Constraints on the use of lifespan-shortening Wolbachia to control dengue fever

Dengue fever, a viral disease spread by the mosquito Aedes aegypti, affects 50–100 million people a year in many tropical countries. Because the virus must incubate within mosquito hosts for two weeks before being able to transmit the infection, shortening the lifespan of mosquitoes may curtail dengue transmission. We developed a continuous time reaction-diffusion model of the spatial spread of Wolbachia through a population of A. aegypti. This model incorporates the lifespan-shortening effects of Wolbachia on infected A. aegypti and the fitness advantage to infected females due to cytoplasmic incompatibility (CI). We found that local establishment of the Wolbachia infection can occur if the fitness advantage due to CI exceeds the fitness reduction due to lifespan-shortening effects, in accordance with earlier results concerning fecundity reduction. However, spatial spread is possible only if the fitness advantage due to CI is twice as great as the fitness reduction due to lifespan shortening effects. Moreover, lifespan-shortening and fecundity-reduction can have different effects on the speed of wave-retreat. Using data from the literature, we estimated all demographic parameters for infected and uninfected mosquitoes and computed the velocities of spread of infection. Our most optimistic estimates suggest that the spatial spread of lifespan-shortening Wolbachia may be so slow that efficient spatial spread would require a prohibitively large number of point releases. However, as these estimates of demographic parameters may not accurately reflect natural conditions, further research is necessary to corroborate these predictions.     

Huntingtin phosphorylation sites mapped by mass spectrometry. Modulation of cleavage and toxicity

Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.     

Cell-to-Cell Spread of the RNA Interference Response Suppresses Semliki Forest Virus (SFV) Infection of Mosquito Cell Cultures and Cannot Be Antagonized by SFV

In their vertebrate hosts, arboviruses such as Semliki Forest virus (SFV) (Togaviridae) generally counteract innate defenses and trigger cell death. In contrast, in mosquito cells, following an early phase of efficient virus production, a persistent infection with low levels of virus production is established. Whether arboviruses counteract RNA interference (RNAi), which provides an important antiviral defense system in mosquitoes, is an important question. Here we show that in Aedes albopictus-derived mosquito cells, SFV cannot prevent the establishment of an antiviral RNAi response or prevent the spread of protective antiviral double-stranded RNA/small interfering RNA (siRNA) from cell to cell, which can inhibit the replication of incoming virus. The expression of tombusvirus siRNA-binding protein p19 by SFV strongly enhanced virus spread between cultured cells rather than virus replication in initially infected cells. Our results indicate that the spread of the RNAi signal contributes to limiting virus dissemination.In animals, RNA interference (RNAi) was first described for Caenorhabditis elegans (27). The production or introduction of double-stranded RNA (dsRNA) in cells leads to the degradation of mRNAs containing homologous sequences by sequence-specific cleavage of mRNAs. Central to RNAi is the production of 21- to 26-nucleotide small interfering RNAs (siRNAs) from dsRNA and the assembly of an RNA-induced silencing complex (RISC), followed by the degradation of the target mRNA (23, 84). RNAi is a known antiviral strategy of plants (3, 53) and insects (21, 39, 51). Study of Drosophila melanogaster in particular has given important insights into RNAi responses against pathogenic viruses and viral RNAi inhibitors (31, 54, 83, 86, 91). RNAi is well characterized for Drosophila, and orthologs of antiviral RNAi genes have been found in Aedes and Culex spp. (13, 63).Arboviruses, or arthropod-borne viruses, are RNA viruses mainly of the families Bunyaviridae, Flaviviridae, and Togaviridae. The genus Alphavirus within the family Togaviridae contains several mosquito-borne pathogens: arboviruses such as Chikungunya virus (16) and equine encephalitis viruses (88). Replication of the prototype Sindbis virus and Semliki Forest virus (SFV) is well understood (44, 71, 74, 79). Their genome consists of a positive-stranded RNA with a 5′ cap and a 3′ poly(A) tail. The 5′ two-thirds encodes the nonstructural polyprotein P1234, which is cleaved into four replicase proteins, nsP1 to nsP4 (47, 58, 60). The structural polyprotein is encoded in the 3′ one-third of the genome and cleaved into capsid and glycoproteins after translation from a subgenomic mRNA (79). Cytoplasmic replication complexes are associated with cellular membranes (71). Viruses mature by budding at the plasma membrane (35).In nature, arboviruses are spread by arthropod vectors (predominantly mosquitoes, ticks, flies, and midges) to vertebrate hosts (87). Little is known about how arthropod cells react to arbovirus infection. In mosquito cell cultures, an acute phase with efficient virus production is generally followed by the establishment of a persistent infection with low levels of virus production (9). This is fundamentally different from the cytolytic events following arbovirus interactions with mammalian cells and pathogenic insect viruses with insect cells. Alphaviruses encode host response antagonists for mammalian cells (2, 7, 34, 38).RNAi has been described for mosquitoes (56) and, when induced before infection, antagonizes arboviruses and their replicons (1, 4, 14, 15, 29, 30, 32, 42, 64, 65). RNAi is also functional in various mosquito cell lines (1, 8, 43, 49, 52). In the absence of RNAi, alphavirus and flavivirus replication and/or dissemination is enhanced in both mosquitoes and Drosophila (14, 17, 31, 45, 72). RNAi inhibitors weakly enhance SFV replicon replication in tick and mosquito cells (5, 33), posing the questions of how, when, and where RNAi interferes with alphavirus infection in mosquito cells.Here we use an A. albopictus-derived mosquito cell line to study RNAi responses to SFV. Using reporter-based assays, we demonstrate that SFV cannot avoid or efficiently inhibit the establishment of an RNAi response. We also demonstrate that the RNAi signal can spread between mosquito cells. SFV cannot inhibit cell-to-cell spread of the RNAi signal, and spread of the virus-induced RNAi signal (dsRNA/siRNA) can inhibit the replication of incoming SFV in neighboring cells. Furthermore, we show that SFV expression of a siRNA-binding protein increases levels of virus replication mainly by enhancing virus spread between cells rather than replication in initially infected cells. Taken together, these findings suggest a novel mechanism, cell-to-cell spread of antiviral dsRNA/siRNA, by which RNAi limits SFV dissemination in mosquito cells.     

