bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermal ablation of tumor cells with antibody-functionalized single-walled carbon nanotubes
Authors: Chakravarty Pavitra , Marches Radu , Zimmerman Neil S. , Swafford Austin D.-E. , Bajaj Pooja , Musselman Inga H. , Pantano Paul , Draper Rockford K. , Vitetta Ellen S.
Published: June 2008

Single-walled carbon nanotubes (CNTs) emit heat when they absorb energy from near-infrared (NIR) light. Tissue is relatively transparent to NIR, which suggests that targeting CNTs to tumor cells, followed by noninvasive exposure to NIR light, will ablate tumors within the range of NIR. In this study, we demonstrate the specific binding of antibody-coupled CNTs to tumor cells
in vitro
, followed by their highly specific ablation with NIR light. Biotinylated polar lipids were used to prepare stable, biocompatible, noncytotoxic CNT dispersions that were then attached to one of two different neutralite avidin-derivatized mAbs directed against either human CD22 or CD25. CD22
+
CD25
−
Daudi cells bound only CNTs coupled to the anti-CD22 mAb; CD22
−
CD25
+
activated peripheral blood mononuclear cells bound only to the CNTs coupled to the anti-CD25 mAb. Most importantly, only the specifically targeted cells were killed after exposure to NIR light.
Stress reduction through consolation in chimpanzees
Authors: Fraser Orlaith N. , Stahl Daniel , Aureli Filippo
Published: June 2008

Consolation, i.e., postconflict affiliative interaction directed from a third party to the recipient of aggression, is assumed to have a stress-alleviating function. This function, however, has never been demonstrated. This study shows that consolation in chimpanzees reduces behavioral measures of stress in recipients of aggression. Furthermore, consolation was more likely to occur in the absence of reconciliation, i.e., postconflict affiliative interaction between former opponents. Consolation therefore may act as an alternative to reconciliation when the latter does not occur. In the debate about empathy in great apes, evidence for the stress-alleviating function of consolation in chimpanzees provides support for the argument that consolation could be critical behavior. Consistent with the argument that relationship quality affects their empathic responses, we found that consolation was more likely between individuals with more valuable relationships. Chimpanzees may thus respond to distressed valuable partners by consoling them, thereby reducing their stress levels, especially in the absence of reconciliation.
Copy number of FCGR3B, which is associated with systemic lupus erythematosus, correlates with protein expression and immune complex uptake
Authors: Willcocks Lisa C. , Lyons Paul A. , Clatworthy Menna R. , Robinson James I. , Yang Wanling , Newland Stephen A. , Plagnol Vincent , McGovern Naomi N. , Condliffe Alison M. , Chilvers Edwin R. , Adu Dwomoa , Jolly Elaine C. , Watts Richard , Lau Yu Lung , Morgan Ann W. , Nash Gerard , Smith Kenneth G.C.
Published: June 2008

Copy number (CN) variation (CNV) has been shown to be common in regions of the genome coding for immune-related genes, and thus impacts upon polygenic autoimmunity. Low CN of FCGR3B has recently been associated with systemic lupus erythematosus (SLE). FcγRIIIb is a glycosylphosphatidylinositol-linked, low affinity receptor for IgG found predominantly on human neutrophils. We present novel data demonstrating that both in a family with FcγRIIIb-deficiency and in the normal population, FCGR3B CNV correlates with protein expression, with neutrophil uptake of and adherence to immune complexes, and with soluble serum FcγRIIIb. Reduced FcγRIIIb expression is thus likely to contribute to the impaired clearance of immune complexes, which is a feature of SLE, explaining the association between low FCGR3B CNV and SLE that we have confirmed in a Caucasian population. In contrast, antineutrophil cytoplasmic antibody–associated systemic vasculitis (AASV), a disease not associated with immune complex deposition, is associated with high FCGR3B CN. Thus, we define a role for FCGR3B CNV in immune complex clearance, a function that may explain why low FCGR3B CNV is associated with SLE, but not AASV. This is the first report of an association between disease-related gene CNV and variation in protein expression and function that may contribute to autoimmune disease susceptibility.
Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages
Authors: Sierra Jose Rafael , Corso Simona , Caione Luisa , Cepero Virna , Conrotto Paolo , Cignetti Alessandro , Piacibello Wanda , Kumanogoh Atsushi , Kikutani Hitoshi , Comoglio Paolo Maria , Tamagnone Luca , Giordano Silvia
Published: June 2008

Increased evidence suggests that cancer-associated inflammation supports tumor growth and progression. We have previously shown that semaphorin 4D (Sema4D), a ligand produced by different cell types, is a proangiogenic molecule that acts by binding to its receptor, plexin B1, expressed on endothelial cells (Conrotto, P., D. Valdembri, S. Corso, G. Serini, L. Tamagnone, P.M. Comoglio, F. Bussolino, and S. Giordano. 2005. Blood. 105:4321–4329). The present work highlights the role of Sema4D produced by the tumor microenvironment on neoplastic angiogenesis. We show that in an environment lacking Sema4D, the ability of cancer cells to generate tumor masses and metastases is severely impaired. This condition can be explained by a defective vascularization inside the tumor. We demonstrate that tumor-associated macrophages (TAMs) are the main cells producing Sema4D within the tumor stroma and that their ability to produce Sema4D is critical for tumor angiogenesis and vessel maturation. This study helps to explain the protumoral role of inflammatory cells of the tumor stroma and leads to the identification of an angiogenic molecule that might be a novel therapeutic target.
The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction
Authors: Varga-Szabo David , Braun Attila , Kleinschnitz Christoph , Bender Markus , Pleines Irina , Pham Mirko , Renné Thomas , Stoll Guido , Nieswandt Bernhard
Published: June 2008

