Isophthalate MOFs: Impact of Linker Functionalization on Gas Sorption and Catalytic Activity † (United States)

    Junghans, Ulrike; Kobalz, Merten; Erhart, Oliver; Preißler, Hannes; Lincke, Jörg; Möllmer, Jens; Krautscheid, Harald; Gläser, Roger


    The synthesis and characterization of an isomorphous series of copper-containing microporous metal-organic frameworks (MOFs) based on triazolyl isophthalate linkers with the general formula ∞3[Cu4(μ3-OH)2(R1-R2-trz-ia)3(H2O)x] are presented. Through size adjustment of the alkyl substituents R1 and/or R2 at the linker, the impact of linker functionalization on structure-property relationships was studied. Due to the arrangement of the substituents towards the cavities, the porosity (pore fraction 28%–39%), as well as the pore size can be adjusted by the size of the substituents of the triazole ring. Thermal analysis and temperature-dependent PXRD studies reveal a thermal stability of the MOFs up to 230 °C due to increasing framework stability through fine-tuning of the linker substitution pattern. Adsorption of CO2 (298 K) shows a decreasing maximum loading with increasing steric demand of the substituents of the triazole ring. Furthermore, the selective oxidation of cyclohexene with tert-butyl hydroperoxide (TBHP) is studied over the MOFs at 323 K in liquid chloroform. The catalytic activity increases with the steric demand of the substituents. Additionally, these isomorphous MOFs exhibit considerable robustness under oxidizing conditions confirmed by CO2 adsorption studies, as well as by the catalytic selective oxidation experiments. PMID:28772698

  4. Accurate and Sensitive Detection of Plasmodium Species in Humans by Use of the Dihydrofolate Reductase-Thymidylate Synthase Linker Region▿ † (United States)

    Tanomsing, Naowarat; Imwong, Mallika; Theppabutr, Sasikrit; Pukrittayakamee, Sasithon; Day, Nicholas P. J.; White, Nicholas J.; Snounou, Georges


    A nested-PCR protocol based on the linker region of the Plasmodium dihydrofolate reductase-thymidylate synthase gene (dhfr-ts) was developed. This provides highly sensitive specific detection and identification of the five parasite species that infect humans. PMID:20702666

  5. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells

    DEFF Research Database (Denmark)

    Rasch, Morten G; Lund, Ida K.; Illemann, Martin


    . No immunoreactivity was observed when the antibody was probed against skin wound material from MMP-9 deficient mice. In conclusion, we have generated and purified two proteolytically active recombinant murine MMP-9 protein constructs, which are critical reagents for future cancer drug discovery studies....... MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full...

  6. Unique gating properties of C. elegans ClC anion channel splice variants are determined by altered CBS domain conformation and the R-helix linker. (United States)

    Dave, Sonya; Sheehan, Jonathan H; Meiler, Jens; Strange, Kevin


    All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-β-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two α-helices and three β-strands arranged as β1-α1-β2-β3-α2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form α2 of the second CBS domain (CBS2). We demonstrate that interchanging α2 between CLH-3a and CLH-3b interchanges their gating properties. The "R-helix" of ClC proteins forms part of the ion-conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the "R-helix linker". C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that α2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in α2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-like gating. We postulate that the R-helix linker interacts with CBS2 α2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.

  7. Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating. (United States)

    Murayama, Takashi; Kurebayashi, Nagomi; Oba, Toshiharu; Oyamada, Hideto; Oguchi, Katsuji; Sakurai, Takashi; Ogawa, Yasuo


    The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

  8. Unusual and highly tunable missing-linker defects in zirconium metal-organic framework UiO-66 and their important effects on gas adsorption. (United States)

    Wu, Hui; Chua, Yong Shen; Krungleviciute, Vaiva; Tyagi, Madhusudan; Chen, Ping; Yildirim, Taner; Zhou, Wei


    UiO-66 is a highly important prototypical zirconium metal-organic framework (MOF) compound because of its excellent stabilities not typically found in common porous MOFs. In its perfect crystal structure, each Zr metal center is fully coordinated by 12 organic linkers to form a highly connected framework. Using high-resolution neutron power diffraction technique, we found the first direct structural evidence showing that real UiO-66 material contains significant amount of missing-linker defects, an unusual phenomenon for MOFs. The concentration of the missing-linker defects is surprisingly high, ∼10% in our sample, effectively reducing the framework connection from 12 to ∼11. We show that by varying the concentration of the acetic acid modulator and the synthesis time, the linker vacancies can be tuned systematically, leading to dramatically enhanced porosity. We obtained samples with pore volumes ranging from 0.44 to 1.0 cm(3)/g and Brunauer-Emmett-Teller surface areas ranging from 1000 to 1600 m(2)/g, the largest values of which are ∼150% and ∼60% higher than the theoretical values of defect-free UiO-66 crystal, respectively. The linker vacancies also have profound effects on the gas adsorption behaviors of UiO-66, in particular CO2. Finally, comparing the gas adsorption of hydroxylated and dehydroxylated UiO-66, we found that the former performs systematically better than the latter (particularly for CO2) suggesting the beneficial effect of the -OH groups. This finding is of great importance because hydroxylated UiO-66 is the practically more relevant, non-air-sensitive form of this MOF. The preferred gas adsorption on the metal center was confirmed by neutron diffraction measurements, and the gas binding strength enhancement by the -OH group was further supported by our first-principles calculations.

  9. Information, polarization and term length in democracy

    DEFF Research Database (Denmark)

    Schultz, Christian


    This paper considers term lengths in a representative democracy where the political issue divides the population on the left-right scale. Parties are ideologically different and better informed about the consequences of policies than voters are. A short term length makes the government more accou...... the uncertainty is large and parties are not very polarized. Partisan voters always prefer a long term length. When politicians learn while in office a long term length becomes more attractive for swing voters....

  10. 28 CFR 551.4 - Hair length. (United States)


    ... to an inmate hair care services which comply with applicable health and sanitation requirements. ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Hair length. 551.4 Section 551.4 Judicial... Hair length. (a) The Warden may not restrict hair length if the inmate keeps it neat and clean. (b)...

  11. String matching with variable length gaps

    DEFF Research Database (Denmark)

    Bille, Philip; Gørtz, Inge Li; Vildhøj, Hjalte Wedel


    We consider string matching with variable length gaps. Given a string T and a pattern P consisting of strings separated by variable length gaps (arbitrary strings of length in a specified range), the problem is to find all ending positions of substrings in T that match P. This problem is a basic...

  12. Mutations at opposite ends of the DIII/S4-S5 linker of sodium channel NaV1.7 produce distinct pain disorders

    Directory of Open Access Journals (Sweden)

    Tyrrell Lynda


    Full Text Available Abstract Background Two groups of gain-of-function mutations in sodium channel NaV1.7, which are expressed in dorsal root ganglion (DRG neurons, produce two clinically-distinct pain syndromes - inherited erythromelalgia (IEM and paroxysmal extreme pain disorder (PEPD. IEM is characterized by intermittent burning pain and skin redness in the feet or hands, triggered by warmth or mild exercise, while PEPD is characterized by episodes of rectal, ocular and mandibular pain accompanied with skin flushing, triggered by bowel movement and perianal stimulation. Most of the IEM mutations are located within channel domains I and II, while most of the PEPD mutations are located within domains III and IV. The structural dichotomy parallels the biophysical effects of the two types of mutations, with IEM mutations shifting voltage-dependence of NaV1.7 activation in a hyperpolarized direction, and PEPD mutations shifting fast-inactivation of NaV1.7 in a depolarized direction. While four IEM and four PEPD mutations are located within cytoplasmic linkers joining segments 4 and 5 (S4-S5 linkers in the different domains (IEM: domains I and II; PEPD: domains III and IV, no S4-S5 linker has been reported to house both IEM and PEPD mutations thus far. Results We have identified a new IEM mutation P1308L within the C-terminus of the DIII/S4-S5 linker of NaV1.7, ten amino acids from a known PEPD mutation V1298F which is located within the N-terminus of this linker. We used voltage-clamp to compare the biophysical properties of the two mutant channels and current-clamp to study their effects on DRG neuron excitability. We confirm that P1308L and V1298F behave as prototypical IEM and PEPD mutations, respectively. We also show that DRG neurons expressing either P1308L or V1298F become hyperexcitable, compared to DRG neurons expressing wild-type channels. Conclusions Our results provide evidence for differential roles of the DIII/S4-S5 linker N- and C-termini in channel

  13. 基于信任列表的可信第三方跨域认证模型%A Trusted Third Party Inter-Domain Authentication Model Based on Trust Lists

    Institute of Scientific and Technical Information of China (English)

    颜海龙; 喻建平; 胡强; 冯纪强


    目前国内已有31家获得电子认证服务许可的第三方认证机构(certification authority,CA),跨CA的信任和验证日益迫切.针对该问题,提出了一种基于信任列表的可信第三方跨域认证模型.该模型依托公钥基础设施(public key infrastructure,PKI)所提供的数字证书等安全服务,通过引入新的信任机制更好地管理和控制了可信根证书列表,既避免了传统信任列表模型的诸多缺点,又能够有效实现多CA互信互认.为支持这一跨信任域模型的实际运行,设计了相应的工作流程和多CA认证方案,并开发了多CA应用支撑模块,以及就其中的关键技术问题进行了详细论述.分析表明,该模型能够让应用系统灵活地动态兼容不同CA所颁发的数字证书,且在认证效率、安全性、实用性以及应用改造等方面均具有明显优势.%At present, there are thirty-one third-party certification authorities ( CAs) obtaining a license of electronic certification services in China. The trust and verification schemes among CAs are becoming an increasingly urgent problem. In view of this problem, a trusted third party inter-domain authentication model based on trust lists is proposed in this paper. To overcome the shortcomings of traditional trust lists model, the proposed model utilizes digital certificate and other security services provided by public key infrastructure. It is better to manage and control the trusted root certificate lists by using a new trust mechanism. Furthermore, the proposed model can effectively realize multi-CA mutual trust and mutual recognition. For the practical running of the new model, we design its work-flow, multi-CA authentication scheme and multi-CA application supporting system. In addition, some key technology problems are discussed in detail. Analysis shows that the proposed model can facilitate application systems' dynamic compatibility to the digital certificates issued by different

  14. Polydopamine Thin Films as Protein Linker Layer for Sensitive Detection of Interleukin-6 by Surface Plasmon Enhanced Fluorescence Spectroscopy. (United States)

    Toma, Mana; Tawa, Keiko


    Polydopamine (PDA) thin films are introduced to the surface modification of biosensor surfaces utilizing surface plasmon enhanced fluorescence spectroscopy (SPFS) as the linker layer of capture antibody on to the sensor surfaces. The capture antibody can be directly attached to the sensor surface without using any coupling agent by functionalizing the gold sensor surface with PDA thin films. The PDA coating is performed by a single-step preparation process by applying the dopamine solution on the sensor surface, which requires an extremely short incubation time (10 min). The real-time in situ measurement of the adsorption kinetics of the capture antibody onto the PDA-coated sensor surface is studied by surface plasmon resonance (SPR) spectroscopy. It reveals that the immobilization of capture antibody immediately occurs after introduction of a solution containing capture antibody, and the sensor surface is fully covered with the capture antibody. The sensitive detection of the cytokine marker interleukin-6 (IL-6) is performed by SPFS using a sandwich assay format with fluorescently labeled detection antibody. The sensor chips functionalized by PDA chemistry exhibited sensitive sensor responses with low nonspecific adsorption of the detection antibody onto the sensor surface. The detection limit of IL-6 with the developed SPFS biosensor is determined to be 2 pg/mL (100 fM), which is within the range of the diagnostic criteria. Our observation elucidates the remarkable utility of PDA coatings for chemical modification of the metallic sensor surfaces by a simple, brief, and inexpensive manner.

  15. Facile linker free growth of CdS nanoshell on 1-D ZnO: Solar cell application (United States)

    Kamble, Archana; Sinha, Bhavesh; Chung, Kookchae; More, Anup; Vanalakar, Sharad; Hong, Chang Woo; Kim, Jin Hyeok; Patil, Pramod


    One dimensional type-II core/shell heterostructures are widely employed in solar cells because of their adventitious role in both light absorption and charge separation. Here we report a facile two step chemical approach to synthesizing ZnO/CdS core/shell nanorod arrays. ZnO nanorods (ZNR) with a high aspect ratio were grown using a hydrothermal technique where a uniform CdS shell was deposited using a facile, linker free, one pot, Hexamethylenetetramine (HMTA) based reflux technique for the first time. Though the reflux technique is quite similar to the chemical bath deposition technique (CBD), we obtained more uniform CdS coating and improved solar cell performance with the ZnO/CdS heterostructure compared to CBD-grown ZnO/CdS heterostructures. To obtain a conformal coating of CdS, we optimized the CdS deposition time. Formation of pure phase ZnO/CdS core/shell heterostructure was confirmed by high resolution transmission electron microscopy and X-ray photoelectron spectroscopy (XPS) depth analysis. Improved solar cell performance of 1.23% was obtained for ZnO/CdS core/shell structures with ZnS surface treatment. [Figure not available: see fulltext.

