bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypothermia for Stroke: Call to Action 2010
Authors: Macleod Malcolm R. , Petersson Jesper , Norrving Bo , Hacke Werner , Dirnagl Ulrich , Wagner Markus , Schwab Stefan ,   on behalf of the participants of the European Hypothermia Stroke Research Workshop – Brussels, 25 January 2010 and endorsed by the European Stroke Network, European Stroke Organisation, European Stroke Research Network for Hypothermia, Society for Crybiology, Stroke Alliance for Europe, World Stroke Organisation and the European Space Agency
Published: November 2010

The European Hypothermia Stroke Research Workshop was held in January 2010, in response to the alarming prospects of a significant increase of stroke expected in the coming years globally. Considering that a minority of patients (around 10%) are currently eligible for thrombolytic treatment, there is a need for an efficacious, cost-effective novel therapy that can be implemented broadly within European health care systems. Accordingly, the primary objective of the workshop was the definition of a research agenda aiming to assess the therapeutic benefits of hypothermia in patients with acute ischaemic stroke. The meeting was organised by the European Stroke Research Network for Hypothermia (EuroHyp) and attended by the representatives of World Stroke Organisation, European Stroke Organisation, Stroke Alliance for Europe, Society for Cryobiology and other organisations – specifically the European Space Agency, and small- and medium-sized enterprises based in EU member states. The participants adopted the ‘Hypothermia for Stroke – Call to Action 2010’, a declaration specifying the priorities for hypothermia research in acute ischaemic stroke. The research programme outlined – a clinical study programme designed to identify and validate therapeutic cooling as a novel treatment providing benefit to a large number of stroke patients–contains a well-integrated series of Phase II studies aiming to refine the intervention (depth, duration, and mode of cooling; antishivering strategy; patient selection) and a pivotal Phase III clinical trial. The proposed integrated Phase II and III clinical study programme would test the effectiveness of this optimised intervention, and would allow the development of evidence-based Clinical Practice Guidelines describing the optimal use of therapeutic hypothermia as a treatment strategy for stroke.
ITPA gene variant protects against anemia induced by pegylated interferon‐α and ribavirin therapy for Japanese patients with chronic hepatitis C
Authors: Sakamoto Naoya , Tanaka Yasuhito , Nakagawa Mina , Yatsuhashi Hiroshi , Nishiguchi Shuhei , Enomoto Nobuyuki , Azuma Seishin , Nishimura‐Sakurai Yuki , Kakinuma Sei , Nishida Nao , Tokunaga Katsushi , Honda Masao , Ito Kiyoaki , Mizokami Masashi , Watanabe Mamoru
Published: October 2010

Aim:  Host genetic variants leading to inosine triphosphatase (ITPA) deficiency, a condition not thought to be clinically important, protect against hemolytic anemia in chronic hepatitis C patients receiving ribavirin. In this study, we evaluated the clinical significance of ITPA variants in Japanese hepatitis C patients who were treated with pegylated interferon plus ribavirin.

Methods:  In this multicenter retrospective cross‐sectional study, 474 hepatitis C patients were enrolled who were treated with pegylated interferon plus ribavirin in four geographically different hospitals in Japan. Patients were grouped according to hemoglobin decline of more than 3 g/dL at week 4. Two single nucleotide polymorphisms (SNP) within or adjacent to the ITPA gene (rs6051702, rs1127354) were genotyped.

Results:  A functional SNP, rs1127354, within the ITPA exon was strongly associated with protection against anemia with only one (0.8%) in 129 patients with the ITPA minor variant A developing severe anemia (P = 5.9 × 10−20). For rs6051702, which had significant association in European‐Americans, significant but weak association with severe hemoglobin reduction was found in Japanese (P = 0.009). In patients excluding genotype 1b and high viral load, those with the ITPA minor variant A achieved significantly higher sustained viral response rate than those with the major variant (CC) (96% vs 70%, respectively, P = 0.0066).