Platelet activation and aggregation are essential to limit posttraumatic blood loss at sites of vascular injury but also contributes to arterial thrombosis, leading to myocardial infarction and stroke. Agonist-induced elevation of [Ca2+]i is a central step in platelet activation, but the underlying mechanisms are not fully understood. A major pathway for Ca2+ entry in nonexcitable cells involves receptor-mediated release of intracellular Ca2+ stores, followed by activation of store-operated calcium (SOC) channels in the plasma membrane. Stromal interaction molecule 1 (STIM1) has been identified as the Ca2+ sensor in the endoplasmic reticulum (ER) that activates Ca2+ release–activated channels in T cells, but its role in mammalian physiology is unknown. Platelets express high levels of STIM1, but its exact function has been elusive, because these cells lack a normal ER and Ca2+ is stored in a tubular system referred to as the sarcoplasmatic reticulum. We report that mice lacking STIM1 display early postnatal lethality and growth retardation. STIM1-deficient platelets have a marked defect in agonist-induced Ca2+ responses, and impaired activation and thrombus formation under flow in vitro. Importantly, mice with STIM1-deficient platelets are significantly protected from arterial thrombosis and ischemic brain infarction but have only a mild bleeding time prolongation. These results establish STIM1 as an important mediator in the pathogenesis of ischemic cardio- and cerebrovascular events.
Memory-efficient dynamic programming backtrace and pairwise local sequence alignment
Authors: Newberg Lee A.
Published: June 2008

Motivation: A backtrace through a dynamic programming algorithm's intermediate results in search of an optimal path, or to sample paths according to an implied probability distribution, or as the second stage of a forward–backward algorithm, is a task of fundamental importance in computational biology. When there is insufficient space to store all intermediate results in high-speed memory (e.g. cache) existing approaches store selected stages of the computation, and recompute missing values from these checkpoints on an as-needed basis.

Results: Here we present an optimal checkpointing strategy, and demonstrate its utility with pairwise local sequence alignment of sequences of length 10 000.

Availability: Sample C++-code for optimal backtrace is available in the Supplementary Materials.

Contact:  leen@cs.rpi.edu

Supplementary information:  Supplementary data is available at Bioinformatics online.
Identification of two distinct bovine parainfluenza virus type 3 genotypes
Authors: Horwood Paul Francis , Gravel Jennifer Lillian , Mahony Timothy John
Published: June 2008

The partial gene sequencing of the matrix (M) protein from seven clinical isolates of bovine parainfluenza virus type 3 (BPIV-3), and the complete sequencing of a representative isolate (Q5592) was completed in this study. Nucleotide sequence analysis was initiated because of the failure of in-house BPIV-3 RT-PCR methods to yield expected products for four of the isolates. Phylogenetic reconstructions based on the nucleotide sequences for the M-protein and the entire genome, using all of the available BPIV-3 nucleotide sequences, demonstrated that there were two distinct BPIV-3 genotypes (BPIV-3a and BPIV-3b). These newly identified genotypes have implications for the development of BPIV-3 molecular detection methods and may also impact on BPIV-3 vaccine formulations.
Rice black streaked dwarf virus P9-1, an α-helical protein, self-interacts and forms viroplasms in vivo
Authors: Zhang Chaozheng , Liu Yueyong , Liu Liyue , Lou Zhiyong , Zhang Hongyan , Miao Hongqin , Hu Xuebo , Pang Yanping , Qiu Bingsheng
Published: June 2008

Replication and assembly of viruses from the family Reoviridae are thought to take place in discrete cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. Rice black streaked dwarf virus (RBSDV) P9-1, a non-structural protein, has been confirmed to accumulate in these intracellular viroplasms in infected plants and insects. However, little is known about its exact function. In this study, P9-1 of RBSDV-Baoding was expressed in Escherichia coli as a His-tagged fusion protein and analysed using biochemical and biophysical techniques. Mass spectrometry and circular dichroism spectroscopy studies showed that P9-1 was a thermostable, α-helical protein with a molecular mass of 41.804 kDa. A combination of gel-filtration chromatography, chemical cross-linking and a yeast two-hybrid assay was used to demonstrate that P9-1 had the intrinsic ability to self-interact and form homodimers in vitro and in vivo. Furthermore, when transiently expressed in Arabidopsis protoplasts, P9-1 formed large, discrete viroplasm-like structures in the absence of infection or other RBSDV proteins. Taken together, these results suggest that P9-1 is the minimal viral component required for viroplasm formation and that it plays an important role in the early stages of the virus life cycle by forming intracellular viroplasms that serve as the sites of virus replication and assembly.
The promiscuous evolutionary history of the family Bromoviridae
Authors: Codoñer Francisco M. , Elena Santiago F.
Published: June 2008

