bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Multiplex Reverse Transcription-PCR Typing of Influenza A and B Virus, and Subtyping of Influenza A Virus into H1, 2, 3, 5, 7, 9, N1 (Human), N1 (Animal), N2, and N7, Including Typing of Novel Swine Origin Influenza A (H1N1) Virus, during the 2009 Outbreak in Milwaukee, Wisconsin
Authors: He Jie , Bose Michael E. , Beck Eric T. , Fan Jiang , Tiwari Sagarika , Metallo Jacob , Jurgens Lisa A. , Kehl Sue C. , Ledeboer Nathan , Kumar Swati , Weisburg William , Henrickson Kelly J.
Published: July 2009

A large outbreak of novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) infection in Milwaukee, WI, occurred in late April 2009. We had recently developed a rapid multiplex reverse transcription-PCR enzyme hybridization assay (FluPlex) to determine the type (A or B) and subtype (H1, H2, H3, H5, H7, H9, N1 [human], N1 [animal], N2, or N7) of influenza viruses, and this assay was used to confirm the diagnoses for the first infected patients in the state. The analytical sensitivity was excellent at 1.5 to 116 copies/reaction, or 10
−3
to 10
−1
50% tissue culture infective doses/ml. The testing of all existing hemagglutinin and neuraminidase subtypes of influenza A virus and influenza B virus (41 influenza virus strains) and 24 common respiratory pathogens showed only one low-level H3 cross-reaction with an H10N7 avian strain and only at 5.2 × 10
6
copies/reaction, not at lower concentrations. Comparisons of the FluPlex results with results from multiple validated in-house molecular assays, CDC-validated FDA-approved assays, and gene sequencing demonstrated 100% positive agreement for the typing of 179 influenza A viruses and 3 influenza B viruses, the subtyping of 110 H1N1 (S-OIV; N1 [animal]), 62 H1N1 (human), and 6 H3N2 (human) viruses, and the identification of 24 negative clinical samples and 100% negative agreement for all viruses tested except H1N1 (human) (97.7%). The small number of false-positive H1N1 (human) samples most likely represent increased sensitivity over that of other in-house assays, with four of four results confirmed by the CDC's influenza virus subtyping assay. The FluPlex is a rapid, inexpensive, sensitive, and specific method for the typing and subtyping of influenza viruses and demonstrated outstanding utility during the first 2 weeks of an S-OIV infection outbreak. Methods for rapid detection and broad subtyping of influenza viruses, including animal subtypes, are needed to address public concern over the emergence of pandemic strains. Attempts to automate this assay are ongoing.
Comparative Evaluation of the AccuProbe Group B Streptococcus Culture Test, the BD GeneOhm Strep B Assay, and Culture for Detection of Group B Streptococci in Pregnant Women
Authors: Scicchitano Lisa M. , Bourbeau Paul P.
Published: July 2009

We compared a rigorous culture method with the Gen-Probe AccuProbe Group B Streptococcus Culture Test (APGB) and the BD GeneOhm StrepB assay (GOSB) for the detection of group B streptococci (GBS) from an 18- to 24-h LIM broth. Culture (95.3%) and GOSB (95.3%) were more sensitive than APGB (86.5%) for the detection of GBS.
Molecular Characterization of
Capnocytophaga canimorsus
and Other Canine
Capnocytophaga
spp. and Assessment by PCR of Their Frequencies in Dogs
Authors: van Dam Alje P. , van Weert Angela , Harmanus Celine , Hovius K. Emiel , Claas Eric C. J. , Reubsaet Frans A. G.
Published: July 2009

Capnocytophaga canimorsus
can be a virulent pathogen, whereas
C. cynodegmi
is of low virulence. Heterogeneity within these species, their frequency in dogs, and pathogenicity factors are largely unknown. Strains from blood cultures from patients presumptively identified as
C. canimorsus
(
n
= 25) and as
C. cynodegmi
by
rrs
analysis (
n
= 4), blood cultures from dogs (
n
= 8), blood cultures from cats (
n
= 2), and cultures from swabs from dog mouths (
n
= 53) were analyzed. PCR-restriction fragment length polymorphism (PCR-RFLP), a species-specific PCR on
rpoB
, and
rrs
sequencing were used. All 29 strains from human blood cultures could be grouped into three PCR-RFLP types. One included the
C. canimorsus
type strain, and the other types were closely related. Two canine strains were
C. canimorsus
and grouped into the least common RLFP pattern group. Five were
C. cynodegmi
and clustered with the reference strain. One canine and both feline strains were distinct. Four human strains that presumptively had been identified as
C. cynodegmi
by RNA gene sequence analysis clustered with the
C. canimorsus
strains by both PCR-RFLP and the sequence-specific PCR of the
rpoB
gene.
C. canimorsus
DNA was present in 73% (range, 61 to 85%) of dogs' mouths, and
C. cynodegmi
DNA was present in 96% (range, 94 to 100%) of dogs' mouths. As defined by
rpoB
PCR-RFLP and by PCRs using specific primers, all strains from human blood were
C. canimorsus.
The sequencing of
rrs
genes suggested the presence of different gene copies in a few strains, indicating that the method is less appropriate for species identification. Both species are present in the majority of dogs. Additional
Capnocytophaga
species occur in dogs' and cats' mouths.
Conjunctivitis with Regional Lymphadenopathy in a Trainee Microbiologist
Authors: Alcolea Adela , Suarez María Jesús , Lizasoain Manuel , Tejada Pilar , Chaves Fernando , Palenque Elia
Published: July 2009