  16. Demonstration of Improved Charge Transfer in Graphene/Au Nanorods Plasmonic Hybrids Stabilized by Benzyl Thiol Linkers

    Directory of Open Access Journals (Sweden)

    Giuseppe Valerio Bianco


    Full Text Available Hybrids based on graphene decorated with plasmonic gold (Au nanostructures are being investigated as possible materials combination to add to graphene functionalities of tunable plasmon resonance and enhanced absorption at selected wavelength in the visible-near-infrared region of the spectrum. Here, we report a solution drop-casting approach for fabricating stable hybrids based on chemical vapor deposition (CVD graphene and Au nanorods, which are able to activate effective charge transfer from graphene. We demonstrate that CVD graphene functionalization by benzyl thiol (BZT provides the linker to strong anchoring, via S-Au bonds, Au nanorods to graphene. Optical measurements by spectroscopic ellipsometry give evidence of the introduction of plasmon resonances at 1.85 and 2.25 eV in the Au nanorods/BZT/graphene hybrids, which enable surface enhanced Raman scattering (SERS detection. Furthermore, an effective electron transfer from graphene to Au nanorods, resulting in an enhancement of p-type doping of graphene with a consequent decrease of its sheet resistance, is probed by Raman spectroscopy and corroborated by electrical measurements.

  17. Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity.

    Directory of Open Access Journals (Sweden)

    Wenyan Xie

    Full Text Available Human parainfluenza virus type 3 (HPIV3 can cause severe respiratory tract diseases in infants and young children, but no licensed vaccines or antiviral agents are currently available for treatment. Fusing the viral and target cell membranes is a prerequisite for its entry into host cells and is directly mediated by the fusion (F protein. Although several domains of F are known to have important effects on regulating the membrane fusion activity, the roles of the DI-DII linker (residues 369-374 of the HPIV3 F protein in the fusogenicity still remains ill-defined. To facilitate our understanding of the role of this domain might play in F-induced cell-cell fusion, nine single mutations were engineered into this domain by site-directed mutagenesis. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated F proteins. These mutants were analyzed for membrane fusion activity, cell surface expression, and interaction between F and HN protein. Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F. All of them resulted in a significant reduction in fusogenic activity in all steps of membrane fusion. Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface. Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F.

  18. Introducing RGD peptides on PHBV films through PEG-containing cross-linkers to improve the biocompatibility. (United States)

    Wang, Yan-Yan; Lü, Lan-Xin; Shi, Jun-Cai; Wang, Hai-Feng; Xiao, Zhong-Dang; Huang, Ning-Ping


    Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, has been a good candidate of biomaterial employed in tissue engineering. However, the PHBV film is hydrophobic and has no recognition sites for cell attachment. In this study, PHBV films are activated by ammonia plasma treatment to produce amino groups on the surface, followed by sequential reactions with a heterobifunctional cross-linker containing a segment of poly(ethylene glycol) (PEG) and further with RGD-containing peptides. XPS analyses of modified surfaces after each reaction step reveal that the RGD-containing peptides have been covalently grafted onto PHBV films. The result of cell viability assay indicates that the RGD-modified PHBV films exhibit a distinctly improved cellular compatibility. Moreover, according to the results of serum adsorption tests by optical waveguide lightmode spectroscopy (OWLS) and fibrinogen adsorption tests by enzyme-linked immunosorbent assay (ELISA) on unmodified and modified PHBV surfaces, the introduced PEG chains can significantly decrease the nonspecific adsorption of proteins from serum and fibrinogen from plasma, thus decreasing the risk of thrombus formation and improving the blood compatibility of implanted materials.

  19. Lying-down metallic single-walled carbon nanotubes as efficient linkers for metalloprotein-based nanodevices. (United States)

    Baldacchini, Chiara; Cannistraro, Salvatore


    Metalloproteins recently emerged as good candidates for signal transduction in bionanodevices, but the feasibility of such novel devices is strongly connected to the achievement of an efficient charge transport between single metalloproteins and metal electrodes. In this work, we propose the use of metallic single-walled carbon nanotubes as efficient linkers between metalloproteins and metal surfaces. By means of a conductive atomic force microscopy investigation, we compare the conduction across single yeast cytochrome c molecules covalently bound both to bare gold and to functionalized metallic single-walled carbon nanotubes lying on gold. At comparable forces applied by the microscope tip (i.e., comparable physical contact), the measured current is higher when a metallic single-walled carbon nanotubes is in between the metalloprotein and the gold surface. The analysis of the single molecule current responses by means of a non-resonant tunneling transport model suggests that the increasing in the conduction is due both to the strong electronic conjugation existing at the nanotubes/gold interface and to the participation of the nanotube electronic bands to the charge transport.

  20. Highly porous metal-organic framework containing a novel organosilicon linker--a promising material for hydrogen storage. (United States)

    Wenzel, Stephanie E; Fischer, Michael; Hoffmann, Frank; Fröba, Michael


    The synthesis and characterization of the new metal-organic framework PCN-12-Si (isoreticular to PCN-12) is reported. PCN-12-Si comprises dicopper paddle-wheel units located at the vertices of a cuboctahedron, which are connected by the new linker 5,5'-(dimethylsilanediyl)diisophthalate (dmsdip). The microporous MOF has a high specific surface area of S(BET) = 2430 m(2) g(-1) and a high specific micropore volume of V(p) = 0.93 cm(3) g(-1) (p/p(0) = 0.18). The activated form of PCN-12-Si shows a remarkable hydrogen storage capacity. Volumetric low pressure hydrogen physisorption isotherms at 77 K reveal an uptake of 2.6 wt % H(2) at 1 bar. Furthermore, results of theoretical GCMC simulations of hydrogen adsorption are presented. The simulated low pressure isotherm is in excellent agreement with the experimental one. Simulations for the high pressure regime predict an excess hydrogen uptake of 4.8 wt % at 30 bar, which corresponds to an absolute amount adsorbed of 5.5 wt %. In addition, the potential field of H(2) inside PCN-12-Si was derived from the simulations and analyzed in detail, providing valuable insights concerning the preferred adsorption sites on an atomic scale.

  1. Solid phase synthesis of mitochondrial triphenylphosphonium-vitamin E metabolite using a lysine linker for reversal of oxidative stress.

    Directory of Open Access Journals (Sweden)

    Mohanad Mossalam

    Full Text Available Mitochondrial targeting of antioxidants has been an area of interest due to the mitochondria's role in producing and metabolizing reactive oxygen species. Antioxidants, especially vitamin E (α-tocopherol, have been conjugated to lipophilic cations to increase their mitochondrial targeting. Synthetic vitamin E analogues have also been produced as an alternative to α-tocopherol. In this paper, we investigated the mitochondrial targeting of a vitamin E metabolite, 2,5,7,8-tetramethyl-2-(2'-carboxyethyl-6-hydroxychroman (α-CEHC, which is similar in structure to vitamin E analogues. We report a fast and efficient method to conjugate the water-soluble metabolite, α-CEHC, to triphenylphosphonium cation via a lysine linker using solid phase synthesis. The efficacy of the final product (MitoCEHC to lower oxidative stress was tested in bovine aortic endothelial cells. In addition the ability of MitoCEHC to target the mitochondria was examined in type 2 diabetes db/db mice. The results showed mitochondrial accumulation in vivo and oxidative stress decrease in vitro.

  2. Optimization and characterization of biomolecule immobilization on silicon substrates using (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde linker (United States)

    Gunda, Naga Siva Kumar; Singh, Minashree; Norman, Lana; Kaur, Kamaljit; Mitra, Sushanta K.


    In the present work, we developed and optimized a technique to produce a thin, stable silane layer on silicon substrate in a controlled environment using (3-aminopropyl)triethoxysilane (APTES). The effect of APTES concentration and silanization time on the formation of silane layer is studied using spectroscopic ellipsometry and Fourier transform infrared spectroscopy (FTIR). Biomolecules of interest are immobilized on optimized silane layer formed silicon substrates using glutaraldehyde linker. Surface analytical techniques such as ellipsometry, FTIR, contact angle measurement system, and atomic force microscopy are employed to characterize the bio-chemically modified silicon surfaces at each step of the biomolecule immobilization process. It is observed that a uniform, homogenous and highly dense layer of biomolecules are immobilized with optimized silane layer on the silicon substrate. The developed immobilization method is successfully implemented on different silicon substrates (flat and pillar). Also, different types of biomolecules such as anti-human IgG (rabbit monoclonal to human IgG), Listeria monocytogenes, myoglobin and dengue capture antibodies were successfully immobilized. Further, standard sandwich immunoassay (antibody-antigen-antibody) is employed on respective capture antibody coated silicon substrates. Fluorescence microscopy is used to detect the respective FITC tagged detection antibodies bound to the surface after immunoassay.

  3. Optimization and characterization of biomolecule immobilization on silicon substrates using (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde linker

    Energy Technology Data Exchange (ETDEWEB)

    Gunda, Naga Siva Kumar [Department of Mechanical Engineering, University of Alberta, Edmonton, Canada T6G 2G8 (Canada); Singh, Minashree [Department of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada T6G 1C9 (Canada); Norman, Lana [Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada T6G 2V4 (Canada); Kaur, Kamaljit [Department of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada T6G 1C9 (Canada); Mitra, Sushanta K., E-mail: [Department of Mechanical Engineering, University of Alberta, Edmonton, Canada T6G 2G8 (Canada)


    In the present work, we developed and optimized a technique to produce a thin, stable silane layer on silicon substrate in a controlled environment using (3-aminopropyl)triethoxysilane (APTES). The effect of APTES concentration and silanization time on the formation of silane layer is studied using spectroscopic ellipsometry and Fourier transform infrared spectroscopy (FTIR). Biomolecules of interest are immobilized on optimized silane layer formed silicon substrates using glutaraldehyde linker. Surface analytical techniques such as ellipsometry, FTIR, contact angle measurement system, and atomic force microscopy are employed to characterize the bio-chemically modified silicon surfaces at each step of the biomolecule immobilization process. It is observed that a uniform, homogenous and highly dense layer of biomolecules are immobilized with optimized silane layer on the silicon substrate. The developed immobilization method is successfully implemented on different silicon substrates (flat and pillar). Also, different types of biomolecules such as anti-human IgG (rabbit monoclonal to human IgG), Listeria monocytogenes, myoglobin and dengue capture antibodies were successfully immobilized. Further, standard sandwich immunoassay (antibody–antigen–antibody) is employed on respective capture antibody coated silicon substrates. Fluorescence microscopy is used to detect the respective FITC tagged detection antibodies bound to the surface after immunoassay.

  4. Phosphonate self-assembled monolayers as organic linkers in solid-state quantum dot sensetized solar cells

    KAUST Repository

    Ardalan, Pendar


    We have employed X-ray photoelectron spectroscopy (XPS), ultraviolet-visible (UV-vis) spectroscopy, infrared (IR) spectroscopy, water contact angle (WCA) measurements, ellipsometry, and electrical measurements to study the effects of self-assembled monolayers (SAMs) with phosphonic acid headgroups on the bonding and performance of cadmium sulfide (CdS) solid-state quantum dot sensitized solar cells (QDSSCs). ∼2 to ∼6 nm size CdS quantum dots (QDs) were grown on the SAM-passivated TiO2 surfaces by successive ionic layer adsorption and reaction (SILAR). Our results show differences in the bonding of the CdS QDs at the TiO2 surfaces with a SAM linker. Moreover, our data indicate that presence of a SAM increases the CdS uptake on TiO2 as well as the performance of the resulting devices. Importantly, we observe ∼2 times higher power conversion efficiencies in the devices with a SAM compared to those that lack a SAM. © 2010 IEEE.

  5. Inheritance of telomere length in a bird.

    Directory of Open Access Journals (Sweden)

    Thorsten Horn

    Full Text Available Telomere dynamics are intensively studied in human ageing research and epidemiology, with many correlations reported between telomere length and age-related diseases, cancer and death. While telomere length is influenced by environmental factors there is also good evidence for a strong heritable component. In human, the mode of telomere length inheritance appears to be paternal and telomere length differs between sexes, with females having longer telomeres than males. Genetic factors, e.g. sex chromosomal inactivation, and non-genetic factors, e.g. antioxidant properties of oestrogen, have been suggested as possible explanations for these sex-specific telomere inheritance and telomere length differences. To test the influence of sex chromosomes on telomere length, we investigated inheritance and sex-specificity of telomere length in a bird species, the kakapo (Strigops habroptilus, in which females are the heterogametic sex (ZW and males are the homogametic (ZZ sex. We found that, contrary to findings in humans, telomere length was maternally inherited and also longer in males. These results argue against an effect of sex hormones on telomere length and suggest that factors associated with heterogamy may play a role in telomere inheritance and sex-specific differences in telomere length.

  6. Data on face-to-face contacts in an office building suggests a low-cost vaccination strategy based on community linkers

    CERN Document Server

    Génois, Mathieu; Fournet, Julie; Panisson, André; Bonmarin, Isabelle; Barrat, Alain


    Empirical data on contacts between individuals in social contexts play an important role in the information of models describing human behavior and how epidemics spread in populations. Here, we analyze data on face-to-face contacts collected in an office building. The statistical properties of contacts are similar to other social situations, but important differences are observed in the contact network structure. In particular, the contact network is strongly shaped by the organization of the offices in departments, which has consequences in the design of accurate agent-based models of epidemic spread. We then consider the contact network as a potential substrate for infectious disease spread and show that its sparsity tends to prevent outbreaks of fast-spreading epidemics. Moreover, we define three typical behaviors according to the fraction $f$ of links each individual shares outside its own department: residents, wanderers and linkers. Linkers ($f\\sim 50%$) act as bridges in the network and have large betw...