Recombination and segment reassortment are important contributors to the standing genetic variation of RNA viruses and are often involved in the genesis of new, emerging viruses. This study explored the role played by these two processes in the evolutionary radiation of the plant virus familyBromoviridae. The evolutionary history of this family has been explored previously using standard molecular phylogenetic methods, but incongruences have been found among the trees inferred from different gene sequences. This would not be surprising if RNA exchange was a common event, as it is well known that recombination and reassortment of genomes are poorly described by standard phylogenetic methods. In an attempt to reconcile these discrepancies, this study first explored the extent of segment reassortment and found that it was common at the origin of the bromoviruses and cucumoviruses and at least at the origin of alfalfa mosaic virus, American plum line pattern virus and citrus leaf rugose virus. Secondly, recombination analyses were performed on each of the three genomic RNAs and it was found that recombination was very common in members of the generaBromovirus,CucumovirusandIlarvirus. Several cases of recombination involving species from different genera were also identified. Finally, a phylogenetic network was constructed reflecting these genetic exchanges. The network confirmed the taxonomic status of the different genera within the family, despite the phylogenetic noise introduced by genetic exchange.
Restoration of the antiviral activity of 3′-azido-3′-deoxythymidine (AZT) against AZT-resistant human immunodeficiency virus by delivery of engineered thymidylate kinase to T cells
Authors: Lavie Arnon , Su Ying , Ghassemi Mahmood , Novak Richard M. , Caffrey Michael , Sekulic Nikolina , Monnerjahn Christian , Konrad Manfred , Cook James L.
Published: June 2008

Emergence of antiviral drug resistance is a major challenge to human immunodeficiency virus (HIV) therapy. The archetypal example of this problem is loss of antiviral activity of the nucleoside analogue 3′-azido-3′-deoxythymidine (AZT), caused by mutations in reverse transcriptase (RT), the viral polymerase. AZT resistance results from an imbalance between rates of AZT-induced proviral DNA chain termination and RT-induced excision of the chain-terminating nucleotide. Conversion of the AZT prodrug from its monophosphorylated to diphosphorylated form by human thymidylate kinase (TMPK) is inefficient, resulting in accumulation of the monophosphorylated AZT metabolite (AZT-MP) and a low concentration of the active triphosphorylated metabolite (AZT-TP). We reasoned that introduction of an engineered, highly active TMPK into T cells would overcome this functional bottleneck in AZT activation and thereby shift the balance of AZT activity sufficiently to block replication of formerly AZT-resistant HIV. Molecular engineering was used to link highly active, engineered TMPKs to the protein transduction domain of Tat for direct cell delivery. Combined treatment of HIV-infected T cells with AZT and these cell-permeable, engineered TMPKs restored AZT-induced repression of viral production. These results provide an experimental basis for the development of new strategies to therapeutically increase the intracellular concentrations of active nucleoside analogue metabolites as a means to overcome emerging drug resistance.
The UL15 protein of herpes simplex virus type 1 is necessary for the localization of the UL28 and UL33 proteins to viral DNA replication centres
Authors: Higgs Martin R. , Preston Valerie G. , Stow Nigel D.
Published: June 2008

The UL15, UL28 and UL33 proteins of herpes simplex virus type 1 (HSV-1) are thought to comprise a terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. Immunofluorescence studies confirmed that shortly after infection with wild-type HSV-1 these three proteins localize to viral DNA replication compartments within the nucleus, identified by the presence of the single-stranded DNA-binding protein, ICP8. In cells infected with either UL28- or UL33-null mutants, the other two terminase proteins also co-localized with ICP8. In contrast, neither UL28 nor UL33 was detectable in replication compartments following infection with a UL15-null mutant, although Western blot analysis showed they were present in normal amounts in the infected cells. Provision of UL15 in a complementing cell line restored the ability of all three proteins to localize to replication compartments. These data indicate that UL15 plays a key role in localizing the terminase complex to DNA replication compartments, and that it can interact independently with UL28 and UL33.
Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep
Authors: Li Hong , Cunha Cristina W. , Davies Christopher J. , Gailbreath Katherine L. , Knowles Donald P. , Oaks J. Lindsay , Taus Naomi S.
Published: June 2008

Ovine herpesvirus 2 (OvHV-2), a rhadinovirus in the subfamilyGammaherpesvirinae, is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal lymphoproliferative disease primarily of ruminants worldwide. Inability to propagate the virusin vitrohas made it difficult to study OvHV-2 replication. Aerosol inoculation of sheep with OvHV-2 from nasal secretions collected from naturally infected sheep during shedding episodes results in infection of naive sheep, providing an excellent system to study OvHV-2 initial replication in the natural host. In this study, we showed that OvHV-2 delivered through the nasal route by nebulization resulted in infection in all lambs, but no infection was established in any lambs after intravenous or intraperitoneal injection. In nebulized lambs, while it was not detected initially in any other tissues, OvHV-2 DNA became detectable in the lung at 3 days post-infection (p.i.), increased to about 900 copies per 50 ng DNA at 5 days p.i., reached peak levels (∼7500 copies) at 7 days p.i., and then declined to an average of 800 copies at 9 days p.i. Transcripts of OvHV-2 open reading frame 25 (coding for the capsid protein), an indicator of virus replication, were only detected in lung tissues, and were positively correlated with OvHV-2 DNA levels in the lungs. In addition, selected immune response genes were also highly expressed in the lung at 5 and 7 days p.i. The data indicate that lung is the primary replication site for OvHV-2 during initial infection in sheep and suggest that viral replication is promptly controlled by a host defence mechanism.
Characterization of mosquito-adapted West Nile virus
Authors: Ciota Alexander T. , Lovelace Amy O. , Jia Yongqing , Davis Lauren J. , Young David S. , Kramer Laura D.
Published: June 2008