We report a case of conjunctival tuberculosis in a trainee microbiologist caused by direct inoculation. The resident strain was analyzed by DNA fingerprinting, and an identical pattern was found in an isolate from sputum handled by the resident. After 6 months of treatment, the patient was cured.
Mapping of Functional Domains in Herpesvirus Saimiri Complement Control Protein Homolog: Complement Control Protein Domain 2 Is the Smallest Structural Unit Displaying Cofactor and Decay-Accelerating Activities
Authors: Singh Akhilesh K. , Yadav Viveka Nand , Pyaram Kalyani , Mullick Jayati , Sahu Arvind
Published: July 2009

Herpesvirus saimiri encodes a functional homolog of human regulator-of-complement-activation proteins named CCPH that inactivates complement by accelerating the decay of C3 convertases and by serving as a cofactor in factor I-mediated inactivation of their subunits C3b and C4b. Here, we map the functional domains of CCPH. We demonstrate that short consensus repeat 2 (SCR2) is the minimum domain essential for classical/lectin pathway C3 convertase decay-accelerating activity as well as for factor I cofactor activity for C3b and C4b. Thus, CCPH is the first example wherein a single SCR domain has been shown to display complement regulatory functions.
Human Ubc9 Contributes to Production of Fully Infectious Human Immunodeficiency Virus Type 1 Virions
Authors: Jaber Tareq , Bohl Christopher R. , Lewis Gentry L. , Wood Charles , West John T. , Weldon Robert A.
Published: July 2009

Ubc9 was identified as a cellular protein that interacts with the Gag protein of Mason-Pfizer monkey virus. We show here that Ubc9 also interacts with the human immunodeficiency virus type 1 (HIV-1) Gag protein and that their interaction is important for virus replication. Gag was found to colocalize with Ubc9 predominantly at perinuclear puncta. While cells in which Ubc9 expression was suppressed with RNA interference produced normal numbers of virions, these particles were 8- to 10-fold less infectious than those produced in the presence of Ubc9. The nature of this defect was assayed for dependence on Ubc9 during viral assembly, trafficking, and Env incorporation. The Gag-mediated assembly of virus particles and protease-mediated processing of Gag and Gag-Pol were unchanged in the absence of Ubc9. However, the stability of the cell-associated Env glycoprotein was decreased and Env incorporation into released virions was altered. Interestingly, overexpression of the Ubc9
trans
-dominant-negative mutant C93A, which is a defective E2-SUMO-1 conjugase, suggests that this activity may not be required for interaction with Gag, virion assembly, or infectivity. This finding demonstrates that Ubc9 plays an important role in the production of infectious HIV-1 virions.
The Human Papillomavirus (HPV) E6* Proteins from High-Risk, Mucosal HPVs Can Direct Degradation of Cellular Proteins in the Absence of Full-Length E6 Protein
Authors: Pim David , Tomaić Vjekoslav , Banks Lawrence
Published: July 2009

The E6 oncoproteins from high-risk mucosotrophic human papillomaviruses (HPVs) target a range of cellular proteins for proteasome-mediated degradation. Apart from the tumor suppressor p53 and proapoptotic Bcl-2 family member Bak, many targets contain class 1 PDZ domains and are involved in cell junction stability and signaling. The targeting mechanism is considered to function by the E6 protein acting as an adaptor molecule linking a cellular ubiquitin ligase to the target protein. In each case, whether the target is the p53 tumor suppressor or a member of the group of PDZ domain-containing targets, this mechanism relies on a direct interaction between E6 and its cellular target. This study focuses on the impact of the HPV type 18 (HPV-18) E6*I protein on the stability of Akt, Dlg, MAGI-1, MAGI-2, and Scribble. We show that HPV-18 E6* expression can downregulate the expression levels of Akt, Dlg, and Scribble in the absence of full-length HPV-18 E6 protein. The reduction in Dlg levels by E6* is independent of transcription and does not require a direct interaction between the two proteins although the proteasome pathway is involved. Further, we provide evidence that activation of certain signal transduction pathways has a profound effect on the targeting of Dlg by E6* and suggest that high-risk HPV E6 oncoproteins can target certain substrates both directly and indirectly through the E6* proteins and may cooperate in their degradation.
Kaposi's Sarcoma-Associated Herpesvirus Encodes a Viral Deubiquitinase
Authors: González Carlos M. , Wang Ling , Damania Blossom
Published: July 2009