  7. Highly Porous Zirconium Metal–Organic Frameworks with β-UH 3 -like Topology Based on Elongated Tetrahedral Linkers

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xin; Zhang, Xu; Johnson, Jacob A.; Chen, Yu-Sheng; Zhang, Jian


    Two non-interpenetrated zirconium metal–organic frameworks (Zr-MOFs), NPF-200 and NPF-201, were synthesized via the assembly of elongated tetrahedral linkers with Zr6 and Zr8 clusters. They represent the first examples of MOFs to have the β-UH3-like, 4,12,12T1 topology. Upon activation, NPF-200 exhibits the largest BET surface area (5463 m2 g–1) and void volume (81.6%) among all MOFs formed from tetrahedral ligands. Composed of negative-charged boron-centered tetrahedral linkers, NPF-201 is an anionic Zr-MOF which selectively uptakes photoactive [Ru(bpy)3]2+ for heterogeneous photo-oxidation of thioanisole.

  8. Factors determining the photovoltaic performance of a CdSe quantum dot sensitized solar cell: the role of the linker molecule and of the counter electrode (United States)

    Mora-Seró, Iván; Giménez, Sixto; Moehl, Thomas; Fabregat-Santiago, Francisco; Lana-Villareal, Teresa; Gómez, Roberto; Bisquert, Juan


    Colloidal CdSe quantum dots (QDs) of different sizes, prepared by a solvothermal route, have been employed as sensitizers of nanostructured TiO2 electrode based solar cells. Three different bifunctional linker molecules have been used to attach colloidal QDs to the TiO2 surface: mercaptopropionic acid (MPA), thioglycolic acid (TGA), and cysteine. The linker molecule plays a determinant role in the solar cell performance, as illustrated by the fact that the incident photon to charge carrier generation efficiency (IPCE) could be improved by a factor of 5-6 by using cysteine with respect to MPA. The photovoltaic properties of QD sensitized electrodes have been characterized for both three-electrode and closed two-electrode solar cell configurations. For three-electrode measurement a maximum power conversion efficiency near 1% can be deduced, but this efficiency is halved in the closed cell configuration mainly due to the decrease of the fill factor (FF).

  9. Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development. (United States)

    Zhu, Minghua; Shen, Shudan; Liu, Yan; Granillo, Olivia; Zhang, Weiguo


    It has been proposed that upon T cell activation, linker for activation of T cells (LAT), a transmembrane adaptor protein localized to lipid rafts, orchestrates formation of multiprotein complexes and activates signaling cascades in lipid rafts. However, whether lipid rafts really exist or function remains controversial. To address the importance of lipid rafts in LAT function, we generated a fusion protein to target LAT to nonraft fractions using the transmembrane domain from a nonraft protein, linker for activation of X cells (LAX). Surprisingly, this fusion protein functioned well in TCR signaling. It restored MAPK activation, calcium flux, and NFAT activation in LAT-deficient cells. To further study the function of this fusion protein in vivo, we generated transgenic mice that express this protein. Analysis of these mice indicated that it was fully capable of replacing LAT in thymocyte development and T cell function. Our results demonstrate that LAT localization to lipid rafts is not essential during normal T cell activation and development.

  10. Synthesis and anti-HIV activity of some [Nucleoside Reverse Transcriptase Inhibitor]-C5'-linker-[Integrase Inhibitor] heterodimers as inhibitors of HIV replication. (United States)

    Sugeac, Elena; Fossey, Christine; Ladurée, Daniel; Schmidt, Sylvie; Laumond, Geraldine; Aubertin, Anne-Marie


    Selected for their expected ability to inhibit HIV replication, a series of eight heterodimers containing a Nucleoside Reverse Transcriptase Inhibitor (NRTI) and an Integrase Inhibitor (INI), bound by a linker, were designed and synthesized. For the NRTIs, d4U, d2U and d4T were chosen. For the INIs, 4-[1-(4-fluorobenzyl)-1H-pyrrol-2-yl]-2,4-dioxobutyric acid (6) and 4-(3,5-dibenzyloxyphenyl)-2,4-dioxobutyric acid (9) (belonging to the beta-diketo acids class) were chosen. The conjugation of the two different inhibitors (NRTI and INI) was performed using an amino acid (glycine or beta-alanine) as a cleavable linker.

  11. Influence of Tuned Linker Functionality on Modulation of Magnetic Properties and Relaxation Dynamics in a Family of Six Isotypic Ln2 (Ln = Dy and Gd) Complexes. (United States)

    Mukherjee, Soumya; Lu, Jingjing; Velmurugan, Gunasekaran; Singh, Shweta; Rajaraman, Gopalan; Tang, Jinkui; Ghosh, Sujit K


    A coordination complex family comprising of six new dinuclear symmetric lanthanide complexes, namely, [Ln2(Lx)2(L')2(CH3OH)2]·yG (H2Lx: three related yet distinct Schiff-base linkers; x = 1-3, according to the nomenclature of the Schiff-base linker employed herein. HL': 2,6-dimethoxyphenol. yG refers to crystallographically assigned guest solvent species in the respective complexes; y = number of solvent molecules; Ln(III) = Dy/Gd) were isolated employing a mixed-ligand strategy stemming out of a strategic variation of the functionalities introduced among the constituent Schiff-base linkers. The purposeful introduction of three diverse auxiliary groups with delicate differences in their electrostatic natures affects the local anisotropy and magnetic coupling of Ln(III) ion-environment in the ensuing Ln2 dinuclear complexes, consequentially resulting into distinctly dynamical magnetic behaviors among the investigated new-fangled family of isotypic Ln2 complexes. Among the entire family, subtle alterations in the chemical moieties render two of the Dy2 analogues to behave as single molecule magnets, while the other Dy2 congener merely exhibits slow relaxation of the magnetization. The current observation marks one of the rare paradigms, wherein magnetic behavior modulation was achieved by virtue of the omnipresent influence of subtly tuned linker functionalities among the constituent motifs of the lanthanide nanomagnets. To rationalize the observed difference in the magnetic coupling, density functional theory and ab initio calculations (CASSCF/RASSI-SO/POLY_ANISO) were performed on all six complexes. Subtle difference in the bond angles leads to difference in the J values observed for Gd2 complexes, while difference in the tunnel splitting associated with the structural alterations lead to variation in the magnetization blockade in the Dy2 complexes.

  12. A competing hydrophobic tug on L596 to the membrane core unlatches S4-S5 linker elbow from TRP helix and allows TRPV4 channel to open. (United States)

    Teng, Jinfeng; Loukin, Stephen H; Anishkin, Andriy; Kung, Ching


    We have some generalized physical understanding of how ion channels interact with surrounding lipids but few detailed descriptions on how interactions of particular amino acids with contacting lipids may regulate gating. Here we discovered a structure-specific interaction between an amino acid and inner-leaflet lipid that governs the gating transformations of TRPV4 (transient receptor potential vanilloid type 4). Many cation channels use a S4-S5 linker to transmit stimuli to the gate. At the start of TRPV4's linker helix is leucine 596. A hydrogen bond between the indole of W733 of the TRP helix and the backbone oxygen of L596 secures the helix/linker contact, which acts as a latch maintaining channel closure. The modeled side chain of L596 interacts with the inner lipid leaflet near the polar-nonpolar interface in our model-an interaction that we explored by mutagenesis. We examined the outward currents of TRPV4-expressing Xenopus oocyte upon depolarizations as well as phenotypes of expressing yeast cells. Making this residue less hydrophobic (L596A/G/W/Q/K) reduces open probability [Po; loss-of-function (LOF)], likely due to altered interactions at the polar-nonpolar interface. L596I raises Po [gain-of-function (GOF)], apparently by placing its methyl group further inward and receiving stronger water repulsion. Molecular dynamics simulations showed that the distance between the levels of α-carbons of H-bonded residues L596 and W733 is shortened in the LOFs and lengthened in the GOFs, strengthening or weakening the linker/TRP helix latch, respectively. These results highlight that L596 lipid attraction counteracts the latch bond in a tug-of-war to tune the Po of TRPV4.

  13. Role of glycine residues highly conserved in the S2-S3 linkers of domains I and II of voltage-gated calcium channel alpha(1) subunits. (United States)

    Teng, Jinfeng; Iida, Kazuko; Ito, Masanori; Izumi-Nakaseko, Hiroko; Kojima, Itaru; Adachi-Akahane, Satomi; Iida, Hidetoshi


    The pore-forming component of voltage-gated calcium channels, alpha(1) subunit, contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6). We have shown previously that a Gly residue in the S2-S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2-S3 linkers of domains I and II, which correspond positionally to the Gly in the S2-S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2-S3 linkers of domains I and II of Ca(v)1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Ca(v)1.2s; G182E, G182Q, G579E, G579Q, and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current-voltage relationships of the four mutants were the same as those of wild-type Ca(v)1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly(182) in domain I is involved in the membrane trafficking or surface expression of alpha(1) subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Ca(v)1.2, although G579Q showed a normal VDI, implying that Gly(579) in domain II is involved in the regulation of VDI and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for alpha(1) subunit to become functional.

  14. Ionic Liquid-Derived Imidazolium Cation Linkers for the Layer-by-Layer Assembly of Polyoxometalate-MWCNT Composite Electrodes with High Power Capability. (United States)

    Genovese, Matthew; Lian, Keryn


    Imidazolium cations derived from ionic liquids were demonstrated as effective linker molecules for the layer-by-layer (LbL) deposition of polyoxometalates (POMs) to increase the charge storage of multi-walled carbon nanotube (MWCNT) electrodes. MWCNTs modified with GeMo12O40(4-) (GeMo12) via an imidazolium cation linker demonstrated highly reversible redox reactions and a capacitance of 84 F cm(-3), close to 4 times larger than bare CNT. Compared to CNT-GeMo12 composites fabricated with a conventional polyelectrolyte linker poly(diallyldimethylammonium chloride), (PDDA), the imidazolium cations resulted in lower POM loading, but higher conductivity and in turn superior performance at fast charge-discharge conditions. A polymerized imidazolium linker (PIL) was also synthesized based on the ethyl-vinyl-imidazolium monomer. CNT-GeMo12 composites fabricated with this PIL achieved high POM loading comparable to PDDA, while still maintaining the good conductivity and high rate capabilities shown by the monomer imidazolium units. The high conductivity imparted by the PIL is especially valuable for the fabrication of multilayer POM composites. Dual-layer GeMo12 O40(4-)-SiMo12O40(4-) (GeMo12-SiMo12) electrodes built with this PIL demonstrated a combined contribution of the individual POMs resulting in a capacitance of 191 F cm(-3), over nine times larger than bare MWCNT. The PIL dual layer composites also maintained 72% of this capacitance at a fast rate of 2 V s(-1), compared to just over 50% retention for similar electrodes fabricated with PDDA.

  15. Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design. (United States)

    Digiammarino, Enrico L; Harlan, John E; Walter, Karl A; Ladror, Uri S; Edalji, Rohinton P; Hutchins, Charles W; Lake, Marc R; Greischar, Amy J; Liu, Junjian; Ghayur, Tariq; Jakob, Clarissa G


    The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.

  16. The role of H1 linker histone subtypes in preserving the fidelity of elaboration of mesendodermal and neuroectodermal lineages during embryonic development. (United States)

    Nguyen, Giang D; Gokhan, Solen; Molero, Aldrin E; Yang, Seung-Min; Kim, Byung-Ju; Skoultchi, Arthur I; Mehler, Mark F


    H1 linker histone proteins are essential for the structural and functional integrity of chromatin and for the fidelity of additional epigenetic modifications. Deletion of H1c, H1d and H1e in mice leads to embryonic lethality by mid-gestation with a broad spectrum of developmental alterations. To elucidate the cellular and molecular mechanisms underlying H1 linker histone developmental functions, we analyzed embryonic stem cells (ESCs) depleted of H1c, H1d and H1e subtypes (H1-KO ESCs) by utilizing established ESC differentiation paradigms. Our study revealed that although H1-KO ESCs continued to express core pluripotency genes and the embryonic stem cell markers, alkaline phosphatase and SSEA1, they exhibited enhanced cell death during embryoid body formation and during specification of mesendoderm and neuroectoderm. In addition, we demonstrated deregulation in the developmental programs of cardiomyocyte, hepatic and pancreatic lineage elaboration. Moreover, ectopic neurogenesis and cardiomyogenesis occurred during endoderm-derived pancreatic but not hepatic differentiation. Furthermore, neural differentiation paradigms revealed selective impairments in the specification and maturation of glutamatergic and dopaminergic neurons with accelerated maturation of glial lineages. These impairments were associated with deregulation in the expression profiles of pro-neural genes in dorsal and ventral forebrain-derived neural stem cell species. Taken together, these experimental observations suggest that H1 linker histone proteins are critical for the specification, maturation and fidelity of organ-specific cellular lineages derived from the three cardinal germ layers.

