bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Determinants of Autoproteolysis of the
Haemophilus influenzae
Hap Autotransporter
Authors: Kenjale Roma , Meng Guoyu , Fink Doran L. , Juehne Twyla , Ohashi Tomoo , Erickson Harold P. , Waksman Gabriel , St. Geme Joseph W.
Published: August 2009

Haemophilus influenzae
is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The
H. influenzae
Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the Hap
S
passenger domain, leaving the Hap
β
C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with
H. influenzae
colonization.
Association of Malaria-Induced Murine Pregnancy Failure with Robust Peripheral and Placental Cytokine Responses
Authors: Poovassery Jayakumar , Moore Julie M.
Published: August 2009

Malarial infection in nonimmune pregnant women is a major risk factor for pregnancy failure. The biological mechanisms that underlie malaria-associated fetal loss, however, are poorly understood.Plasmodium chabaudiAS infection during early pregnancy results in midgestational embryonic loss in naive C57BL/6 mice. To define the immunopathogenesis of this malaria-induced pregnancy compromise, cytokine production in plasma, spleen, and placenta cell culture supernatants during the first 11 days of infection and gestation was studied. In infected pregnant mice, systemic interleukin-1β and both systemic and splenic gamma interferon levels were elevated relative to those in uninfected pregnant mice, and gamma interferon was also robustly produced within the placenta 1 to 2 days before malaria-induced fetal loss. Although circulating tumor necrosis factor production was not affected by pregnancy or infection, circulating soluble tumor necrosis factor receptor II was highest in infected pregnant mice, particularly those undergoing abortion, but decreased at the placental level preceding abortion. Systemic levels of interleukin-10 were also high in infected mice at this time point, but this cytokine was not detected at the placental level. Histological examination revealed that trophoblast giant cells of aborting mice phagocytosed infected red blood cells and hemozoin. Furthermore, in vitro-cultured trophoblast cells isolated from embryos on day 7 of gestation phagocytosedP. chabaudiAS-infected red blood cells and secreted tumor necrosis factor. These results suggest that systemic and placenta-level proinflammatory antimalarial immune responses, in the absence of adequate and sustained counterregulatory mechanisms, contribute to pregnancy loss in this model.
Ce-Duox1/BLI-3 Generates Reactive Oxygen Species as a Protective Innate Immune Mechanism in
Caenorhabditis elegans
Authors: Chávez Violeta , Mohri-Shiomi Akiko , Garsin Danielle A.
Published: August 2009

Caenorhabditis elegans
was recently developed as a model system to study both pathogen virulence mechanisms and host defense responses. We previously demonstrated that
C. elegans
produces reactive oxygen species (ROS) in response to exposure to the important gram-positive nosocomial pathogen
Enterococcus faecalis
. We also presented evidence of oxidative stress and upregulation of stress responses after exposure to the pathogen. As in mammalian systems, this new work shows that production of ROS for innate immune functions occurs via an NADPH oxidase. Specifically, reducing expression of a dual oxidase, Ce-Duox1/BLI-3, causes a decrease in ROS production in response to
E. faecalis
. We also present evidence that reduction of expression of Ce-Duox1/BLI-3 increases susceptibility to this pathogen, specifically when expression is reduced in the intestine and the hypodermis. Ce-Duox1/BLI-3 was previously characterized as having a role in cuticle cross-linking. Two
C. elegans
mutants with point mutations in the peroxidase domain that exhibit severe cuticle defects were discovered to be unaffected in ROS production or pathogen susceptibility. These results demonstrate an important biological role for the peroxidase domain in cuticle cross-linking that is unrelated to ROS production. To further demonstrate the protective effects of the pathogen-induced ROS production, we show that antioxidants that scavenge ROS increase the sensitivity of the nematode to the infection, in stark contrast to their longevity-promoting effects under nonpathogenic conditions. In conclusion, we postulate that the generation of ROS by NADPH oxidases in the barrier epithelium is an ancient, highly conserved innate immune defense mechanism.
Identification of a Modular Pathogenicity Island That Is Widespread among Urease-Producing Uropathogens and Shares Features with a Diverse Group of Mobile Elements
Authors: Flannery Erika L. , Mody Lona , Mobley Harry L. T.
Published: August 2009

Pathogenicity islands (PAIs) are a specific group of genomic islands that contribute to genomic variability and virulence of bacterial pathogens. Using a strain-specific comparative genomic hybridization array, we report the identification of a 94-kb PAI, designated ICE
Pm1
, that is common to
Proteus mirabilis
,
Providencia stuartii
, and
Morganella morganii
. These organisms are highly prevalent etiologic agents of catheter-associated urinary tract infections (caUTI), the most common hospital acquired infection. ICE
Pm1
carries virulence factors that are important for colonization of the urinary tract, including a known toxin (
Proteus
toxic agglutinin) and the high pathogenicity island of
Yersinia
spp. In addition, this PAI shares homology and gene organization similar to the PAIs of other bacterial pathogens, several of which have been classified as mobile integrative and conjugative elements (ICEs). Isolates from this study were cultured from patients with caUTI and show identical sequence similarity at three loci within ICE
Pm1
, suggesting its transfer between bacterial genera. Screening for the presence of ICE
Pm1
among
P. mirabilis
colonizing isolates showed that ICE
Pm1
is more prevalent in urine isolates compared to
P. mirabilis
strains isolated from other body sites (
P
< 0.0001), further suggesting that it contributes to niche specificity and is positively selected for in the urinary tract.
A Periplasmic Thioredoxin-Like Protein Plays a Role in Defense against Oxidative Stress in
Neisseria gonorrhoeae
Authors: Achard Maud E. S. , Hamilton Amanda J. , Dankowski Tarek , Heras Begoña , Schembri Mark S. , Edwards Jennifer L. , Jennings Michael P. , McEwan Alastair G.
Published: August 2009