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically linked to Kaposi's sarcoma, primary effusion lymphomas, and multicentric Castleman's disease. Like other herpesviruses, KSHV can exist in either a lytic or a latent phase during its life cycle. We report that the lytic protein encoded by KSHV open reading frame 64 (Orf64) is a viral deubiquitinase (DUB) enzyme capable of deubiquitinating cellular proteins in vitro and in vivo. Orf64 DUB activity is effective against lysine 48 (K48)- and lysine 63 (K63)-linked ubiquitin chains. Thus, KSHV Orf64 is a viral DUB that does not show specificity toward K48 or K63 ubiquitin linkages. Orf64 DUB activity lies within the first 205 residues of the protein, and deubiquitination is dependent on a cysteine at position 29, since mutation of this residue ablated this activity. Cell fractionation studies revealed that the N terminus and the full-length protein localized to both the nuclear and cytoplasmic compartments. The function of Orf64 was tested by short interfering RNA (siRNA) knockdown studies on latently infected cells that were induced into lytic replication. We found that depletion of Orf64 by siRNA resulted in decreased viral lytic transcription and lytic protein expression. These experiments indicate that Orf64 plays a role in KSHV lytic replication.
Tomato Bushy Stunt Virus
Recombination Guided by Introduced MicroRNA Target Sequences
Authors: Kumar Pavan , Uratsu Sandra , Dandekar Abhaya , Falk Bryce W.
Published: July 2009

Previously we described
Tomato bushy stunt virus
(TBSV) vectors, which retained their capsid protein gene and were engineered with magnesium chelatase (ChlH) and phytoene desaturase (PDS) gene sequences from
Nicotiana benthamiana
. Upon plant infection, these vectors eventually lost the inserted sequences, presumably as a result of recombination. Here, we modified the same vectors to also contain the plant miR171 or miR159 target sequences immediately 3′ of the silencing inserts. We inoculated
N. benthamiana
plants and sequenced recombinant RNAs recovered from noninoculated upper leaves. We found that while some of the recombinant RNAs retained the microRNA (miRNA) target sites, most retained only the 3′ 10 and 13 nucleotides of the two original plant miRNA target sequences, indicating in planta miRNA-guided RNA-induced silencing complex cleavage of the recombinant TBSV RNAs. In addition, recovered RNAs also contained various fragments of the original sequence (ChlH and PDS) upstream of the miRNA cleavage site, suggesting that the 3′ portion of the miRNA-cleaved TBSV RNAs served as a template for negative-strand RNA synthesis by the TBSV RNA-dependent RNA polymerase (RdRp), followed by template switching by the RdRp and continued RNA synthesis resulting in loss of nonessential nucleotides.
Diverse Cross-Reactive Potential and Vβ Gene Usage of an Epitope-Specific Cytotoxic T-Lymphocyte Population in Monkeys Immunized with Diverse Human Immunodeficiency Virus Type 1 Env Immunogens
Authors: Hulot Sandrine L. , Seaman Michael S. , Sen Pritha , Autissier Patrick A. , Manuel Edwin R. , Letvin Norman L.
Published: July 2009

An ideal human immunodeficiency virus type 1 (HIV-1) vaccine would elicit potent cellular and humoral immune responses that recognize diverse strains of the virus. In the present study, combined methodologies (flow cytometry, Vβ repertoire analysis, and complementarity-determining region 3 sequencing) were used to determine the clonality of CD8+T lymphocytes taking part in the recognition of variant epitope peptides elicited in Mamu-A*01-positive rhesus monkeys immunized with vaccines encoding diverse HIV-1 envelopes (Envs). Monkeys immunized with clade B Envs generated CD8+T lymphocytes that cross-recognized both clade B- and clade C-p41A epitope peptides using a large degree of diversity in Vβ gene usage. However, with two monkeys immunized with clade C Env, one monkey exhibited p41A-specific cytotoxic T-lymphocytes (CTL) with the capacity for cross-recognition of variant epitopes, while the other monkey did not. These studies demonstrate that the cross-reactive potential of variant p41A epitope peptide-specific CTL populations can differ between monkeys that share the same restricting major histocompatibility complex class I molecule and receive the same vaccine immunogens.
Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling
Authors: Cornillez-Ty Cromwell T. , Liao Lujian , Yates John R. , Kuhn Peter , Buchmeier Michael J.
Published: July 2009

The severe acute respiratory syndrome coronavirus (SARS-CoV) generates 16 nonstructural proteins (nsp's) through proteolytic cleavage of a large precursor protein. Although several nsp's exhibit catalytic activities that are important for viral replication and transcription, other nsp's have less clearly defined roles during an infection. In order to gain a better understanding of their functions, we attempted to identify host proteins that interact with nsp's during SARS-CoV infections. For nsp2, we identified an interaction with two host proteins, prohibitin 1 (PHB1) and PHB2. Our results suggest that nsp2 may be involved in the disruption of intracellular host signaling during SARS-CoV infections.
Growth-Promoting Properties of Epstein-Barr Virus EBER-1 RNA Correlate with Ribosomal Protein L22 Binding
Authors: Houmani Jennifer L. , Davis Christopher I. , Ruf Ingrid K.
Published: July 2009

