bluuebunny/crossref_metadata_2025 huggingface dataset:
High expression of angiotensin‐converting enzyme and angiotensin‐converting enzyme 2 in preservation injury after liver transplantation in rats
Authors: Xia Chun‐Yan , Li Ling , Liu Hui‐Min , Cong Wen‐Ming
Published: August 2009

Aim:  To explore the possible role of local renin‐angiotensin system (RAS) in preservation injury (PI) after liver transplantation by studying expression of angiotensin‐converting enzyme (ACE) and angiotensin‐converting enzyme 2 (ACE2) in the transplanted liver of rats and its relationship with PI.

Methods:  The animals receiving liver transplantation were assigned to cold preservation group (CP group) and non‐cold preservation group (NCP group). The sham‐operation group was used as the control. The severity of PI was assessed by histology. The mRNA and protein expressions of ACE and ACE2 were detected by real‐time PCR, Western blot, respectively. Tissue hypoxia was assessed by pimonidazole staining.

Results:  Various degrees of tissue injury were observed after liver transplantation, especially in CP group. ACE2 mRNA and protein expressions in the transplantation groups were elevated significantly compared with those of the control group (P < 0.01, P < 0.05), and higher in CP group than those in NCP group (P < 0.05). There was a close positive correlation between PI and mRNA expression of ACE and ACE2. Positive pimonidazole staining distributed around the hepatic central vein, and became darker and more extensive with deterioration of PI.

Conclusion:  ACE2 was closely related to tissue hypoxia due to CP‐induced PI of the transplanted liver, and ACE may aggravate the inflammation in PI. Local RAS may play an important role in PI of the transplanted liver.
Spinal Endocannabinoids and CB
1
Receptors Mediate C-Fiber–Induced Heterosynaptic Pain Sensitization
Authors: Pernía-Andrade Alejandro J. , Kato Ako , Witschi Robert , Nyilas Rita , Katona István , Freund Tamás F. , Watanabe Masahiko , Filitz Jörg , Koppert Wolfgang , Schüttler Jürgen , Ji Guangchen , Neugebauer Volker , Marsicano Giovanni , Lutz Beat , Vanegas Horacio , Zeilhofer Hanns Ulrich
Published: August 2009

Plastic Pain Perception

Drugs and endocannabinoids acting on cannabinoid (CB) receptors have potential in the treatment of certain types of pain. In the spinal cord they are believed to suppress nociception, the perception of pain and noxious stimuli.

Pernia-Andrade
et al.

(p.
760
) now find that endocannabinoids, which are released in spinal cord by noxious stimulation, may promote rather than inhibit nociception by acting on CB1 receptors. Endocannabinoids not only depress transmission at excitatory synapses in the spinal cord, but also block the release of inhibitory neurotransmitters, thereby facilitating nociception.
Synaptic Integration in Tuft Dendrites of Layer 5 Pyramidal Neurons: A New Unifying Principle
Authors: Larkum Matthew E. , Nevian Thomas , Sandler Maya , Polsky Alon , Schiller Jackie
Published: August 2009

Fine Dendrites Fire Differently

The pyramidal neuron is the basic computational unit in the brain cortex. Its distal tuft dendrite is heavily innervated by horizontal fibers coursing through cortical layer-I providing long-range corticocortical and thalamocortical associational input.

Larkum
et al.

(p.
756
) investigated whether the apical tuft dendrites of layer-5 neocortical pyramidal neurons, like basal dendrites, generate
n
-methyl-
d
-aspartate (NMDA) spikes using two-photon–guided direct dendritic recording, glutamate uncaging, and modeling. NMDA spikes could be evoked in the distal tuft dendrites, while Ca
2+
spikes could be triggered at the bifurcation points. Block of the hyperpolarization-activated current enhanced these NMDA spikes. Thus, the generation of NMDA spikes is a general principle of thin, basal, and tuft dendrites.
Generalized Models Reveal Stabilizing Factors in Food Webs
Authors: Gross Thilo , Rudolf Lars , Levin Simon A. , Dieckmann Ulf
Published: August 2009

Untangling Food Webs

The factors affecting the stability of food webs are important in conservation and ecological restoration.

Gross
et al.

(p.
747
) used a generalized modeling approach to evaluate billions of replicates of food webs in order to reveal the properties that stabilize (or destabilize) food webs. Variability in the strength of trophic links between predator and prey strength affected stability in different ways depending on the size of the web—stabilizing only in relatively small food webs and destabilizing in larger ones. Universal topological rules were extracted for the patterns of network links that enhance food-web stability.
The Genetic Architecture of Maize Flowering Time
Authors: Buckler Edward S. , Holland James B. , Bradbury Peter J. , Acharya Charlotte B. , Brown Patrick J. , Browne Chris , Ersoz Elhan , Flint-Garcia Sherry , Garcia Arturo , Glaubitz Jeffrey C. , Goodman Major M. , Harjes Carlos , Guill Kate , Kroon Dallas E. , Larsson Sara , Lepak Nicholas K. , Li Huihui , Mitchell Sharon E. , Pressoir Gael , Peiffer Jason A. , Rosas Marco Oropeza , Rocheford Torbert R. , Romay M. Cinta , Romero Susan , Salvo Stella , Villeda Hector Sanchez , Sofia da Silva H. , Sun Qi , Tian Feng , Upadyayula Narasimham , Ware Doreen , Yates Heather , Yu Jianming , Zhang Zhiwu , Kresovich Stephen , McMullen Michael D.
Published: August 2009

Codifying Maize Modifications

Maize, one of our most important crop species, has been the target of genetic investigation and experimentation for more than 100 years. Crossing two inbred lines tends to result in “better” offspring, in a process known as heterosis. Attempts to map the genetic loci that control traits important for farming have been made, but few have been successful (see the Perspective by

Mackay

).