Thioredoxin-like proteins of the TlpA/ResE/CcmG subfamily are known to face the periplasm in gram-negative bacteria. Using the
tlpA
gene of
Bradyrhizobium japonicum
as a query, we identified a locus (NGO1923) in
Neisseria gonorrhoeae
that encodes a thioredoxin-like protein (NG_TlpA). Bioinformatics analysis indicated that the predicted NG_TlpA protein contained a cleavable signal peptide at the N terminus, and secondary structure analysis identified a thioredoxin fold with a helical insertion (∼25 residues), similar to that found in
B. japonicum
TlpA but absent in cytoplasmic thioredoxins. Biochemical characterization of a recombinant form of NG_TlpA revealed a standard redox potential (E
0
′) of −206 mV. This property and the observation that the oxidized form of the protein exhibited greater thermal stability than the reduced species indicated that NG_TlpA is a reducing thioredoxin and not an oxidizing thiol-disulfide oxidoreductase like DsbA. The thioredoxin activity of NG_TlpA was confirmed in an insulin disulfide reduction assay. A
tlpA
mutant of
N. gonorrhoeae
strain 1291 was found to be highly sensitive to oxidative killing by paraquat and hydrogen peroxide, indicating an antioxidant role for the NG_TlpA in this bacterium. The
tlpA
mutant also exhibited reduced intracellular survival in human primary cervical epithelial cells.
Herpes Simplex Virus Type 1 Production Requires a Functional ESCRT-III Complex but Is Independent of TSG101 and ALIX Expression
Authors: Pawliczek Tobias , Crump Colin M.
Published: August 2009

Herpes simplex virus type 1 (HSV-1) acquires its mature virus envelope by budding into the lumen of cytoplasmic membranous compartments carrying the viral glycoproteins. In a cellular context, a budding process with identical topology occurs during the formation of intraluminal vesicles in multivesicular bodies. The cellular machinery that mediates this budding process is composed of four protein complexes termed endosomal sorting complexes required for transport (ESCRTs) and several associated proteins, including the ATPase VPS4. We have recently shown that functional VPS4 is specifically required for the cytoplasmic envelopment of HSV-1. We now demonstrate that, consistent with a role of VPS4 in virus envelopment, dominant-negative ESCRT-III proteins potently block HSV-1 production. Retroviruses are known to recruit the ESCRT machinery by small peptide motifs termed late domains. These late domains interact with various ESCRT components and thereby promote ESCRT recruitment. The best-characterized late-domain interacting ESCRT proteins are ALIX and TSG101. The presence of potential ALIX and TSG101 binding sequence motifs in various structural HSV-1 proteins suggested a functional role of these proteins in HSV-1 envelopment. We therefore used a set of dominant-negative proteins, as well as RNA interference, to characterize the contribution of ALIX and TSG101 to HSV-1 production. Interestingly, despite the strict requirement for a functional ESCRT-III complex, our data suggest that HSV-1 production is independent of ALIX and TSG101 expression. In line with these data, we also find that ESCRT-III proteins and VPS4A/B are specifically incorporated into mature HSV-1 virions.
Biological Signature Characteristics of Primary Isolates from Human Immunodeficiency Virus Type 1 Group O in Ex Vivo Human Tonsil Histocultures
Authors: Geuenich Silvia , Kaderali Lars , Allespach Ina , Sertel Serkan , Keppler Oliver T.
Published: August 2009

Human immunodeficiency virus type 1 (HIV-1) group M viruses have achieved a global distribution, while HIV-1 group O viruses are endemic only in particular regions of Africa. Here, we evaluated biological characteristics of group O and group M viruses in ex vivo models of HIV-1 infection. The replicative capacity and ability to induce CD4 T-cell depletion of eight group O and seven group M primary isolates were monitored in cultures of human peripheral blood mononuclear cells and tonsil explants. Comparative and longitudinal infection studies revealed HIV-1 group-specific activity patterns: CCR5-using (R5) viruses from group M varied considerably in their replicative capacity but showed similar levels of cytopathicity. In contrast, R5 isolates from group O were relatively uniform in their replicative fitness but displayed a high and unprecedented variability in their potential to deplete CD4 T cells. Two R5 group O isolates were identified that cause massive depletion of CD4 T cells, to an extent comparable to CXCR4-using viruses and not documented for any R5 isolate from group M. Intergroup comparisons found a five- to eightfold lower replicative fitness of isolates from group O than for isolates from group M yet a similar overall intrinsic pathogenicity in tonsil cultures. This study establishes biological ex vivo characteristics of HIV-1 group O primary isolates. The current findings challenge the belief that a grossly reduced replicative fitness or inherently impaired cytopathicity of viruses from this group underlies their low global prevalence.
A Gammaherpesvirus Ubiquitin-Specific Protease Is Involved in the Establishment of Murine Gammaherpesvirus 68 Infection
Authors: Gredmark-Russ Sara , Isaacson Marisa K. , Kattenhorn Lisa , Cheung Evelyn J. , Watson Nicki , Ploegh Hidde L.
Published: August 2009

