DCV

Mossoró, Brazil
Mossoró, Brazil
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Torres S.B.,Rural University | de Paiva E.P.,DCV | de Almeida J.P.N.,DCV | Benedito C.P.,Rural University | Carvalho S.M.C.,DCV
Revista Ciencia Agronomica | Year: 2015

The tests of germination and vigour are essential components of the quality control process of seed producers. Therefore, the aim of this research was to study the methodology of the test of electrical conductivity, verifying its efficiency in identifying different levels of vigour in batches of coriander seed. To do this, four cultivars were used (Português, Super Verdão, Tabocas and Verdão), each represented by four batches of seed, which were initially subjected to evaluations of moisture content, germination, germination first count and seedling emergence. The test of electrical conductivity was carried out on 25 to 50 seeds soaked in 50 and 75 mL of distilled water at 25 °C for 2, 8 and 24 hours. The test of electrical conductivity is efficient to evaluate the physiological potential of coriander seeds when carried out with 50 seeds immersed in 50 mL of distilled water at 25 °C, after soaking for two hours.


HCV NS5A interacts with viral and cellular proteins, spurring investigations into its role(s) in the virus life cycle5. NS5A crystallizes as a dimer in two forms (reviewed in ref. 6) hypothesized to assemble into helical polymers with alternating crystallographic interfaces7, 8, 9. The pM potency of the NS5A inhibitor DCV (Extended Data Fig. 1), which is unprecedented for antiviral agents10, triggered an investigation of the mechanism of DCV inhibition. We estimated the amount of NS5A protein to be approximately 10.6 fg per cell or ~189 nM (Extended Data Fig. 2). This amount of NS5A is significantly lower than other reported values11, 12, probably owing to differences in the status of replicon cells maintained in different laboratories. On the basis of our calculations, the ratio of NS5A to DCV in cells is approximately 47,000:1 (Extended Data Fig. 2). This ratio suggests that a small number of inhibitor molecules can impact the function of a large number of NS5A protein molecules, consistent with published observations that NS5A can dimerize in cells, and may form oligomers8, 13. On the basis of these findings we developed a working model for NS5A inhibitor action: NS5A proteins interact with each other, and a single bound inhibitor perturbs the function of an NS5A oligomer, disrupting the formation of the replication complex or the function of NS5A within the replication complex and amplifying the inhibitory effect. A methodology combining chemical and classical genetics was used to test this working model. A biotinylated compound, BMS-671 (Extended Data Fig. 1)10, inhibited GT-1b wild-type and resistant (Y93H) replicons with the dramatically different half-maximum effective concentration (EC ) values of 33 nM and ~7,400 nM, respectively, an ~224-fold difference (Extended Data Fig. 3a). However, the ability of BMS-671 to bind wild-type and resistant NS5A was very similar (compare BMS-671-bound wild-type and resistant (Y93H) NS5A; Extended Data Fig. 3a)13. Moreover, the binding of BMS-671 was competed by the NS5A inhibitor BMS-665 (Extended Data Fig. 1), but only an ~3-fold difference in binding affinity was observed between the wild-type and Y93H variant (3.9 versus 1.4 μM or 7.8 versus 2.8 μM; Extended Data Fig. 3b, c). The discrepancy between potency and binding affinity prompted us to probe the underlying mechanism. We speculated that an NS5A inhibitor such as DCV might bind to resistant NS5A without triggering inhibition, and that a second inhibitor added to the system could deliver one of two functional outcomes: (1) compete with DCV for binding and have no impact on potency; or (2) bind to an adjacent or nearby NS5A that has undergone a conformational change promoted by DCV binding, and enhance potency. Pairs of inhibitors were screened to determine whether either competition or enhancement of activity could be detected. A combination experiment with pairs of NS5A inhibitors is illustrated in Fig. 1a. In light of the potent EC of DCV on GT-1a wild-type replicons (0.033 nM), EC values >100 nM were considered inactive in this study14. The second inhibitor, referred to as a synergist (Syn), is inactive alone against both wild-type and resistant variants but can greatly enhance the potency of DCV against resistant variants. Specifically, DCV exhibits an EC of 0.033 nM against wild-type GT-1a, but has no activity towards a GT-1a Y93N mutant (EC 339 nM; Fig. 1b). The synergist Syn-395 (Extended Data Fig. 1) is inactive towards both wild type and Y93N (EC 214 nM and 215 nM, respectively; Extended Data Fig. 4). However, in the presence of Syn-395, the potency of DCV against the Y93N variant is greatly enhanced. For example, no inhibition of Y93N was observed at 40 nM Syn-395, but in the presence of 40 nM Syn-395 the potency of DCV against Y93N is enhanced by approximately 2,600-fold, with the EC value shifting from 339 nM to 0.13 nM (Fig. 1b). A similar synergistic effect was observed in a reciprocal experiment (Extended Data Fig. 4). The synergistic effects on potency encouraged us to test the impact of the combination of DCV and Syn-395 on the DCV resistance barrier using an in vitro colony elimination assay (Extended Data Fig. 5a)15. Consistent with its EC , DCV (30 nM) reduced wild-type GT-1a colony formation dramatically but 100 nM Syn-395 had little or no effect. However, a combination of 10 nM DCV and 100 nM Syn-395 completely eliminated replicons. Genotypic and phenotypic analysis revealed linked substitutions (M28T–Q30R, M28T–S38F or M28T–Q30R–S38F) with higher level resistance in 100% of the replicon cells surviving treatment with the DCV plus Syn-395 combination, indicating that the combination has an enhanced resistance barrier compared to DCV alone. To investigate the synergistic effect further, a more biologically relevant system—an infectious virus assay—was used to monitor viral kinetics (Fig. 1c). In the absence of compounds (dimethylsulfoxide (DMSO) control), infectious GT-1a HCV replicated well, with a gradual decrease of luciferase (Luc) signal due to the death of infected cells. Similarly, Syn-395 (150 nM) has no apparent inhibitory activity towards wild-type virus. In contrast, infected cells treated with 250 nM DCV (mean trough plasma concentration recorded in phase 1 studies after a single daily dose of 60 mg) displayed viral kinetics similar to those observed in clinical studies with DCV monotherapy: a rapid initial viral decline (>3 log ) (ref. 10), indicated by a decreasing Luc signal