Buckler
et al.

(p.
714
) and

McMullen
et al.

(p.
737
) produced a genomic map of maize that relates recombination to genome structure. Even tremendous adaptations in very diverse species were produced by numerous, small additive steps. Differences in flowering time in maize among inbred lines were not caused by a few genes with large effects, but by the cumulative effects of numerous quantitative trait loci—each of which has only a small impact on the trait.
Genetic Properties of the Maize Nested Association Mapping Population
Authors: McMullen Michael D. , Kresovich Stephen , Villeda Hector Sanchez , Bradbury Peter , Li Huihui , Sun Qi , Flint-Garcia Sherry , Thornsberry Jeffry , Acharya Charlotte , Bottoms Christopher , Brown Patrick , Browne Chris , Eller Magen , Guill Kate , Harjes Carlos , Kroon Dallas , Lepak Nick , Mitchell Sharon E. , Peterson Brooke , Pressoir Gael , Romero Susan , Rosas Marco Oropeza , Salvo Stella , Yates Heather , Hanson Mark , Jones Elizabeth , Smith Stephen , Glaubitz Jeffrey C. , Goodman Major , Ware Doreen , Holland James B. , Buckler Edward S.
Published: August 2009

Codifying Maize Modifications

Maize, one of our most important crop species, has been the target of genetic investigation and experimentation for more than 100 years. Crossing two inbred lines tends to result in “better” offspring, in a process known as heterosis. Attempts to map the genetic loci that control traits important for farming have been made, but few have been successful (see the Perspective by

Mackay

).

Buckler
et al.

(p.
714
) and

McMullen
et al.

(p.
737
) produced a genomic map of maize that relates recombination to genome structure. Even tremendous adaptations in very diverse species were produced by numerous, small additive steps. Differences in flowering time in maize among inbred lines were not caused by a few genes with large effects, but by the cumulative effects of numerous quantitative trait loci—each of which has only a small impact on the trait.
The C-Ala Domain Brings Together Editing and Aminoacylation Functions on One tRNA
Authors: Guo Min , Chong Yeeting E. , Beebe Kirk , Shapiro Ryan , Yang Xiang-Lei , Schimmel Paul
Published: August 2009

Transfer RNA Fix

Accurate protein synthesis requires that transfer RNAs (tRNAs) must be acylated with the correct amino acid. Mistranslation of serine or glycine for alanine apparently presents a particular challenge and is corrected by an editing domain of alanyl-tRNA synthetases (AlaRSs).

Guo
et al.

(p.
744
) now show that aside from the aminoacylation and editing domains, a third domain, C-Ala, plays a key role in AlaRS function. It forms a nucleic acid–binding motif that promotes binding of both aminoacylation and editing domains to tRNA
Ala
. The coupling of these two functions was likely important in the evolution of AlaRSs.
Looking Forward in Pharmaceutical Process Chemistry
Authors: Davies Ian W. , Welch Christopher J.
Published: August 2009

Core activities of process chemistry involve the synthesis of drug candidates at a scale that supports clinical evaluations and the creation of a manufacturing process that is safe, robust, and cost effective. Innovative problem solving has always been a key element in process chemistry and will be increasingly important for addressing emerging challenges in science, globalization, and commoditization. The acquisition and creation of platform chemical technologies that enable rapid problem solving through the use of high-throughput experimentation will continue to be an important focus going forward. Linking and deploying platforms throughout the discovery-development continuum is expected to enable drug discovery. Increasing collaboration with academia will also become more important for accessing technical solutions and providing the next generation of innovative problem solvers.
Imagining Our Ancient Future

Knossos and the Prophets of Modernism
by Cathy Gere

University of Chicago Press, Chicago, 2009. 287 pp. $27.50, £19. ISBN 9780226289533.
Authors: Forth Christopher E.
Published: August 2009

Gere explores how Arthur Evans's often-fanciful interpretations of Minoan civilization (as pacifist, matriarchal, and sexually free), religion, and architecture influenced 20th-century European writers, artists, and intellectuals.
C3PO, an Endoribonuclease That Promotes RNAi by Facilitating RISC Activation
Authors: Liu Ying , Ye Xuecheng , Jiang Feng , Liang Chunyang , Chen Dongmei , Peng Junmin , Kinch Lisa N. , Grishin Nick V. , Liu Qinghua
Published: August 2009

RNA Wars

During RNA interference (RNAi), the Dicer endonuclease generates small interfering (si)RNAs that, with the help of the protein R2D2, are loaded into the siRNA-induced silencing complex (RISC). Using siRNAs as guides, RISC, and specifically its Argonaute subunit, targets complementary RNAs for destruction. In order to identify other components of the RISC complex,

Liu
et al.