  17. Surface expression and subunit specific control of steady protein levels by the Kv7.2 helix A-B linker.

    Directory of Open Access Journals (Sweden)

    Paloma Aivar

    Full Text Available Kv7.2 and Kv7.3 are the main components of the neuronal voltage-dependent M-current, which is a subthreshold potassium conductance that exerts an important control on neuronal excitability. Despite their predominantly intracellular distribution, these channels must reach the plasma membrane in order to control neuronal activity. Thus, we analyzed the amino acid sequence of Kv7.2 to identify intrinsic signals that may control its surface expression. Removal of the interlinker connecting helix A and helix B of the intracellular C-terminus produces a large increase in the number of functional channels at the plasma membrane. Moreover, elimination of this linker increased the steady-state amount of protein, which was not associated with a decrease of protein degradation. The magnitude of this increase was inversely correlated with the number of helix A - helix B linkers present in the tetrameric channel assemblies. In contrast to the remarkable effect on the amount of Kv7.2 protein, removal of the Kv7.2 linker had no detectable impact on the steady-state levels of Kv7.3 protein.

  18. A new bis-tetraamine ligand with a chromophoric 4-(9-anthracenyl)-2,6-dimethylpyridinyl linker for glyphosate and ATP sensing. (United States)

    Pouessel, Jacky; Abada, Sabah; Le Bris, Nathalie; Elhabiri, Mourad; Charbonnière, Loïc J; Tripier, Raphaël


    The synthesis of a new linear bis-tetraamine ligand L1, based on two 1,4,8,11-tetraazaundecane units grafted at the 2 and 6 positions of a pyridinyl linker substituted by an anthracenyl fluorophore in the para position, is described and anion complexation studies of L1 with anionic substrates are reported. The protonation pattern and the study of the binding properties of L1 in an aqueous medium with two anionic substrates, the nucleotide adenosine triphosphate (ATP) and the herbicide glyphosate (N-(phosphonomethyl)glycine, PMG), were investigated by means of potentiometry, NMR spectroscopy and absorption and emission spectroscopic techniques. To decipher the impact of the chromophoric linker on the complexation process and to highlight its optical properties, a comparison is established with its previously reported analog L2 devoid of the anthracenyl group. The results unambiguously show that the protonation and complexation properties are preserved despite the presence of the bulky linker, allowing for the use of L1 as a fluorescent sensor for ATP and PMG.

  19. A VL-linker-VH Orientation Dependent Single Chain Variable Antibody Fragment Against Rabies Virus G Protein with Enhanced Neutralizing Potency in vivo. (United States)

    Cheng, Yue; Li, Zhuang; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge


    Lethal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragment (scFv), which is composed of a variable heavy chain (VH) and variable light chain (VL) connected by a peptide linker, may be developed as alternative to RIG for neutralizing rabies virus (RV). However, our previously constructed scFv (FV57S) with the (NH2) VH-linker-VL (COOH) orientation showed a lower neutralizing potency than its parent RIG. This orientation may inhibit FV57S from refolding into an intact and correct conformation. Therefore, the RFV57S protein with a VL-linker-VH orientation was constructed based on FV57S. A HIS tag was incorporated to aid in purification and detection of RFV57S and FV57S. However, abilities of RFV57S and FV57S to bind with the anti-HIS tag mAb were different. Therefore, a novel direct ELISA was established by utilizing a biotin-labeled truncated glycoprotein of RV. Although with similar stability and in vitro neutralizing potency as FV57S, RFV57S showed enhanced binding ability, affinity and in vivo protective efficacy against lethal dose of RV. Our studies support the feasibility of developing a scFv with reversed orientation and provide a novel method for evaluating the binding ability, stability and affinity of engineered antibodies recognizing linear epitope.

  20. The Influence of Linker Geometry on Uranyl Complexation by Rigidly-Linked Bis(3-hydroxy-N-methyl-pyridin-2-one)

    Energy Technology Data Exchange (ETDEWEB)

    Szigethy, Geza; Raymond, Kenneth


    A series of bis(3-hydroxy-N-methyl-pyridin-2-one) ligands was synthesized, and their respective uranyl complexes were characterized by single crystal X-ray diffraction analyses. These structures were inspected for high-energy conformations and evaluated using a series of metrics to measure co-planarity of chelating moieties with each other and the uranyl coordination plane, as well as to measure coordinative crowding about the uranyl dication. Both very short (ethyl, 3,4-thiophene and o-phenylene) and very long ({alpha},{alpha}{prime}-m-xylene and 1,8-fluorene) linkers provide optimal ligand geometries about the uranyl cation, resulting in planar, unstrained molecular arrangements. The planarity of the rigid linkers also suggests there is a degree of pre-organization for a planar coordination mode that is ideal for uranyl-selective ligand design. Comparison of intramolecular N{sub amide}-O{sub phenolate} distances and {sup 1}H NMR chemical shifts of amide protons supports earlier results that short linkers provide the optimal geometry for intramolecular hydrogen bonding.

  1. The role of H1 linker histone subtypes in preserving the fidelity of elaboration of mesendodermal and neuroectodermal lineages during embryonic development.

    Directory of Open Access Journals (Sweden)

    Giang D Nguyen

    Full Text Available H1 linker histone proteins are essential for the structural and functional integrity of chromatin and for the fidelity of additional epigenetic modifications. Deletion of H1c, H1d and H1e in mice leads to embryonic lethality by mid-gestation with a broad spectrum of developmental alterations. To elucidate the cellular and molecular mechanisms underlying H1 linker histone developmental functions, we analyzed embryonic stem cells (ESCs depleted of H1c, H1d and H1e subtypes (H1-KO ESCs by utilizing established ESC differentiation paradigms. Our study revealed that although H1-KO ESCs continued to express core pluripotency genes and the embryonic stem cell markers, alkaline phosphatase and SSEA1, they exhibited enhanced cell death during embryoid body formation and during specification of mesendoderm and neuroectoderm. In addition, we demonstrated deregulation in the developmental programs of cardiomyocyte, hepatic and pancreatic lineage elaboration. Moreover, ectopic neurogenesis and cardiomyogenesis occurred during endoderm-derived pancreatic but not hepatic differentiation. Furthermore, neural differentiation paradigms revealed selective impairments in the specification and maturation of glutamatergic and dopaminergic neurons with accelerated maturation of glial lineages. These impairments were associated with deregulation in the expression profiles of pro-neural genes in dorsal and ventral forebrain-derived neural stem cell species. Taken together, these experimental observations suggest that H1 linker histone proteins are critical for the specification, maturation and fidelity of organ-specific cellular lineages derived from the three cardinal germ layers.

  2. Surface expression and subunit specific control of steady protein levels by the Kv7.2 helix A-B linker. (United States)

    Aivar, Paloma; Fernández-Orth, Juncal; Gomis-Perez, Carolina; Alberdi, Araitz; Alaimo, Alessandro; Rodríguez, Manuel S; Giraldez, Teresa; Miranda, Pablo; Areso, Pilar; Villarroel, Alvaro


    Kv7.2 and Kv7.3 are the main components of the neuronal voltage-dependent M-current, which is a subthreshold potassium conductance that exerts an important control on neuronal excitability. Despite their predominantly intracellular distribution, these channels must reach the plasma membrane in order to control neuronal activity. Thus, we analyzed the amino acid sequence of Kv7.2 to identify intrinsic signals that may control its surface expression. Removal of the interlinker connecting helix A and helix B of the intracellular C-terminus produces a large increase in the number of functional channels at the plasma membrane. Moreover, elimination of this linker increased the steady-state amount of protein, which was not associated with a decrease of protein degradation. The magnitude of this increase was inversely correlated with the number of helix A - helix B linkers present in the tetrameric channel assemblies. In contrast to the remarkable effect on the amount of Kv7.2 protein, removal of the Kv7.2 linker had no detectable impact on the steady-state levels of Kv7.3 protein.

  3. Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method. (United States)

    Duhaime, Melissa B; Deng, Li; Poulos, Bonnie T; Sullivan, Matthew B


    Metagenomics generates and tests hypotheses about dynamics and mechanistic drivers in wild populations, yet commonly suffers from insufficient (metagenomics analyses include linear amplification for deep sequencing (LADS), which requires more DNA than is normally available, linker-amplified shotgun libraries (LASLs), which is prohibitively low throughput, and whole-genome amplification, which is significantly biased and thus non-quantitative. Here, we adapt the LASL approach to next generation sequencing by offering an alternate polymerase for challenging samples, developing a more efficient sizing step, integrating a 'reconditioning PCR' step to increase yield and minimize late-cycle PCR artefacts, and empirically documenting the quantitative capability of the optimized method with both laboratory isolate and wild community viral DNA. Our optimized linker amplification method requires as little as 1 pg of DNA and is the most precise and accurate available, with G + C content amplification biases less than 1.5-fold, even for complex samples as diverse as a wild virus community. While optimized here for 454 sequencing, this linker amplification method can be used to prepare metagenomics libraries for sequencing with next-generation platforms, including Illumina and Ion Torrent, the first of which we tested and present data for here.

  4. Single particle electron microscopy analysis of the bovine anion exchanger 1 reveals a flexible linker connecting the cytoplasmic and membrane domains.

    Directory of Open Access Journals (Sweden)

    Jiansen Jiang

    Full Text Available Anion exchanger 1 (AE1 is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO₂ transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO. We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.

  5. Axial Globe Length in Congenital Ptosis. (United States)

    Takahashi, Yasuhiro; Kang, Hyera; Kakizaki, Hirohiko


    To compare axial globe length between affected and unaffected sides in patients with unilateral congenital ptosis. This prospective observational study included 37 patients (age range: 7 months to 58 years). The axial globe length, margin reflex distance-1 (MRD-1), and refractive power were measured. The axial globe length difference was calculated by subtracting the axial globe length on the unaffected side from that of the affected side. The relationships among axial globe length differences, MRD-1 on the affected sides, and patient ages were analyzed using multiple regression analysis. No significant differences were found in the axial globe length between sides (P = .677). The axial globe length difference was 0.17 ± 0.30 mm (mean ± standard deviation), and two patients (5.4%), aged 32 to 57 years, showed axial globe length more than 0.67 mm longer (corresponding to a refractive power of 2 diopters) on the affected side compared to the unaffected side. The multiple regression model between axial globe length difference, patient age, and MRD-1 on the affected sides was less appropriate (YAGL = 0.003XAGE-0.048XMRD-1 +0.112; r = 0.338; adjusted r2 = 0.062; P = .127). The cylindrical power was greater on the affected side (P = .046), although the spherical power was not different between sides (P = .657). No significant difference was identified in the axial globe length between sides, and only 5% of non-pediatric patients showed an axial globe length more than 0.67 mm longer on the affected side. Congenital ptosis may have little effect on axial globe length elongation, and the risk of axial myopia-induced anisometropic amblyopia may be low in patients with unilateral congenital ptosis. Copyright 2015, SLACK Incorporated.

  6. Diabetes mellitus and the eye: axial length


    Huntjens, B.; O’Donnell, C.


    Background and aims: The refractive error of the eye is dependent on its axial length. Refractive error is known to fluctuate significantly in poorly controlled diabetic patients. Recently it has been reported that human eyes fluctuate in axial length during the day. However, this change is not detectable in all subjects, suggesting physiological influences such as diet. The purpose of this study was to investigate fluctuations in axial length and blood glucose levels (BGLs) in diabetic patie...

  7. Fibonacci Lengths of Certain Nilpotent 2-Groups

    Institute of Scientific and Technical Information of China (English)



    In this paper, we study two classes of 2-generated 2-groups of nilpotency class 2 classified by Kluempen in 2002 and also a class of finite 2-groups of high nilpotency class for their Fibonacci lengths.Their involvement in certain interesting sequences of Tribonacci numbers gives us some explicit formulas for the Fibonacci lengths and this adds to the small class of finite groups for which the Fibonacci length are known.

  8. Large Slip Length over a Nanopatterned Surface

    Institute of Scientific and Technical Information of China (English)

    LI Ding; DI Qin-Feng; LI Jing-Yuan; QIAN Yue-Hong; FANG Hai-Ping


    A thermodynamic method is employed to analyse the slip length of hydrophobic nanopatterned surface.The maximal slip lengths with respect to the hydrophobicity of the nanopatterned surface are computed.It is found that the slip length reaches more than 50μm if the nanopatterned surfaces have a contact angle larger than 160°.Such results are expected to find extensive applications in micro-channels and helpful to understand recent experimental observations of the slippage of nanopatterned surfaces.

  9. Dither Cavity Length Controller with Iodine Locking

    Directory of Open Access Journals (Sweden)

    Lawson Marty


    Full Text Available A cavity length controller for a seeded Q-switched frequency doubled Nd:YAG laser is constructed. The cavity length controller uses a piezo-mirror dither voltage to find the optimum length for the seeded cavity. The piezo-mirror dither also dithers the optical frequency of the output pulse. [1]. This dither in optical frequency is then used to lock to an Iodine absorption line.