West Nile virus (WNV), a mosquito-borne flavivirus, has significantly expanded its geographical and host range since its 1999 introduction into North America. The underlying mechanisms of evolution of WNV and other arboviruses are still poorly understood. Studies evaluating virus adaptation and fitness in relevantin vivosystems are largely lacking. In order to evaluate the capacity for host-specific adaptation and the genetic correlates of adaptationin vivo, this study measured phenotypic and genotypic changes in WNV resulting from passage inCulex pipiensmosquitoes. An increase in replicative ability of WNV inC. pipienswas attained for the two lineages of WNV tested. This adaptation for replication in mosquitoes did not result in a replicative cost in chickens, but did decrease cell-to-cell spread of virus in vertebrate cell culture. Genetic analyses of one mosquito-adapted lineage revealed a total of nine consensus nucleotide substitutions with no accumulation of a significant mutant spectrum. These results differed significantly from previousin vitrostudies. When St Louis encephalitis virus (SLEV), a closely related flavivirus, was passaged inC. pipiens, moderately attenuated growth inC. pipienswas observed for two lineages tested. These results suggest that significant differences in the capacity for mosquito adaptation may exist between WNV and SLEV, and demonstrate that further comparative studies in relevantin vivosystems will help elucidate the still largely unknown mechanisms of arboviral adaptation in ecologically relevant hosts.
Efficient dengue virus (DENV) infection of human muscle satellite cells upregulates type I interferon response genes and differentially modulates MHC I expression on bystander and DENV-infected cells
Authors: Warke Rajas V. , Becerra Aniuska , Zawadzka Agatha , Schmidt Diane J. , Martin Katherine J. , Giaya Kris , Dinsmore Jonathan H. , Woda Marcia , Hendricks Gregory , Levine Tracy , Rothman Alan L. , Bosch Irene
Published: June 2008

Dengue virus (DENV) is a mosquito-borne flavivirus that causes an acute febrile disease in humans, characterized by musculoskeletal pain, headache, rash and leukopenia. The cause of myalgia during DENV infection is still unknown. To determine whether DENV can infect primary muscle cells, human muscle satellite cells were exposed to DENVin vitro. The results demonstrated for the first time high-efficiency infection and replication of DENV in human primary muscle satellite cells. Changes in global gene expression were also examined in these cells following DENV infection using Affymetrix GeneChip analysis. The differentially regulated genes belonged to two main functional categories: cell growth and development, and antiviral type I interferon (IFN) response genes. Increased expression of the type I IFN response genes for tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), melanoma-derived antigen 5 (MDA-5), IFN-γ-inducible protein 10 (IP-10), galectin 3 soluble binding protein (LGals3BP) and IFN response factor 7 (IRF7) was confirmed by quantitative RT-PCR. Furthermore, higher levels of cell-surface-bound intracellular adhesion molecule-1 (ICAM-1) and soluble ICAM-1 in the cell-culture medium were detected following DENV infection. However, DENV infection impaired the ability of the infected cells in the culture medium to upregulate cell-surface expression of MHC I molecules, suggesting a possible mechanism of immune evasion by DENV. The findings of this study warrant further clinical research to identify whether muscle cells are targets for DENV infection during the acute stage of the diseasein vivo.
Structural and functional characterization of the 5′ region of subgenomic RNA5 of cucumber mosaic virus
Authors: Thompson Jeremy R. , Buratti Emanuele , de Wispelaere Mélissanne , Tepfer Mark
Published: June 2008

The uncapped and ORF-less subgenomic RNA5 is produced in subgroup II strains of cucumber mosaic virus (CMV), but not in subgroup I strains. Its initiation nucleotide (nt 1903) is in a 21 nt conserved sequence (Box1) that is absent in CMV subgroup I. Putative non-coding RNA structural elements surrounding Box1 in the plus and minus strand were identified in silico and by in vitro RNase probing. Four main stem–loop structures (SLM, SLL, SLK and SLJ) were identified between nt 1887 and 1999 of isolate R-CMV (subgroup II), with notable differences within SLM and SLL between the two strands. Mutation of a stem–loop within SLM, even when the predicted wild-type structure was maintained, showed significant reduction in RNA5 levels in planta. Three mutants containing 3–4 nt substitutions between positions −39 and +49 showed significantly reduced levels of RNA5, while another similar mutant at positions 80–83 had RNA5 levels comparable to wild-type. Deletion of Box1 resulted in similar levels of RNA3 and 4 as wild-type, while eliminating RNA5. Insertion of Box1 into a subgroup I isolate was not sufficient to produce RNA5. However, in a mutant with an additional 21 nt of R-CMV 3′ of Box1 (positions −1 to +41), low levels of RNA5 were detected. Taken together, these results have identified regions of the viral genome responsible for RNA5 production and in addition provide strong evidence for the existence of newly identified conserved structural elements in the 5′ part of the 3′ untranslated region.
Human, porcine and bovine rotaviruses in Slovenia: evidence of interspecies transmission and genome reassortment
Authors: Steyer Andrej , Poljšak-Prijatelj Mateja , Barlič-Maganja Darja , Marin Jožica
Published: June 2008