(p.
750
) reconstituted the core RISC activity, using purified
Drosophila
Dicer, R2D2, and Ago-2. The protein C3PO (component 3 promoter of RISC), which consists of heterodimer of Translin and Translin-associated factor X (Trax), was found to enhance RISC activity in this system, and in vivo, with the Trax endonuclease activity activating RISC through the removal of siRNA passenger strand cleavage products.
Entropy Landscape of Phase Formation Associated with Quantum Criticality in Sr
3
Ru
2
O
7
Authors: Rost A. W. , Perry R. S. , Mercure J.-F. , Mackenzie A. P. , Grigera S. A.
Published: August 2009

Mapping Out an Entropic Landscape

Quantum critical points are continuous phase transitions occurring near absolute zero displaying interesting properties that may have valuable applications. Experimental thermodynamic information on quantum critical systems is sparse, partly because the better known systems are tuned using hydrostatic pressure, and thermodynamic measurements are difficult to perform under such conditions.

Rost
et al.

(p.
1360
, published online 6 August 2009; see the Perspective by

Fisk

) built and calibrated bespoke apparatus for a thermodynamic study of strontium ruthenate using a magnetic field as the tuning parameter. The specific heat and magnetocaloric measuremements were combined to map an “entropy landscape” of the quantum criticality and phase formation. This technique should offer a way to probe other materials to reveal unusual properties, such as novel metallic states and superconductivity.
Future Energy Institutes
Authors: Falkowski Paul G. , Goodman Robert M.
Published: August 2009

The landmark energy bill wending its way through Congress this summer seeks to reduce emissions of carbon dioxide and other greenhouse gases and supports the development of alternative energies, including solar and wind power. It's a bill that aims to create both a “green” U.S. economy and a sustainable environment. At this critical juncture, America must take an equally sustainable view toward investing in the brainpower required to confront the world's complex energy issues.
Designer Curvature
Authors: Liu Yan , Yan Hao
Published: August 2009

Complex nanostructures can be created by systematically tuning the bending and twisting of synthetic DNA assemblies.
Faulty Risk Analysis Puts Biofacility Plan in Jeopardy
Authors: Bhattacharjee Yudhijit
Published: August 2009

A damning report from the Government Accountability Office accuses the U.S. Department of Homeland Security of relying on a flawed risk analysis to select Manhattan, Kansas, as the site for its planned $450 million National Bio and Agro-Defense Facility.
Key Malaria Parasite Likely Evolved From Chimp Version
Authors: Cohen Jon
Published: August 2009

An international research team has uncovered compelling evidence that the malaria parasite,
Plasmodium falciparum
, evolved from a relative called
P. reichenowi
that infects chimpanzees.
Space Telescope's Chief Scientist Recalls How Hubble Was Saved
Authors: Bhattacharjee Yudhijit
Published: August 2009

As he prepares to retire, David Leckrone reflects on the rescue effort that kept the observatory in orbit and on the prospects for humans in space.
Can Gravity and Quantum Particles Be Reconciled After All?
Authors: Cho Adrian
Published: August 2009

One group of intrepid theorists thinks the answer to that question may be “yes,” and if they're right, one argument in favor of string theory unravels.
Putting Chemicals on a Path To Better Risk Assessment
Authors: Stokstad Erik
Published: August 2009

Industry and regulators are embracing new technologies to move beyond slow, expensive, and perplexing animal tests.
Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays
Authors: Verma Anita , Ngundi Miriam M. , Meade Bruce D. , De Pascalis Roberto , Elkins Karen L. , Burns Drusilla L.
Published: August 2009

Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fcγ) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcγ receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fcγ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcγ receptor blocking monoclonal antibodies, we found that at least three murine Fcγ receptor classes, IIB, III, and IV, can contribute to Fcγ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcγ receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output.
Age-Dependent Association between Low Frequency of CD27/CD28 Expression on pp65 CD8+T Cells and Cytomegalovirus Replication after Transplantation
Authors: Cantisán Sara , Torre-Cisneros Julián , Lara Rosario , Rodríguez-Benot Alberto , Santos Francisco , Gutiérrez-Aroca Juan , Gayoso Inmaculada , González-Padilla Marcelino , Casal Manuel , Rivero Antonio , Solana Rafael
Published: August 2009

In this cross-sectional study of 42 solid organ transplant recipients, the association of human cytomegalovirus (HCMV) replication and age with the phenotype of the HCMV-specific CD8+T cells was analyzed by using the CMV pp65 HLA-A*0201 pentamer. A correlation between the proportion of CD28−HCMV-specific CD8+T cells and age was observed in patients without HCMV replication (r= 0.50;P= 0.02) but not in patients with HCMV replication (r= −0.05;P= 0.83), a finding which differs from that observed for total CD8+T cells. Within the group of patients younger than 50 years of age, patients with HCVM replication after transplantation had higher percentages of CD28−HCMV-specific CD8+T cells (85.6 compared with 58.7% for patients without HCMV replication;P= 0.004) and CD27−HCMV-specific CD8+T cells (90.7 compared with 68.8% for patients without HCMV replication;P= 0.03). However, in patients older than age 50 years, a high frequency of these two subpopulations was observed in patients both with and without previous HCMV replication (for CD28−HCMV-specific CD8+T cells, 84.4 and 80.9%, respectively [P= 0.39]; for CD27−HCMV-specific CD8+T cells 86.6 and 81.5%, respectively [P= 0.16]). In conclusion, the present study shows that in the group of recipients younger than age 50 years, HCMV replication after transplantation is associated with a high percentage of CD27−and CD28−HCMV-specific CD8+T cells. These results suggest that the increased percentage of CD27−or CD28−HCMV-specific subsets can be considered a biomarker of HCMV replication in solid organ transplant recipients younger than age 50 years but not in older patients. Further studies are necessary to define the significance of these changes in HCMV-associated clinical complications posttransplantation.
Genetic Evolution of H9 Subtype Influenza Viruses from Live Poultry Markets in Shanghai, China
Authors: Ge Fei-Fei , Zhou Jin-Ping , Liu Jian , Wang Jian , Zhang Wei-Yi , Sheng Li-Ping , Xu Feng , Ju Hou-Bing , Sun Quan-Yun , Liu Pei-Hong
Published: August 2009