Structural Model for Phenylalkylamine Binding to L-type Calcium Channels

Phenylalkylamines (PAAs), a major class of L-type calcium channel (LTCC) blockers, have two aromatic rings connected by a flexible chain with a nitrile substituent. Structural aspects of ligand-channel interactions remain unclear. We have built a KvAP-based model of LTCC and used Monte Carlo energy minimizations to dock devapamil, verapamil, gallopamil, and other PAAs. The PAA-LTCC models have the following common features: (i) the meta-methoxy group in ring A, which is proximal to the nitrile group, accepts an H-bond from a PAA-sensing Tyr_IIIS6; (ii) the meta-methoxy group in ring B accepts an H-bond from a PAA-sensing Tyr_IVS6; (iii) the ammonium group is stabilized at the focus of P-helices; and (iv) the nitrile group binds to a Ca²⁺ ion coordinated by the selectivity filter glutamates in repeats III and IV. The latter feature can explain Ca²⁺ potentiation of PAA action and the presence of an electronegative atom at a similar position of potent PAA analogs. Tyr substitution of a Thr in IIIS5 is known to enhance action of devapamil and verapamil. Our models predict that the para-methoxy group in ring A of devapamil and verapamil accepts an H-bond from this engineered Tyr. The model explains structure-activity relationships of PAAs, effects of LTCC mutations on PAA potency, data on PAA access to LTCC, and Ca²⁺ potentiation of PAA action. Common and class-specific aspects of action of PAAs, dihydropyridines, and benzothiazepines are discussed in view of the repeat interface concept.L-type calcium channels (LTCCs)² are targets for different drugs. Benzo(thi)azepines (BTZs), dihydropyridines (DHPs), and phenylalkylamines (PAAs) constitute the three major classes of the LTCC ligands (for reviews, see Refs. 1 and 2). All of these ligands bind to overlapping binding sites in the pore-forming domain of the α₁ subunit, but each class demonstrates unique characteristics of action. Depending on their chemical structure, DHPs act as agonists or antagonists (3). All known PAAs and BTZs are antagonists, but they have different access pathways to their binding sites: external for BTZs (4, 5) and predominantly internal for PAAs (6). Clinical use of verapamil in treatments of hypertension and arrhythmias (7) had stimulated intensive electrophysiological, mutational, and pharmacological studies involving PAAs.The pore-forming domain of LTCC includes the pore-lining inner helices S6, the outer helices S5, and the P-loops from all four repeats of the α₁ subunit. According to mutational analyses, the PAA-binding site is located in the interface between repeats III and IV. In particular, residues in transmembrane helices IIIS5, IIIS6, and IVS6 and P-loops of repeats III and IV contribute to binding of PAAs (8–14).Structure-activity relationships of PAAs were intensively studied (15–17). A common feature of potent PAAs is the presence of two methoxylated aromatic rings (named A and B). The rings are connected by a flexible alkylamine chain with a nitrile and an isopropyl group at the chiral tetrasubstituted carbon atom, which is proximal to ring A. Ring B is proximal to the amino group (see Fig. 1).Open in a separate windowFIGURE 1.Structural formulae of PAAs.Despite the fact that some specific contacts between functional groups of PAAs and PAA-sensing residues (residues that, when mutated, affect action of PAAs) have been proposed (10, 14), the flexibility of the ligands did not allow the characterization of the binding mode and the general pattern of ligand-channel interactions. In the absence of such knowledge, it is hardly possible to provide a molecular basis for structure-activity relationships. The problem is further complicated by the dependence of PAA action on the functional state of the channel, the ionic environment, the transmembrane voltage, and other