The Epstein-Barr virus (EBV)-encoded RNAs, EBER-1 and EBER-2, are highly abundant noncoding nuclear RNAs expressed during all forms of EBV latency. The EBERs have been shown to impart significant tumorigenic potential upon EBV-negative Burkitt lymphoma (BL) cells and to contribute to the growth potential of other B-cell lymphoma-, gastric carcinoma-, and nasopharyngeal carcinoma-derived cell lines. However, the mechanisms underlying this EBER-dependent enhancement of cell growth potential remain to be elucidated. Here we focused on the known interaction between EBER-1 and the cellular ribosomal protein L22 and the consequences of this interaction with respect to the growth-promoting properties of the EBERs. L22, a component of 60S ribosomal subunits, binds three sites on EBER-1, and a substantial fraction of available L22 is relocalized from nucleoli to the nucleoplasm in EBV-infected cells. To investigate the hypothesis that EBER-1-mediated relocalization of L22 in EBV-infected cells is critical for EBER-dependent functions, we investigated whether EBER-1 expression is necessary and sufficient for nucleoplasmic retention of L22. Following demonstration of this, we utilized RNA-protein binding assays and fluorescence localization studies to demonstrate that mutation of the L22 binding sites on EBER-1 prevents L22 binding and inhibits EBER-1-dependent L22 relocalization. Finally, the in vivo consequence of preventing L22 relocalization in EBER-expressing cells was examined in soft agar colony formation assays. We demonstrate that BL cells expressing mutated EBER-1 RNAs rendered incapable of binding L22 have significantly reduced capacity to enhance cell growth potential relative to BL cells expressing wild-type EBERs.
Dominant CD8+T-Lymphocyte Responses Suppress Expansion of Vaccine-Elicited Subdominant T Lymphocytes in Rhesus Monkeys Challenged with Pathogenic Simian-Human Immunodeficiency Virus
Authors: Manuel Edwin R. , Yeh Wendy W. , Seaman Michael S. , Furr Kathryn , Lifton Michelle A. , Hulot Sandrine L. , Autissier Patrick , Letvin Norman L.
Published: July 2009

Emerging data suggest that a cytotoxic T-lymphocyte response against a diversity of epitopes confers greater protection against a human immunodeficiency virus/simian immunodeficiency virus infection than does a more focused response. To facilitate the creation of vaccine strategies that will generate cellular immune responses with the greatest breadth, it will be important to understand the mechanisms employed by the immune response to regulate the relative magnitudes of dominant and nondominant epitope-specific cellular immune responses. In this study, we generated dominant Gag p11C- and subdominant Env p41A-specific CD8+T-lymphocyte responses inMamu-A*01+rhesus monkeys through vaccination with plasmid DNA and recombinant adenovirus encoding simian-human immunodeficiency virus (SHIV) proteins. Infection of vaccinatedMamu-A*01+rhesus monkeys with a SHIV Gag Δp11C mutant virus generated a significantly increased expansion of the Env p41A-specific CD8+T-lymphocyte response in the absence of secondary Gag p11C-specific CD8+T-lymphocyte responses. These results indicate that the presence of the Gag p11C-specific CD8+T-lymphocyte response following virus challenge may exert suppressive effects on primed Env p41A-specific CD8+T-lymphocyte responses. These findings suggest that immunodomination exerted by dominant responses during SHIV infection may diminish the breadth of recall responses primed during vaccination.
Role of the Cytoplasmic Tail Amino Acid Sequences of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein in Virion Incorporation, Cell Fusion, and Pathogenicity
Authors: Kim Shin-Hee , Yan Yongqi , Samal Siba K.
Published: July 2009

To determine the role of amino acid sequences of the hemagglutinin-neuraminidase (HN) cytoplasmic tail in Newcastle disease virus (NDV) replication and pathogenicity, we generated recombinant NDVs with a deletion or point mutation in the N-terminal cytoplasmic tail. The first 2-amino-acid deletion in the cytoplasmic tail did not affect the biological characteristics of NDV. However, a 4-amino-acid deletion and the substitution of alanine for serine at position 6 affected cell fusion, pathogenicity, and colocalization of the HN and M proteins of NDV, indicating that these residues of the HN cytoplasmic tail are critical for its specific incorporation into virions.
Intracellular Localization of the Pseudorabies Virus Large Tegument Protein pUL36
Authors: Möhl Britta S. , Böttcher Sindy , Granzow Harald , Kuhn Jana , Klupp Barbara G. , Mettenleiter Thomas C.
Published: July 2009

Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the
Herpesviridae
, complex with pUL37, and form part of the capsid-associated “inner” tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.
Chimeric L1-L2 Virus-Like Particles as Potential Broad-Spectrum Human Papillomavirus Vaccines
Authors: Schellenbacher Christina , Roden Richard , Kirnbauer Reinhard
Published: July 2009

The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freund's adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freund's adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.
Genetic and Pharmacologic Alteration of Cathepsin Expression Influences Reovirus Pathogenesis
Authors: Johnson Elizabeth M. , Doyle Joshua D. , Wetzel J. Denise , McClung R. Paul , Katunuma Nobuhiko , Chappell James D. , Washington M. Kay , Dermody Terence S.
Published: July 2009

The cathepsin family of endosomal proteases is required for proteolytic processing of several viruses during entry into host cells. Mammalian reoviruses utilize cathepsins B (Ctsb), L (Ctsl), and S (Ctss) for disassembly of the virus outer capsid and activation of the membrane penetration machinery. To determine whether cathepsins contribute to reovirus tropism, spread, and disease outcome, we infected 3-day-old wild-type (wt),Ctsb−/−,Ctsl−/−, andCtss−/−mice with the virulent reovirus strain T3SA+. The survival rate ofCtsb−/−mice was enhanced in comparison to that of wt mice, whereas the survival rates ofCtsl−/−andCtss−/−mice were diminished. Peak titers at sites of secondary replication in all strains of cathepsin-deficient mice were lower than those in wt mice. Clearance of the virus was delayed inCtsl−/−andCtss−/−mice in comparison to the levels for wt andCtsb−/−mice, consistent with a defect in cell-mediated immunity in mice lacking cathepsin L or S. Cathepsin expression was dispensable for establishment of viremia, but cathepsin L was required for maximal reovirus growth in the brain. Treatment of wt mice with an inhibitor of cathepsin L led to amelioration of reovirus infection. Collectively, these data indicate that cathepsins B, L, and S influence reovirus pathogenesis and suggest that pharmacologic modulation of cathepsin activity diminishes reovirus disease severity.
Zanamivir-Resistant Influenza Viruses with a Novel Neuraminidase Mutation
Authors: Hurt Aeron C. , Holien Jessica K. , Parker Michael , Kelso Anne , Barr Ian G.
Published: July 2009

The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.
Similarities and Differences in Antagonism of Neuron Alpha/Beta Interferon Responses by Venezuelan Equine Encephalitis and Sindbis Alphaviruses
Authors: Yin Jun , Gardner Christina L. , Burke Crystal W. , Ryman Kate D. , Klimstra William B.
Published: July 2009

Venezuelan equine encephalitis virus (VEEV) is highly virulent in adult laboratory mice, while Sindbis virus (SINV) is avirulent regardless of dose or inoculation route, dependent upon functioning alpha/beta interferon (IFN-α/β) responses. We have examined each virus' resistance to and/or antagonism of IFN-α/β responses in neurons, a cell type targeted by both viruses in mice, by infecting IFN-α/β-treated or untreated primary cultures with viruses or virus-derived replicons that lacked the structural proteins. Priming with IFN-α/β prior to infection revealed that VEEV replication and progeny virion production were resistant to an established antiviral state while those of SINV were more sensitive. Postinfection IFN-α/β treatment revealed that phosphorylation of STAT1 and STAT2 was partially blocked by infection with either virus, dependent upon expression of nonstructural proteins (nsP), but not structural proteins (sP). However, inhibition of STAT phosphorylation by VEEV replicons was not correlated with inhibition of IFN-stimulated gene (ISG) mRNA induction, yet ISG induction was inhibited when sP were present. Host translation was inhibited by VEEV nsP even when cells were pretreated with IFN-α/β. SINV blocked ISG induction and translation, associated with nsP-mediated shutoff of macromolecular synthesis, but both activities were sensitive to IFN-α/β pretreatment. We conclude that both VEEV and SINV limit ISG induction in infected neurons through shutoff of host transcription and translation but that inhibition by VEEV is more resistant to IFN-α/β priming. Likewise, both viruses inhibit IFN receptor-initiated signaling, although the effect upon host responses is not clear. Finally, VEEV appears to be more resistant to effectors of the preestablished antiviral state.
Small Molecule Targets Env for Endoplasmic Reticulum-Associated Protein Degradation and Inhibits Human Immunodeficiency Virus Type 1 Propagation
Authors: Jejcic Alenka , Daniels Robert , Goobar-Larsson Laura , Hebert Daniel N. , Vahlne Anders
Published: July 2009

Human immunodeficiency virus type 1 (HIV-1) is dependent on its envelope glycoprotein (Env) to bind, fuse, and subsequently infect a cell. We show here that treatment of HIV-1-infected cells with glycyl-prolyl-glycine amide (GPG-NH2), dramatically reduced the infectivity of the released viral particles by decreasing their Env incorporation. The mechanism of GPG-NH2was uncovered by examining Env expression and maturation in treated cells. GPG-NH2treatment was found to affect Env by significantly decreasing its steady-state levels, its processing into gp120/gp41, and its mass by inducing glycan removal in a manner dependent on its native signal sequence and the proteasome. Therefore, GPG-NH2negatively impacts Env maturation, facilitating its targeting for endoplasmic reticulum-associated protein degradation, where Env is deglycosylated en route to its degradation. These findings illustrate that nontoxic drugs such as GPG-NH2, which can selectively target glycoproteins to existing cellular degradation pathways, may be useful for pathogen therapy.
Fusion genes in epithelial neoplasia
Authors: Zhang H , Oliveira A M
Published: July 2009