  10. TCR triggering by pMHC ligands tethered on surfaces via poly(ethylene glycol depends on polymer length.

    Directory of Open Access Journals (Sweden)

    Zhengyu Ma

    Full Text Available Antigen recognition by T cells relies on the interaction between T cell receptor (TCR and peptide-major histocompatibility complex (pMHC at the interface between the T cell and the antigen presenting cell (APC. The pMHC-TCR interaction is two-dimensional (2D, in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol (PEG polymers of different lengths, thereby gradually increasing the ligands' range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.

  11. New length operator for loop quantum gravity

    CERN Document Server

    Ma, Yongge; Yang, Jinsong


    An alternative expression for the length operator in loop quantum gravity is presented. The operator is background-independent, symmetric, positive semi-definite, and well-defined on the kinematical Hilbert space. The expression for the regularized length operator can moreover be understood both from a simple geometrical perspective as the average of a formula relating the length to area, volume and flux operators, and also consistently as the result of direct substitution of the densitized triad operator with the functional derivative operator into the regularized expression of the length. Both these derivations are discussed, and the origin of an undetermined overall factor in each case is also elucidated.

  12. On the attached length of orifices (United States)

    Komkin, A. I.; Mironov, M. A.; Yudin, S. I.


    The attached length of orifices in reactive mufflers has been estimated based on numerical calculations by the finite-element method. The numerical results for a diaphragm in a duct are compared with the theoretical data obtained by Rayleigh, Fock, Karal, and Ingard. The dependence of the attached length on the diaphragm thickness is given. The results obtained are generalized for the case in which the orifice is a Helmholtz resonator neck. The effect of the resonator length on the attached length of the neck is analyzed.

  13. Mouse gestation length is genetically determined.

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    Stephen A Murray

    Full Text Available BACKGROUND: Preterm birth is an enormous public health problem, affecting over 12% of live births and costing over $26 billion in the United States alone. The causes are complex, but twin studies support the role of genetics in determining gestation length. Despite widespread use of the mouse in studies of the genetics of preterm birth, there have been few studies that actually address the precise natural gestation length of the mouse, and to what degree the timing of labor and birth is genetically determined. METHODOLOGY/PRINCIPAL FINDINGS: To further develop the mouse as a genetic model of preterm birth, we developed a high-throughput monitoring system and measured the gestation length in 15 inbred strains. Our results show an unexpectedly wide variation in overall gestation length between strains that approaches two full days, while intra-strain variation is quite low. Although litter size shows a strong inverse correlation with gestation length, genetic difference alone accounts for a significant portion of the variation. In addition, ovarian transplant experiments support a primary role of maternal genetics in the determination of gestation length. Preliminary analysis of gestation length in the C57BL/6J-Chr#(A/J/NaJ chromosome substitution strain (B.A CSS panel suggests complex genetic control of gestation length. CONCLUSIONS/SIGNIFICANCE: Together, these data support the role of genetics in regulating gestation length and present the mouse as an important tool for the discovery of genes governing preterm birth.

  14. Stomach Chitinase from Japanese Sardine Sardinops melanostictus: Purification, Characterization, and Molecular Cloning of Chitinase Isozymes with a Long Linker. (United States)

    Kawashima, Satoshi; Ikehata, Hiroki; Tada, Chihiro; Ogino, Tomohiro; Kakizaki, Hiromi; Ikeda, Mana; Fukushima, Hideto; Matsumiya, Masahiro


    Fish express two different chitinases, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), in the stomach. AFCase-1 and AFCase-2 have different degradation patterns, as fish efficiently degrade chitin ingested as food. For a comparison with the enzymatic properties and the primary structures of chitinase isozymes obtained previously from the stomach of demersal fish, in this study, we purified chitinase isozymes from the stomach of Japanese sardine Sardinops melanostictus, a surface fish that feeds on plankton, characterized the properties of these isozymes, and cloned the cDNAs encoding chitinases. We also predicted 3D structure models using the primary structures of S. melanostictus stomach chitinases. Two chitinase isozymes, SmeChiA (45 kDa) and SmeChiB (56 kDa), were purified from the stomach of S. melanostictus. Moreover, two cDNAs, SmeChi-1 encoding SmeChiA, and SmeChi-2 encoding SmeChiB were cloned. The linker regions of the deduced amino acid sequences of SmeChi-1 and SmeChi-2 (SmeChi-1 and SmeChi-2) are the longest among the fish stomach chitinases. In the cleavage pattern groups toward short substrates and the phylogenetic tree analysis, SmeChi-1 and SmeChi-2 were classified into AFCase-1 and AFCase-2, respectively. SmeChi-1 and SmeChi-2 had catalytic domains that consisted of a TIM-barrel (β/α)₈-fold structure and a deep substrate-binding cleft. This is the first study showing the 3D structure models of fish stomach chitinases.

  15. Whole genome amplification for CGH analysis: Linker-adapter PCR as the method of choice for difficult and limited samples. (United States)

    Pirker, Christine; Raidl, Maria; Steiner, Elisabeth; Elbling, Leonilla; Holzmann, Klaus; Spiegl-Kreinecker, Sabine; Aubele, Michaela; Grasl-Kraupp, Bettina; Marosi, Christine; Micksche, Michael; Berger, Walter


    Comparative genomic hybridization (CGH) is a powerful method to investigate chromosomal imbalances in tumor cells. However, DNA quantity and quality can be limiting factors for successful CGH analysis. The aim of this study was to investigate the applicability of degenerate oligonucleotide-primed PCR (DOP-PCR) and a recently developed linker-adapter-mediated PCR (LA-PCR) for whole genome amplification for use in CGH, especially for difficult source material. We comparatively analyzed DNA of variable quality derived from different cell/tissue types. Additionally, dilution experiments down to the DNA content of a single cell were performed. FISH and/or classical cytogenetic analyses were used as controls. In the case of high quality DNA samples, both methods were equally suitable for CGH. When analyzing very small amounts of these DNA samples (equivalent to one or a few human diploid cells), DOP-PCR-CGH, but not LA-PCR-CGH, frequently produced false-positive signals (e.g., gains in 1p and 16p, and losses in chromosome 4q). In case of formalin-fixed paraffin-embedded tissues, success rates by LA-PCR-CGH were significantly higher as compared to DOP-PCR-CGH. DNA of minor quality frequently could be analyzed correctly by LA-PCR-CGH, but was prone to give false-positive and/or false-negative results by DOP-PCR-CGH. LA-PCR is superior to DOP-PCR for amplification of DNA for CGH analysis, especially in the case of very limited or partly degraded source material. Copyright 2004 Wiley-Liss, Inc

  16. Preparation and in vivo evaluation of a novel stabilized linker for {sup 211}At labeling of protein

    Energy Technology Data Exchange (ETDEWEB)

    Talanov, Vladimir S. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)]. E-mail:; Garmestani, Kayhan [Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Regino, Celeste A.S. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Milenic, Diane E. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Plascjak, Paul S. [Department of Nuclear Medicine, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Waldmann, Thomas A. [Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Brechbiel, Martin W. [Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)]. E-mail:


    Significant improvement of in vivo stability of {sup 211}At-labeled radioimmunoconjugates achieved upon employment of a recently reported new linker, succinimidyl N-2-(4-[{sup 211}At]astatophenethyl)succinamate (SAPS), prompted additional studies of its chemistry. The {sup 211}At radiolabeling of succinimidyl N-2-(4-tributylstannylphenethyl)succinamate (1) was noted to decline after storage at -15{sup o}C for greater than 6 months. Compound 1 was found to degrade via a ring closure reaction with the formation of N-2-(4-tributylstannylphenethyl)succinimide (3), and a modified procedure for the preparation of 1 was developed. The N-methyl structural analog of 1, succinimidyl N-2-(4-tributylstannylphenethyl)-N-methyl succinamate (SPEMS), was synthesized to investigate the possibility of improving the stability of reagent-protein linkage chemistry. Radiolabeling of SPEMS with {sup 211}At generates succinimidyl N-2-(4-[{sup 211}At]astatophenethyl)-N-methyl succinamate (Methyl-SAPS), with yields being consistent for greater than 1 year. Radiolabelings of 1 and SPEMS with {sup 125}I generated succinimidyl N-2-(4-[{sup 125}I]iodophenethyl)succinamate (SIPS) and succinimidyl N-2-(4-[{sup 125}I]iodophenethyl)-N-methyl succinamate (Methyl-SIPS), respectively, and showed no decline in yields. Methyl-SAPS, SAPS, Methyl-SIPS and SIPS were conjugated to Herceptin for a comparative assessment in LS-174T xenograft-bearing mice. The conjugates of Herceptin with Methyl-SAPS or Methyl-SIPS demonstrated immunoreactivity equivalent to if not superior to the SAPS and SIPS paired analogs. The in vivo studies also revealed that the N-methyl modification resulted in a superior statinated product.

  17. Large scale load of phosphotungstic acid on multiwalled carbon nanotubes with a grafted poly(4-vinylpyridine) linker

    Energy Technology Data Exchange (ETDEWEB)

    Yuan Junhua, E-mail: [College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004 (China); Jin Xiaoli; Li Na; Chen Jianrong; Miao Jigen [College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004 (China); Zhang Qixian [State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Niu Li, E-mail: [State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China); Song Jixia [Jilin City Institute of Testing on Product Quality, Jilin 132013 (China)


    Highlights: > Carbon nanotubes was grafted with poly(4-vinylpyridine). > Phosphotungstic acid was deposited onto carbon nanotubes with this polymer linker. > This deposition is highly uniform and large scale. > These phosphotungstic acid/carbon nanotubes hybrids can be used as nitrite sensor. > Its linear concentration is 1.2 to 17.5 {mu}M with a detection limit of 0.2 {mu}M. - Abstract: Multiwalled carbon nanotubes (MWCNTs) were grafted with poly(4-vinylpyridine) (PV4P) in aqueous solution by in situ free radical polymerization of 4-vinylpyridine. The as-prepared PV4P-g-MWCNTs hybrids can load phosphotungstic acid (PW) on a large scale by electrostatic interaction, which was characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The as-prepared PWs/PV4P-g-MWCNTs hybrids were modified onto a carbon glassy electrode. Cyclic voltammograms (CVs) show that the electrochemical behavior of the modified electrode follows a four-one-electron surface-confined process of Keggin-type PWs. The modified electrode can be used as a nitrite sensor. The comparison of CVs shows that the response current of nitrite reduction at the electrode modified with PWs/PV4P-g-MWCNTs hybrids is 15 times higher than that of PWs/MWCNTs hybrids in a control experiment at 0.65 V (vs. AgCl). The amperometric i-t curve for the electrode modified with PWs/PV4P-g-MWCNTs hybrids exhibits a linear concentration of NO{sub 2}{sup -} ranged from 1.2 to 17.5 {mu}M with a detection limit of 0.2 {mu}M.

  18. Studies on synthesis and activation mechanism of mitomycin dimers connected by 1,2-dithiolane and diol linkers. (United States)

    Kim, Hyoung Rae; Kim, Jae Jin; Park, Jung Jae; Lee, Sang Hyup


    We report the synthetic and mechanistic studies on a new cyclic disulfide mitomycin dimer, 7-N,7'-N'-(1″,2″-dithiolanyl-3″,5″-dimethylenyl)bismitomycin C (8), and a diol mitomycin dimer, 7-N,7'-N'-(2″,4″-dihydroxy-1″,5″-pentanediyl)bismitomycin C (9). Mitomycin 8 is a dimer connected by a 1,2-dithiolane (a five-membered cyclic disulfide) linker, and was specifically designed to undergo nucleophilic activation and double DNA alkylations leading to efficient production of DNA interstrand cross-link (DNA ISC) adducts. Disulfide cleavage in 8 would generate two thiol groups that could serve as probes to activate two mitomycin rings. At first, the target mitomycin 8 was synthesized using mitomycin A (1) and the key intermediate, cyclic disulfide (10), which was prepared through a seven-step synthetic sequence. Diol mitomycin 9 was also synthesized from 1 and diamine salt 13. Next, kinetic studies using solvolysis reaction revealed that the activation rates of 8 were much higher than those of 9 and mitomycin C (2) under nucleophilic conditions provided by Et(3)P presumably due to the presence of a cyclic disulfide unit in 8. These findings led us to propose a nucleophilic activation pathway for 8. Then, DNA ISC experiments further revealed that the levels of DNA ISC caused by 8 in the presence of Et(3)P were much higher (97%) than those by 9 (5%) and 2 (4%). More importantly, mitomycin 8 underwent much faster activation and produced slightly higher levels of DNA ISC than the previously reported mitomycins 5-7. Overall, we concluded that 8 was highly efficient for both nucleophilic activation and corresponding DNA ISC formation, and that this differentiation came from the crucial function of the cyclic disulfide unit in 8.