Conclusion:  ITPA SNP, rs1127354, is confirmed to be a useful predictor of ribavirin‐induced anemia in Japanese patients. Patients with the ITPA minor variant A (∼27%) have an advantage in pegylated interferon plus ribavirin‐based therapies, due to expected adherence of ribavirin doses, resulting in a higher viral clearance rate.
Computationally Efficient Finite Element Evaluation of Natural Patellofemoral Mechanics
Authors: Fitzpatrick Clare K. , Baldwin Mark A. , Rullkoetter Paul J.
Published: October 2010

Finite element methods have been applied to evaluate in vivo joint behavior, new devices, and surgical techniques but have typically been applied to a small or single subject cohort. Anatomic variability necessitates the use of many subject-specific models or probabilistic methods in order to adequately evaluate a device or procedure for a population. However, a fully deformable finite element model can be computationally expensive, prohibiting large multisubject or probabilistic analyses. The aim of this study was to develop a group of subject-specific models of the patellofemoral joint and evaluate trade-offs in analysis time and accuracy with fully deformable and rigid body articular cartilage representations. Finite element models of eight subjects were used to tune a pressure-overclosure relationship during a simulated deep flexion cycle. Patellofemoral kinematics and contact mechanics were evaluated and compared between a fully deformable and a rigid body analysis. Additional eight subjects were used to determine the validity of the rigid body pressure-overclosure relationship as a subject-independent parameter. There was good agreement in predicted kinematics and contact mechanics between deformable and rigid analyses for both the tuned and test groups. Root mean square differences in kinematics were less than 0.5 deg and 0.2 mm for both groups throughout flexion. Differences in contact area and peak and average contact pressures averaged 5.4%, 9.6%, and 3.8%, respectively, for the tuned group and 6.9%, 13.1%, and 6.4%, respectively, for the test group, with no significant differences between the two groups. There was a 95% reduction in computational time with the rigid body analysis as compared with the deformable analysis. The tuned pressure-overclosure relationship derived from the patellofemoral analysis was also applied to tibiofemoral (TF) articular cartilage in a group of eight subjects. Differences in contact area and peak and average contact pressures averaged 8.3%, 11.2%, and 5.7% between rigid and deformable analyses in the tibiofemoral joint. As statistical, probabilistic, and optimization techniques can require hundreds to thousands of analyses, a viable platform is crucial to component evaluation or clinical applications. The computationally efficient rigid body platform described in this study may be integrated with statistical and probabilistic methods and has potential clinical application in understanding in vivo joint mechanics on a subject-specific or population basis.
Alleviating Cancer Drug Toxicity by Inhibiting a Bacterial Enzyme
Authors: Wallace Bret D. , Wang Hongwei , Lane Kimberly T. , Scott John E. , Orans Jillian , Koo Ja Seol , Venkatesh Madhukumar , Jobin Christian , Yeh Li-An , Mani Sridhar , Redinbo Matthew R.
Published: November 2010

Blocking Interfering Microbes

Irinotecan is a widely used anticancer pro-drug that is converted in the liver into the active form, but when it gets into the gut, the normally benign microbial flora can convert it into the toxic form, which kills the rapidly multiplying gut epithelium as it would kill rapidly dividing tumor cells, and thus causes diarrhea.Wallaceet al.(p.831; see the Perspective byPatel and Kaufmann) used high-throughput screening to identify inhibitors that target the offending bacterial enzyme, β-glucuronidase, without killing the bacteria or affecting orthologous mammalian enzymes. Crystal structures revealed the molecular basis of selectivity, and in vivo studies showed that an inhibitor protected mice from irinotecan-induced toxicity.
Transient Middle Eocene Atmospheric CO
2
and Temperature Variations
Authors: Bijl Peter K. , Houben Alexander J. P. , Schouten Stefan , Bohaty Steven M. , Sluijs Appy , Reichart Gert-Jan , Sinninghe Damsté Jaap S. , Brinkhuis Henk
Published: November 2010

The Dependable Warmer

During the middle of the Eocene, about 40 million years ago, a transient warming event interrupted the long-term cooling trend that had been in progress for the previous 10 million years.

Bijl
et al.