Recurrent chromosomal rearrangements with the formation of fusion genes have been traditionally associated with haematological and mesenchymal neoplasms. This view has cultivated the erroneous impression that the oncogenic mechanisms present in these tumours are fundamentally different from those found in epithelial neoplasms. In spite of the fact that, as a group, most epithelial tumours apparently show a higher degree of chromosomal instability, a careful and critical analysis of relatively older findings and several more recent discoveries has shown that some common mechanistic denominators occur in all forms of neoplasms independent of their line of differentiation. The aim of this review is to briefly review and discuss the occurrence, mechanisms and biological relevance of chromosomal rearrangements and fusion genes in epithelial neoplasia, with a special emphasis in thyroid, head and neck, kidney, breast, prostate and lung neoplasms.
Characterisation of organisational issues in paediatric clinical ethics consultation: a qualitative study
Authors: Opel D J , Wilfond B S , Brownstein D , Diekema D S , Pearlman R A
Published: July 2009

Background:

The traditional approach to resolving ethics concerns may not address underlying organisational issues involved in the evolution of these concerns. This represents a missed opportunity to improve quality of care “upstream”. The purpose of this study was to understand better which organisational issues may contribute to ethics concerns.

Methods:

Directed content analysis was used to review ethics consultation notes from an academic children’s hospital from 1996 to 2006 (N  =  71). The analysis utilised 18 categories of organisational issues derived and modified from published quality improvement protocols.

Results:

Organisational issues were identified in 68 of the 71 (96%) ethics consult notes across a range of patient settings and reasons for consultation. Thirteen of the 18 categories of organisational issues were identified and there was a median of two organisational issues per consult note. The most frequently identified organisational issues were informal organisational culture (eg, collective practices and approaches to situations with ethical dimensions that are not guided by policy), policies and procedures (eg, staff knows policy and/or procedural guidelines for an ethical concern but do not follow it) and communication (eg, communication about critical information, orders, or hand-offs repeatedly does not occur among services).

Conclusions:

Organisational issues contribute to ethical concerns that result in clinical ethics consults. Identifying and addressing organisational issues such as informal culture and communication may help decrease the recurrence of future similar ethics concerns.
“They just don’t get it!” When family disagrees with expert opinion
Authors: Ho A
Published: July 2009

The notions of “expert” and “expertise” imply that some people have more credibility than others on certain matters. While expert authority is often taken for granted, there are questions as to whether expert power in some cases can be a form of epistemic oppression. Informed by bedside disagreements between family and clinicians as well as feminist discussions of epistemic oppression, this paper argues for a commitment to epistemic humility and the adoption of a two-way collaborative approach between clinicians and families that can help to enhance professionals’ own understanding of their theoretical framework and also promote responsive patient care.
Factors influencing attitudes towards medical confidentiality among Swiss physicians
Authors: Elger B S
Published: July 2009

Medical confidentiality is a core concept of professionalism and should be an integral part of pregraduate and postgraduate medical education. The aim of our study was to define the factors influencing attitudes towards patient confidentiality in everyday situations in order to define the need for offering further education to various subgroups of physicians. All internists and general practitioners who were registered members of the association of physicians in Geneva or who were working in the department of internal medicine or in the medical polyclinic of the University Hospital of Geneva in 2004 received a standardised questionnaire. Physicians were asked to indicate for seven vignettes whether a violation of confidentiality had occurred and whether the violation was not important, important or serious (scores 1–3; no violation  =  0). 508 completed questionnaires were returned (participation rate 55%). Physicians who had worked in the hospital for more than 20 years identified violations of confidentiality more often than physicians with less hospital experience. Binary logistic regression showed that ethics education, total years of professional experience, being an internist, having a private practice, the length of working in private practice and gender were factors associated with correct identification of violations and their severity. However, each factor played a specific role only for single cases or a small number of situations (Cronbach α <0.6). Postgraduate education programs on confidentiality should be offered to a wide range of physicians and should address specific hypothetical situations in which there is a risk of avoidable breaches of confidentiality.
Federal provider conscience regulation: unconscionable
Authors: Card R
Published: July 2009