H9N2 influenza viruses have become established and maintain long-term endemicity in poultry. The complete genomes of seven avian H9N2 influenza viruses were characterized. These seven influenza virus isolates were obtained from live poultry markets in Shanghai, China, in 2002 and from 2006 to 2008. Genetic analysis revealed that all seven isolates had an RSSR motif at the cleavage site of hemagglutinin (HA), indicating low pathogenicity in chickens. Phylogenetic analyses indicated that the seven avian H9N2 viruses belonged to the lineage represented by Duck/Hong Kong/Y280/97 (H9N2), a virus belonging to the Chicken/Beijing/1/94-like (H9N2) lineage, and that they are all quadruple reassortants consisting of genes from different lineages. The six internal genes of the isolates possessed H5N1-like sequences, indicating that they were reassortants of H9 and H5 viruses. All of the viruses had nonstructural (as well as HA and neuraminidase) genes derived from the Duck/Hong Kong/Y280/97-like virus lineage but also had other genes of mixed avian virus origin, including genes similar to those of H5N1 viruses (Gs/GD-like). The infected chickens showed no signs of disease. These results show the genetic and biological diversity of H9N2 viruses in Shanghai and support their potential role as pandemic influenza agents.
Molecular Surveillance and Population Structure Analysis of Methicillin-Susceptible and Methicillin-Resistant
Staphylococcus aureus
in High-Risk Wards
Authors: Sergi Simona , Donnarumma Francesca , Mastromei Giorgio , Goti Emanuele , Nicoletti Pierluigi , Pecile Patrizia , Cecconi Daniela , Mannino Roberta , Fanci Rosa , Bosi Alberto , Bartolozzi Benedetta , Casalone Enrico
Published: August 2009

In this study we report the results of analysis of 253 isolates of
Staphylococcus aureus
(132 methicillin [meticillin]-resistant
S. aureus
[MRSA] isolates and 121 methicillin-susceptible
S. aureus
[MSSA] isolates) from 209 patients admitted to 18 high-risk wards of six hospitals located in Florence, Italy, over an 8-month period during which a program of epidemiological surveillance of hospital-acquired infections was conducted. The majority (69%) of the 87 reported
S. aureus
infections were caused by MRSA. No outbreak events have been reported. All the isolates were typed by amplified fragment length polymorphism (AFLP), and AFLP profiles were analyzed in order to define similarity groups. The discriminatory power of AFLP is very high with MSSA (Simpson index of diversity [
D
], 95.9%), whereas its resolution capability with MRSA (
D
, 44.7%) is hampered by the well-known high clonality of these populations (the main MRSA group accounted for 74% of the MRSA isolates). Combining AFLP, improved by visual inspection of polymorphisms, with multiplex PCR greatly increases MRSA resolution (
D
, 85.5%), resolving the MRSA population to a level that is one of the highest reported in the literature. Widespread and sporadic clones of MSSA and MRSA were identified, and their diffusion in the different hospitals and wards over the surveillance period was studied. The understanding of MSSA and MRSA population structures should be the starting point for the design of a more rational surveillance program for
S. aureus
species, maximizing benefits and reducing the cost of infection control strategies.
Atypical
Aspergillus flavus
Isolates Associated with Chronic Azole Therapy
Authors: Brandt Mary E. , Gade Lalitha , McCloskey Cindy B. , Balajee S. Arunmozhi
Published: August 2009

A case of chronic sinus disease due to morphologically atypical
Aspergillus flavus
is described. Multiple fungal isolates sporulated poorly or not at all, displaying unusual color and microscopic morphology, including the absence of typical vesicles and phialides, which caused the isolates to resemble several other fungal genera superficially. The patient received multiple antifungal therapies over at least 10 years with various azole drugs, including voriconazole, itraconazole, and posaconazole. We speculate that this lengthy exposure to azole antifungal drugs may have caused or promoted the atypical morphology seen in these isolates.
Serological Monitoring of Progression of Alveolar Echinococcosis with Multiorgan Involvement by Use of Recombinant Em18
Authors: Ishikawa Yuji , Sako Yasuhito , Itoh Sonoyo , Ohtake Takaaki , Kohgo Yutaka , Matsuno Takeo , Ohsaki Yoshinobu , Miyokawa Naoyuki , Nakao Minoru , Nakaya Kazuhiro , Ito Akira
Published: August 2009