factors. For example, it is generally believed that PAAs bind to the open/inactivated channels with higher affinities than to the closed state (for review, see Ref 1). However, the molecular basis for this state dependence is unclear.Lipkind and Fozzard (18) docked devapamil in a KcsA-based homology model of the L-type Ca²⁺ channel. They suggested an angular conformation of the drug, with ring B extended into the III/IV repeat interface and ring A in the central cavity. They also suggested that the protonated amino group of devapamil interacts directly with the selectivity filter glutamates. This model explains the effect of some mutations, particularly those in the P-loops and IVS6. However, other important aspects of PAA action such as the role of the nitrile group, the Ca²⁺ potentiation effect, and the effects of mutations in IIIS6 and IIIS5 remain unexplained.The gap between the amount of experimental data on PAA action and the level of understanding of the atomic level mechanisms necessitates further studies. In the absence of x-ray structures of Ca²⁺ channels, molecular modeling is the only available approach to address the structural aspects of PAA-LTCC interactions. Recently, we proposed molecular models for the action of other classes of L-type channel ligands. In the BTZ-LTCC models (19), the main body of the ligands binds in the repeat interface, whereas the amino group protrudes into the inner pore, where it is stabilized by nucleophilic C-terminal ends of the pore helices. In the DHP-LTCC models (20), the ligands also bind in the interface between repeats III and IV, whereas the moieties that differ between agonist and antagonists extend to the pore. Both models suggest direct interactions between the ligands and a Ca²⁺ ion bound to the selectivity filter glutamates in repeats III and IV.In this work, we elaborate molecular models for PAA·LTCC complexes that agree with a large body of experimental data. We further discuss common and different aspects of action of different ligands on LTCC and propose that certain aspects of the ligand action may be relevant to other P-loop channels.     

Simultaneous analysis of physiological and electrical output changes in an operating microbial fuel cell with Shewanella oneidensis

Changes in metabolism and cellular physiology of facultative anaerobes during oxygen exposure can be substantial, but little is known about how these changes connect with electrical current output from an operating microbial fuel cell (MFC). A high‐throughput voltage based screening assay (VBSA) was used to correlate current output from a MFC containing Shewanella oneidensis MR‐1 to carbon source (glucose or lactate) utilization, culture conditions, and biofilm coverage over 250 h. Lactate induced an immediate current response from S. oneidensis MR‐1, with both air‐exposed and anaerobic anodes throughout the duration of the experiments. Glucose was initially utilized for current output by MR‐1 when cultured and maintained in the presence of air. However, after repeated additions of glucose, the current output from the MFC decreased substantially while viable planktonic cell counts and biofilm coverage remained constant suggesting that extracellular electron transfer pathways were being inhibited. Shewanella maintained under an anaerobic atmosphere did not utilize glucose consistent with literature precedents. Operation of the VBSA permitted data collection from nine simultaneous S. oneidensis MR‐1 MFC experiments in which each experiment was able to demonstrate organic carbon source utilization and oxygen dependent biofilm formation on a carbon electrode. These data provide the first direct evidence of complex cellular responses to electron donor and oxygen tension by Shewanella in an operating MFC at select time points. Biotechnol. Bioeng. 2009;103: 524–531. Published 2009 Wiley Periodicals, Inc.     