between days 0 and 3, followed by an increasing Luc signal due to the emergence of resistance in the presence of DCV (viral breakthrough). Genotypic analysis of the breakthrough virus population showed the emergence of variants (Y93N and Y93R) conferring a relatively high level of resistance (>10,000-fold) to DCV (ref. 14 and R.A.F., unpublished observations). However, the combination of 250 nM DCV and 150 nM Syn-395 completely suppressed HCV replication. Both compounds were removed at day 12. No viral rebound was observed at days 14, 16 or 18, indicating that HCV was eradicated (Fig. 1c). The effectiveness of the DCV and Syn-395 cooperative interaction was further investigated by removing DCV on day 23 from the DCV-only treated cultures, when breakthrough (resistant) virus titre was close to baseline level. At day 25, DCV alone, Syn-395 alone or DCV plus Syn-395 were added back to the infected cells (Fig. 1c). Even though DCV and Syn-395 monotherapy yielded no detectable anti-HCV activity, DCV plus Syn-395 yielded an HCV decline of ~3 log , with kinetics similar to the initial viral decline. This is a clear illustration of the cooperative interaction between DCV and Syn-395 on DCV-resistant NS5A, resulting in the inhibition of HCV replication. Another pair of compounds, BMS-393 (ref. 16) and Syn-776 (Extended Data Fig. 1), with acceptable pharmacokinetic profiles in mice was used to test for synergy in vivo. The in vitro virology profile of this pair is shown in Extended Data Fig. 5b (see also ref. 16). PXB mice17 were treated with BMS-393 and/or Syn-776 in combination or singly at levels determined to be tolerable (Extended Data Fig. 6a, b). We observed a pattern of activity with a recognizable phenotype: BMS-393 plus Syn-776 (0.4 mg kg−1 plus 15 mg kg−1) yielded a delay in viral rebound compared to either compound alone (Extended Data Fig. 6c). A higher barrier to resistance was supported by the genotypic analysis of variants that emerged during treatment (Extended Data Fig. 6c and Extended Data Table 1). To determine whether a synergistic combination (DCV and Syn-395) has enhanced potency against additional genotypes (beyond GT-1) and resistant variants, a panel of GT-1a, -1b, -2a and -3a resistant variants that have been observed frequently in vitro and in clinical studies10, 18, 19 was investigated (Fig. 2). DCV EC values against this panel of GT-1a resistant variants range from 12.5 to >1,000 nM; the presence of 40 nM Syn-395 enhanced the values to <0.2 nM. Similarly, DCV EC values against GT-1b, GT-2a (J6) and GT-3a resistant variants were enhanced from 326, 18.7 and >1,000 nM to 0.66, 0.12 and 1.03 nM, respectively (Fig. 2). This finding suggests that the feature(s) of NS5A that promotes the synergistic effects characterized extensively for GT-1a is conserved across genotypes. In addition, when Syn-395 was tested in combination with a different NS5A inhibitor, ledipasvir (LDV; also known as GS-5885), a synergistic effect was observed, except for GT-1b L31V–Y93H (Extended Data Fig. 7). To determine whether a synergistic combination of NS5A inhibitors can be as effective as a combination of direct-acting antiviral agents (DAAs) targeting different HCV proteins, a head-to-head comparison was performed with individual DAAs that have achieved a high sustained viral response as triple combination therapy in phase II clinical studies: DCV 3DAA contains DCV plus an NS3 protease inhibitor (asunaprevir (ASV)) plus an NS5B polymerase inhibitor (beclabuvir (BCV))20. Combinations that replace either ASV or BCV with Syn-395 were compared to the DCV 3DAA combination in GT-1a replicon elimination studies. Each of the triple combinations was effective at eliminating the HCV replicon (Extended Data Fig. 8), suggesting that an NS5A synergist could replace either the protease or the polymerase inhibitor in a triple combination. The broad genotype coverage observed with NS5A synergy combinations enhances the potential value of this approach for new HCV therapies. Several models of NS5A inhibitor–protein complexes can explain the synergistic interactions between pairs of NS5A inhibitors. The model illustrated in Fig. 3 is supported crystallographically. Domain I of NS5A has been crystallized in different forms7, 8, 9 and the structure of the amino-terminal amphipathic α-helix has been solved by NMR21. The structure of the NS5A domain I monomer found in each crystallographic form is highly conserved but presents a unique symmetrical dimer interface. These forms are hypothesized to assemble into a polymer network by alternating crystallographic interfaces8, 9. The structure of DCV is symmetrical and has the greatest chemical complementarity when docked across the dimer interface reported previously7 (Fig. 3a). In a model based on chemical shape matching and resistance mapping, DCV binds along the dimer ridge (defined by Y93, Q62, F37, F37′, Q62′ and Y93′) and between and under the 28–35 loops to disrupt the natural packing of the NS5A N termini. The complementary effect of DCV and Syn-395 requires communication between two or more binding sites that cooperatively inhibit NS5A function. In this case, the dimer model was incorporated into an NS5A helical polymer (Fig. 3b) with the NS5A N termini extending from the helix axis and radiating out along the helical groove to position the NS4B/NS5A cleavage sites near the surface. Within the uninhibited NS5A polymer model, the hydrophobic residues from the N termini (28–35) pack along the floor of the helical axis defined by F37, Q62 and Y93. As in the dimer model, DCV binds along the NS5A helical axis, under and between the 28–35 loops, to disrupt the natural packing. However, in the NS5A polymer model, the inhibitor-induced perturbation affects the NS5A dimer in which DCV is bound and is also transmitted along the helical axis through P29–P35 loop interactions (Fig. 3b) to inhibit multiple NS5A proteins. Resistance mutations, which are typically smaller amino acids (L31V, Y93H), partially compensate for the DCV-induced disturbance and are able to restore HCV replication function in the presence of bound inhibitor(s). Syn-395 binding, either adjacent to or a few dimers to either side of DCV, potentiates the effect of DCV to introduce a conformational change that resensitizes the resistant NS5A towards inhibition (Fig. 3c). Consistent with this hypothesis, only specific pairs of NS5A compounds exhibited this unparalleled synergistic effect. Synergy was not observed for BMS-313, a diastereomer of DCV (Extended Data Fig. 1) that is inactive against wild-type and resistant GT-1a replicons (Extended Data Fig. 9) at Syn-395 concentrations ≤40 nM.


