Diet composition and consumption rate in round goby (<Emphasis Type="BoldItalic">Neogobius melanostomus</Emphasis>) in its expansion phase in the Trent River,Ontario

We assessed density, gut fullness and prey composition of round goby (Neogobius melanostomus) from three areas in the Trent River (Ontario) representing areas of initial introduction and subsequent expansion. Round goby had been present at the area of original introduction since 2003, and by 2007/2008, their range had expanded upstream and downstream into the outermost reaches sampled in the study. Catch per unit angling effort in nearshore sites indicated that round goby density in the area of original introduction was more than double their density in the upstream expansion area and nearly three times the density in the downstream expansion area. Gut fullness index was lower in gobies from the area of original introduction than for those at the upstream and downstream edges of their expanded range. The most dramatic difference in diet composition was with dreissenids, where large gobies (≥70 mm) occupying the area of original introduction had almost no dreissenid biomass in their guts, whereas dreissenids were the predominant prey type in gobies occupying the two expansion areas. Post-hoc zebra mussel density in the area of original introduction was an order of magnitude lower than in the two expansion areas which, combined with the differences in stomach fullness and prey composition, suggest that local, density-related reduction of this prey type was occurring in the river.     

Synthesis,binding and bioactivity of γ-methylene γ-lactam ecdysone receptor ligands: Advantages of QSAR models for flexible receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or ‘attractors’. We describe the synthesis, in vitro binding and selected in vivo toxicity data for γ-methylene γ-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.     

Diversity versus disparity and the radiation of modern cetaceans

Modern whales are frequently described as an adaptive radiation spurred by either the evolution of various key innovations (such as baleen or echolocation) or ecological opportunity following the demise of archaic whales. Recent analyses of diversification rate shifts on molecular phylogenies raise doubts about this interpretation since they find no evidence of increased speciation rates during the early evolution of modern taxa. However, one of the central predictions of ecological adaptive radiation is rapid phenotypic diversification, and the tempo of phenotypic evolution has yet to be quantified in cetaceans. Using a time-calibrated molecular phylogeny of extant cetaceans and a morphological dataset on size, we find evidence that cetacean lineages partitioned size niches early in the evolutionary history of neocetes and that changes in cetacean size are consistent with shifts in dietary strategy. We conclude that the signature of adaptive radiations may be retained within morphological traits even after equilibrium diversity has been reached and high extinction or fluctuations in net diversification have erased any signature of an early burst of diversification in the structure of the phylogeny.     

Comparative Genomic Analysis of 60 Mycobacteriophage Genomes: Genome Clustering, Gene Acquisition, and Gene Size

Mycobacteriophages are viruses that infect mycobacterial hosts. Expansion of a collection of sequenced phage genomes to a total of 60—all infecting a common bacterial host—provides further insight into their diversity and evolution. Of the 60 phage genomes, 55 can be grouped into nine clusters according to their nucleotide sequence similarities, 5 of which can be further divided into subclusters; 5 genomes do not cluster with other phages. The sequence diversity between genomes within a cluster varies greatly; for example, the 6 genomes in Cluster D share more than 97.5% average nucleotide similarity with one another. In contrast, similarity between the 2 genomes in Cluster I is barely detectable by diagonal plot analysis. In total, 6858 predicted open-reading frames have been grouped into 1523 phamilies (phams) of related sequences, 46% of which possess only a single member. Only 18.8% of the phams have sequence similarity to non-mycobacteriophage database entries, and fewer than 10% of all phams can be assigned functions based on database searching or synteny. Genome clustering facilitates the identification of genes that are in greatest genetic flux and are more likely to have been exchanged horizontally in relatively recent evolutionary time. Although mycobacteriophage genes exhibit a smaller average size than genes of their host (205 residues compared with 315), phage genes in higher flux average only 100 amino acids, suggesting that the primary units of genetic exchange correspond to single protein domains.     