(p.
819
; see the Perspective by

Pearson

) constructed records of sea surface temperature and atmospheric CO
2
concentrations across the warming period. It appears that vast amounts of CO
2
were injected into the atmosphere, and a sea surface temperature increase of as much a 6°C accompanied the atmospheric CO
2
rise.
Collaborative Non-Self Recognition System in S-RNase–Based Self-Incompatibility
Authors: Kubo Ken-ichi , Entani Tetsuyuki , Takara Akie , Wang Ning , Fields Allison M. , Hua Zhihua , Toyoda Mamiko , Kawashima Shin-ichi , Ando Toshio , Isogai Akira , Kao Teh-hui , Takayama Seiji
Published: November 2010

Dissecting Self-Incompatibility

Although the pollen may be available for a flower to fertilize itself, molecular determinants on the pollen and the pistil prevent inbreeding in a process termed self-incompatibility. In the
Petunia
self-incompatibility, if male determinants (F-box proteins) on pollen are recognized by a female ribonuclease determinant on the pistil, the pollen tube is killed when its ribosomal RNA is digested. Outcrossed fertilizations can occur because of allelic diversity in the female that fails to recognize its male counterparts; however, the genetic diversity of the ribonuclease gene is greater than that of the known
F-box
gene.

Kubo
et al.

(p.
796
; see the Perspective by

Indriolo and Goring

) have discovered that there are several related
F-box
genes in
Petunia
, each of which brings its own allelic diversity to bear—thus, increasing the variety of potential mating partners.
Suppression of Antitumor Immunity by Stromal Cells Expressing Fibroblast Activation Protein–α
Authors: Kraman Matthew , Bambrough Paul J. , Arnold James N. , Roberts Edward W. , Magiera Lukasz , Jones James O. , Gopinathan Aarthi , Tuveson David A. , Fearon Douglas T.
Published: November 2010

Tumor Vaccination Success

Vaccination with tumor-specific antigens is one of several attempted therapies seeking to harness the immune system, but—unfortunately—this strategy has been unsuccessful, possibly because of the immunosuppressive properties of the tumor microenvironment.

Kraman
et al.

(p.
827
; see the Perspective by

Schreiber and Rowley

) have identified immunosuppressive cells of mesenchymal origin in mice comprising 2% of the tumor stromal cell population. They were identified by expression of the fibroblast activation protein–α. Deletion of these cells in lung or pancreatic cancers in mice allowed successful therapeutic vaccination against the tumors, which was dependent on the adaptive immune system and the cytokines interferon-γ and tumor necrosis factor–α. These findings reveal that multiple cell types contribute to the immunosuppressive tumor microenvironment and will inform therapeutic cancer vaccine design.
Awakening Immunity
Authors: Schreiber Hans , Rowley Donald A.
Published: November 2010

The elimination of specific stromal cells allows the immune system to suppress the growth of solid malignant tumors.
Thermal Enhancement with Optically Activated Gold Nanoshells Sensitizes Breast Cancer Stem Cells to Radiation Therapy
Authors: Atkinson Rachel L. , Zhang Mei , Diagaradjane Parmeswaran , Peddibhotla Sirisha , Contreras Alejandro , Hilsenbeck Susan G. , Woodward Wendy A. , Krishnan Sunil , Chang Jenny C. , Rosen Jeffrey M.
Published: October 2010

Local hyperthermia with gold nanoshells sensitizes cancer stem cells to radiation treatment in mouse and human breast cancer preclinical models.
ICOStomizing Immunotherapies with T
H
17
Authors: Garaude Johan , Blander J. Magarian
Published: October 2010

Tilting the balance between spurring or suppressing the expansion of selected T
H
17 cells may be a versatile key to tailoring immunotherapies.
An Expanded Tumor Playing Field
Authors: Alumkal Joshi
Published: October 2010

Tumors promote DNA damage at distant sites by boosting chronic inflammation.
Licensed to Enter
Authors: Cheng Elaine
Published: October 2010

A protein transduction domain peptide enhances the cellular uptake of Foxp3 protein for the treatment of autoimmune and allergic disorders.
Prospective Survey of (1→3)-β-
d
-Glucan and Its Relationship to Invasive Candidiasis in the Surgical Intensive Care Unit Setting
Authors: Mohr John F. , Sims Charles , Paetznick Victor , Rodriguez Jose , Finkelman Malcolm A. , Rex John H. , Ostrosky-Zeichner Luis
Published: November 2010

