Impact of the HIV-1 env genetic context outside HR1-HR2 on resistance to the fusion inhibitor enfuvirtide and viral infectivity in clinical isolates

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1-HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1-HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1-HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1-HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy.     

Hydromineral regulation in the hydrothermal vent crab Bythograea thermydron.

This study investigates the salinity tolerance and the pattern of osmotic and ionic regulation of Bythograea thermydron Williams, 1980, a brachyuran crab endemic to the deep-sea hydrothermal vent habitat. Salinities of 33 per thousand-35 per thousand were measured in the seawater surrounding the captured specimens. B. thermydron is a marine stenohaline osmoconformer, which tolerates salinities ranging between about 31 per thousand and 42 per thousand. The time of osmotic adaptation after a sudden decrease in external salinity is about 15-24 h, which is relatively short for a brachyuran crab. In the range of tolerable salinities, it exhibits an iso-osmotic regulation, which is not affected by changes in hydrostatic pressure, and an iso-ionic regulation for Na(+) and Cl(-). The hemolymph Ca(2+) concentration is slightly hyper-regulated, K(+) concentration is slightly hyper-hypo-regulated, and Mg(2+) concentration is strongly hypo-regulated. These findings probably reflect a high permeability of the teguments to water and ions. In addition to limited information about salinity around hydrothermal vents, these results lead to the hypothesis that B. thermydron lives in a habitat of stable seawater salinity. The osmoconformity of this species is briefly discussed in relation to its potential phylogeny.     

Molecular phylogeny and evolution of host-plant use in conifer seed chalcids in the genus Megastigmus (Hymenoptera: Torymidae)

Abstract. Phylogenetic relationships amongst Megastigmus species (Chalcidoidea: Torymidae) associated with conifer seeds were inferred from DNA sequence data. Twenty‐nine species of seed chalcids were analysed using two different genes, cytochrome b (mitochondrial DNA) and the D2 domain of the 28S ribosomal DNA. Maximum‐parsimony and maximum‐likelihood analyses showed that taxa formed two monophyletic groups, one clade comprising all species associated with Cupressaceae and Taxodiaceae hosts with the exception of Chamaecyparis, and the other clade composed of species associated with Pinaceae. Species infesting Cupressaceae and Taxodiaceae seemed to be specialized to particular host genera or even to be species specific, which was consistent with a taxonomic radiation following initial host adaptation. By contrast, Megastigmus species associated with Pinaceae appeared capable of shifting onto different congeneric species or even onto a new host genus, with their evolution apparently less constrained by plant association. We hypothesized that the Megastigmus group associated with Pinaceae may have a much higher invasive potential than that related to Cupressaceae. The study also confirmed the presence of invasive Nearctic species in the Palaearctic, and demonstrated the existence of a cryptic species complex.     

Flash photolysis using a light emitting diode: an efficient, compact, and affordable solution

Flash photolysis has become an essential technique for dynamic investigations of living cells and tissues. This approach offers several advantages for instantly changing the concentration of bioactive compounds outside and inside living cells with high spatial resolution. Light sources for photolysis need to deliver pulses of high intensity light in the near UV range (300-380 nm), to photoactivate a sufficient amount of molecules in a short time. UV lasers are often required as the light source, making flash photolysis a costly approach. Here we describe the use of a high power 365 nm light emitting diode (UV LED) coupled to an optical fiber to precisely deliver the light to the sample. The ability of the UV LED light source to photoactivate several caged compounds (CMNB-fluorescein, MNI-glutamate, NP-EGTA, DMNPE-ATP) as well as to evoke the associated cellular Ca(2+) responses is demonstrated in both neurons and astrocytes. This report shows that UV LEDs are an efficient light source for flash photolysis and represent an alternative to UV lasers for many applications. A compact, powerful, and low-cost system is described in detail.     

Ultrasonic wave spectroscopy study of sugar oligomers and polysaccharides in aqueous solutions: the hydration length concept

The determination of apparent persistence length and radius of gyration of maltodextrins in water is achievable through high-resolution ultrasonic spectroscopy measurements. Classical hydration number for those carbohydrates is characteristic of an apparent persistence degree of polymerisation of the polymer. A force-field based molecular modeling of a 10DP malto-oligomer allows measurement of the corresponding length for the lowest energetic conformation in solution. A good agreement between the apparent radii of gyration determined by this technique and the freely rotating polymer chain model is found with radii of gyration calculated from the intrinsic viscosity.     

