Sequence Analysis of In Vivo Defective Interfering-Like RNA of Influenza A H1N1 Pandemic Virus

Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.     

The antimicrobial activity of lapachol and its thiosemicarbazone and semicarbazone derivatives

Lapachol was chemically modified to obtain its thiosemicarbazone and semicarbazone derivatives. These compounds were tested for antimicrobial activity against several bacteria and fungi by the broth microdilution method. The thiosemicarbazone and semicarbazone derivatives of lapachol exhibited antimicrobial activity against the bacteria Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentrations (MICs) of 0.05 and 0.10 µmol/mL, respectively. The thiosemicarbazone and semicarbazone derivatives were also active against the pathogenic yeast Cryptococcus gattii (MICs of 0.10 and 0.20 µmol/mL, respectively). In addition, the lapachol thiosemicarbazone derivative was active against 11 clinical isolates of Paracoccidioides brasiliensis, with MICs ranging from 0.01-0.10 µmol/mL. The lapachol-derived thiosemicarbazone was not cytotoxic to normal cells at the concentrations that were active against fungi and bacteria. We synthesised, for the first time, thiosemicarbazone and semicarbazone derivatives of lapachol. The MICs for the lapachol-derived thiosemicarbazone against S. aureus, E. faecalis, C. gattii and several isolates of P. brasiliensis indicated that this compound has the potential to be developed into novel drugs to treat infections caused these microbes.     

New records of the chaetiferous leech-like annelid Paracanthobdella livanowi (Epshtein, 1966) (Annelida: Clitellata: Acanthobdellida) from Kamchatka, Russia

Acanthobdellidans are unique in their organisation and phylogenetic relationships due to having transitional characters that combine features of oligochaetous and achaetous annelids. Alongside the relatively well-studied Acanthobdella peledina Grube, 1851, there is another member of the group, Paracanthobdella livanowi (Epshtein, 1966), with five rows of chaetae and an anterior sucker. It appears that the anterior sucker is weakly developed in small juveniles but acquires a deep cavity in adults. Smaller individuals of P. livanowi can be distinguished from A. peledina, which does not possess an anterior sucker, by the varying breadth of their chaetae. The mid-body segment consists of two doubled annuli in juveniles and is quadri-annulate in large individuals. In Kamchatka freshwaters, hosts of P. livanowi mostly include Salvelinus spp. and more rarely Gasterosteus aculeatus, Oncorhynchus mykiss and O. kisutch. New information on the distribution and the biology of P. livanowi is presented.     

Absence of a Context‐General Behavioural Syndrome in a Solitary Predator

The correlation of seemingly unrelated behaviours into behavioural syndromes has been established in a wide range of species and taxa. However, most studies report on short‐term behavioural correlations without insight into individual consistency or temporal stability of the behavioural syndrome. Here, we examine the individual repeatability of single behaviours, and the presence and temporal stability of a context‐general behavioural syndrome in a solitary piscivorous predator, the pike (Esox lucius). Behavioural measurements on the same individuals were quantified independently through time and across three contexts: activity in the presence of a competitor, exploration of a novel environment and boldness under predation risk. There was no indication of a temporally stable behavioural syndrome, consisting of boldness, activity and exploration, nor were individuals consistent in the separate behaviours, contradicting the general assertion of its taxonomic prevalence. Furthermore, the study did not provide support for size or growth‐dependent behaviour in this size‐dimorphic species in conditions of limited food availability. The study highlights the importance of independent multiple observations of individual behaviour across time or contexts when measuring behavioural repeatability and covariation.     

Anti-influenza active and low toxic N-phenyl-substituted β-amidoamidines structurally related to natural antibiotic amidinomycin

A set of racemic N-phenyl-substituted β-amidoamidines hydrochlorides 4, which are structurally related to natural antiviral agent amidinomycin (1), was synthesized in four steps starting from methacryloyl anilide (5). In the final step of the synthetic route, an uncommon monoacylation of β-aminoamidine 8 at the less reactive β-phenylamino-group took place. To rationalize this result, a mechanism which involves initial acylation at the more active amidine-function followed by intramolecular acyl-group transfer to β-phenylamino-group was suggested. All three β-amidoamidines 4d–f bearing long linear aliphatic chain (from n-C₈H₁₇ to n-C₁₂H₂₅) revealed significant in vitro activity against influenza A virus (H3N2) and modest cytotoxicity. The in vitro antiviral potency of 4d,e is 6–20 times greater than that of commercial rimantadine with lower EC₅₀ values and higher therapeutic index. The non-toxic in vivo compounds 4d–f showed a beneficial protective effect in influenza A (H3N2) infected mice.     

Novel thiol- and thioether-containing amino acids: cystathionine and homocysteine families

Natural l-homocysteine and l,l-cystathionine, along with a series of unnatural analogues, have been prepared from l-aspartic and l-glutamic acid. Manipulation of the protected derivatives provided ω-iodoamino acids, which were used in thioalkylation reactions of sulfur nucleophiles, such as the ester of l-cysteine and potassium thioacetate.     

Advanced electron paramagnetic resonance on the catalytic iron–sulfur cluster bound to the CCG domain of heterodisulfide reductase and succinate: quinone reductase

Heterodisulfide reductase (Hdr) is a key enzyme in the energy metabolism of methanogenic archaea. The enzyme catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes M (CoM-SH) and B (CoB-SH). Cleavage of CoM-S-S-CoB at an unusual FeS cluster reveals unique substrate chemistry. The cluster is fixed by cysteines of two cysteine-rich CCG domain sequence motifs (CX_31–39CCX_35–36CXXC) of subunit HdrB of the Methanothermobacter marburgensis HdrABC complex. We report on Q-band (34 GHz) ⁵⁷Fe electron-nuclear double resonance (ENDOR) spectroscopic measurements on the oxidized form of the cluster found in HdrABC and in two other CCG-domain-containing proteins, recombinant HdrB of Hdr from M. marburgensis and recombinant SdhE of succinate: quinone reductase from Sulfolobus solfataricus P2. The spectra at 34 GHz show clearly improved resolution arising from the absence of proton resonances and polarization effects. Systematic spectral simulations of 34 GHz data combined with previous 9 GHz data allowed the unambiguous assignment of four ⁵⁷Fe hyperfine couplings to the cluster in all three proteins. ¹³C Mims ENDOR spectra of labelled CoM-SH were consistent with the attachment of the substrate to the cluster in HdrABC, whereas in the other two proteins no substrate is present. ⁵⁷Fe resonances in all three systems revealed unusually large ⁵⁷Fe ENDOR hyperfine splitting as compared to known systems. The results infer that the cluster’s unique magnetic properties arise from the CCG binding motif.     

Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-¹³C] acetate does not label α carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-¹³C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA–CO coupling in the backbone should improve the resolution of NCACX spectra.