Murine gammaherpesvirus 68 (MHV-68) contains a ubiquitin (Ub)-specific cysteine protease (USP) domain embedded within the large tegument protein ORF64, as do all other herpesviruses. The biological role of this protease is still unclear, but for the alphaherpesvirus Marek's disease virus, its USP is involved in T-cell lymphoma formation. We here study the role of the MHV-68 USP, encoded by ORF64. By constructing a mutant virus with a single cysteine-to-alanine replacement in the active site of ORF64, we demonstrate that the USP activity of ORF64 is abolished. The mutant virus replicates less efficiently in vitro, and plaque size is reduced compared to that of a revertant virus. Electron microscopy of infected cells did not reveal any obvious differences in virion morphogenesis or differences in egress for the mutant and revertant viruses. Intraperitoneal infection of C57/BL6 mice demonstrates that the mutant virus is generally cleared by day 7, indicating a role for the USP in the persistence of MHV-68 infection or efficient replication. However, the USP activity in MHV-68 is unlikely to be involved in the establishment of latency or reactivation, since we observed no significant difference in viral DNA genome copy number in the spleen or in the number of cells that reactivate MHV-68 from latency. Our results for MHV-68 ORF64 are consistent with an enzymatic function of the tegument protein that is beneficial to the virus during acute infection, particularly in vivo.
DC-SIGN Mediates Cell-Free Infection and Transmission of Human T-Cell Lymphotropic Virus Type 1 by Dendritic Cells
Authors: Jain Pooja , Manuel Sharrón L. , Khan Zafar K. , Ahuja Jaya , Quann Kevin , Wigdahl Brian
Published: August 2009

Despite the susceptibility of dendritic cells (DCs) to human T-cell lymphotropic virus type 1 (HTLV-1) infection and the defined role of these cells in disease pathogenesis, the mechanisms of viral binding to DCs have not been fully delineated. Recently, a glucose transporter, GLUT-1, heparan sulfate proteoglycans (HSPGs), and neuropilin-1 (NRP-1) were demonstrated to facilitate HTLV-1 entry into T cells. DCs express their own array of antigen receptors, the most important being the DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin (DC-SIGN) with respect to retrovirus binding. Consequently, the role of DC-SIGN and other HTLV-1 attachment factors was analyzed in viral binding, transmission, and productive infection using monocyte-derived DCs (MDDCs), blood myeloid DCs, and B-cell lines expressing DC-SIGN. The relative expression of DC-SIGN, GLUT-1, HSPGs, and NRP-1 first was examined on both DCs and B-cell lines. Although the inhibition of these molecules reduced viral binding, HTLV-1 transmission from DCs to T cells was mediated primarily by DC-SIGN. DC-SIGN also was shown to play a role in the infection of MDDCs as well as model B-cell lines. The HTLV-1 infection of MDDCs also was achieved in blood myeloid DCs following the enhancement of virus-induced interleukin-4 production and subsequent DC-SIGN expression in this cell population. This study represents the first comprehensive analysis of potential HTLV-1 receptors on DCs and strongly suggests that DC-SIGN plays a critical role in HTLV-1 binding, transmission, and infection, thereby providing an attractive target for the development of antiretroviral therapeutics and microbicides.
The Pre-Transmembrane Domain of the
Autographa californica
Multicapsid Nucleopolyhedrovirus GP64 Protein Is Critical for Membrane Fusion and Virus Infectivity
Authors: Li Zhaofei , Blissard Gary W.
Published: August 2009

The envelope glycoprotein, GP64, of the baculovirus
Autographa californica
multicapsid nucleopolyhedrovirus (AcMNPV) is a class III viral fusion protein that mediates pH-triggered membrane fusion during virus entry. Viral fusion glycoproteins from many viruses contain a short region in the ectodomain and near the transmembrane domain, referred to as the pre-transmembrane (PTM) domain. In some cases, the PTM domain is rich in aromatic amino acids and plays an important role in membrane fusion. Although the 23-amino-acid (aa) PTM domain of AcMNPV GP64 lacks aromatic amino acids, we asked whether this region might also play a significant role in membrane fusion. We generated alanine scanning and single and multiple amino acid substitutions in the GP64 PTM domain. We specifically focused on amino acid positions conserved between baculovirus GP64 and thogotovirus GP75 proteins, as well as hydrophobic and charged amino acids. For each PTM-modified construct, we examined trimerization, cell surface localization, and membrane fusion activity. Membrane merger and pore formation were also examined. We identified eight aa positions that are important for membrane fusion activity. Critical positions were not clustered in the linear sequence but were distributed throughout the PTM domain. While charged residues were not critical or essential, three hydrophobic amino acids (L465, L476, and L480) played an important role in membrane fusion activity and appear to be involved in formation of the fusion pore. We also asked whether selected GP64 constructs were capable of rescuing a
gp64
null AcMNPV virus. These studies suggested that several conserved residues (T463, G460, G462, and G474) were not required for membrane fusion but were important for budding and viral infectivity.
VP5* Rearranges when Rotavirus Uncoats
Authors: Yoder Joshua D. , Trask Shane D. , Vo Phuoc T. , Binka Mawuena , Feng Ningguo , Harrison Stephen C. , Greenberg Harry B. , Dormitzer Philip R.
Published: August 2009