The multifaceted roles of the receptor tyrosine kinase ROS in development and cancer

The proto-oncogene receptor tyrosine kinase ROS was originally discovered through the identification of oncogenic variants isolated from tumors. These discoveries spearheaded a body of work aimed at elucidating the function of this evolutionarily conserved receptor in development and cancer. Through genetic and biochemical approaches, progress in the characterization of ROS points to distinctive roles in the program of epithelial cell differentiation during the development of a variety of organs. Although substantial, these advances remain hampered by the absence of an identified ligand, making ROS one of the last two remaining orphan receptor tyrosine kinases. Recent studies on the oncogenic activation of ROS as a result of different chromosomal rearrangements found in brain and lung cancers have shed light on the molecular mechanisms underlying ROS transforming activities. ROS and its oncogenic variants therefore constitute clinically relevant targets for cancer therapeutic intervention. This review highlights the various roles that this receptor plays in multiple system networks in normalcy and disease and points to future directions towards the elucidation of ROS function in the context of ligand identification, signaling pathways and clinical applications.     

Docking of calcium ions in proteins with flexible side chains and deformable backbones

A method of docking Ca²⁺ ions in proteins with flexible side chains and deformable backbones is proposed. The energy was calculated with the AMBER force field, implicit solvent, and solvent exposure-dependent and distance-dependent dielectric function. Starting structures were generated with Ca²⁺ coordinates and side-chain torsions sampled in 1000 Å³ cubes centered at the experimental Ca²⁺ positions. The energy was Monte Carlo-minimized. The method was tested on fourteen Ca²⁺-binding sites. For twelve Ca²⁺-binding sites the root mean square (RMS) deviation of the apparent global minimum from the experimental structure was below 1.3 and 1.7 Å for Ca²⁺ ions and side-chain heavy atoms, respectively. Energies of multiple local minima correlate with the RMS deviations from the X-ray structures. Two Ca²⁺-binding sites at the surface of proteinase K were not predicted, because of underestimation of Ca²⁺ hydration energy by the implicit-solvent method.     

Regulated expression of inhibitor of apoptosis protein 3 in the rat corpus luteum

We sought to investigate the role inhibitor of apoptosis proteins (IAPs) play in the life cycle of the corpus luteum (CL) of the rat. We isolated two clones with amino acid homology to rat IAP2 (BIRC 3) and three to rat IAP3 (rIAP3; BIRC 4). The expression of rIAP3 mRNA was examined in the rat CL during and after pregnancy, in Day 8 pregnant rats after 24-h treatment of gonadotropin-releasing hormone-agonist (GnRH-Ag), and in a CL organ culture model of spontaneous apoptosis in the absence of tropic support with and without superoxide dismutase. We used real-time RT-PCR to quantitate rIAP3 mRNA expression. Interestingly, a significant reduction in rIAP3 levels was seen at the time of CL regression in the course of natural pregnancy and the GnRH-Ag model. Surprisingly, rIAP3 mRNA levels in the CL organ culture model of spontaneous apoptosis failed to show significant changes, although TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end-labeling) reaction showed 30%-40% of the cells undergoing DNA fragmentation after 2 h in culture. In situ hybridization revealed that rIAP3 expression was localized to the cytoplasm of luteal and granulosa cells. These data clearly demonstrate both the presence of IAPs in the rat CL and the regulation of rIAP3 during in vivo apoptotic cell death, indicating a role for IAPs in the maintenance of CL function and demise.     

Apoptosis: a link between cancer genetics and chemotherapy

Defects in apoptosis underpin both tumorigenesis and drug resistance, and because of these defects chemotherapy often fails. Understanding the molecular events that contribute to drug-induced apoptosis, and how tumors evade apoptotic death, provides a paradigm to explain the relationship between cancer genetics and treatment sensitivity and should enable a more rational approach to anticancer drug design and therapy.