A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms

Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.A novel coronavirus (CoV), severe acute respiratory syndrome-CoV (SARS-CoV), is the causal agent of severe acute respiratory syndrome, which afflicted thousands of people worldwide in 2002 and 2003 (10, 39). SARS-CoV is an enveloped, single- and positive-strand RNA virus that encodes four major structural proteins: S, spike glycoprotein (GP); E, envelope protein; M, membrane glycoprotein; and N, nucleocapsid protein (46, 55). Similar to other coronaviruses, the S glycoprotein of the virus mediates the initial attachment of the virus to host cell receptors, angiotensin-converting enzyme 2 (ACE2) (44) and/or DC-SIGNR (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-related molecule; also CD209L or L-SIGN[liver/lymph node-SIGN]) (32) and subsequent fusion of the viral and cellular membranes to allow viral entry into susceptible target cells. The S glycoprotein of SARS-CoV (SARS-S) is a 1,255-amino-acid (aa) type I membrane glycoprotein (46) with 23 potential N-linked glycosylation sites (55). The S glycoproteins of some coronaviruses are translated as a large polypeptide that is subsequently proteolytically cleaved into two functional subunits, S1 (harboring the receptor-binding domain [RBD]) and S2 (containing the membrane fusion domains) (1, 31, 51), during biogenesis, but others are not. The S glycoprotein on mature SARS-CoV virions does not appear to be cleaved (50, 61), but sequence alignments with other coronavirus S glycoproteins allow definition of S1 and S2 regions (46, 55). More recently, studies showed the proteolysis of the S glycoprotein of SARS-CoV on mature virions by cathepsin L (CTSL) (28, 59), as well as trypsin (43, 61) and factor Xa (11), suggesting that a critical cleavage event may occur during cell entry rather than during virion biogenesis.Mannose-binding lectin (MBL; also known as mannose-binding or mannan-binding protein [MBP]) is a Ca²⁺-dependent (C-type) serum lectin that plays an important role in innate immunity by binding to carbohydrates on the surface of a wide range of pathogens (including bacteria, viruses, fungi, and protozoa) (8, 14, 18), where it activates the complement system or acts directly as an opsonin (30, 40, 52). In order to activate the complement system, MBL must be in complex with a group of MBL-associated serine proteases (MASPs), MASP-1, -2, and -3. Currently, only the role of MASP-2 in complement activation has been clearly defined (65). The MBL-MASP-2 complex cleaves C4 and C2 to form C3 convertase (C4bC2a), which, in turn, activates the downstream complement cascade. MBL is a pattern recognition molecule (9), and surface recognition is mediated through its C-terminal carbohydrate recognition domains (CRDs), which are linked to collagenous stems by a short coiled-coil of alpha-helices. MBL is a mixture of oligomers assembled from subunits that are formed from three identical polypeptide chains (9) and usually has two to six clusters of CRDs. Within each of the clusters, the carbohydrate-binding sites have a fixed orientation, which allows selective recognition of patterns of carbohydrate residues on the surfaces of a wide range of microorganisms (8, 14, 18). The concentration of MBL in the serum varies greatly and is affected by mutations of the promoter and coding regions of the human MBL gene (45). MBL deficiency is associated with susceptibility to various infections, as well as autoimmune, metabolic, and cardiovascular diseases, although MBL-deficient individuals are generally healthy (13, 37, 67).There are conflicting results with regard to the role of MBL in SARS-CoV infection (29, 42, 72, 73). While the association of MBL gene polymorphisms with susceptibility to SARS-CoV infection was reported in some studies (29, 73), Yuan et al. demonstrated that there were no significant differences in MBL genotypes and allele frequencies among SARS patients and controls (72). Ip et al. observed binding to, and inhibition of, SARS-CoV by MBL (29). However, in other studies, no binding of MBL to purified SARS-CoV S glycoprotein was detected (42).In this study, retroviral particles pseudotyped with SARS-S and in vitro assays were used to characterize the role of MBL in SARS-CoV infection. The data indicated that MBL selectively bound to SARS-S and mediated inhibition of viral infection in susceptible cell lines. Moreover, we identified a single N-linked glycosylation site, N330, on SARS-S that is critical for the specific interactions with MBL.     