Two cases of alveolar echinococcosis (AE) with multiple-organ involvement (the liver, lungs, and bone) were monitored by imaging and serology for 20 years. Resection of the bone lesion was complete in one case but incomplete in the other case. Albendazole treatment was markedly to moderately effective against hepatic and pulmonary AE lesions in both cases, whereas it had almost no effect against the bone lesion in one case. The results of the serological tests with recombinant Em18 antigen coincided with the clinical findings in each case. An enzyme-linked immunosorbent assay for the detection of immunoglobulin G (IgG) responses, especially IgG4 responses, is expected to be a real-time indicator of the dynamics of active AE.
Early Steps in Cell Infection by Parvoviruses: Host-Specific Differences in Cell Receptor Binding but Similar Endosomal Trafficking
Authors: Harbison Carole E. , Lyi Sangbom Michael , Weichert Wendy S. , Parrish Colin R.
Published: August 2009

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are closely related parvoviruses that differ in their host ranges for cats and dogs. Both viruses bind their host transferrin receptor (TfR), enter cells by clathrin-mediated endocytosis, and traffic with that receptor through endosomal pathways. Infection by these viruses appears to be inefficient and slow, with low numbers of virions infecting the cell after a number of hours. Species-specific binding to TfR controls viral host range, and in this study FPV and strains of CPV differed in the levels of cell attachment, uptake, and infection in canine and feline cells. During infection, CPV particles initially bound and trafficked passively on the filopodia of canine cells while they bound to the cell body of feline cells. That binding was associated with the TfR as it was disrupted by anti-TfR antibodies. Capsids were taken up from the cell surface with different kinetics in canine and feline cells but, unlike transferrin, most did not recycle. Capsids labeled with fluorescent markers were seen in Rab5-, Rab7-, or Rab11-positive endosomal compartments within minutes of uptake, but reached the nucleus. Constitutively active or dominant negative Rab mutants changed the intracellular distribution of capsids and affected the infectivity of virus in cells.
Tissue-Spanning Redox Gradient-Dependent Assembly of Native Human Papillomavirus Type 16 Virions
Authors: Conway Michael J. , Alam Samina , Ryndock Eric J. , Cruz Linda , Christensen Neil D. , Roden Richard B. S. , Meyers Craig
Published: August 2009

Papillomavirus capsids are composed of 72 pentamers reinforced through inter- and intrapentameric disulfide bonds. Recent research suggests that virus-like particles and pseudovirions (PsV) can undergo a redox-dependent conformational change involving disulfide interactions. We present here evidence that native virions exploit a tissue-spanning redox gradient that facilitates assembly events in the context of the complete papillomavirus life cycle. DNA encapsidation and infectivity titers are redox dependent in that they can be temporally modulated via treatment of organotypic cultures with oxidized glutathione. These data provide evidence that papillomavirus assembly and maturation is redox-dependent, utilizing multiple steps within both suprabasal and cornified layers.
Testing an Electrostatic Interaction Hypothesis of Hepatitis B Virus Capsid Stability by Using an In Vitro Capsid Disassembly/Reassembly System
Authors: Newman Margaret , Chua Pong Kian , Tang Fan-Mei , Su Pei-Yi , Shih Chiaho
Published: August 2009

To test a previously coined “charge balance hypothesis” of human hepatitis B virus (HBV) capsid stability, we established an in vitro disassembly and reassembly system using bacterially expressed HBV capsids. Capsid disassembly can be induced by micrococcal nuclease digestion of encapsidated RNA. HBV core protein (HBc) mutants containing various amounts of arginine were constructed by serial truncations at the C terminus. Capsids containing smaller amounts of arginine (HBc 149, 154, and 157) remained intact after micrococcal nuclease digestion by native gel electrophoresis. Capsids containing larger amounts of arginine (HBc 159, 164, 169, and 171) exhibited reduced and more diffuse banding intensity and slightly upshifted mobility (HBc 159 and 164). Capsids containing the largest amounts of arginine (HBc 173, 175, and 183), as well as HBc 167, exhibited no detectable banding signal, indicating loss of capsid integrity or stability. Interestingly, capsid reassembly can be induced by polyanions, including oligonucleotides, poly-glutamic acid, and nonbiological polymer (polyacrylic acid). In contrast, polycations (polylysine and polyethylenimine) and low-molecular-weight anions (inositol triphosphate) induced no capsid reassembly. Results obtained by gel assay were confirmed by electron microscopy. Reassembled capsids comigrated with undigested parental capsids on agarose gels and cosedimented with undigested capsids by sucrose gradient ultracentrifugation. Taken together, the results indicate that HBV capsid assembly and integrity depend on polyanions, which probably can help minimize intersubunit charge repulsion caused mainly by arginine-rich domain III or IV in close contact. The exact structure of polyanions is not important for in vitro capsid reassembly. A large amount of independent experimental evidence for this newly coined “electrostatic interaction hypothesis” is discussed.
Activation of MDA5 Requires Higher-Order RNA Structures Generated during Virus Infection
Authors: Pichlmair Andreas , Schulz Oliver , Tan Choon-Ping , Rehwinkel Jan , Kato Hiroki , Takeuchi Osamu , Akira Shizuo , Way Michael , Schiavo Giampietro , Reis e Sousa Caetano
Published: August 2009