Non-culture-based diagnostic strategies are needed for diagnosing invasive candidiasis (IC). We evaluated serial serum (1→3)-β-
d
-glucan (BG) levels in patients in the surgical trauma intensive care unit (SICU) patients with clinical evidence of IC. Serum samples from patients admitted to the SICU for a minimum of 3 days were collected twice weekly and analyzed for BG by using a Fungitell kit with a positive cutoff of ≥80 pg/ml. Diagnosis of IC was done using a set of predefined and validated clinical practice-based criteria. A total of 57 patients consented to participate and were enrolled. The median ICU stay was 16 days (range, 3 to 51). A total of 14 of 57 (25%) false positives were observed in the first sample (ICU day 3) and, overall, 73% of the day 3 samples had higher BG levels than subsequent samples. On the date of clinical diagnosis of IC, the sensitivity of a positive BG for identifying invasive candidiasis was 87%, with a 73% specificity. In patients with evidence of IC, the median BG value was significantly higher than those without evidence of IC (171 versus 48 pg/ml,
P
= 0.02), respectively. In the three patients with proven IC, BG was detected 4 to 8 days prior to diagnosis. BG serum detection may be a useful tool to aid in the early diagnosis of IC in SICU patients, particularly after day 3 and in patients with at least two positive samples drawn several days apart. Elevated BG levels within the first 3 days need to be further characterized.
Inverse Association between Lancefield Group G
Streptococcus
Colonization and Sore Throat in Slum and Nonslum Settings in Brazil
Authors: Tartof Sara Yee , Farrimond Frances , de Matos Juliana Arruda , Reis Joice Neves , Ramos Regina Terse Trindade , Andrade Aurelio Nei , dos Reis Mitermayer Galvão , Riley Lee Woodland
Published: November 2010

Group G
Streptococcus
has been implicated as a causative agent of pharyngitis in outbreak situations, but its role in endemic disease remains elusive. We found an unexpected inverse association of
Streptococcus dysgalactiae
subsp.
equisimilis
colonization and sore throat in a study of 2,194 children of 3 to 15 years of age in Salvador, Brazil.
Modification of Nonstructural Protein 1 of Influenza A Virus by SUMO1
Authors: Xu Ke , Klenk Christoph , Liu Bin , Keiner Bjoern , Cheng Jinke , Zheng Bo-Jian , Li Li , Han Qinglin , Wang Chen , Li Tianxian , Chen Ze , Shu Yuelong , Liu Jinhua , Klenk Hans-Dieter , Sun Bing
Published: November 2010

Nonstructural protein 1 (NS1) is one of the major factors resulting in the efficient infection rate and high level of virulence of influenza A virus. Although consisting of only approximately 230 amino acids, NS1 has the ability to interfere with several systems of the host viral defense. In the present study, we demonstrate that NS1 of the highly pathogenic avian influenza A/Duck/Hubei/L-1/2004 (H5N1) virus interacts with human Ubc9, which is the E2 conjugating enzyme for sumoylation, and we show that SUMO1 is conjugated to H5N1 NS1 in both transfected and infected cells. Furthermore, two lysine residues in the C terminus of NS1 were identified as SUMO1 acceptor sites. When the SUMO1 acceptor sites were removed by mutation, NS1 underwent rapid degradation. Studies of different influenza A virus strains of human and avian origin showed that the majority of viruses possess an NS1 protein that is modified by SUMO1, except for the recently emerged swine-origin influenza A virus (S-OIV) (H1N1). Interestingly, growth of a sumoylation-deficient WSN virus mutant was retarded compared to that of wild-type virus. Together, these results indicate that sumoylation enhances NS1 stability and thus promotes rapid growth of influenza A virus.
Comprehensive Analysis of Frequency and Phenotype of T Regulatory Cells in HIV Infection: CD39 Expression of FoxP3+T Regulatory Cells Correlates with Progressive Disease
Authors: Schulze zur Wiesch Julian , Thomssen Adriana , Hartjen Philip , Tóth Ilona , Lehmann Clara , Meyer-Olson Dirk , Colberg Kristina , Frerk Sebastian , Babikir Dalia , Schmiedel Stefan , Degen Olaf , Mauss Stefan , Rockstroh Jürgen , Staszewski Schlomo , Khaykin Pavel , Strasak Alexander , Lohse Ansgar W. , Fätkenheuer Gerd , Hauber Joachim , van Lunzen Jan
Published: November 2010