Trypsin primes rotavirus for efficient infectivity by cleaving the spike protein, VP4, into VP8* and VP5*. A recombinant VP5* fragment has a trimeric, folded-back structure. Comparison of this structure with virion spikes suggests that a rearrangement, analogous to those of enveloped virus fusion proteins, may mediate membrane penetration by rotavirus during entry. To detect this inferred rearrangement of virion-associated authentic VP5*, we raised conformation-specific monoclonal antibodies against the recombinant VP5* fragment in its putative post-membrane penetration conformation. Using one of these antibodies, we demonstrate that rotavirus uncoating triggers a conformational change in the cleaved VP4 spike to yield rearranged VP5*.
Homodimerization of the Meq Viral Oncoprotein Is Necessary for Induction of T-Cell Lymphoma by Marek's Disease Virus
Authors: Brown Andrew C. , Smith Lorraine P. , Kgosana Lydia , Baigent Susan J. , Nair Venugopal , Allday Martin J.
Published: August 2009

Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B Meq
BZIP/BZIP
) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B Meq
Hom/Hom
). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV.
Theiler's Virus Infection Induces a Predominant Pathogenic CD4+T Cell Response to RNA Polymerase in Susceptible SJL/J Mice
Authors: Jin Young-Hee , Kang Bongsu , Kim Byung S.
Published: August 2009

Theiler's murine encephalomyelitis virus (TMEV)-induced immune-mediated demyelinating disease in susceptible mouse strains has been extensively investigated as a relevant model for human multiple sclerosis. Previous investigations of antiviral T-cell responses focus on immune responses to viral capsid proteins, while virtually nothing is reported on immune responses to nonstructural proteins. In this study, we have identified noncapsid regions recognized by CD4+T cells from TMEV-infected mice using an overlapping peptide library. Interestingly, a greater number of CD4+T cells recognizing an epitope (3D21-36) of the 3D viral RNA polymerase, in contrast to capsid epitopes, were detected in the CNS of TMEV-infected SJL mice, whereas only a minor population of CD4+T cells from infected C57BL/6 mice recognized this region. The effects of preimmunization and tolerization with these epitopes on the development of demyelinating disease indicated that capsid-specific CD4+T cells are protective during the early stages of viral infection, whereas 3D21-36-specific CD4+T cells exacerbate disease development. Therefore, protective versus pathogenic CD4+T-cell responses directed to TMEV appear to be epitope dependent, and the differences in CD4+T-cell responses to these epitopes between susceptible and resistant mice may play an important role in the resistance or susceptibility to virally induced demyelinating disease.
Attenuated Strains of Influenza A Viruses Do Not Induce Degradation of RNA Polymerase II
Authors: Rodriguez Ariel , Pérez-González Alicia , Hossain M. Jaber , Chen Li-Mei , Rolling Thierry , Pérez-Breña Pilar , Donis Ruben , Kochs Georg , Nieto Amelia
Published: August 2009

We have previously shown that infection with laboratory-passaged strains of influenza virus causes both specific degradation of the largest subunit of the RNA polymerase II complex (RNAP II) and inhibition of host cell transcription. When infection with natural human and avian isolates belonging to different antigenic subtypes was examined, we observed that all of these viruses efficiently induce the proteolytic process. To evaluate whether this process is a general feature of nonattenuated viruses, we studied the behavior of the influenza virus strains A/PR8/8/34 (PR8) and the cold-adapted A/Ann Arbor/6/60 (AA), which are currently used as the donor strains for vaccine seeds due to their attenuated phenotype. We have observed that upon infection with these strains, degradation of the RNAP II does not occur. Moreover, by runoff experiments we observe that PR8 has a reduced ability to inhibit cellular mRNA transcription. In addition, a hypervirulent PR8 (hvPR8) variant that multiplies much faster than standard PR8 (lvPR8) in infected cells and is more virulent in mice than the parental PR8 virus, efficiently induces RNAP II degradation. Studies with reassortant viruses containing defined genome segments of both hvPR8 and lvPR8 indicate that PA and PB2 subunits individually contribute to the ability of influenza virus to degrade the RNAP II. In addition, recently it has been reported that the inclusion of PA or PB2 from hvPR8 in lvPR8 recombinant viruses, highly increases their pathogenicity. Together, the data indicate that the capacity of the influenza virus to degrade RNAP II and inhibit the host cell transcription machinery is a feature of influenza A viruses that might contribute to their virulence.
Human Immunodeficiency Virus Type 1 Is Trapped by Acidic but Not by Neutralized Human Cervicovaginal Mucus
Authors: Lai Samuel K. , Hida Kaoru , Shukair Shetha , Wang Ying-Ying , Figueiredo Anna , Cone Richard , Hope Thomas J. , Hanes Justin
Published: August 2009