Recognition of virus presence via RIG-I (retinoic acid inducible gene I) and/or MDA5 (melanoma differentiation-associated protein 5) initiates a signaling cascade that culminates in transcription of innate response genes such as those encoding the alpha/beta interferon (IFN-α/β) cytokines. It is generally assumed that MDA5 is activated by long molecules of double-stranded RNA (dsRNA) produced by annealing of complementary RNAs generated during viral infection. Here, we used an antibody to dsRNA to show that the presence of immunoreactivity in virus-infected cells does indeed correlate with the ability of RNA extracted from these cells to activate MDA5. Furthermore, RNA from cells infected with encephalomyocarditis virus or with vaccinia virus and precipitated with the anti-dsRNA antibody can bind to MDA5 and induce MDA5-dependent IFN-α/β production upon transfection into indicator cells. However, a prominent band of dsRNA apparent in cells infected with either virus does not stimulate IFN-α/β production. Instead, stimulatory activity resides in higher-order structured RNA that contains single-stranded RNA and dsRNA. These results suggest that MDA5 activation requires an RNA web rather than simply long molecules of dsRNA.
Effect of B-Cell Depletion on Viral Replication and Clinical Outcome of Simian Immunodeficiency Virus Infection in a Natural Host
Authors: Gaufin Thaidra , Pattison Melissa , Gautam Rajeev , Stoulig Crystal , Dufour Jason , MacFarland Jeanne , Mandell Daniel , Tatum Coty , Marx Matthew H. , Ribeiro Ruy M. , Montefiori David , Apetrei Cristian , Pandrea Ivona
Published: August 2009

Simian immunodeficiency virus (SIV)-infected African nonhuman primates do not progress to AIDS in spite of high and persistent viral loads (VLs). Some authors consider the high viral replication observed in chronic natural SIV infections to be due to lower anti-SIV antibody titers than those in rhesus macaques, suggesting a role of antibodies in controlling viral replication. We therefore investigated the impact of antibody responses on the outcome of acute and chronic SIVagm replication in African green monkeys (AGMs). Nine AGMs were infected with SIVagm.sab. Four AGMs were infused with 50 mg/kg of body weight anti-CD20 (rituximab; a gift from Genentech) every 21 days, starting from day −7 postinfection up to 184 days. The remaining AGMs were used as controls and received SIVagm only. Rituximab-treated AGMs were successfully depleted of CD20 cells in peripheral blood, lymph nodes (LNs), and intestine, as shown by the dynamics of CD20+and CD79a+cells. There was no significant difference in VLs between CD20-depleted AGMs and control monkeys: peak VLs ranged from 107to 108copies/ml; set-point values were 104to 105SIV RNA copies/ml. Levels of acute mucosal CD4+T-cell depletion were similar for treated and nontreated animals. SIVagm seroconversion was delayed for the CD20-depleted AGMs compared to results for the controls. There was a significant difference in both the timing and magnitude of neutralizing antibody responses for CD20-depleted AGMs compared to results for controls. CD20 depletion significantly altered the histological structure of the germinal centers in the LNs and Peyer's patches. Our results, although obtained with a limited number of animals, suggest that humoral immune responses play only a minor role in the control of SIV viral replication during acute and chronic SIV infection in natural hosts.
Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans
Authors: Kwilas Steven , Liesman Rachael M. , Zhang Liqun , Walsh Edward , Pickles Raymond J. , Peeples Mark E.
Published: August 2009

Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.
Activation of Tumor Suppressor Protein p53 Is Required for Theiler's Murine Encephalomyelitis Virus-Induced Apoptosis in M1-D Macrophages
Authors: Son Kyung-No , Pugazhenthi Subbiah , Lipton Howard L.
Published: August 2009

Theiler's murine encephalomyelitis virus (TMEV) is a highly cytolytic picornavirus that persists in the mouse central nervous system (CNS) largely in macrophages with infection maintained by macrophage-to-macrophage spread. Infected macrophages in the CNS undergo apoptosis. We recently showed that M1-D macrophages infected with the low-neurovirulence TMEV BeAn virus became apoptotic through the mitochondrial pathway that is Bax mediated. Our present analyses of the molecular events and signaling pathway(s) culminating in the mitochondrial outer membrane permeabilization that initiates the caspase cascade and apoptosis of BeAn virus-infected M1-D macrophages revealed activation of p38 mitogen-activated protein kinase by 2 to 3 h postinfection (p.i.), followed by phosphorylation of tumor suppressor protein p53 Ser 15 at 3 to 6 h p.i., stabilizing p53 levels until 6 h p.i. Activated p53 upregulated the transcription of proapoptoticpumaandnoxagenes at 2 to 4 h p.i. and their BH3-only protein expression, followed by the loss of detectable prosurvival Mcl-1 and A1 proteins at 4 to 10 h p.i. Degradation of the prosurvival proteins is known to release Bax, which forms homo-oligomers and translocates into and permeabilizes the mitochondrial outer membrane. Inhibition of phospho-p38 by two specific inhibitors, SB203580 and BIRB796, led to a significant decrease in apoptosis at 10 h p.i., with no effect on virus titers (only SB203580 tested). Together, these data indicate that p53 activation is required for the induction of apoptosis in infected M1-D cells.
Cochaperone Activity of Human Butyrate-Induced Transcript 1 Facilitates Hepatitis C Virus Replication through an Hsp90-Dependent Pathway
Authors: Taguwa Shuhei , Kambara Hiroto , Omori Hiroko , Tani Hideki , Abe Takayuki , Mori Yoshio , Suzuki Tetsuro , Yoshimori Tamotsu , Moriishi Kohji , Matsuura Yoshiharu
Published: August 2009