A surveillance of human, porcine and bovine rotaviruses was carried out in Slovenia in 2004 and 2005. Stool samples were collected from a total of 406 pigs (373 from asymptomatic animals), 132 cattle (126 from asymptomatic animals) and 241 humans (all with diarrhoea), tested for group A rotaviruses using RT-PCR and analysed by sequencing. The aims of the study were to determine the incidence of asymptomatic rotavirus infection in animals, to look for evidence of zoonotic transmission and to detect reassortment among rotaviruses. The rates of asymptomatic shedding of rotaviruses in pigs and cattle were 18.0 % (67/373) and 4.0 % (5/126), respectively. Evidence for zoonotic transmission was detected in one human rotavirus strain, SI-MB6, with the G3P[6] genotype combination, as the nucleotide and predicted amino acid sequences of the VP6, VP7, VP8* and NSP4 genes of strain SI-MB6 and of porcine strains showed high nucleotide and amino acid sequence identity. Two porcine rotavirus strains carried VP7 of probable human origin, suggesting an interspecies reassortment event in the past.
Accumulation of point mutations and reassortment of genomic RNA segments are involved in the microevolution of Puumala hantavirus in a bank vole (Myodes glareolus) population
Authors: Razzauti Maria , Plyusnina Angelina , Henttonen Heikki , Plyusnin Alexander
Published: June 2008

The genetic diversity of Puumala hantavirus (PUUV) was studied in a local population of its natural host, the bank vole (Myodes glareolus). The trapping area (2.5×2.5 km) at Konnevesi, Central Finland, included 14 trapping sites, at least 500 m apart; altogether, 147 voles were captured during May and October 2005. Partial sequences of the S, M and L viral genome segments were recovered from 40 animals. Seven, 12 and 17 variants were detected for the S, M and L sequences, respectively; these represent new wild-type PUUV strains that belong to the Finnish genetic lineage. The genetic diversity of PUUV strains from Konnevesi was 0.2–4.9 % for the S segment, 0.2–4.8 % for the M segment and 0.2–9.7 % for the L segment. Most nucleotide substitutions were synonymous and most deduced amino acid substitutions were conservative, probably due to strong stabilizing selection operating at the protein level. Based on both sequence markers and phylogenetic clustering, the S, M and L sequences could be assigned to two groups, ‘A’ and ‘B’. Notably, not all bank voles carried S, M and L sequences belonging to the same group, i.e. SAMALAor SBMBLB. A substantial proportion (8/40, 20 %) of the newly characterized PUUV strains possessed reassortant genomes such as SBMALA, SAMBLBor SBMALB. These results suggest that at least some of the PUUV reassortants are viable and can survive in the presence of their parental strains.
Genes contributing to prion pathogenesis
Authors: Tamgüney Gültekin , Giles Kurt , Glidden David V. , Lessard Pierre , Wille Holger , Tremblay Patrick , Groth Darlene F. , Yehiely Fruma , Korth Carsten , Moore Richard C. , Tatzelt Jörg , Rubinstein Eric , Boucheix Claude , Yang Xiaoping , Stanley Pamela , Lisanti Michael P. , Dwek Raymond A. , Rudd Pauline M. , Moskovitz Jackob , Epstein Charles J. , Cruz Tracey Dawson , Kuziel William A. , Maeda Nobuyo , Sap Jan , Ashe Karen Hsiao , Carlson George A. , Tesseur Ina , Wyss-Coray Tony , Mucke Lennart , Weisgraber Karl H. , Mahley Robert W. , Cohen Fred E. , Prusiner Stanley B.
Published: June 2008

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrPC) to a misfolded, pathogenic isoform (PrPSc). Prion inoculation experiments in mice expressing homologous PrPCmolecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrPC, our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.
EBV LMP2A provides a surrogate pre-B cell receptor signal through constitutive activation of the ERK/MAPK pathway
Authors: Anderson Leah J. , Longnecker Richard
Published: June 2008

Latent membrane protein 2A (LMP2A) of Epstein–Barr virus (EBV) provides developmental and survival signals that mimic those of a B-cell receptor (BCR). Expression of LMP2A during B-cell development results in the ability of B cells to exit the bone marrow in the absence of a BCR and persist in the periphery, where they would normally undergo apoptosis. This study extends the current knowledge of LMP2A function by examining the growth properties of bone marrow B cells from TgE LMP2A mice. Despite the lack of pre-BCR expression, bone marrow B cells from TgE LMP2A mice proliferate and survive in low concentrations of interleukin 7, similar to wild-type cells. Constitutive phosphorylation of ERK/MAPK and PI3K/Akt in TgE LMP2A bone marrow B cells is also reminiscent of signalling through the pre-BCR, altogether demonstrating that LMP2A provides a pre-BCR-like signal to developing B cells.
Implication of p38 mitogen-activated protein kinase isoforms (α, β, γ and δ) in CD4+ T-cell infection with human immunodeficiency virus type I
Authors: Gutierrez-Sanmartin Dolores , Varela-Ledo Eduardo , Aguilera Antonio , Romero-Yuste Susana , Romero-Jung Patricia , Gomez-Tato Antonio , Regueiro Benito J.
Published: June 2008