Poly(A)-Binding Protein 1 Partially Relocalizes to the Nucleus during Herpes Simplex Virus Type 1 Infection in an ICP27-Independent Manner and Does Not Inhibit Virus Replication

Infection of cells by herpes simplex virus type 1 (HSV-1) triggers host cell shutoff whereby mRNAs are degraded and cellular protein synthesis is diminished. However, virus protein translation continues because the translational apparatus in HSV-infected cells is maintained in an active state. Surprisingly, poly(A)-binding protein 1 (PABP1), a predominantly cytoplasmic protein that is required for efficient translation initiation, is partially relocated to the nucleus during HSV-1 infection. This relocalization occurred in a time-dependent manner with respect to virus infection. Since HSV-1 infection causes cell stress, we examined other cell stress inducers and found that oxidative stress similarly relocated PABP1. An examination of stress-induced kinases revealed similarities in HSV-1 infection and oxidative stress activation of JNK and p38 mitogen-activated protein (MAP) kinases. Importantly, PABP relocalization in infection was found to be independent of the viral protein ICP27. The depletion of PABP1 by small interfering RNA (siRNA) knockdown had no significant effect on viral replication or the expression of selected virus late proteins, suggesting that reduced levels of cytoplasmic PABP1 are tolerated during infection.The lytic replication cycle of herpes simplex virus type 1 (HSV-1) can be divided into three phases, immediate-early (IE), early (E), and late (L), that occur in a coordinated sequential gene expression program. IE proteins can regulate E and L gene expression, which produces proteins involved in DNA replication, capsid production, and virion assembly. HSV infection results in host cell shutoff to facilitate the efficient production of viral proteins. First, mRNA is degraded by the virion-associated vhs protein, and then ICP27, a multifunctional regulator of gene expression, inhibits pre-mRNA splicing. As most viral mRNAs are intronless, this abrogates the production of stable cellular mRNAs that can be exported to the cytoplasm and compete for translation with viral mRNAs (44).HSV mRNAs are capped and polyadenylated and so are translated via a normal cap-dependent mechanism. Translation initiation, during which translationally active ribosomes are assembled, is a tightly regulated process (21). Eukaryotic initiation factor 4F (eIF4F) (composed of eIF4E, eIF4G, and eIF4A) that binds the cap at the 5′ end of the mRNA promotes the recruitment of the 40S ribosomal subunit and associated factors, including eIF2-GTP initiator tRNA. The recognition of the start codon then promotes large ribosomal subunit joining. Poly(A)-binding protein 1 (PABP1), which binds and multimerizes on mRNA poly(A) tails, enhances translation initiation through interactions with the eIF4G component of the eIF4F cap-binding complex (20, 29, 32, 51) to circularize the mRNA in a “closed-loop” conformation (24). Key protein-RNA and protein-protein interactions in the translation initiation complex are strengthened by this PABP1-mediated circularization (12).HSV-1 maintains active viral translation in the face of host translational