  19. Interactions between S4-S5 linker and S6 transmembrane domain modulate gating of HERG K+ channels. (United States)

    Tristani-Firouzi, Martin; Chen, Jun; Sanguinetti, Michael C


    Outward movement of the voltage sensor is coupled to activation in voltage-gated ion channels; however, the precise mechanism and structural basis of this gating event are poorly understood. Potential insight into the coupling mechanism was provided by our previous finding that mutation to Lys of a single residue (Asp(540)) located in the S4-S5 linker endowed HERG (human ether-a-go-go-related gene) K(+) channels with the unusual ability to open in response to membrane depolarization and hyperpolarization in a voltage-dependent manner. We hypothesized that the unusual hyperpolarization-induced gating occurred through an interaction between Lys(540) and the C-terminal end of the S6 domain, the region proposed to form the activation gate. Therefore, we mutated six residues located in this region of S6 (Ile(662)-Tyr(667)) to Ala in D540K HERG channels. Mutation of Arg(665), but not the other five residues, prevented hyperpolarization-dependent reopening of D540K HERG channels. Mutation of Arg(665) to Gln or Asp also prevented reopening. In addition, D540R and D540K/R665K HERG reopened in response to hyperpolarization. Together these findings suggest that a single residue (Arg(665)) in the S6 domain interacts with Lys(540) by electrostatic repulsion to couple voltage sensing to hyperpolarization-dependent opening of D540K HERG K(+) channels. Moreover, our findings suggest that the C-terminal ends of S4 and S6 are in close proximity at hyperpolarized membrane potentials.

  20. A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted uncoupler and fluorescent neuro- and nephroprotector. (United States)

    Antonenko, Yuri N; Denisov, Stepan S; Silachev, Denis N; Khailova, Ljudmila S; Jankauskas, Stanislovas S; Rokitskaya, Tatyana I; Danilina, Tatyana I; Kotova, Elena A; Korshunova, Galina A; Plotnikov, Egor Y; Zorov, Dmitry B


    Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. Linking a triphenyl-phosphonium cation to fluorescein through a decyl (C10) spacer yields a fluorescent uncoupler, coined mitoFluo, that selectively accumulates in energized mitochondria (Denisov et al., Chem.Commun. 2014). Proton-transport activity of mitoFluo was tested in liposomes reconstituted with bacteriorhodopsin. To examine the uncoupling action on mitochondria, we monitored mitochondrial membrane potential in parallel with oxygen consumption. Neuro- and nephroprotecting activity was detected by a limb-placing test and a kidney ischemia/reperfusion protocol, respectively. We compared mitoFluo properties with those of its newly synthesized analog having a short (butyl) spacer (C4-mitoFluo). MitoFluo, but not C4-mitoFluo, caused collapse of mitochondrial membrane potential resulting in stimulation of mitochondrial respiration. The dramatic difference in the uncoupling activity of mitoFluo and C4-mitoFluo was in line with the difference in their protonophoric activity on a lipid membrane. The accumulation of mitoFluo in mitochondria was more pronounced than that of C4-mitoFluo. MitoFluo decreased the rate of ROS production in mitochondria. MitoFluo was effective in preventing consequences of brain trauma in rats: it suppressed trauma-induced brain swelling and reduced a neurological deficit. Besides, mitoFluo attenuated acute kidney injury after ischemia/reperfusion in rats. A long alkyl linker was proved mandatory for mitoFluo to be a mitochondria- targeted uncoupler. MitoFluo showed high protective efficacy in certain models of oxidative stress-related diseases. MitoFluo is a candidate for developing therapeutic and fluorescence imaging agents to treat brain and kidney pathologies. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Influence of bonding mode of the linkers in the electronic communication of molecular pairs having dimolybdenum units linked by pseudohalides. (United States)

    Cotton, F Albert; Murillo, Carlos A; Zhao, Qinliang


    Depending on conditions the reactions of [Mo(2)(cis-DAniF)(2)(NCCH(3))(4)](BF(4))(2) (DAniF = N,N'-di-p-anisylformamidinate) with solutions containing thiocyanate anions lead to two compounds: a quadruple-bonded dinuclear species 1, (Bu(n)(4)N)(2)[Mo(2)(cis-DAniF)(2)(NCS)(4)], and a molecular pair 3, [Mo(2)(cis-DAniF)(2)](2)(mu(1,3)-NCS)(4). The latter has a cuboidal structure having two (cis-DAniF)(2)Mo(2)(2+) units, [Mo(2)], with four thiocyanate groups bridging two [Mo(2)] units in an end-to-end fashion in which the N and S atoms serve as the bridging units. On the contrary, the structure of the cyanate isomer, [Mo(2)(cis-DAniF)(2)](2)(mu(1,1)-NCO)(4) (2), shows an end-on binding of the cyanate linkers. Various physical measurements of 2 and its oxidized species 2.PF(6) indicate that there is strong electronic communication between the two dimetal cores. For 1, two reversible oxidation processes were observed in the cyclic voltammogram corresponding to the successive oxidation to 1(+) and an uncommon 1(2+) species that has a triple-bonded Mo(2)(6+) core. DFT calculations indicate that the antibonding character between the Mo-Mo delta orbitals and thiocyanate p orbitals plays a very important role in elevating the HOMO delta orbital energy that allows formation of the dication. A selenium isomer of 3 was also studied. In both the thiocyanate and selenocyanate bridged dimers of dimers, in which the pseudohalide bridges bind the two dimetal units in an end-to-end fashion, long separations between the dimetal units are observed, and these generate very weak electronic interactions.

  2. 23 CFR 658.13 - Length. (United States)


    ... power unit behind the cab and on an over-cab rack. No State shall impose an overall length limitation of... may carry boats on the power unit so long as the length and width restrictions of the vehicles and...) in 23 CFR 658.19. (6) Munitions carriers using dromedary equipment. A truck tractor equipped with...

  3. Length-biased Weighted Maxwell Distribution

    Directory of Open Access Journals (Sweden)

    Kanak Modi


    Full Text Available The concept of length-biased distribution can be employed in development of proper models for life-time data. In this paper, we develop the length-biased form of Weighted Maxwell distribution (WMD. We study the statistical properties of the derived distribution including moments, moment generating function, hazard rate, reverse hazard rate, Shannon entropy and estimation of parameters

  4. Segment lengths influence hill walking strategies. (United States)

    Sheehan, Riley C; Gottschall, Jinger S


    Segment lengths are known to influence walking kinematics and muscle activity patterns. During level walking at the same speed, taller individuals take longer, slower strides than shorter individuals. Based on this, we sought to determine if segment lengths also influenced hill walking strategies. We hypothesized that individuals with longer segments would display more joint flexion going uphill and more extension going downhill as well as greater lateral gastrocnemius and vastus lateralis activity in both directions. Twenty young adults of varying heights (below 155 cm to above 188 cm) walked at 1.25 m/s on a level treadmill as well as 6° and 12° up and downhill slopes while we collected kinematic and muscle activity data. Subsequently, we ran linear regressions for each of the variables with height, leg, thigh, and shank length. Despite our population having twice the anthropometric variability, the level and hill walking patterns matched closely with previous studies. While there were significant differences between level and hill walking, there were few hill walking variables that were correlated with segment length. In support of our hypothesis, taller individuals had greater knee and ankle flexion during uphill walking. However, the majority of the correlations were between tibialis anterior and lateral gastrocnemius activities and shank length. Contrary to our hypothesis, relative step length and muscle activity decreased with segment length, specifically shank length. In summary, it appears that individuals with shorter segments require greater propulsion and toe clearance during uphill walking as well as greater braking and stability during downhill walking.

  5. Influence of mandibular length on mouth opening

    NARCIS (Netherlands)

    Dijkstra, PU; Hof, AL; Stegenga, B; De Bont, LGM


    Theoretically, mouth opening not only reflects the mobility of the temporomandibular joints (TMJs) but also the mandibular length. Clinically, the exact relationship between mouth opening, mandibular length, and mobility of TMJs is unclear. To study this relationship 91 healthy subjects, 59 women an

  6. Extending electronic length frequency analysis in R

    DEFF Research Database (Denmark)

    Taylor, M. H.; Mildenberger, Tobias K.


    Electronic length frequency analysis (ELEFAN) is a system of stock assessment methods using length-frequency (LFQ) data. One step is the estimation of growth from the progression of LFQ modes through time using the von Bertalanffy growth function (VBGF). The option to fit a seasonally oscillating...

  7. Leaf Length Variation in Perennial Forage Grasses

    Directory of Open Access Journals (Sweden)

    Philippe Barre


    Full Text Available Leaf length is a key factor in the economic value of different grass species and cultivars in forage production. It is also important for the survival of individual plants within a sward. The objective of this paper is to discuss the basis of within-species variation in leaf length. Selection for leaf length has been highly efficient, with moderate to high narrow sense heritability. Nevertheless, the genetic regulation of leaf length is complex because it involves many genes with small individual effects. This could explain the low stability of QTL found in different studies. Leaf length has a strong response to environmental conditions. However, when significant genotype × environment interactions have been identified, their effects have been smaller than the main effects. Recent modelling-based research suggests that many of the reported environmental effects on leaf length and genotype × environment interactions could be biased. Indeed, it has been shown that leaf length is an emergent property strongly affected by the architectural state of the plant during significant periods prior to leaf emergence. This approach could lead to improved understanding of the factors affecting leaf length, as well as better estimates of the main genetic effects.

  8. Analysis of ureteral length in adult cadavers

    Directory of Open Access Journals (Sweden)

    Hugo F. F. Novaes


    Full Text Available Introduction In some occasions, correlations between human structures can help planning surgical intra-abdominal interventions. The previous determination of ureteral length helps pre-operatory planning of surgeries, reduces costs of auxiliary exams, the correct choice of double-J catheter with low morbidity and fewer symptoms, and an adequate adhesion to treatment. Objective To evaluate ureteral length in adult cadavers and to analyze its correlation with anthropometric measures. Materials and Methods: From April 2009 to January 2012 we determined ureteral length of adult cadavers submitted to necropsy and obtained the following measures: height, distance from shoulder to wrist, elbow-wrist, xiphoid appendix-umbilicus, umbilicus-pubis, xiphoid appendix-pubis and between iliac spines. We analyzed the correlations between ureteral length and those anthropometric measures. Results We dissected 115 ureters from 115 adult corpses from April 2009 to January 2012. Median ureteral length didn't vary between sexes or according to height. It was observed no correlation among ureteral length and all considered anthropometric measures in all analyzed subgroups and in general population. There were no significant differences between right and left ureteral measures. Conclusions There is no difference of ureteral length in relation to height or gender (male or female. There is no significant correlation among ureteral length and the considered anthropometric measures.

  9. 7 CFR 29.6024 - Length. (United States)


    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Length. 29.6024 Section 29.6024 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... INSPECTION Standards Definitions § 29.6024 Length. The linear measurement of cured tobacco leaves from...

  10. Length contraction in Very Special Relativity

    CERN Document Server

    Dutta, Biplab


    Glashow and Cohen claim that many results of special theory of relativity (SR) like time dilation, relativistic velocity addition, etc, can be explained by using certain proper subgroups, of the Lorentz group, which collectively form the main body of Very special relativity (VSR). They did not mention about length contraction in VSR. Length contraction in VSR has not been studied at all. In this article we calculate how the length of a moving rod contracts in VSR, particularly in the HOM(2) version. The results are interesting in the sense that in general the length contraction formulas in VSR are different from SR but in many cases the two theories predict similar length contraction of moving rods.

  11. Screening length in dusty plasma crystals (United States)

    Nikolaev, V. S.; Timofeev, A. V.


    Particles interaction and value of the screening length in dusty plasma systems are of great interest in dusty plasma area. Three inter-particle potentials (Debye potential, Gurevich potential and interaction potential in the weakly collisional regime) are used to solve equilibrium equations for two dusty particles suspended in a parabolic trap. The inter-particle distance dependence on screening length, trap parameter and particle charge is obtained. The functional form of inter-particle distance dependence on ion temperature is investigated and compared with experimental data at 200-300 K in order to test used potentials applicability to dusty plasma systems at room temperatures. The preference is given to the Yukawa-type potential including effective values of particle charge and screening length. The estimated effective value of the screening length is 5-15 times larger than the Debye length.

  12. Variable length representation in evolutionary electronics. (United States)

    Zebulum, R S; Pacheco, M A; Vellasco, M


    This work investigates the application of variable length representation (VLR) evolutionary algorithms (EAs) in the field of Evolutionary Electronics. We propose a number of VLR methodologies that can cope with the main issues of variable length evolutionary systems. These issues include the search for efficient ways of sampling a genome space with varying dimensionalities, the task of balancing accuracy and parsimony of the solutions, and the manipulation of non-coding segments. We compare the performance of three proposed VLR approaches to sample the genome space: Increasing Length Genotypes, Oscillating Length Genotypes, and Uniformly Distributed Initial Population strategies. The advantages of reusing genetic material to replace non-coding segments are also emphasized in this work. It is shown, through examples in both analog and digital electronics, that the variable length genotype's representation is natural to this particular domain of application. A brief discussion on biological genome evolution is also provided.