This paper argues that the provider conscience regulation recently put into place in the USA is misguided. The rule is too broad in the scope of protection it affords, and its conception of what constitutes assistance in the performance of an objectionable procedure reveals that it is unworkable in practice. Furthermore, the regulation wrongly treats refusal of other reproductive services as on a par with conscientious objection to participation in abortion. Finally, the rule allows providers to refuse even to discuss “objectionable” options with patients and serves to protect discriminatory refusals of medical care. For all of these reasons, this regulation is unwise.
EXPERIMENTAL METHODOLOGIES FOR LARGE-SCALE SYSTEMS: A SURVEY
Authors: GUSTEDT JENS , JEANNOT EMMANUEL , QUINSON MARTIN
Published: July 2009

The increasing complexity of available infrastructures with specific features (caches, hyperthreading, dual core, etc.) or with complex architectures (hierarchical, parallel, distributed, etc.) makes it extremely difficult to build analytical models that allow for a satisfying prediction. Hence, it raises the question on how to validate algorithms if a realistic analytic analysis is not possible any longer. As for some many other sciences, the one answer is experimental validation. Nevertheless, experimentation in Computer Science is a difficult subject that today still opens more questions than it solves: What may an experiment validate? What is a "good experiment"? How to build an experimental environment that allows for "good experiments"? etc. In this paper we will provide some hints on this subject and show how some tools can help in performing "good experiments", mainly in the context of parallel and distributed computing. More precisely we will focus on four main experimental methodologies, namely in-situ (real-scale) experiments (with an emphasis on PlanetLab and Grid'5000), Emulation (with an emphasis on Wrekavoc) benchmarking and simulation (with an emphasis on SimGRID and GridSim). We will provide a comparison of these tools and methodologies from a quantitative but also qualitative point of view.
EFFICIENT COARSE GRAINED PERMUTATION EXCHANGES AND MATRIX MULTIPLICATION
Authors: BOXER LAURENCE
Published: July 2009

We present an algorithm for a permutation exchange operation on a coarse grained parallel computer. Our algorithm is more efficient that a previously published solution to this problem, and enables us to derive an efficient algorithm for matrix multiplication.
THE PAINLEVÉ INTEGRABILITY AND N-SOLITONIC SOLUTION IN TERMS OF THE WRONSKIAN DETERMINANT FOR A VARIABLE-COEFFICIENT VARIANT BOUSSINESQ MODEL OF NONLINEAR WAVES
Authors: WANG MING-ZHEN , GAO YI-TIAN , ZHANG CHENG , MENG XIANG-HUA , YU XIN , XU TAO , FENG QIAN
Published: July 2009

A variable-coefficient variant Boussinesq (VCVB) model describes the propagation of long waves in shallow water, the nonlinear lattice waves, the ion sound waves in plasmas, and the vibrations in a nonlinear string. With the help of symbolic computation, a VCVB model is investigated for its integrability through the Painlevé analysis. Then, by truncating the Painlevé expansion at the constant level term with two singular manifolds, the dependent variable transformations are obtained through which the VCVB model is bilinearized. Furthermore, the corresponding N-solitonic solutions with graphic analysis are given by the Hirota method and Wronskian technique. Additionally, a bilinear Bäcklund transformation is constructed for the VCVB model, by which a sample one-solitonic solution is presented.
A UNIFIED DESCRIPTION OF TIME DEPENDENCE OF INFORMATION ENTROPY PRODUCTION AND FLUX IN THERMAL BROADBAND NOISE-DRIVEN DYNAMICAL SYSTEMS
Authors: MAJEE PRADIP , GOSWAMI GURUPADA , BARIK DEBASHIS , BAG BIDHAN CHANDRA
Published: July 2009

In this paper we have studied the dynamics of thermal broadband noise-driven dynamical system in terms of information entropy at both the nonstationary and stationary states. Here, a unified description of fluctuating force is considered in a thermodynamically closed system. Based on the Fokker–Planck description of stochastic processes and the entropy balance equation, we have calculated the time-dependence of the information entropy production and entropy flux in the presence and absence of nonequilibrium constraint. Our calculation considers how the time evolution of these quantities is affected if the characteristic of noise changes from white to red or green and red to green in a unified scheme.
CONTRIBUTION TO STUDY OF THE EFFECTS OF ADDITION OF PLASTICIZER AND OF AGEING ON THE VISCOELASTIC PROPERTIES OF POLYAMIDE
Authors: HADJADJ L.
Published: July 2009

The purpose of this study is to obtain some information about the viscoelastic properties of the material and to see the effect of the addition of plasticizer on these properties. In particular, we try to study the loss of plasticizer, caused by ageing under conditions of utilization, on the properties of material at the neighborhood of vitreous transition.