To reliably infect a primate model for human immunodeficiency virus (HIV), ∼10,000-fold more virus must be delivered vaginally than intravenously. However, the vaginal mechanisms that help protect against HIV are poorly understood. Here, we report that human cervicovaginal mucus (CVM), obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse more than 1,000-fold more slowly than it does in water. Lactobacilli acidify CVM to pH ∼4 by continuously producing lactic acid. At this acidic pH, we found that lactic acid, but not HCl, abolished the negative surface charge on HIV without lysing the virus membrane. In contrast, in CVM neutralized to pH 6 to 7, as occurs when semen temporarily neutralizes the vagina, HIV maintained its native surface charge and diffused only 15-fold more slowly than it would in water. Thus, methods that can maintain both a high lactic acid content and acidity for CVM during coitus may contribute to both vaginal and penile protection by trapping HIV before it can reach target cells. Our results reveal that CVM likely plays an important but currently unappreciated role in decreasing the rate of HIV sexual transmission.
Crystal Structure of Porcine Reproductive and Respiratory Syndrome Virus Leader Protease Nsp1α
Authors: Sun Yuna , Xue Fei , Guo Yu , Ma Ming , Hao Ning , Zhang Xuejun C. , Lou Zhiyong , Li Xuemei , Rao Zihe
Published: August 2009

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV), a positive-strand RNA virus that belongs to theArteriviridaefamily ofNidovirales, has been identified as the causative agent of PRRS. Nsp1α is the amino (N)-terminal protein in a polyprotein encoded by the PRRSV genome and is reported to be crucial for subgenomic mRNA synthesis, presumably by serving as a transcription factor. Before functioning in transcription, nsp1α proteolytically releases itself from nsp1β. However, the structural basis for the self-releasing and biological functions of nsp1α remains elusive. Here we report the crystal structure of nsp1α of PRRSV (strain XH-GD) in its naturally self-processed form. Nsp1α contains a ZF domain (which may be required for its biological function), a papain-like cysteine protease (PCP) domain with a zinc ion unexpectedly bound at the active site (which is essential for proteolytic self-release of nsp1α), and a carboxyl-terminal extension (which occupies the substrate binding site of the PCP domain). Furthermore, we determined the exact location of the nsp1α self-processing site at Cys-Ala-Met180↓Ala-Asp-Val by use of crystallographic data and N-terminal amino acid sequencing. The crystal structure also suggested an incisself-processing mechanism for nsp1α. Furthermore, nsp1α appears to have a dimeric architecture both in solution and as a crystal, with a hydrophilic groove on the molecular surface that may be related to nsp1α's biological function. Compared with existing structure and function data, our results suggest that PRRSV nsp1α functions differently from other reported viral leader proteases, such as that of foot-and-mouth disease.
Review of current and emerging therapies in acute ischemic stroke
Authors: Novakovic R , Toth G , Purdy P D
Published: August 2009

The statistics for stroke in the USA reads like a familiar ad slogan cited in most papers pertaining to acute ischemic stroke (AIS). Stroke is the third leading cause of death in the USA. While stroke ranks third among all causes of death, behind diseases of the heart and cancer, it is the leading cause of serious long-term disability in the USA.1Approximately 795 000 people, 87% of whom are ischemic, suffer from stroke each year in the USA.2That means that on average, every 40 seconds someone within the USA develops a stroke. For 2009 the combined direct and indirect cost of stroke, from hospitalization and rehabilitation to institutionalization, is estimated at $68.9 billion within the USA.2
Current status of the management of symptomatic intracranial atherosclerotic disease: the rationale for a randomized trial of medical therapy and intracranial stenting
Authors: Fiorella D , Turan T N , Derdeyn C P , Chimowitz M I
Published: August 2009

Stenting and Aggressive Medical Management for Preventing Recurrent stroke in Intracranial Stenosis (SAMMPRIS) is a randomized trial comparing aggressive medical management alone with aggressive medical management in combination with angioplasty and stenting using the Gateway–Wingspan system in patients with symptomatic, high-grade, intracranial stenosis. This trial represents a landmark in the maturation of the field of neurointervention, establishing a foundation for evidenced-based practice. We review the natural history of symptomatic intracranial stenosis when treated medically, the available interventional therapies and the rationale for the design of the SAMMPRIS trial.
Case volumes of intra-arterial and intravenous treatment of ischemic stroke in the USA
Authors: Hirsch J A , Yoo A J , Nogueira R G , Verduzco L A , Schwamm L H , Pryor J C , Rabinov J D , González R G
Published: August 2009

Background and purpose

Ischemic stroke is a major cause of disability and death in the USA. Intravenous tissue plasminogen activator (t-PA) remains underutilized. With the development of newer intra-arterial reperfusion therapies, there is increased opportunity to address the more devastating large-vessel occlusions. We seek to identify the numbers of patients with stroke treated with intravenous and intra-arterial therapies, as well as to estimate the potential number of intra-arterial cases in the foreseeable future.