News Article | October 29, 2016
Site: www.prweb.com

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Yuri L.M.,DCV | Isabele A.G.,Federal University of Rio Grande do Norte | Dantas C.V.S.,Federal University of Rio Grande do Norte | de Brito L.K.F.,Federal University of Rio Grande do Norte | And 2 more authors.
Revista Brasileira de Fruticultura | Year: 2011

To assess the effects of salt on the pineapple MD Gold during the multiplication and rooting phases in vitro, this study evaluated its performance in different concentrations of NaCl in the absence or presence of growth regulators. Pineapple shoots were inoculated on MS solution in the absence and presence of the growth regulators naphthaleneacetic acid (NAA) and 6-benzlaminopurine (BAP) and different concentrations of NaCl (Control - 0, 50, 100 and 150 mM). Monthly, shoots were subcultured and it was analyzed height, number of alive and dead leaves, and the rates of sprouting and rooting. During the multiplication in the absence of NAA and BAP, the NaCl treatments caused significant reduction in growth and development of pineapple shootings, expressed by the height and number of leaves, in the highest dose, which was not observed in the presence of growth regulators. During the first 60 days, an increase in leaf production occurred. However, after 90 days, there was a decrease in average living leaves in the shoots treated with salt. The in vitro cultivation of pineapple in the presence of salt is more efficient in the presence of NAA and BAP, ensuring continued growth, increasing the number of leaves, producing new buds and accelerating the process of rooting.

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