The CD4+ T-cell reduction characteristic of human immunodeficiency virus type 1 (HIV-1) infection is thought to result, in addition to infected T-cell death, mainly from uninfected bystander T-cell apoptosis. Nevertheless, the immunological and virological mechanisms leading to T-cell death during HIV-1 infection are not yet fully understood. In the present study, we analysed the individual implication of the p38 mitogen-activated protein kinase (MAPK) isoforms (p38α, p38β, p38γ and p38δ) during apoptosis induced by HIV-1, taking into account that HIV-1 replication is known to be blocked by p38 inhibitors. For this purpose, we used the SupT1 cell line, where death induced by HIV-1 mainly occurs by uninfected bystander cell apoptosis. A variety of SupT1-based cell lines were constructed constitutively expressing, under the control of cytomegalovirus promoter (PCMV), each dominant-negative (dn) p38 isoform and each wild-type p38 isoform as a control. An enhanced green fluorescent protein marker gene, under the control of the HIV-1 promoter, was inserted in all of them. These cell lines were infected with HIV-1 and analysed by flow cytometry. We found that survival in SupT1-based cell lines infected by HIV-1 was increased by the p38αdn, p38γdn and p38δdn isoforms, but not by the p38βdn isoform. HIV-1 replication was delayed most by p38δdn and to a lesser extent by p38αdn and p38γdn. Moreover, these three isoforms, p38αdn, p38γdn and p38δdn, reduced apoptosis induced by HIV-1. These results suggest that, in SupT1-based cell lines, p38α, p38γ and p38δ, but not p38β, are implicated in both HIV-1 induced replication and apoptosis in infected and uninfected bystander cells.
Immunolocalization of deformed wing virus particles within the mite Varroa destructor
Authors: Santillán-Galicia Ma. Teresa , Carzaniga Raffaella , Ball Brenda V. , Alderson Peter G.
Published: June 2008

Deformed wing virus (DWV) induces wing deformation when bees are infected during their pupal development. Field observations and laboratory experiments suggest that the miteVarroa destructoris a vector of the virus. Moreover, it has been stated that DWV replicates within this mite. In order to understand the role ofV. destructorin the transmission of DWV, the objective of this work was to locate the sites of retention and/or replication of DWV within the mite by immunohistochemistry. There was no evidence that DWV was replicating in the mite as no tissues showed specific antibody binding to DWV. Also, there were no specific structures that could be suggested as retention sites. DWV was found only in the midgut lumen ofV. destructorin structures resembling large, dense spheres, which were presumably faecal pellets.
Functional characterization and subcellular localization of the 16K cysteine-rich suppressor of gene silencing protein of tobacco rattle virus
Authors: Ghazala Walid , Waltermann Angelika , Pilot Ruth , Winter Stephan , Varrelmann Mark
Published: June 2008

The 16 kDa cysteine-rich protein (16K) of tobacco rattle virus (TRV) is known to partially suppress RNA silencing in Drosophila cells. In this study, we show that 16K suppresses RNA silencing in green fluorescent protein (GFP)-transgenic Nicotiana benthamiana plants using an Agrobacterium-mediated transient assay. 16K slightly reduced the accumulation of short interfering RNAs (siRNA) of GFP, suggesting that the protein may interfere with the initiation and/or maintenance of RNA silencing. Deletion of either the N- or C-terminal part of 16K indicated that the entire 16K open reading frame (ORF) is necessary for its silencing suppression function. Pentapeptide insertion scanning mutagenesis (PSM) revealed that only two short regions of 16K tolerated five extra amino acid insertions without considerable reduction in its silencing suppression function. The tolerant regions coincide with sequence variability between tobravirus cysteine-rich proteins, indicating a strong functional and/or structural conservation of TRV 16K. Confocal laser scanning microscopy of transiently expressed 16K fusions to red fluorescent protein (RFP) revealed a predominant cytoplasmic localization and, in addition, a nuclear localization. In contrast, fusions of RFP with the N-terminal region of 16K localized exclusively to the cytoplasm, whereas fusions between RFP and the C-terminal region of 16K displayed an exclusive nuclear localization. Further analysis of 16K-derived peptide fusions demonstrated that the 16K C-terminal region contained at least two functional bipartite nuclear localization signals which were independently capable of nuclear targeting.
Hepatitis A virus protein 2B suppresses beta interferon (IFN) gene transcription by interfering with IFN regulatory factor 3 activation
Authors: Paulmann Dajana , Magulski Thomas , Schwarz Rebecca , Heitmann Lisa , Flehmig Bertram , Vallbracht Angelika , Dotzauer Andreas
Published: June 2008

Hepatitis A virus (HAV) antagonizes the innate immune response by inhibition of retinoic acid-inducible gene I-mediated and melanoma differentiation-associated gene 5-mediated beta interferon (IFN-β) gene expression. This study showed that this is due to an interaction of HAV with mitochondrial antiviral signalling protein (MAVS)-dependent signalling, in which the viral non-structural protein 2B and the protein intermediate 3ABC recently suggested in this context seem to be involved, cooperatively affecting the activities of MAVS and the kinases TANK-binding kinase 1 (TBK1) and the inhibitor of NF-κB kinase ϵ (IKKϵ). In consequence, interferon regulatory factor 3 (IRF-3) is not activated. As IRF-3 is necessary for IFN-βtranscription, inhibition of this factor results in efficient suppression of IFN-βsynthesis. This ability might be of vital importance for HAV, which is an exceptionally slow growing virus sensitive to IFN-β, as it allows the virus to establish infection and maintain virus replication for a longer period of time.
Influence of human papillomavirus type 16 (HPV-16) E2 polymorphism on quantification of HPV-16 episomal and integrated DNA in cervicovaginal lavages from women with cervical intraepithelial neoplasia
Authors: Azizi Naoufel , Brazete Jessica , Hankins Catherine , Money Deborah , Fontaine Julie , Koushik Anita , Rachlis Anita , Pourreaux Karina , Ferenczy Alex , Franco Eduardo ,   François Coutlée for The Canadian Women's HIV Study Group
Published: June 2008