Methods

We performed a literature search to determine case volumes of intravenous t-PA use. We extrapolated the current case volume of intra-arterial stroke therapies from the numbers of cases in which the Merci retrieval device was used. In order to estimate the potential numbers of intra-arterial stroke cases, we characterized the percentage of patients with stroke who received intra-arterial therapy at two leading stroke centers. We applied these percentages to the numbers of patients with stroke seen at the top 100, 200 and 500 stroke centers by volume.

Results

The rate of intravenous t-PA use is 2.4–3.6%, resulting in 15 000–22 000 cases/year in the USA. The estimated case volume of intra-arterial therapies is 3500–7200 in 2006. Based on data from St. Luke's Brain and Stroke Institute and Massachusetts General Hospital, approximately 5–20% of patients with ischemic stroke can be treated with intra-arterial therapies. Extrapolating this to the top 500 stroke centers by volume, the potential number of intra-arterial cases in the USA is 10 400–41 500/year.

Conclusion

Based on the current numbers of intra-arterial cases, our theoretical model identifies a potential for significant growth of this stroke therapy.
Long term clinical and angiographic outcomes with the Wingspan stent for treatment of symptomatic 50–99% intracranial atherosclerosis: single center experience in 51 cases
Authors: Wolfe T J , Fitzsimmons B F , Hussain S I , Lynch J R , Zaidat O O
Published: August 2009

Background and aim

Two independent post-approval registries have reported favorable periprocedural and short term outcomes with the use of the Wingspan stent for treatment of intracranial arterial stenosis. Data on long term clinical and imaging outcomes after Wingspan stent placement are limited.

Methods

All patients treated with the Wingspan stent in a single academic center from January 2006 to February 2008 were identified. Data on stenting indication, severity of stenosis, technical success, re-stenosis and clinical outcome were collected.

Results

51 patients were treated with the Wingspan stent system for a symptomatic intracranial atherosclerotic stenosis of 50–99%. The technical success rate was 98%. The mean pre- and post-stent stenoses were 73 (11)% and 21 (7)%. Any stroke or death within 24 h of the procedure occurred in 1/51 (2%). The frequency of any stroke or death within 30 days or ipsilateral stroke beyond 30 days was 5/51 (10.0%) at a mean follow-up time of 14.6 months (range 8–30). The frequency of ≥ 50% re-stenosis on follow-up imaging was 7/29 (24%) at 8.6 (4.4) months (range 3–20); all were detected on the initial imaging within 3–6 months, and only one was symptomatic.

Conclusion

The use of the Wingspan stent in patients with ≥50% symptomatic intracranial stenosis is associated with good long term clinical outcome. One stroke occurred after the first 30 days, suggesting a significant stabilization of the adverse event rate after the first month.
Incidence and time course of carotid in-stent restenosis in a consecutive series of 295 patients
Authors: Heck D
Published: August 2009

Background and purpose

In-stent restenosis (ISR) is a potentially preventable cause of stroke in carotid artery stent (CAS) patients. Understanding the frequency and timing of ISR would be useful in developing optimal protocols for carotid stent surveillance. The time course and frequency of moderate and severe ISR in our single institution prospective registry of CAS procedures is reported here.

Methods

Data were collected prospectively from 296 consecutive elective CAS procedures. Doppler surveillance was performed at 1, 6 and 12 months and annually thereafter in some cases. Moderate ISR (>50%) was defined as a peak systolic velocity (PSV) > 200 cm/s. Severe ISR (>70%) was defined as PSV > 200 cm/sandend diastolic velocity > 125 cm/s or internal carotid artery/common carotid artery ratio >4. Patients with severe ISR underwent digital subtraction angiography for confirmation and possible retreatment.

Results

Clinical follow-up at 1 year was 98%. Clinical and ultrasound follow-up at 1 month was 100%, at 6 months 96% and at 1 year 91%. The incidence of all ISR at 6 months was 8%; 5% moderate and 3% severe. Two patients had asymptomatic occlusions at 6 months (0.8%). Patients with moderate ISR at 6 months did not progress to severe ISR. There were two strokes caused by stent thrombosis, one acute and one delayed.

Conclusions

Doppler surveillance is important for detecting ISR after CAS procedures. Severe ISR (>70%) should be retreated in most cases as ISR may progress to occlusion. Asymptomatic patients with moderate ISR (50–69%) at 6 months can be safely followed conservatively. Progression of ISR after 6–12 months is uncommon over a 2–3 year period.
Performance and training standards for endovascular ischemic stroke treatment
Authors:   Writing Group for the American Academy of Neurology, AANS/CNS Cerebrovascular Section, Society of NeuroInterventional Surgery, and the Society of Vascular & Interventional Neurology, Meyers P M , Schumacher H C , Alexander M J , Derdeyn C P , Furlan A J , Higashida R T , Moran C J , Tarr R W , Heck D V , Hirsch J A , Jensen M E , Linfante I , McDougall C G , Nesbit G M , Rasmussen P A , Tomsick T A , Wechsler L R , Wilson J R , Zaidat O O
Published: August 2009