There are conflicting data about the frequency and role of regulatory T cells (Tregs) during the course of HIV infection. Peripheral blood of a large cohort of HIV-infected patients (n= 131) at different stages of disease, including 15 long-term nonprogressors and 21 elite controllers, was analyzed to determine the frequency and phenotype of Tregs, defined as CD4+, CD25high, CD127low, FoxP3highcells. A significantly increased relative frequency of Tregs within the CD4+compartment of HIV+patients compared to that of healthy controls (P< 0.0001) was observed. Additionally, the relative frequency of Tregs directly correlated with HIV viral load and inversely with CD4+counts. However, the absolute Treg number was reduced in HIV-infected patients versus healthy controls (P< 0.0001), with the exception of elite controllers (P> 0.05). The loss of absolute Treg numbers coincided with rising markers of immune activation (P< 0.0006). The initiation of antiviral therapy significantly increased absolute Treg numbers (P< 0.0031). We find that the expression of CD39, a newly defined ectonucleotidase with immunomodulatory functions on Tregs, correlated with progressive HIV disease, HIV viral load, and immune activation. Of note, when tested in peripheral blood mononuclear cells of healthy volunteers, thein vitrocapacity to suppress T-cell proliferation was limited to CD4+, CD25high, CD39+T cells. Interestingly, Tregs of elite controllers exhibited not only the highest expression of CCR5, CTLA-4, and ICOS but also the lowest level of CD39. The data presented here reconcile the seemingly contradictory results of previous studies looking at Tregs in HIV and highlight the complexity of Treg-mediated immunoregulation during human viral infections.
Alphacoronavirus Transmissible Gastroenteritis Virus nsp1 Protein Suppresses Protein Translation in Mammalian Cells and in Cell-Free HeLa Cell Extracts but Not in Rabbit Reticulocyte Lysate
Authors: Huang Cheng , Lokugamage Kumari G. , Rozovics Janet M. , Narayanan Krishna , Semler Bert L. , Makino Shinji
Published: November 2010

The nsp1 protein of transmissible gastroenteritis virus (TGEV), an alphacoronavirus, efficiently suppressed protein synthesis in mammalian cells. Unlike the nsp1 protein of severe acute respiratory syndrome coronavirus, a betacoronavirus, the TGEV nsp1 protein was unable to bind 40S ribosomal subunits or promote host mRNA degradation. TGEV nsp1 also suppressed protein translation in cell-free HeLa cell extract; however, it did not affect translation in rabbit reticulocyte lysate (RRL). Our data suggested that HeLa cell extracts and cultured host cells, but not RRL, contain a host factor(s) that is essential for TGEV nsp1-induced translational suppression.
Human Cytomegalovirus UL47 Tegument Protein Functions after Entry and before Immediate-Early Gene Expression
Authors: Bechtel Jill T. , Shenk Thomas
Published: October 2010

The human cytomegalovirus UL47 open reading frame encodes a 110-kDa protein that is a component of the virion tegument. We have constructed a cytomegalovirus mutant, AD
sub
UL47, in which the central portion of the UL47 open reading frame has been replaced by two marker genes. The mutant replicated to titers 100-fold lower than those for wild-type virus after infection at either a high or a low input multiplicity in primary human fibroblasts but was substantially complemented on cells expressing UL47 protein. A revertant virus in which the mutation was repaired, AD
rev
UL47, replicated with wild-type kinetics. Mutant virions lacked UL47 protein and contained reduced amounts of UL48 protein. The mutant was found to be less infectious than wild-type virus, and a defect very early in the replication cycle was observed. Transcription of the viral immediate-early 1 gene was delayed by 8 to 10 h. However, this delay was not the result of a defect in virus entry or of the inability of virion proteins to transactivate the major immediate-early promoter. We also show that the UL47 protein coprecipitated with the UL48 and UL69 tegument proteins and the UL86-encoded major capsid protein. We propose that a UL47-containing complex is involved in the release of viral DNA from the disassembling virus particle and that the loss of UL47 protein causes this process to be delayed.
Protective Effect of Avian Myelomonocytic Growth Factor in Infection with Marek’s Disease Virus
Authors: Djeraba Aouatef , Musset Eugène , Lowenthal John W. , Boyle David B. , Chaussé Anne-Marie , Péloille Michele , Quéré Pascale
Published: October 2010