Using a viscoelastimeter, the study of a polyamide-11 (PA11) at the neighborhood of its temperature of vitreous transition Tv has shown that the increase of the plasticizer rate clearly improves the properties of the material by decreasing its Tv temperature. The comparison of the curves giving the modulus of elasticity E' of the various samples showed that the mechanical properties of the material improve with the increase of the concentration of plasticizer, but ageing reduces this improvement by causing the loss of plasticizer, thus, the temperature range of utilization of material is reduced.
FIELD-INDUCED SPIN EXCITATIONS IN RASHBA-DRESSELHAUS TWO-DIMENSIONAL ELECTRON SYSTEMS PROBED BY SURFACE ACOUSTIC WAVES
Authors: KLEINERT P. , BRYKSIN V. V.
Published: July 2009

A spin-rotation symmetry in spin-orbit coupled two-dimensional electron systems gives rise to a long-lived spin excitation that is robust against short-range impurity scattering. The influence of a constant in-plane electric field on this persistent spin helix is studied. To probe the field-induced eigen-modes of the spin-charge coupled system, a surface acoustic wave is exploited that provides the wave-vector for resonant excitation. The approach takes advantage of methods worked out in the field of space-charge waves. Sharp resonances in the field dependence of the in-plane and out-of-plane magnetization are identified.
ON THE EMERGENCE OF COLLECTIVE ORDER IN SWARMING SYSTEMS: A RECENT DEBATE
Authors: ALDANA M. , LARRALDE H. , VÁZQUEZ B.
Published: July 2009

In this work, we consider the phase transition from ordered to disordered states that occur in the Vicsek model of self-propelled particles. This model was proposed to describe the emergence of collective order in swarming systems. When noise is added to the motion of the particles, the onset of collective order occurs through a dynamical phase transition. Based on their numerical results, Vicsek and his colleagues originally concluded that this phase transition was of second order (continuous). However, recent numerical evidence seems to indicate that the phase transition might be of first order (discontinuous), thus challenging Vicsek's original results. In this work, we review the evidence supporting both aspects of this debate. We also show new numerical results indicating that the apparent discontinuity of the phase transition may in fact be a numerical artifact produced by the artificial periodicity of the boundary conditions.
Regulation of Kir channels in bovine retinal pigment epithelial cells by phosphatidylinositol 4,5-bisphosphate
Authors: Pattnaik Bikash R. , Hughes Bret A.
Published: July 2009

The inwardly rectifying K+(Kir) current in mammalian retinal pigment epithelial (RPE) cells, which is largely mediated by Kir7.1 channels, is stable in cells dialyzed with MgATP but runs down when intracellular ATP is depleted. A potential mechanism for this rundown is a decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) regeneration by ATP-dependent lipid kinases. Here, we used the whole cell voltage-clamp technique to investigate the membrane PIP2dependence of Kir channels in isolated bovine RPE cells. When RPE cells were dialyzed with ATP-free solution containing PIP2(25–50 μM), rundown persisted but was markedly reduced. Removal of Mg2+from the pipette solution also slowed rundown, indicating that elevated intracellular Mg2+concentration contributes to rundown. Cell dialysis with the PIP2scavenger neomycin in MgATP solution diminished Kir current in a voltage-dependent manner, suggesting that it acted at least in part by blocking the Kir channel. Kir current in MgATP-loaded cells was partially inhibited by bath application of quercetin (100 μM), phenylarsine oxide (100 μM), or wortmannin (50 μM), inhibitors of phosphatidylinositol (PI) kinases, and was completely inhibited by cell dialysis with 2 mM adenosine, a PI4 kinase inhibitor. Both LY-294002 (100 μM), an inhibitor of PI3 kinases, and its inactive analog LY-303511 (100 μM) rapidly and reversibly inhibited Kir current, suggesting that these compounds act as direct channel blockers. We conclude that the activity of Kir channels in the RPE is critically dependent on the regeneration of membrane PIP2by PI4 kinases and that this may explain the dependence of these channels on hydrolyzable ATP.
Rab34 and its effector munc13-2 constitute a new pathway modulating protein secretion in the cellular response to hyperglycemia
Authors: Goldenberg Neil M. , Silverman Mel
Published: July 2009

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease requiring dialysis in the Western world. Clinical studies reveal that stringent control of blood glucose levels reduces the risk of most diabetic complications, underscoring the importance of understanding the cellular response to hyperglycemia. Our work identifies a new pathway of potential significance in this response, linking hyperglycemia to the stimulation of constitutive protein secretion via a pathway involving munc13 and rab34. These two proteins have previously been shown to interact at the Golgi via the munc13 homology domain 2 (MHD2). In the present study, using cultured rat mesangial cells (RMC), we show that high glucose-induced upregulation of endogenous munc13-2 increases secretion of the model protein, vesicular stomatitis virus glycoprotein-green fluorescent protein (VSVG-GFP), while small interfering (si)RNA-mediated knockdown of either munc13-2 or rab34 abolishes this effect. Similarly, increased secretion of VSVG-GFP is observed following transfection of HeLa cells with wild-type munc13-2, but not when HeLa cells are transfected with a mutant protein in which the MHD2 domain is deleted. Finally, we show that high glucose-stimulated secretion of fibronectin in RMC is abolished by siRNA knockdown of munc13-2. Collectively, our results demonstrate that the mechanistic basis for our observed high glucose-induced protein secretion is through interaction of munc13 and rab34, indicating a potentially critical role for this newly described pathway in the pathogenesis of DN.