Stroke is the third leading cause of death in the USA, Canada, Europe, and Japan. According to the American Heart Association and the American Stroke Association, there are now 750 000 new strokes that occur each year, resulting in 200 000 deaths, or 1 of every 16 deaths, per year in the USA alone. Endovascular therapy for patients with acute ischemic stroke is an area of intense investigation. The American Stroke Association has given a qualified endorsement of intra-arterial thrombolysis in selected patients. Intra-arterial thrombolysis has been studied in two randomized trials and numerous case series. Although two devices have been granted FDA approval with an indication for mechanical stroke thrombectomy, none of these thrombectomy devices has demonstrated efficacy for the improvement of patient outcomes. The purpose of the present document is to define what constitutes adequate training to perform neuroendovascular procedures in patients with acute ischemic stroke and what performance standards should be adopted to assess outcomes. These guidelines have been written and approved by multiple neuroscience societies which historically have been directly involved in the medical, surgical and endovascular care of patients with acute stroke. The participating member organizations of the Neurovascular Coalition involved in the writing and endorsement of this document are the Society of NeuroInterventional Surgery, the American Academy of Neurology, the American Association of Neurological Surgeons/Congress of Neurological Surgeons Cerebrovascular Section, and the Society of Vascular & Interventional Neurology.
Regression models for public health surveillance data: a simulation study
Authors: Kim H , Kriebel D
Published: August 2009

Objectives:

Poisson regression is now widely used in epidemiology, but researchers do not always evaluate the potential for bias in this method when the data are overdispersed. This study used simulated data to evaluate sources of overdispersion in public health surveillance data and compare alternative statistical models for analysing such data. If count data are overdispersed, Poisson regression will not correctly estimate the variance. A model called negative binomial 2 (NB2) can correct for overdispersion, and may be preferred for analysis of count data. This paper compared the performance of Poisson and NB2 regression with simulated overdispersed injury surveillance data.

Methods:

Monte Carlo simulation was used to assess the utility of the NB2 regression model as an alternative to Poisson regression for data which had several different sources of overdispersion. Simulated injury surveillance datasets were created in which an important predictor variable was omitted, as well as with an incorrect offset (denominator). The simulations evaluated the ability of Poisson regression and NB2 to correctly estimate the true determinants of injury and their confidence intervals.

Results:

The NB2 model was effective in reducing overdispersion, but it could not reduce bias in point estimates which resulted from omitting a covariate which was a confounder, nor could it reduce bias from using an incorrect offset. One advantage of NB2 over Poisson for overdispersed data was that the confidence interval for a covariate was considerably wider with the former, providing an indication that the Poisson model did not fit well.

Conclusion:

When overdispersion is detected in a Poisson regression model, the NB2 model should be fit as an alternative. If there is no longer overdispersion, then the NB2 results may be preferred. However, it is important to remember that NB2 cannot correct for bias from omitted covariates or from using an incorrect offset.
A cross-sectional study among detergent workers exposed to liquid detergent enzymes
Authors: van Rooy F G B G J , Houba R , Palmen N , Zengeni M M , Sander I , Spithoven J , Rooyackers J M , Heederik D J J
Published: August 2009

Objectives:

To investigate sensitisation and respiratory health among workers who produce liquid detergent products and handle liquid detergent enzymes.

Methods:

We performed a cross-sectional study among 109 eligible workers of a detergent products plant. 108 were interviewed for respiratory and allergic symptoms and 106 blood samples were taken from them to examine sensitisation to enzymes. Those sensitised to ⩾1 enzymes were referred for clinical evaluation. Workers and representatives were interviewed to characterise exposure qualitatively and estimate exposure semi-quantitatively. Workers were classified into three exposure groups with varying exposure profiles to enzymes, based on frequency, duration, and level of exposure.

Results:

Workers were exposed to proteases, α-amylase, lipase and cellulase. The highest exposures occurred in the mixing area. Liquid spills with concentrated enzyme preparations and leakage of enzymes during weighing, transportation and filling were causing workplace contaminations and subsequently leading to both dermal and inhalation exposure for workers.

Workers with the highest exposures reported significantly more work-related symptoms of itching nose (prevalence ratio (PR) = 4.2, 95% CI 1.5 to 12.0) and sneezing (PR = 4.0, 95% CI 1.5 to 10.8) and marginally significant more symptoms of wheezing (PR = 2.9, 95% CI 0.9 to 8.7) compared with the least exposed group.

Fifteen workers (14.2%) were sensitised to ⩾1 enzymes. A marginally statistically significant gradient in sensitisation across the exposure categories was found (p = 0.09). There was a clinical case of occupational asthma and two others with probable occupational rhinitis.