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of the replication complex consisting of several host and viral proteins. We have previously reported that human butyrate-induced transcript 1 (hB-ind1) recruits heat shock protein 90 (Hsp90) and FK506-binding protein 8 (FKBP8) to the replication complex through interaction with NS5A. To gain more insights into the biological functions of hB-ind1 in HCV replication, we assessed the potential cochaperone-like activity of hB-ind1, because it has significant homology with cochaperone p23, which regulates Hsp90 chaperone activity. The chimeric p23 in which the cochaperone domain was replaced with the p23-like domain of hB-ind1 exhibited cochaperone activity comparable to that of the authentic p23, inhibiting the glucocorticoid receptor signaling in an Hsp90-dependent manner. Conversely, the chimeric hB-ind1 in which the p23-like domain was replaced with the cochaperone domain of p23 resulted in the same level of recovery of HCV propagation as seen in the authentic hB-ind1 in cells with knockdown of the endogenous hB-ind1. Immunofluorescence analyses revealed that hB-ind1 was colocalized with NS5A, FKBP8, and double-stranded RNA in the HCV replicon cells. HCV replicon cells exhibited a more potent unfolded-protein response (UPR) than the parental and the cured cells upon treatment with an inhibitor for Hsp90. These results suggest that an Hsp90-dependent chaperone pathway incorporating hB-ind1 is involved in protein folding in the membranous web for the circumvention of the UPR and that it facilitates HCV replication.
IRF3 Inhibition by Rotavirus NSP1 Is Host Cell and Virus Strain Dependent but Independent of NSP1 Proteasomal Degradation
Authors: Sen Adrish , Feng Ningguo , Ettayebi Khalil , Hardy Michele E. , Greenberg Harry B.
Published: August 2009

Rotavirus host range restriction forms a basis for strain attenuation although the underlying mechanisms are unclear. In mouse fibroblasts, the inability of rotavirus NSP1 to mediate interferon (IFN) regulatory factor 3 (IRF3) degradation correlates with IFN-dependent restricted replication of the bovine UK strain but not the mouse EW and simian RRV strains. We found that UK NSP1 is unable to degrade IRF3 when expressed in murine NIH 3T3 cells in contrast to the EW and RRV NSP1 proteins. Surprisingly, UK NSP1 expression led to IRF3 degradation in simian COS7 cells, indicating that IRF3 degradation by NSP1 is host cell dependent, a finding further supported using adenovirus-expressed NSP1 from NCDV bovine rotavirus. By expressing heterologous IRF3 proteins in complementary host cells, we found that IRF3 is the minimal host factor constraining NSP1 IRF3-degradative ability. NSP1-mediated IRF3 degradation was enhanced by transfection of double-stranded RNA (dsRNA) in a host cell-specific manner, and in IRF3-dependent positive regulatory domain III reporter assays, NSP1 inhibited IRF3 function in response to pathway activation by dsRNA, TBK-1, IRF3, or constitutively activated IRF3-5D. An interesting observation arising from these experiments is the ability of transiently expressed UK NSP1 to inhibit poly(I:C)-directed IRF3 activity in NIH 3T3 cells in the absence of detectable IRF3 degradation, an unexpected finding since UK virus infection was unable to block IFN secretion, and UK NSP1 expression did not result in suppression of IRF3-directed activation of the pathway. RRV and EW but not UK NSP1 was proteasomally degraded, requiring E1 ligase activity, although NSP1 degradation was not required for IRF3 degradation. Using a chimeric RRV NSP1 protein containing the carboxyl 100 residues derived from UK NSP1, we found that the RRV NSP1 carboxyl 100 residues are critical for its IRF3 inhibition in murine cells but are not essential for NSP1 degradation. Thus, NSP1's ability to degrade IRF3 is host cell dependent and is independent of NSP1 proteasomal degradation.
Reconstitution of Authentic Nanovirus from Multiple Cloned DNAs
Authors: Grigoras Ioana , Timchenko Tatiana , Katul Lina , Grande-Pérez Ana , Vetten Heinrich-Josef , Gronenborn Bruno
Published: August 2009

We describe a new plant single-stranded DNA (ssDNA) virus, a nanovirus isolate originating from the faba bean in Ethiopia. We applied rolling circle amplification (RCA) to extensively copy the individual circular DNAs of the nanovirus genome. By sequence analyses of more than 208 individually cloned genome components, we obtained a representative sample of eight polymorphic swarms of circular DNAs, each about 1 kb in size. From these heterogeneous DNA populations after RCA, we inferred consensus sequences of the eight DNA components of the virus genome. Based on the distinctive molecular and biological properties of the virus, we propose to consider it a new species of the genus
Nanovirus
and to name it faba bean necrotic stunt virus (FBNSV). Selecting a representative clone of each of the eight DNAs for transfer by T-DNA plasmids of
Agrobacterium tumefaciens
into
Vicia faba
plants, we elicited the development of the typical FBNSV disease symptoms. Moreover, we showed that the virus thus produced was readily transmitted by two different aphid vector species,
Aphis craccivora
and
Acyrthosiphon pisum
. This represents the first reconstitution of a fully infectious and sustainably insect-transmissible nanovirus from its cloned DNAs and provides compelling evidence that the genome of a legume-infecting nanovirus is typically comprised of eight distinct DNA components.
Baculovirus Transduction of Mesenchymal Stem Cells Triggers the Toll-Like Receptor 3 Pathway
Authors: Chen Guan-Yu , Shiah Hsiao-Chiao , Su Hung-Ju , Chen Chi-Yuan , Chuang Yung-Jen , Lo Wen-Hsin , Huang Jie-Len , Chuang Ching-Kuang , Hwang Shiaw-Min , Hu Yu-Chen
Published: August 2009

