Regulated cleavages at the West Nile virus NS4A-2K-NS4B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the NS4A protein

A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.     

Low-Cost Motility Tracking System (LOCOMOTIS) for Time-Lapse Microscopy Applications and Cell Visualisation

Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6×. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8×). In preliminary cell culture experiments using our system, velocities (mean µm/min ± SE) of 0.81±0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17±0.004 (MDA-MB-231 breast cancer cells), 1.24±0.006 (SC5 mouse Sertoli cells) and 2.21±0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers.     

Structural determinants of specificity and catalytic mechanism in mammalian 25-kDa thiamine triphosphatase

Background

Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates.

Methods

Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase.

Results

Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine–tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds.

Conclusions

The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals.

General significance

We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins.     

Lipoplex thermodynamics: determination of DNA-cationic lipoid interaction energies

An experimental study of the cationic lipid-DNA binding affinity is presented. The binding free energy was determined by monitoring lipoplex dissociation under conditions of increasing salt concentration. The primary procedure was based on the extent of quenching by energy transfer of fluorophores on DNA molecules by fluorophore on a lipid as these molecules came into close association in the lipoplex. Titration calorimetry on the Dickerson dodecamer was also done, with results that were in agreement with the fluorescence data. Measurements on short oligonucleotides allowed estimation of the binding energy per nucleotide. The binding free energy is approximately 0.6 kcal/mole nucleotide for the Dickerson dodecamer and declines for longer oligonucleotides. The entropy gained upon complex formation is approximately 1 entropy unit per released counterion. The method was applied to long DNA molecules (herring and lambda-phage DNA) and revealed that complete dissociation occurs at 750 mM NaCl. Likely contributions of macromolecular desolvation and DNA flexibility to the binding energy are discussed.     

Extracellular vesicles released from mesenchymal stromal cells stimulate bone growth in osteogenesis imperfecta

Background

Systemic infusion of mesenchymal stromal cells (MSCs) has been shown to induce acute acceleration of growth velocity in children with osteogenesis imperfecta (OI) despite minimal engraftment of infused MSCs in bones. Using an animal model of OI we have previously shown that MSC infusion stimulates chondrocyte proliferation in the growth plate and that this enhanced proliferation is also observed with infusion of MSC conditioned medium in lieu of MSCs, suggesting that bone growth is due to trophic effects of MSCs. Here we sought to identify the trophic factor secreted by MSCs that mediates this therapeutic activity.

Adenosine receptors regulate exosome production

Exosomes, small-sized extracellular vesicles, carry components of the purinergic pathway. The production by cells of exosomes carrying this pathway remains poorly understood. Here, we asked whether type 1, 2A, or 2B adenosine receptors (A₁Rs, A_2ARs, and A_2BRs, respectively) expressed by producer cells are involved in regulating exosome production. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from wildtype, A₁R^?/?, A_2AR^?/?, and A_2BR^?/? rats, and exosome production was quantified under normal or metabolic stress conditions. Exosome production was also measured in various cancer cells treated with pharmacologic agonists/antagonists of A₁Rs, A_2ARs, and A_2BRs in the presence or absence of metabolic stress or cisplatin. Functional activity of exosomes was determined in Jurkat cell apoptosis assays. In PGVSMCs, A₁R and A_2AR constrained exosome production under normal conditions (p?=?0.0297; p?=?0.0409, respectively), and A₁R, A_2AR, and A_2BR constrained exosome production under metabolic stress conditions. Exosome production from cancer cells was reduced (p?=?0.0028) by the selective A_2AR agonist CGS 21680. These exosomes induced higher levels of Jurkat apoptosis than exosomes from untreated cells or cells treated with A₁R and A_2BR agonists (p?=?0.0474). The selective A_2AR antagonist SCH 442416 stimulated exosome production under metabolic stress or cisplatin treatment, whereas the selective A_2BR antagonist MRS 1754 reduced exosome production. Our findings indicate that A_2ARs suppress exosome release in all cell types examined, whereas effects of A₁Rs and A_2BRs are dependent on cell type and conditions. Pharmacologic targeting of cancer with A_2AR antagonists may inadvertently increase exosome production from tumor cells.

     

The emergent macrophyte Montrichardia linifera (Arruda) Schott (Alismatales: Araceae), a rekindled old friend from the Pacific Slope of lower Central America and western Colombia

The genus Montrichardia are among the most remarkable emergent macrophytes in tropical wetlands. It occurs exclusively in the Neotropics and contains two living species, M. linifera (Arruda) Schott and M. arborescens (L.) Schott. Montrichardia linifera has been reported mainly in the Amazon basin (southern Venezuela to Guyana, Brazil, Ecuador and Peru), whereas M. arborescens occurs in Central America (Mexico to Panama), the Lesser Antilles and northern South America. Based on our review of herbarium specimens from Central America and Colombia, as well as field documentation in Panama, we hereby provide the first report of the occurrence of M. linifera in Panama, Central America and western Colombia. Furthermore, this finding represents the first record on the Pacific Slope for this species in the Neotropics. The habitat, ecology, life-form, taxonomic remarks and morphological characteristics of the species are presented, discussed and illustrated. Additionally, a key for the species of the genus Montrichardia is included; we also provide a discussion about the helophyte concept for the genus Montrichardia.     

From natural woodlands to cultivated land: diversity of fruit‐feeding butterflies and beetles in the mid‐Zambezi valley,northern Zimbabwe

We tested the effect of cultivation on butterfly (Nymphalidae: Charaxes) and beetle (Coleoptera: Scarabaeidae: Cetoniinae) species richness and abundance along a cultivation intensification gradient. Results showed significant differences in species richness and abundance between natural woodlands and cultivated landscapes with larger differences in areas of high cultivation intensity. The results indicate that natural woodland clearing for cultivation purposes has negative impacts on arthropod diversity, a situation more severe in highly intensified cultivated areas. Our results imply that mosaics of different land‐use units, each in a different phase of clearance‐cultivation‐abandonment‐recovery‐clearance cycle could counter the negative effects of cultivation intensity on arthropod diversity.