shutoff. Infection activates protein kinase R (PKR), which phosphorylates eIF2α, resulting in translation inhibition. However, HSV-1 ICP34.5 redirects protein phosphatase 1α to reverse eIF2α phosphorylation, abrogating the block to translation (17, 38). In addition, the HSV-1 US11 protein inhibits PKR and may also block PKR-mediated eIF2α phosphorylation (40, 42). HSV-1 infection also enhances eIF4F assembly in quiescent cells by the phosphorylation and proteasome-mediated degradation of the eIF4E-binding protein (4E-BP), which, when hypophosphorylated, can negatively regulate eIF4F complex formation (54). However, ICP6 may also contribute to eIF4F assembly by binding to eIF4G (55). Finally, ICP6 is required for Mnk-1 phosphorylation of eIF4E, but the mechanisms behind this remain unclear (54). ICP27 has also been implicated in translation regulation during HSV infection (6, 8, 10, 30) and may also activate p38 mitogen-activated protein (MAP) kinase that can phosphorylate eIF4E (16, 59).PABP1 appears to be a common cellular target of RNA and DNA viruses. PABP1 can undergo proteolysis, intracellular relocalization, or modification of its interaction with other translation factors in response to infection. For example, poliovirus induces host cell shutoff by cleaving PABP1, thus disrupting certain PABP1-containing complexes (28, 29). The rotavirus NSP3 protein can displace PABP1 from translation initiation complexes (41). However, NSP3 also interacts with a cellular protein, RoXaN, which is required to relocate PABP1 to the nucleus (13). Similarly, the Kaposi''s sarcoma herpesvirus (KSHV) SOX protein plays a role in relocating PABP1, its cofactor in cellular mRNA decay, to the nucleus (33). Although steady-state levels of PABP1 are highest in the cytoplasm of normal cells, where it has cytoplasmic functions, it is a nucleocytoplasmic shuttling protein (1). However, it is unclear how PABP1 enters or exits the nucleus, as it contains neither a canonical nuclear export nor an import signal.Here we describe the loss of PABP1 from cap-binding complexes and the partial relocation of PABP1 to the nucleus in HSV-1-infected cells in a time-dependent manner. Relocation is specific for PABP1, as other translation factors remained in the cytoplasm. Cells undergo stress during HSV-1 infection, and analysis of a variety of cell stresses revealed that PABP relocalization was also observed upon oxidative stress. Paxillin, a potential PABP1 nuclear chaperone, was phosphorylated, and the paxillin-PABP1 interaction was reduced during virus infection. However, the interaction was weak and cell type dependent, indicating that other effectors of PABP1 relocation in the infected cell must exist. Recently, the HSV-1 ICP27 protein was suggested to alter the PABP1 cellular location (6). However, infections with ICP27-null mutant viruses clearly demonstrated that ICP27 is not required for PABP1 nuclear relocation in the context of infection. Although HSV-1 mRNAs are translated by a normal cap-dependent mechanism known to be enhanced by PABP1, small interfering RNA (siRNA) knockdown of PABP1 indicated that at late times of infection, the translation of certain virus late proteins tolerates very low levels of PABP1.     