Human mesenchymal stem cells (hMSCs) can be genetically modified with viral vectors and hold promise as a cell source for regenerative medicine, yet how hMSCs respond to viral vector transduction remains poorly understood, leaving the safety concerns unaddressed. Here, we explored the responses of hMSCs against an emerging DNA viral vector, baculovirus (BV), and discovered that BV transduction perturbed the transcription of 816 genes associated with five signaling pathways. Surprisingly, Toll-like receptor-3 (TLR3), a receptor that generally recognizes double-stranded RNA, was apparently upregulated by BV transduction, as confirmed by microarray, PCR array, flow cytometry, and confocal microscopy. Cytokine array data showed that BV transduction triggered robust secretion of interleukin-6 (IL-6) and IL-8 but not of other inflammatory cytokines and beta interferon (IFN-β). BV transduction activated the signaling molecules (e.g., Toll/interleukin-1 receptor domain-containing adaptor-inducing IFN-β, NF-κB, and IFN regulatory factor 3) downstream of TLR3, while silencing the
TLR3
gene with small interfering RNA considerably abolished cytokine expression and promoted cell migration. These data demonstrate, for the first time, that a DNA viral vector can activate the TLR3 pathway in hMSCs and lead to a cytokine expression profile distinct from that in immune cells. These findings underscore the importance of evaluating whether the TLR3 signaling cascade plays roles in the immune response provoked by other DNA vectors (e.g., adenovirus). Nonetheless, BV transduction barely disturbed surface marker expression and induced only transient and mild cytokine responses, thereby easing the safety concerns of using BV for hMSCs engineering.
Application of Bioluminescence Imaging to the Prediction of Lethality in Vaccinia Virus-Infected Mice
Authors: Zaitseva Marina , Kapnick Senta M. , Scott John , King Lisa R. , Manischewitz Jody , Sirota Lev , Kodihalli Shantha , Golding Hana
Published: August 2009

To find an alternative endpoint for the efficacy of antismallpox treatments, bioluminescence was measured in live BALB/c mice following lethal challenge with a recombinant WR vaccinia virus expressing luciferase. Intravenous vaccinia immunoglobulin treatments were used to confer protection on a proportion of animals. Using known lethality outcomes in 200 animals and total fluxes recorded daily in live animals, we performed univariate receiver operating characteristic (ROC) curve analysis to assess whether lethality can be predicted based on bioluminescence. Total fluxes in the spleens on day 3 and in the livers on day 5 generated accurate predictive models; the area under the ROC curve (AUC) was 0.91. Multiple logistic regression analysis utilizing a linear combination of six measurements: total flux in the liver on days 2, 3, and 5; in the spleen on days 1 and 3; and in the nasal cavity on day 4 generated the most accurate predictions (AUC = 0.96). This model predicted lethality in 90% of animals with only 10% of nonsurviving animals incorrectly predicted to survive. Compared with bioluminescence, ROC analysis with 25% and 30% weight loss as thresholds accurately predicted survival on day 5, but lethality predictions were low until day 9. Collectively, our data support the use of bioimaging for lethality prediction following vaccinia virus challenge and for gaining insight into protective mechanisms conferred by vaccines and therapeutics.
A B-Box 2 Surface Patch Important for TRIM5α Self-Association, Capsid Binding Avidity, and Retrovirus Restriction
Authors: Diaz-Griffero Felipe , Qin Xu-rong , Hayashi Fumiaki , Kigawa Takanori , Finzi Andres , Sarnak Zoe , Lienlaf Maritza , Yokoyama Shigeyuki , Sodroski Joseph
Published: August 2009

TRIM5α is a tripartite motif (TRIM) protein that consists of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. The TRIM5α
rh
protein from rhesus monkeys recognizes the human immunodeficiency virus type 1 (HIV-1) capsid as it enters the host cell and blocks virus infection prior to reverse transcription. HIV-1-restricting ability can be eliminated by disruption of the B-box 2 domain. Changes in the TRIM5α
rh
B-box 2 domain have been associated with alterations in TRIM5α
rh
turnover, the formation of cytoplasmic bodies and higher-order oligomerization. We present here the nuclear magnetic resonance structure of the TRIM5 B-box 2 domain and identify an unusual hydrophobic patch (cluster 1) on the domain surface. Alteration of cluster 1 or the flanking arginine 121 resulted in various degrees of inactivation of HIV-1 restriction, in some cases depending on compensatory changes in other nearby charged residues. For this panel of TRIM5α
rh
B-box 2 mutants, inhibition of HIV-1 infection was strongly correlated with higher-order self-association and binding affinity for capsid complexes but not with TRIM5α
rh
half-life or the formation of cytoplasmic bodies. Thus, promoting cooperative TRIM5α
rh
interactions with the HIV-1 capsid represents a major mechanism whereby the B-box 2 domain potentiates HIV-1 restriction.