Fauna of an unsaturated karstic zone in Central Slovenia: two new species of Harpacticoida (Crustacea: Copepoda), Elaphoidella millennii n. sp. and E. tarmani n. sp., their ecology and morphological adaptations

The unsaturated zone in fissured (= karstic) aquifers continues to be a source of new species of Harpacticoida (Crustacea: Copepoda). The first species were discovered about 70 years ago in the Škocjanske Jame Cave in Slovenia. Intensive sampling of percolating water in caves there over the last 20 years has yielded several new species, some of them well adapted to that environment. The most recent studies revealed that such a specialised fauna is also present in other regions of Europe, South and North America, and Asia. In Europe, three genera belonging to the order Harpacticoida are characteristic of the unsaturated karstic zone: Morariopsis, Paramorariopsis and Elaphoidella. In this article, two highly specialised species of Elaphoidella are described. A detailed analysis of their ecology and morphological adaptations along with other species of the genus Elaphoidella from Slovenia is included, and comparisons are made with the epikarstic genera Morariopsis and Paramorariopsis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: S. I. Dodson     

Probing of exosites leads to novel inhibitor scaffolds of HCV NS3/4A proteinase

Background

Hepatitis C is a treatment-resistant disease affecting millions of people worldwide. The hepatitis C virus (HCV) genome is a single-stranded RNA molecule. After infection of the host cell, viral RNA is translated into a polyprotein that is cleaved by host and viral proteinases into functional, structural and non-structural, viral proteins. Cleavage of the polyprotein involves the viral NS3/4A proteinase, a proven drug target. HCV mutates as it replicates and, as a result, multiple emerging quasispecies become rapidly resistant to anti-virals, including NS3/4A inhibitors.

Methodology/Principal Findings

To circumvent drug resistance and complement the existing anti-virals, NS3/4A inhibitors, which are additional and distinct from the FDA-approved telaprevir and boceprevir α-ketoamide inhibitors, are required. To test potential new avenues for inhibitor development, we have probed several distinct exosites of NS3/4A which are either outside of or partially overlapping with the active site groove of the proteinase. For this purpose, we employed virtual ligand screening using the 275,000 compound library of the Developmental Therapeutics Program (NCI/NIH) and the X-ray crystal structure of NS3/4A as a ligand source and a target, respectively. As a result, we identified several novel, previously uncharacterized, nanomolar range inhibitory scaffolds, which suppressed of the NS3/4A activity in vitro and replication of a sub-genomic HCV RNA replicon with a luciferase reporter in human hepatocarcinoma cells. The binding sites of these novel inhibitors do not significantly overlap with those of α-ketoamides. As a result, the most common resistant mutations, including V36M, R155K, A156T, D168A and V170A, did not considerably diminish the inhibitory potency of certain novel inhibitor scaffolds we identified.

Conclusions/Significance

Overall, the further optimization of both the in silico strategy and software platform we developed and lead compounds we identified may lead to advances in novel anti-virals.     

Inhibition of SAH-hydrolase activity during seed germination leads to deregulation of flowering genes and altered flower morphology in tobacco

Developmental processes are closely connected to certain states of epigenetic information which, among others, rely on methylation of chromatin. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are key cofactors of enzymes catalyzing DNA and histone methylation. To study the consequences of altered SAH/SAM levels on plant development we applied 9-(S)-(2,3-dihydroxypropyl)-adenine (DHPA), an inhibitor of SAH-hydrolase, on tobacco seeds during a short phase of germination period (6 days). The transient drug treatment induced: (1) dosage-dependent global DNA hypomethylation mitotically transmitted to adult plants; (2) pleiotropic developmental defects including decreased apical dominance, altered leaf and flower symmetry, flower whorl malformations and reduced fertility; (3) dramatic upregulation of floral organ identity genes NTDEF, NTGLO and NAG1 in leaves. We conclude that temporal SAH-hydrolase inhibition deregulated floral genes expression probably via chromatin methylation changes. The data further show that plants might be particularly sensitive to accurate setting of SAH/SAM levels during critical developmental periods.     

Nonuniform size distribution of nascent globin peptides, evidence for pause localization sites, and a cotranslational protein-folding model

Examination of nascent globin peptides accumulatingin vitro during globin synthesis in rabbit reticulocyte lysates was carried out. A view was supported that nonrandom distribution of codons with different usage frequencies in mRNA may determine the messenger's translation kinetics. Regions of reduced translation of - and -globin polypeptide chains were localized, and the cotranslational protein-folding model suggested previously was substantiated. An active conjunction of synthesis and folding of proteins was proposed as one of the main destinations of a translation nonuniformity.     

Autoregulation of root nodule formation: signals of both symbiotic partners studied in a split-root system of Vicia sativa subsp. nigra

Inhibition of root nodule formation on leguminous plants by already induced or existing root nodules is called autoregulation of root nodule formation (AUT). Optimal conditions for AUT were determined using a split-root technique newly developed for Vicia sativa subsp. nigra. Infection of a root A with nodulating Rhizobium leguminosarum bv. viciae bacteria systemically inhibited nodulation of a spatially separated root B inoculated 2 days later with the same bacteria. This treatment gives complete AUT (total absence of nodules on root B). Only partial AUT of root B was obtained by incubation of root A with mitogenic nodulation (Nod) factors or with a noninfective strain producing normal mitogenic Nod factors. Nonmitogenic Nod factors did not evoke AUT. We identified two systemic plant signals induced by Rhizobium bacteria. Signal 1 (at weak buffering) was correlated with sink formation in root A and induced acidification of B-root medium. This signal is induced by treatment of root A with (i) nodulating rhizobia, (ii) mitogenic Nod factors, (iii) nonmitogenic Nod factors, or (iv) the cytokinin zeatin. Signal 2 (at strong buffering) could only be evoked by treatment with nodulating rhizobia or with mitogenic Nod factors. Most probably, this signal represents the specific AUT signal. Induction of complete AUT appears to require actively dividing nodule cells in nodule primordia, nodule meristems, or both of root A.     

Real-time monitoring of rolling-circle amplification using a modified molecular beacon design

We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2′-O-Me-RNA to prevent 3′ exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.     

Effects of NS2B-NS3 protease and furin inhibition on West Nile and Dengue virus replication

West Nile virus (WNV) and Dengue virus (DENV) replication depends on the viral NS2B-NS3 protease and the host enzyme furin, which emerged as potential drug targets. Modification of our previously described WNV protease inhibitors by basic phenylalanine analogs provided compounds with reduced potency against the WNV and DENV protease. In a second series, their decarboxylated P1-trans-(4-guanidino)cyclohexylamide was replaced by an arginyl-amide moiety. Compound 4-(guanidinomethyl)-phenylacetyl-Lys-Lys-Arg-NH₂ inhibits the NS2B-NS3 protease of WNV with an inhibition constant of 0.11?µM. Due to the similarity in substrate specificity, we have also tested the potency of our previously described multibasic furin inhibitors. Their further modification provided chimeric inhibitors with additional potency against the WNV and DENV proteases. A strong inhibition of WNV and DENV replication in cell culture was observed for the specific furin inhibitors, which reduced virus titers up to 10,000-fold. These studies reveal that potent inhibitors of furin can block the replication of DENV and WNV.