Epitope-Specific Regulatory CD4 T Cells Reduce Virus-Induced Illness while Preserving CD8 T-Cell Effector Function at the Site of Infection

The role of epitope-specific regulatory CD4 T cells in modulating CD8 T-cell-mediated immunopathology during acute viral infection has not been well defined. In the murine model of respiratory syncytial virus (RSV) infection, CD8 T cells play an important role in both viral clearance and immunopathology. We have previously characterized two RSV epitope-specific CD4 T-cell responses with distinct phenotypic properties. One of them, the IA^bM₂₀₉-specific subset, constitutively expresses FoxP3 and modulates CD8 T-cell function in vitro. We show here that the IA^bM₂₀₉-specific CD4 T-cell response regulates CD8 T-cell function in vivo and is associated with diminished RSV-induced illness without affecting viral clearance at the site of infection. Achieving the optimal balance of regulatory and effector T-cell function is an important consideration for designing future vaccines.A subset of CD4 T cells with regulatory function (Treg) has been shown to play an important role in modulating adaptive immune responses. Natural Tregs are characterized by the expression of FoxP3 and participate in reducing the activation of CD8 T-cell responses in peripheral lymphoid organs (11, 20, 35). This modulation can diminish the ability of adaptive immune responses to control systemic infections (4). However, the presence of natural regulatory CD4 T cells can have a beneficial effect on immune-mediated pathology, particularly at the site of infection. Tregs have been shown to limit pulmonary inflammation and lung injury induced by pneumocystis infection (29) and to modulate herpes simplex virus-induced inflammatory lesions of the eye (46). Natural Tregs also reduce the symptoms of West Nile virus infections in both humans and mice; Treg-deficient mice were more likely to develop lethal infection (25). Viral infection can also induce antigen-specific CD4 T cells that express FoxP3 (27), and their role in protective immunity and immunopathology needs more detailed investigation.T lymphocytes are key components of adaptive immunity against respiratory syncytial virus (RSV) infection. Children with T-cell deficiencies have delayed virus clearance and are more susceptible to fatal RSV infection (10, 18). The absence of T cells infiltrating into lung is associated with fatal RSV infections in children without recognized underlying disease (49). In the murine model, CD8 T cells play a major role in RSV clearance, presumably through direct cytotoxicity to infected cells and the generation of immunocompetent molecules (2, 15, 43); depletion of CD8 T cells in mice results in delayed viral clearance (14). The CD8 T-cell response also induces immunopathology in primary infection of mice (15, 32, 48). Transferring high doses of CD8 T cells facilitates virus clearance but also causes hemorrhagic pneumonia and enhanced disease (6, 14). These studies demonstrate that while CD8 T cells are required for viral clearance, they are responsible for immunopathology. We have described the pattern of CD8 T-cell responses that occur in mice that are the F₁ hybrid (H-2^d/b) between BALB/c (H-2^d) and C57BL/6 (H-2^b) (39). These mice respond both to the well-characterized K^dM2₈₂ epitope (24) and to a more recently described D^bM₁₈₇ epitope (38). Both CD8 T-cell responses are dominant in the parent strains but assume a hierarchy (K^dM2₈₂ > D^bM₁₈₇) in the F₁ hybrid (39). This model infection allows the analysis of factors that determine T-cell response hierarchy and provides multiple endpoints for the assessment of factors that modify or regulate CD8 T-cell responses.We recently described epitope-specific CD4 T-cell responses distinguished by novel major histocompatibility complex (MHC) class II tetramers in RSV infection. The IA^bM₂₀₉-specific CD4 T cells have a high frequency of FoxP3 expression and suppress RSV-specific CD8 T-cell cytokine production in vitro (27). To investigate the regulatory role of IA^bM₂₀₉-specific CD4 T cells in vivo, we sought to determine how immunizing mice with the CD4 T-cell epitope peptide M₂₀₉ would affect the RSV-specific CD8 T-cell response. We show here that the IA^bM₂₀₉-specific CD4 T cells have a regulatory effect on the dominant CD8 T-cell response to RSV infection, reducing both the magnitude and effector cytokine production in peripheral lymphoid organs while allowing effector functions at the site of infection to clear virus with normal kinetics. Viral clearance was thus achieved with less illness.     

Development of an active elbow flexion simulator to evaluate joint kinematics with the humerus in the horizontal position

In-vitro simulation of active joint motion is useful to evaluate rehabilitation protocols and surgical procedures in the laboratory prior to their application in patients. To date, simulated active elbow flexion has been reliably achieved and well established only in the dependent position (humerus vertical with hand down). We have developed and evaluated the performance of a new elbow motion simulator capable of active flexion in the dependent, varus, valgus and horizontal positions. Muscle loading and motion control were achieved via a combination of motors and actuators attached to relevant tendons. Simulated active flexion was compared to passive flexion in terms of repeatability, motion pathways and joint laxity. The joint kinematics of active flexion were significantly more repeatable than passive flexion (p<0.05). Active flexion reduced varus–valgus joint laxity by 29% (supinated p<0.05) and 26% (pronated p<0.05) compared to passive flexion. Greater repeatability of simulated active flexion suggests that this mode of in-vitro testing should increase statistical power and decrease required sample sizes.     

Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism

Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34–318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34–318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34–318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34–318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.