Integrated human papillomavirus type 16 (HPV-16) viral loads are currently estimated by quantification with real-time PCR of HPV-16 E6 (RT-E6 and HPV-16 PG) and E2 (RT-E2-1) DNA. We assessed the influence of HPV-16 E2 polymorphism on quantification of integrated HPV-16 DNA in anogenital specimens. HPV-16 E2 was sequenced from 135 isolates (123 from European and 12 from non-European lineages). An assay targeting conserved HPV-16 E2 sequences (RT-E2-2) was optimized and applied with RT-E6 and RT-E2-1 on 139 HPV-16-positive cervicovaginal lavages collected from 74 women [58 human immunodeficiency virus (HIV)-seropositive and 16 HIV-seronegative]. Ratios of HPV-16 copies measured with RT-E2-2 and RT-E2-1 obtained with African 2 (median=3.23, range=1.92–3.49) or Asian–American (median=3.78, range=1.47–37) isolates were greater than those obtained with European isolates (median=1.02, range=0.64–1.80; P<0.02 for each comparison). The distribution of HPV-16 E2 copies measured in 139 samples with RT-E2-2 (median=6150) and RT-E2-1 (median=8960) were different (P<0.0001). The risk of high-grade cervical intraepithelial neoplasia (CIN-2,3) compared with women without CIN was increased with higher HPV-16 total [odds ratio (OR)=2.17, 95 % confidence interval (CI)=1.11–4.23], episomal (OR=2.14, 95 % CI=1.09–4.19), but not for HPV-16 integrated viral load (OR=1.71, 95 % CI=0.90–3.26), after controlling for age, race, CD4 count, HIV and HPV-16 polymorphism. The proportion of samples with an E6/E2 ratio >2 in women without squamous intraepithelial lesion (7 of 35) was similar to that of women with CIN-2,3 (5 of 11, P=0.24) or CIN-1 (5 of 14, P=0.50). HPV-16 E2 polymorphism was a significant factor that influenced measures of HPV-16 integrated viral load.
Virological characterization of the hepatitis C virus JFH-1 strain in lymphocytic cell lines
Authors: Murakami Kyoko , Kimura Toshiro , Osaki Motonao , Ishii Koji , Miyamura Tatsuo , Suzuki Tetsuro , Wakita Takaji , Shoji Ikuo
Published: June 2008

While hepatocytes are the major site of hepatitis C virus (HCV) infection, a number of studies have suggested that HCV can replicate in lymphocytes. However, in vitro culture systems to investigate replication of HCV in lymphocytic cells are severely limited. Robust HCV culture systems have been established using the HCV JFH-1 strain and Huh-7 cells. To gain more insights into the tissue tropism of HCV, we investigated the infection, replication, internal ribosome entry site (IRES)-dependent translation and polyprotein processing of the HCV JFH-1 strain in nine lymphocytic cell lines. HCV JFH-1 failed to infect lymphocytes and replicate, but exhibited efficient polyprotein processing and IRES-dependent translation in lymphocytes as well as in Huh-7 cells. Our results suggest that lymphocytic cells can support HCV JFH-1 translation and polyprotein processing, but may lack some host factors essential for HCV JFH-1 infection and replication.
Diversity of begomoviruses associated with mosaic disease of cultivated cassava (Manihot esculenta Crantz) and its wild relative (Manihot glaziovii Müll. Arg.) in Uganda
Authors: Sserubombwe W. S. , Briddon R. W. , Baguma Y. K. , Ssemakula G. N. , Bull S. E. , Bua A. , Alicai T. , Omongo C. , Otim-Nape G. W. , Stanley J.
Published: June 2008

Cassava (Manihot esculenta) growing in Uganda during 2001–2002 has been screened for the presence of begomoviruses using PCR-RFLP, cloning full-length genomic components and nucleotide sequence analysis. In contrast with a recent survey in neighbouring Kenya, which identified three distinct strains of East African cassava mosaic virus (EACMV, EACMV-UG and EACMV-KE2) as well as East African cassava mosaic Zanzibar virus and the new species East African cassava mosaic Kenya virus, only EACMV-UG and, to a lesser extent, African cassava mosaic virus (ACMV) were found associated with cassava in Uganda. The integrity of the cloned genomic components of representative virus isolates was confirmed by demonstrating their infectivity in Nicotiana benthamiana and cassava using biolistic inoculation, providing a convenient means to screen cassava varieties for disease resistance. Both EACMV-UG and ACMV were also associated with Manihot glaziovii. Infectivity studies using cloned components confirmed that viruses from one host could infect the other, suggesting that this wild relative of cassava might be a reservoir host for the disease. The relatively low level of diversity of begomoviruses associated with cassava mosaic disease in Uganda is consistent with reports that EACMV-UG has displaced other begomovirus species and strains during the recent epidemic that swept through the country.
Use of synthetic peptides to represent surface-exposed epitopes defined by neutralizing dengue complex- and flavivirus group-reactive monoclonal antibodies on the native dengue type-2 virus envelope glycoprotein
Authors: Falconar Andrew K. I.
Published: June 2008

The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393–401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101–109 sequence containing 105-C by G substitution and the 393–401 sequence are good candidates for diagnostic assays and cross-protection experiments.
Sequence-specific cleavage of hepatitis C virus RNA by DNAzymes: inhibition of viral RNA translation and replication
Authors: Roy Swagata , Gupta Nidhi , Subramanian Nithya , Mondal Tanmoy , Banerjea Akhil Chandra , Das Saumitra
Published: June 2008