  13. Kidney Length in Normal Korean Children

    Energy Technology Data Exchange (ETDEWEB)

    Kim, In One; Cheon, Jung Eun [Seoul National University College of Medicine, Seoul (Korea, Republic of); Lee, Young Seok [Dankook University College of Medicine, Cheonan (Korea, Republic of); Lee, Sun Wha [Ewha Womans University College of Medicine, Seoul (Korea, Republic of); Kim, Ok Hwa [Ajou University College of Medicine, Suwon (Korea, Republic of); Kim, Ji Hye [Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Kim, Hong Dae [Kangnam Sacred Heart Hospital, Hanllym University College of Medicine, Seoul (Korea, Republic of); Sim, Jung Suk [Mothers Clinic, Bundang, Seongnam (Korea, Republic of)


    Renal length offers important information to detect or follow-up various renal diseases. The purpose of this study was to determine the kidney length of normal Korean children in relation to age, height, weight, body surface area (BSA), and body mass index (BMI). Children between 1 month and 15 years of age without urological abnormality were recruited. Children below 3rd percentile and over 97th percentile for height or weight were excluded. Both renal lengths were measured in the prone position three times and then averaged by experienced radiologists. The mean length and standard deviation for each age group was obtained, and regression equation was calculated between renal length and age, weight, height, BSA, and BMI, respectively. Renal length was measured in 550 children. Renal length grows rapidly until 24 month, while the growth rate is reduced thereafter. The regression equation for age is: renal length (mm) = 45.953 + 1.064 x age (month, {<=} 24 months) (R2 = 0.720) or 62.173 + 0.203 x age (months, > 24 months) (R2 = 0.711). The regression equation for height is: renal length (mm) = 24.494 + 0.457 x height (cm) (R2 = 0.894). The regression equation for weight is: renal length (mm) = 38.342 + 2.117 x weight (kg, {<=}18 kg) (R2 = 0.852) or 64.498 + 0.646 x weight (kg, > 18 kg) (R2 = 0.651). The regression equation for BSA is: renal length (mm) = 31.622 + 61.363 x BSA (m2, {<=} 0.7) (R2 = 0.857) or 52.717 + 29.959 x BSA (m2, > 0.7) (R2 = 0.715). The regression equation for BMI is: renal length (mm) = 44.474 + 1.163 x BMI (R2 = 0.079). This study provides data on the normal renal length and its association with age, weight, height, BSA and BMI. The results of this study will guide the detection and follow-up of renal diseases in Korean children

  14. Backbone amide linker strategy

    DEFF Research Database (Denmark)

    Shelton, Anne Pernille Tofteng; Jensen, Knud Jørgen


    amino acid residue by reductive amination. This can be used as a general approach for the introduction of other C-terminal modifications as well as functionalities, such as fluorophors. The second step is an acylation of a secondary amine, followed by standard Fmoc-based solid-phase synthesis...

  15. Inproved grade length limitation of freeways

    Institute of Scientific and Technical Information of China (English)

    XING En-hui; PEI Yu-long


    A method of ascertaining grade length limitation of freeways in mountain terrain is presented. The relationship models between 15th percentile speeds and grades were built through the surveys and analyses of operation speeds on 7 typical sections of 3 freeways in mountain terrain. Using 15th percentile and 85th percentile as speed limitations, the methods of determining admitted velocities were put forward according to the grades. Deceleration distances of longitudinal slopeways were analyzed utilizing the theories of vehicle. According to the results of analysis, grade length limitation was loosed. Finally the values of grade length limitation for freeways were put forward. The results could be used as references for freeway design.

  16. Scattering lengths of calcium and barium isotopes

    Energy Technology Data Exchange (ETDEWEB)

    Dammalapati, U.; Willmann, L.; Knoop, S. [Kernfysisch Versneller Instituut (KVI), University of Groningen, Zernikelaan 25, 9747 AA Groningen (Netherlands); LaserLaB Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam (Netherlands)


    We have calculated the s-wave scattering length of all the even isotopes of calcium (Ca) and barium (Ba) in order to investigate the prospect of Bose-Einstein condensation (BEC). For Ca we have used an accurate molecular potential based on detailed spectroscopic data. Our calculations show that Ca does not provide other isotopes alternative to the recently Bose condensed {sup 40}Ca that suffers strong losses because of a very large scattering length. For Ba we show by using a model potential that the even isotopes cover a broad range of scattering lengths, opening the possibility of BEC for at least one of the isotopes.

  17. The tree length of an evolving coalescent

    CERN Document Server

    Pfaffelhuber, Peter; Weisshaupt, Heinz


    A well-established model for the genealogy of a large population in equilibrium is Kingman's coalescent. For the population together with its genealogy evolving in time, this gives rise to a time-stationary tree-valued process. We study the sum of the branch lengths, briefly denoted as tree length, and prove that the (suitably compensated) sequence of tree length processes converges, as the population size tends to infinity, to a limit process with cadlag paths, infinite infinitesimal variance, and a Gumbel distribution as its equilibrium.

  18. Stylish lengths: Mate choice in flowers

    Indian Academy of Sciences (India)

    B T Ramesha; M D Yetish; G Ravikanth; K N Ganeshaiah; Jaboury Ghazoul; R Uma Shaanker


    The styles of flowers may represent an arena for pollen competition in the race to fertilize ovules. Accordingly, selection should favour a longer ‘race’ to better discriminate among variable pollen by increasing style length. Sampling across a taxonomically diverse range of wild and outcrossed species, we found that the distribution of style lengths within plants were skewed towards longer styles, as predicted. In self-pollinated domesticated species, where discrimination among pollen is less important, we found no such pattern. We conclude that style length is under directional selection towards longer styles as a mechanism for mate choice among pollen of variable quality.

  19. Bunch Length Measurements in SPEAR3

    Energy Technology Data Exchange (ETDEWEB)

    Corbett, W.J.; Fisher, A.; Huang, X.; Safranek, J.; Sebek, J.; /SLAC; Lumpkin, A.; /Argonne; Sannibale, F.; /LBL, Berkeley; Mok, W.; /Unlisted


    A series of bunch length measurements were made in SPEAR3 for two different machine optics. In the achromatic optics the bunch length increases from the low-current value of 16.6ps rms to about 30ps at 25ma/bunch yielding an inductive impedance of -0.17{Omega}. Reducing the momentum compaction factor by a factor of {approx}60 [1] yields a low-current bunch length of {approx}4ps rms. In this paper we review the experimental setup and results.

  20. CPS Trawl Life History Length Frequency Data (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Length distribution of a subset of individuals from a species (mainly non-target) caught during SWFSC-FRD fishery independent trawl surveys of coastal pelagic...

  1. Impact of Backbone Tether Length and Structure on the Electrochemical Performance of Viologen Redox Active Polymers

    Energy Technology Data Exchange (ETDEWEB)

    Burgess, Mark; Chénard, Etienne; Hernández-Burgos, Kenneth; Nagarjuna, Gavvalapalli; Assary, Rajeev S.; Hui, Jingshu; Moore, Jeffrey S.; Rodríguez-López, Joaquín


    The design of chemically stable and electrochemically reversible redox active polymers (RAPs) is of great interest for energy storage technologies. Particularly, RAPs are new players for flow batteries relying on a size-exclusion based mechanism of electrolyte separation, but few studies have provided detailed molecular understanding of redox polymers in solution. Here, we use a systematic molecular design approach to investigate the impact of linker and redox-pendant electronic interactions on the performance of viologen RAPs. We used scanning electrochemical microscopy, cyclic voltammetry, bulk electrolysis, temperature-dependent absorbance, and spectroelectrochemistry to study the redox properties, charge transfer kinetics, and self-exchange of electrons through redox active dimers and their equivalent polymers. Stark contrast was observed between the electrochemical properties of viologen dimers and their corresponding polymers. Electron self-exchange kinetics in redox active dimers that only differ by their tether length and rigidity influences their charge transfer properties. Predictions from the Marcus Hush theory were consistent with observations in redox active dimers, but they failed to fully capture the behavior of macromolecular systems. For example, polymer bound viologen pendants, if too close in proximity, do not retain chemical reversibility. In contrast to polymer films, small modifications to the backbone structure decisively impact the bulk electrolysis of polymer solutions. This first comprehensive study highlights the careful balance between electronic interactions and backbone rigidity required to design RAPs with superior electrochemical performance.

  2. Cytoplasmic dynein binding, run length, and velocity are guided by long-range electrostatic interactions. (United States)

    Li, Lin; Alper, Joshua; Alexov, Emil


    Dyneins are important molecular motors involved in many essential biological processes, including cargo transport along microtubules, mitosis, and in cilia. Dynein motility involves the coupling of microtubule binding and unbinding to a change in the configuration of the linker domain induced by ATP hydrolysis, which occur some 25 nm apart. This leaves the accuracy of dynein stepping relatively inaccurate and susceptible to thermal noise. Using multi-scale modeling with a computational focusing technique, we demonstrate that the microtubule forms an electrostatic funnel that guides the dynein's microtubule binding domain (MTBD) as it finally docks to the precise, keyed binding location on the microtubule. Furthermore, we demonstrate that electrostatic component of the MTBD's binding free energy is linearly correlated with the velocity and run length of dynein, and we use this linearity to predict the effect of mutating each glutamic and aspartic acid located in MTBD domain to alanine. Lastly, we show that the binding of dynein to the microtubule is associated with conformational changes involving several helices, and we localize flexible hinge points within the stalk helices. Taken all together, we demonstrate that long range electrostatic interactions bring a level of precision to an otherwise noisy dynein stepping process.

  3. Length and coverage of inhibitory decision rules

    KAUST Repository

    Alsolami, Fawaz


    Authors present algorithms for optimization of inhibitory rules relative to the length and coverage. Inhibitory rules have a relation "attribute ≠ value" on the right-hand side. The considered algorithms are based on extensions of dynamic programming. Paper contains also comparison of length and coverage of inhibitory rules constructed by a greedy algorithm and by the dynamic programming algorithm. © 2012 Springer-Verlag.

  4. Process for fabricating continuous lengths of superconductor (United States)

    Kroeger, Donald M.; List, III, Frederick A.


    A process for manufacturing a superconductor. The process is accomplished by depositing a superconductor precursor powder on a continuous length of a first substrate ribbon, overlaying a continuous length of a second substrate ribbon on said first substrate ribbon, and applying sufficient pressure to form a bound layered superconductor precursor between said first substrate ribbon and said second substrates ribbon. The layered superconductor precursor is then heat treated to form a super conductor layer.

  5. Fragment Length of Circulating Tumor DNA.

    Directory of Open Access Journals (Sweden)

    Hunter R Underhill


    Full Text Available Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively. Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively. Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

  6. Fragment Length of Circulating Tumor DNA. (United States)

    Underhill, Hunter R; Kitzman, Jacob O; Hellwig, Sabine; Welker, Noah C; Daza, Riza; Baker, Daniel N; Gligorich, Keith M; Rostomily, Robert C; Bronner, Mary P; Shendure, Jay


    Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134-144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132-145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA.

  7. Derived length for arbitrary topological spaces

    Directory of Open Access Journals (Sweden)

    A. J. Jayanthan


    Full Text Available The notion of derived length is as old as that of ordinal numbers itself. It is also known as the Cantor-Bendixon length. It is defined only for dispersed (that is scattered spaces. In this paper this notion has been extended in a natural way for all topological spaces such that all its pleasing properties are retained. In this process we solve a problem posed by V. Kannan. ([1] Page 158.

  8. Reduced fetal telomere length in gestational diabetes.

    Directory of Open Access Journals (Sweden)

    Jian Xu

    Full Text Available Gestational diabetes mellitus (GDM is an important complication of pregnancy that poses significant threats to women and their offspring. Telomere length shortens as cellular damage increases and is associated with metabolic diseases. Telomere length in fetal leucocytes was determined in 82 infants of women with GDM (N = 82 and 65 normal pregnant women (N = 65. Women with preeclampsia (N = 45 and gestational hypertension (N = 23 were also studied. In the GDM group, telomere length was significantly shorter than normal pregnancy (P = 0.028, but there were no significant differences in fetal telomere length between preeclampsia and normal pregnancy (P = 0.841 and between gestational hypertension and normal pregnancy (P = 0.561. Regression analysis revealed that fetal telomere length was significantly associated with intrauterine exposure to GDM (P = 0.027 after adjustment for maternal age, gestational age at delivery, birth weight and fetal gender. Shortened telomere length may increase the risk of metabolic diseases in adulthood of GDM offspring.

  9. Classifying stages of third molar development: crown length as a predictor for the mature root length. (United States)

    Altalie, Salem; Thevissen, Patrick; Willems, Guy


    Multiple tooth development staging techniques were reported based on arbitrarily set borderlines between succeeding stages. Anatomic tooth features or predictions of future tooth part dimensions were described to identify the thresholds between the established stages. The need to predict mature tooth dimensions, while the tooth considered is still in development, is a drawback to use this staging technique for dental age estimations. Using the fully mature crown length as a predictor for the future root length could provide a tool for undisputable staging. The aim of this study was first to measure the crown and root length of fully mature third molars and second to investigate whether the crown length could be used as a predictor of the root length, in order to classify the observed root length as a proportion of the future mature root. The crown and root lengths of all present third molars were digitally measured on dental panoramic radiographs of 1,000 subjects. The included subjects were equally distributed in gender, and their age ranged between 22 and 40 years. Two occlusal borders, the cement enamel junction and the root apices, were defined as landmarks for standardized measurements. Regression models with root length as response and crown length as predictor were established and revealed low R (2) and high RMSE values. Due to the small explained variance by the prediction models and the high variation in prediction errors, the observed crown length cannot be used to predict the final root length of a developing third molar.