Conclusions:

Workers exposed to liquid detergent enzymes are at risk of developing sensitisation (14%) and respiratory allergy.
Domoic Acid—A Neurotoxic Amino Acid Produced by the Marine Diatom Nitzschia pungens in Culture
Authors: Rao D. V. Subba , Quilliam M. A. , Pocklington R.
Published: August 2009

During late 1987, an outbreak of poisoning resulting from the ingestion of cultivated blue mussels (Mytilus edulis) from a localized area in eastern Canada (Cardigan Bay, Prince Edward Island) was associated with massive blooms of Nitzschia pungens, a widely distributed diatom not previously known to produce toxins; human fatalities resulted. Here we provide proof that the causative agent, domoic acid, is indeed produced by this diatom. Although no domoic acid could be detected (<2 ng∙mL−1) in culture medium (FE) prepared from Cardigan River water, it was found in cultures of Nitzschia pungens grown in this medium at concentrations ranging from 0.03 to 0.8 pg∙cell−1 in various separate cultures harvested for chemical analysis 7–68 d after inoculation.
Response of Wild Rainbow (Salmo gairdneri) and Cutthroat Trout (S. clarki) to Stocked Rainbow Trout in Fertile and infertile Streams
Authors: Petrosky C E. , Bjornn T. C.
Published: August 2009

Wild rainbow trout (Salmo gairdneri) and cutthroat trout (S. clarki) were unaffected by stocking of catchable-size rainbow trout in two Idaho streams, except at the highest stocking rates, and even then the effects were limited. In the infertile stream, stocking 50 or 150 trout per section (doubling or tripling the density) did not reduce the abundance of wild cutthroat trout. Wild trout abundance declined at a faster rate in an unreplicated section stocked with 500 trout than in unstocked sections. In the fertile stream, stocking 50 or 100 hatchery trout in sections containing 26–120 similar-sized wild trout did not increase the dispersion or reduce the abundance, growth, or survival rates of wild rainbow trout. When we stocked 400 trout (100 on four dates) in sections containing 32–53 tagged wild trout of similar size, the summer mortality rate of wild trout was higher in stocked than in unstocked sections; the other parameters were not significantly different.
Functional Maturity in Small Male Snow Crabs (Chionoecetes opilio)
Authors: Ennis G. P. , Hooper R. G. , Taylor D. M.
Published: August 2009

The mean size of male snow crabs (Chionoecetes opilio) in sexual pairs during the annual spring breeding migration to shallow water in Bonne Bay, Newfoundland, decreased from 118.6 mm carapace width (CW) in 1983 to 100.3 mm in 1987. This decrease is due to an increase in males <95 mm CW participating from 1.5% in 1983 to 32.3% in 1987. This change appears to have resulted from a reduced abundance of commercial size [Formula: see text] males due to a rapid development of an illegal fishery on this previously unfished population and, as a consequence, less competition between males for possession of females. The percentages of spermathecae containing new spermatophores for females paired with males <95 mm CW (67%) and those with males [Formula: see text] (79%) were not significantly different. We assumed that each female with new spermatophores had recently mated with the male with which it was paired. Observations on selected pairs in captivity showed that males <95 mm CW are capable of mating with both primiparous and muitiparous females. Our results indicate that small mature males can replace large males in breeding activity in a snow crab population. We conclude that in the male-only snow crab fishery in Atlantic Canada with a minimum legal size of 95 mm CW, population reproductive potential is maintained at a high level despite high exploitation rates on males [Formula: see text].
Contaminant Effects on Ovarian Development in English Sole (Parophrys vetulus) from Puget Sound, Washington
Authors: Johnson Lyndal L. , Casillas Edmundo , Collier Tracy K. , McCain Bruce B. , Varanasi Usha
Published: August 2009

In a study evaluating the effects of exposure to xenobiotic compounds on ovarian development in English sole (Parophrys vetulus), prespawning females were sampled from four sites in Puget Sound, Washington, during the 1986 and 1987 spawning seasons. Two sampling sites had high concentrations of xenobiotic compounds in the sediment, while the other sites were less contaminated. The following factors associated with ovarian maturation were measured: ovarian developmental stage, ovarian atresia, gonadosomatic index, plasma estradiol, and plasma vitellogenin as estimated from alkali-labile phosphorus. Contaminant exposure was assessed by measuring concentrations of fluorescent aromatic compounds in the bile, hepatic aryl hydrocarbon hydroxylase (AHH) activity, and hepatic polychlorinated biphenyl levels, and liver tissue was examined histologically for the presence of suspected toxicopathic lesions. Female English sole from the heavily contaminated sites were significantly less likely to undergo gonadal recrudescence and had lower mean levels of plasma estradiol than females from the less contaminated sites. The risk of inhibited gonadal recrudescence was significantly increased in sole with elevated hepatic AHH activity, and AHH activity was also significantly negatively correlated with plasma estradiol level. These findings suggest that contaminant exposure may interfere with ovarian development in female English sole.
Behaviour and Microhabitat of Young Atlantic Salmon (Salmo salar) during Winter
Authors: Cunjak Richard A.
Published: August 2009

Underwater observations at two sites along a small Nova Scotian river were carried out between December and April (water temperature range = 0.5–7.0 °C) to describe the winter microhabitat of young Atlantic salmon (Salmo salar). Salmon (5–15 cm fork length) were consistently found hiding beneath rocks (mean diameter = 16.8–23.0 cm) in riffle-run habitats where mean water depths were 40.9–48.9 cm and mean water velocities were 38.7–45.7 cm∙s−1. Many of the salmon were found overwintering within redd excavations. "Home stones" were distributed closer to midstream than to river banks and where sediment compaction was minimal. Monthly collections of fish (ages 1 and 2) indicated that feeding continued over winter. The data suggest a nocturnal activity pattern and photonegative response by young salmon during winter.