DNAzyme (Dz) molecules have been shown to be highly efficient inhibitors of virus replication. Hepatitis C virus RNA translation is mediated by an internal ribosome entry site (IRES) element located mostly in the 5′ untranslated region (UTR), the mechanism of which is fundamentally different from cap-dependent translation of cellular mRNAs, and thus an attractive target for designing antiviral drugs. Inhibition of HCV IRES-mediated translation has drastic consequences for the replication of viral RNA as well. We have designed several Dzs, targeting different regions of HCV IRES specific for 1b and also sequences conserved across genotypes. The RNA cleavage and translation inhibitory activities of these molecules were tested in a cell-free system and in cell culture using transient transfections. The majority of Dzs efficiently inhibited HCV IRES-mediated translation. However, these Dz molecules did not show significant inhibition of coxsackievirus B3 IRES-mediated translation or cap-dependent translation of reporter gene, showing high level of specificity towards target RNA. Also, Northern blot hybridization analysis showed significant cleavage of HCV IRES by the Dz molecules in Huh7 cells transiently transfected with the HCV–FLuc monocistronic construct. Interestingly, one of the Dzs was more effective against genotype1b, whereas the other showed significant inhibition of viral RNA replication in Huh7 cells harbouring a HCV 2a monocistronic replicon. As expected, mutant-Dz failed to cleave RNA and inhibit HCV RNA translation, showing the specificity of inhibition. Taken together, these findings suggest that the Dz molecule can be used as selective and effective inhibitor of HCV RNA replication, which can be explored further for development of a potent therapeutic agent against HCV infection.
The molecular basis of mouse adaptation by human enterovirus 71
Authors: Chua Beng Hooi , Phuektes Patchara , Sanders Sharon A. , Nicholls Philip K. , McMinn Peter C.
Published: June 2008

A mouse-adapted strain of human enterovirus 71 (HEV71) was selected by serial passage of a HEV71 clinical isolate (HEV71-26M) in Chinese hamster ovary (CHO) cells (CHO-26M) and in newborn BALB/c mice (MP-26M). Despite improved growth in CHO cells, CHO-26M did not show increased virulence in newborn BALB/c mice compared with HEV71-26M. By contrast, infection of newborn mice with MP-26M resulted in severe disease of high mortality. Skeletal muscle was the primary site of replication in mice for both viruses. However, MP-26M infection induced severe necrotizing myositis, whereas CHO-26M infection caused only mild inflammation. MP-26M was also isolated from whole blood, heart, liver, spleen and brain of infected mice. CHO-26M harboured a single mutation within the open reading frame (ORF), resulting in an amino acid substitution of K149→I in the VP2 capsid protein; two further ORF mutations that resulted in amino acid substitutions were identified in MP-26M, located within the VP1 capsid protein (G145→E) and the 2C protein (K216→R). Infectious cDNA clone-derived mutant virus populations containing the mutations identified in CHO-26M and MP-26M were generated in order to study the molecular basis of CHO cell and mouse adaptation. The VP2 (K149→I) change was responsible only for improved growth in CHO cells and did not lead to increased virulence in mice. Of the two amino acid substitutions identified in MP-26M, the VP1 (G145→E) mutation alone was sufficient to increase virulence in mice to the level observed in MP-26M-infected mice.
Characterization of nuclear localization signals of the prototype foamy virus integrase
Authors: An Dog Gn , Hyun Usok , Shin Cha-Gyun
Published: June 2008

To analyse the potential karyophilic activity of prototype foamy viruses (PFVs), we expressed the PFV integrase (IN) and its mutants as fusion proteins with enhanced green fluorescence protein. The subcellular localization of the fusion proteins was investigated by fluorescence microscopy. The PFV IN was found to be karyophilic and targeted the fusion protein to the nucleus. Mutational analyses demonstrated that the PFV IN contains a potent but non-transferable nuclear localization signal (NLS) in its C-terminal domain and contains five arginine and lysine residues between amino acids 308 and 329 that are critical for its NLS function.
Induction of lung adenocarcinoma in transgenic mice expressing activated EGFR driven by the SP‐C promoter
Authors: Ohashi Kadoaki , Rai Kammei , Fujiwara Yoshiro , Osawa Masahiro , Hirano Seiki , Takata Katsuyoshi , Kondo Eisaku , Yoshino Tadashi , Takata Minoru , Tanimoto Mitsune , Kiura Katsuyuki
Published: June 2008

To investigate the role of an activating epidermal growth factor receptor (EGFR) mutation in lung cancer, we generated transgenic mice expressing the delE748‐A752 mutant version of mouse EGFR driven by the SP‐C promoter, which is equivalent to the delE746‐A750 mutation found in lung cancer patients. Strikingly, the mice invariably developed multifocal lung adenocarcinomas of varying sizes at between 5 and 6 weeks of age, and they died from tumor progression approximately 2 months later if left untreated. Daily oral administration of the EGFR tyrosine kinase inhibitor (TKI) gefitinib (5 mg/kg/day) reduced the total and phosphorylation levels of EGFR to those in wild‐type mouse lung tissue; in addition, it abrogated tumor growth within 1 week and prolonged survival to >30 weeks. Interestingly, phosphorylated ErbB2, ErbB3, and thyroid transcriptional factor‐1 increased in the transgenic mice compared with those in wild‐type mice. They might play some roles in tumors progression in the transgenic mice. This model will be useful for studying the mechanisms of carcinogenesis, chemoprevention, and acquired resistance to EGFR TKIs in lung cancer patients carrying activating EGFR mutations. (Cancer Sci 2008; 99: 1747–1753)