  10. Dissociation Behavior of a TEMPO-Active Ester Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) in Negative ESI-MS (United States)

    Hage, Christoph; Ihling, Christian H.; Götze, Michael; Schäfer, Mathias; Sinz, Andrea


    We have synthesized a homobifunctional amine-reactive cross-linking reagent, containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) and a benzyl group (Bz), termed TEMPO-Bz-linker, to derive three-dimensional structural information of proteins. The aim for designing this novel cross-linker was to facilitate the mass spectrometric analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). In an initial study, we had investigated the fragmentation behavior of TEMPO-Bz-derivatized peptides upon collision activation in (+)-electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) experiments. In addition to the homolytic NO-C bond cleavage FRIPS pathway delivering the desired odd-electron product ions, an alternative heterolytic NO-C bond cleavage, resulting in even-electron product ions mechanism was found to be relevant. The latter fragmentation route clearly depends on the protonation of the TEMPO-Bz-moiety itself, which motivated us to conduct (-)-ESI-MS, CID-MS/MS, and MS3 experiments of TEMPO-Bz-cross-linked peptides to further clarify the fragmentation behavior of TEMPO-Bz-peptide molecular ions. We show that the TEMPO-Bz-linker is highly beneficial for conducting FRIPS in negative ionization mode as the desired homolytic cleavage of the NO-C bond is the major fragmentation pathway. Based on characteristic fragments, the isomeric amino acids leucine and isoleucine could be discriminated. Interestingly, we observed pronounced amino acid side chain losses in cross-linked peptides if the cross-linked peptides contain a high number of acidic amino acids.

  11. The Domain II S4-S5 Linker in Nav1.9: A Missense Mutation Enhances Activation, Impairs Fast Inactivation, and Produces Human Painful Neuropathy. (United States)

    Han, Chongyang; Yang, Yang; de Greef, Bianca T A; Hoeijmakers, Janneke G J; Gerrits, Monique M; Verhamme, Camiel; Qu, Jian; Lauria, Giuseppe; Merkies, Ingemar S J; Faber, Catharina G; Dib-Hajj, Sulayman D; Waxman, Stephen G


    Painful small fiber neuropathy is a challenging medical condition with no effective treatment. Non-genetic causes can be identified in one half of the subjects. Gain-of-function variants of sodium channels Nav1.7 and Nav1.8 have recently been associated with painful small fiber neuropathy. More recently, mutations of sodium channel Nav1.9 have been linked to human pain disorders, with two gain-of-function mutations found in patients with painful small fiber neuropathy. Here we report a novel Nav1.9 mutation, a glycine 699 substitution by arginine (G699R) in the domain II S4-S5 linker, identified in a patient with painful small fiber neuropathy. In this study, we assayed the mutant channels by voltage-clamp in superior cervical ganglion neurons, which do not produce endogenous Nav1.8 or Nav1.9 currents, and provide a novel platform where Nav1.9 is expressed at relatively high levels. Voltage-clamp analysis showed that the mutation hyperpolarizes (-10.1 mV) channel activation, depolarizes (+6.3 mV) steady-state fast inactivation, slows deactivation, and enhances ramp responses compared with wild-type Nav1.9 channels. Current-clamp analysis showed that the G699R mutant channels render dorsal root ganglion neurons hyperexcitable, via depolarized resting membrane potential, reduced current threshold and increased evoked firing. These observations show that the domain II S4-S5 linker plays an important role in the gating of Nav1.9 and demonstrates that a mutation in this linker is linked to a common pain disorder.

  12. Semi-interpenetrating solid polymer electrolyte based on thiol-ene cross-linker for all-solid-state lithium batteries (United States)

    Suk, Jungdon; Lee, Yu Hwa; Kim, Do Youb; Kim, Dong Wook; Cho, Song Yun; Kim, Ji Man; Kang, Yongku


    We developed highly promising solid polymer electrolytes (SPEs) based on a novel cross-linker containing star-shaped phosphazene with poly(ethylene oxide) (PEO) branches with very high ionic conductivity (7.6 × 10-4 S cm-1), improved mechanical stability, and good electrochemical stability for all-solid-state lithium batteries. In particular, allyl groups were introduced at the ends of the cross-linker in order to overcome the easy self-polymerization of existing cross-linking acrylate end groups. A novel semi-interpenetrating network (semi-IPN) SPE was prepared by in-situ radical polymerization of a precursor solution containing lithium salt, poly(ethylene glycol) dimethyl ether as a plasticizer, and a mixture of pentaerythritol tetrakis(3-mercaptopropionate) and a synthesized hexakis(allyloxy)cyclotriphosphazene (thiol-ene PAL) as the cross-linker. Batteries employing LiFePO4 as the cathode, lithium foil as the anode, and the SPE thin film as the electrolyte were assembled and tested. At ambient temperature, the initial discharge capacity was 147 mAh/g at 0.1 °C and 132 mAh/g at 0.5 °C, and 97% of the capacity was retained at the 100th cycle. All-solid-state pouch-package lithium cells assembled with the SPEs exhibited stable electrochemical performance, even under a severely wrinkled state. These outstanding properties of SPEs based on thiol-ene PAL demonstrate feasibility for practical battery applications with improved reliability and safety.

  13. Integrated Workflow for Structural Proteomics Studies Based on Cross-Linking/Mass Spectrometry with an MS/MS Cleavable Cross-Linker. (United States)

    Arlt, Christian; Götze, Michael; Ihling, Christian H; Hage, Christoph; Schäfer, Mathias; Sinz, Andrea


    Cross-linking combined with mass spectrometry (MS) has evolved as an alternative strategy in structural biology for characterizing three-dimensional structures of protein assemblies and for mapping protein-protein interactions. Here, we describe an integrated workflow for an automated identification of cross-linked products that is based on the use of a tandem mass spectrometry (MS/MS) cleavable cross-linker (containing a 1,3-bis-(4-oxo-butyl)-urea group, BuUrBu) generating characteristic doublet patterns upon fragmentation. We evaluate different fragmentation methods available on an Orbitrap Fusion mass spectrometer for three proteins and an E. coli cell lysate. An updated version of the dedicated software tool MeroX was employed for a fully automated identification of cross-links. The strength of our cleavable cross-linker is that characteristic patterns of the cross-linker as well as backbone fragments of the connected peptides are already observed at the MS/MS level, eliminating the need for conducting MS(3) or sequential CID (collision-induced dissociation)- and ETD (electron transfer dissociation)-MS/MS experiments. This makes our strategy applicable to a broad range of mass spectrometers with MS/MS capabilities. For purified proteins and protein complexes, our workflow using CID-MS/MS acquisition performs with high confidence, scoring cross-links at 0.5% false discovery rate (FDR). The cross-links provide structural insights into the intrinsically disordered tetrameric tumor suppressor protein p53. As a time-consuming manual inspection of cross-linking data is not required, our workflow will pave the way for making the cross-linking/MS approach a routine technique for structural proteomics studies.

  14. Dissociation Behavior of a TEMPO-Active Ester Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) in Negative ESI-MS (United States)

    Hage, Christoph; Ihling, Christian H.; Götze, Michael; Schäfer, Mathias; Sinz, Andrea


    We have synthesized a homobifunctional amine-reactive cross-linking reagent, containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) and a benzyl group (Bz), termed TEMPO-Bz-linker, to derive three-dimensional structural information of proteins. The aim for designing this novel cross-linker was to facilitate the mass spectrometric analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). In an initial study, we had investigated the fragmentation behavior of TEMPO-Bz-derivatized peptides upon collision activation in (+)-electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) experiments. In addition to the homolytic NO-C bond cleavage FRIPS pathway delivering the desired odd-electron product ions, an alternative heterolytic NO-C bond cleavage, resulting in even-electron product ions mechanism was found to be relevant. The latter fragmentation route clearly depends on the protonation of the TEMPO-Bz-moiety itself, which motivated us to conduct (-)-ESI-MS, CID-MS/MS, and MS3 experiments of TEMPO-Bz-cross-linked peptides to further clarify the fragmentation behavior of TEMPO-Bz-peptide molecular ions. We show that the TEMPO-Bz-linker is highly beneficial for conducting FRIPS in negative ionization mode as the desired homolytic cleavage of the NO-C bond is the major fragmentation pathway. Based on characteristic fragments, the isomeric amino acids leucine and isoleucine could be discriminated. Interestingly, we observed pronounced amino acid side chain losses in cross-linked peptides if the cross-linked peptides contain a high number of acidic amino acids.

  15. Synthesis of monooxime-monocarbamoyl bispyridinium compounds bearing (E)-but-2-ene linker and evaluation of their reactivation activity against tabun- and paraoxon-inhibited acetylcholinesterase. (United States)

    Musilek, Kamil; Holas, Ondrej; Kuca, Kamil; Jun, Daniel; Dohnal, Vlastimil; Opletalova, Veronika; Dolezal, Martin


    Six AChE monooxime-monocarbamoyl reactivators with an (E)-but-2-ene linker were synthesized using modification of currently known synthetic pathways. Their potency to reactivate AChE inhibited by the nerve agent tabun and insecticide paraoxon was tested in vitro. The reactivation efficacies of pralidoxime, HI-6, obidoxime, K048, K075 and the newly prepared reactivators were compared. According to the results obtained, one reactivator seems to be promising against tabun-inhibited AChE and two reactivators against paraoxon-inhibited AChE. The best results were obtained for bisquaternary substances with at least one oxime group in position four.

  16. A highly stable MOF with a rod SBU and a tetracarboxylate linker: unusual topology and CO2 adsorption behaviour under ambient conditions. (United States)

    Li, Ru-Jin; Li, Mian; Zhou, Xiao-Ping; Li, Dan; O'Keeffe, Michael


    A Mn-based rod metal-organic framework (MOF), ROD-6, with a new lrk net is synthesized. It represents an unusual type of MOF topology containing both a 1-periodic secondary building unit (rod SBU) and a polytopic linker (here 1,3,6,8-tetrakis(p-benzoic acid)pyrene), and also exhibits high stability (up to 485 °C) and unusual CO2 uptake behaviour and selectivity despite the lack of strong interacting sites, demonstrated by the low and increasing heat of adsorption.

  17. FMO3-LCMO study of electron transfer coupling matrix element and pathway: Application to hole transfer between two triptophanes through cis- and trans-polyproline-linker systems

    CERN Document Server

    Kitoh-Nishioka, Hirotaka


    The linear-combination of fragment molecular orbitals with three-body correction (FMO3-LCMO) is examined for electron transfer (ET) coupling matrix elements and ET pathway analysis, with application to hole transfer between two triptophanes bridged by cis- and trans-polyproline linker conformations. A projection to the minimal-valence-plus-core FMO space was found to give sufficient accuracy with significant reduction of computational cost while avoiding the problem of linear dependence of FMOs stemming from involvement of bond detached atoms.

  18. FMO3-LCMO study of electron transfer coupling matrix element and pathway: Application to hole transfer between two tryptophans through cis- and trans-polyproline-linker systems (United States)

    Kitoh-Nishioka, Hirotaka; Ando, Koji


    The linear-combination of fragment molecular orbitals with three-body correction (FMO3-LCMO) is examined for electron transfer (ET) coupling matrix elements and ET pathway analysis, with application to hole transfer between two tryptophans bridged by cis- and trans-polyproline linker conformations. A projection to the minimal-valence-plus-core FMO space was found to give sufficient accuracy with significant reduction of computational cost while avoiding the problem of linear dependence of FMOs stemming from involvement of bond detached atoms.

  19. KCNQ1 Channels Voltage Dependence through a Voltage-dependent Binding of the S4-S5 Linker to the Pore Domain*



    Voltage-dependent potassium (Kv) channels are tetramers of six transmembrane domain (S1–S6) proteins. Crystallographic data demonstrate that the tetrameric pore (S5–S6) is surrounded by four voltage sensor domains (S1–S4). One key question remains: how do voltage sensors (S4) regulate pore gating? Previous mutagenesis data obtained on the Kv channel KCNQ1 highlighted the critical role of specific residues in both the S4-S5 linker (S4S5L) and S6 C terminus (S6T). From these data, we hypothesiz...

  20. Purification and SAXS analysis of the integrin linked kinase, PINCH, parvin (IPP heterotrimeric complex.

    Directory of Open Access Journals (Sweden)

    Amy L Stiegler

    Full Text Available The heterotrimeric protein complex containing the integrin linked kinase (ILK, parvin, and PINCH proteins, termed the IPP complex, is an essential component of focal adhesions, where it interacts with many proteins to mediate signaling from integrin adhesion receptors. Here we conduct a biochemical and structural analysis of the minimal IPP complex, comprising full-length human ILK, the LIM1 domain of PINCH1, and the CH2 domain of α-parvin. We provide a detailed purification protocol for IPP and show that the purified IPP complex is stable and monodisperse in solution. Using small-angle X-ray scattering (SAXS, we also conduct the first structural characterization of IPP, which reveals an elongated shape with dimensions 120×60×40 Å. Flexibility analysis using the ensemble optimization method (EOM is consistent with an IPP complex structure with limited flexibility, raising the possibility that inter-domain interactions exist. However, our studies suggest that the inter-domain linker in ILK is accessible and we detect no inter-domain contacts by gel filtration analysis. This study provides a structural foundation to understand the conformational restraints that govern the IPP complex.