Probing plasmid partition with centromere-based incompatibility

Low-copy number plasmids of bacteria rely on specific centromeres for regular partition into daughter cells. When also present on a second plasmid, the centromere can render the two plasmids incompatible, disrupting partition and causing plasmid loss. We have investigated the basis of incompatibility exerted by the F plasmid centromere, sopC, to probe the mechanism of partition. Measurements of the effects of sopC at various gene dosages on destabilization of mini-F, on repression of the sopAB operon and on occupancy of mini-F DNA by the centromere-binding protein, SopB, revealed that among mechanisms previously proposed, no single one fully explained incompatibility. sopC on multicopy plasmids depleted SopB by titration and by contributing to repression. The resulting SopB deficit is proposed to delay partition complex formation and facilitate pairing between mini-F and the centromere vector, thereby increasing randomization of segregation. Unexpectedly, sopC on mini-P1 exerted strong incompatibility if the P1 parABS locus was absent. A mutation preventing the P1 replication initiation protein from pairing (handcuffing) reduced this strong incompatibility to the level expected for random segregation. The results indicate the importance of kinetic considerations and suggest that mini-F handcuffing promotes pairing of SopB-sopC complexes that can subsequently segregate as intact aggregates.     

Virtual screening to enrich a compound collection with CDK2 inhibitors using docking, scoring, and composite scoring models

Docking programs can generate subsets of a compound collection with an increased percentage of actives against a target (enrichment) by predicting their binding mode (pose) and affinity (score), and retrieving those with the highest scores. Using the QXP and GOLD programs, we compared the ability of six single scoring functions (PLP, Ligscore, Ludi, Jain, ChemScore, PMF) and four composite scoring models (Mean Rank: MR, Rank-by-Vote: Vt, Bayesian Statistics: BS and PLS Discriminant Analysis: DA) to separate compounds that are active against CDK2 from inactives. We determined the enrichment for the entire set of actives (IC50 < 10 microM) and for three activity subsets. In all cases, the enrichment for each subset was lower than for the entire set of actives. QXP outperformed GOLD at pose prediction, but yielded only moderately better enrichments. Five to six scoring functions yielded good enrichments with GOLD poses, while typically only two worked well with QXP poses. For each program, two scoring functions generally performed better than the others (Ligscore2 and Ludi for GOLD; QXP and Jain for QXP). Composite scoring functions yielded better results than single scoring functions. The consensus approaches MR and Vt worked best when separating micromolar inhibitors from inactives. The statistical approaches BS and DA, which require training data, performed best when distinguishing between low and high nanomolar inhibitors. The key observation that all hit rate profiles for all four activity intervals for all scoring schemes for both programs are significantly better than random, is evidence that docking can be successfully applied to enrich compound collections.     

General synthesis and biological evaluation of alpha-1-C-substituted derivatives of fagomine (2-deoxynojirimycin-alpha-C-glycosides)

A general synthesis of alpha-1-C-substituted derivatives of fagomine (2-deoxynojirimycin-alpha-C-glycosides) by ring-opening reactions of an aziridine with various heteroatomic nucleophiles, including thiol, amine, alcohol, carboxylate and phosphate, is described. The nine-step reaction sequence proceeded in an overall yield of 14-28% from tri-O-benzyl-D-glucal. Biological evaluation of alpha-1-C-substituted derivatives of fagomine, of the 2-deoxy analog of alpha-homonojirimycin 19 and its 1,N-anhydro derivative 22 as glycosidase inhibitors is reported. The glycosyl phosphate mimetic 15k was found to display no inhibitory activity towards glycogen phosphorylase b and phosphoglucomutase.     

Membrane topology of loop 13-14 of the Na+/glucose cotransporter (SGLT1): a SCAM and fluorescent labelling study

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na+/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES(-) and MTSET(+) had no effect on either cotransport or Na+ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K(i)(Pz) and K(m)(alphaMG) for wtSGLT1 and for cysteine mutants at positions 588, 605-608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K(i)(Pz) values than wtSGLT1 with minimal changes in K(m)((alpha)MG), the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.