Marek’s disease virus (MDV) is a herpesvirus that induces T lymphomas in chickens. The aim of this study was to assess the role of the macrophage activator chicken myelomonocytic growth factor (cMGF) in controlling MDV infection. B13/B13 chickens, which are highly susceptible to MD, were either treated with cMGF delivered via a live fowlpox virus (fp/cMGF) or treated with the parent vector (fp/M3) or were left as untreated controls. Seven days later, when challenged with the very virulent RB-1B strain of MDV, the spleens of chickens treated with fp/cMGF showed increased expression of the inducible nitric oxide synthase (iNOS) gene compared to those of control chickens and fp/M3-treated chickens. Increased iNOS gene expression was also accompanied by greater induction of gamma interferon and macrophage inflammatory protein (K203) gene expression, both possible activators of iNOS. fp/cMGF treatment also increased the number of monocytes and systemic NO production in contrast to fp/M3 treatment. Even though cMGF treatment was unable to prevent death for the chickens, it did prolong their survival time, and viremia and tumor incidence were greatly reduced. In addition, cMGF treatment improved the partial protection induced by vaccination with HVT (herpesvirus isolated from turkeys) against RB-1B, preventing 100% mortality (versus 66% with vaccination alone) and greatly reducing tumor development. Treatment with fp/M3 did not have such effects. These results suggest that cMGF may play multiple roles in protection against MD. First, it may enhance the innate immune response by increasing the number and activity of monocytes and macrophages, resulting in increased NO production. Second, it may enhance the acquired immune response, indicated by its ability to enhance vaccine efficacy.
Inhibition of Interferons by Ectromelia Virus
Authors: Smith Vincent P. , Alcami Antonio
Published: October 2010

Ectromelia virus (EV) is an orthopoxvirus (OPV) that causes mousepox, a severe disease of laboratory mice. Mousepox is a useful model of OPV infection because EV is likely to be a natural mouse pathogen, unlike its close relatives vaccinia virus (VV) and variola virus. Several studies have highlighted the importance of mouse interferons (IFNs) in resistance to and recovery from EV infection, but little is known of the anti-IFN strategies encoded by the virus itself. We have determined that 12 distinct strains and isolates of EV encode soluble, secreted receptors for IFN-γ (vIFN-γR) and IFN-α/β (vIFN-α/βR) that are homologous to those identified in other OPVs. We demonstrate for the first time that the EV vIFN-γR has the unique ability to inhibit the biological activity of mouse IFN-γ. The EV vIFN-α/βR was a potent inhibitor of human and mouse IFN-α and human IFN-β but, surprisingly, was unable to inhibit mouse IFN-β. The replication of all of the EVs included in our study and of cowpox virus was more resistant than VV to the antiviral effects induced in mouse L-929 cells by IFN-α/β and IFN-γ. Sequencing studies showed that this EV resistance is likely to be partly mediated by the double-stranded-RNA-binding protein encoded by an intact EV homolog of the VV E3L gene. The absence of a functional K3L gene, which encodes a viral eIF-2α homolog, in EV suggests that the virus encodes a novel mechanism to counteract the IFN response. These findings will facilitate future studies of the role of viral anti-IFN strategies in mousepox pathogenesis. Their significance in the light of earlier data on the role of IFNs in mousepox is discussed.
A Six-Nucleotide Segment within the 3′ Untranslated Region of Hibiscus Chlorotic Ringspot Virus Plays an Essential Role in Translational Enhancement
Authors: Koh Dora Chin-Yen , Liu D. X. , Wong Sek-Man
Published: October 2010

RNA plant viruses use various translational regulatory mechanisms to control their gene expression. Translational enhancement of viral mRNAs that leads to higher levels of protein synthesis from specific genes may be essential for the virus to successfully compete for cellular translational machinery. The control elements have yet to be analyzed for members of the genus
Carmovirus
, a small group of plant viruses with positive-sense RNA genomes. In this study, we examined the 3′ untranslated region (UTR) of hibiscus chlorotic ringspot virus (HCRSV) genomic RNA (gRNA) and subgenomic RNA (sgRNA) for its role in the translational regulation of viral gene expression. The results showed that the 3′ UTR of HCRSV significantly enhanced the translation of several open reading frames on gRNA and sgRNA and a viral gene in a bicistronic construct with an inserted internal ribosome entry site. Through deletion and mutagenesis studies of both the bicistronic construct and full-length gRNA, we demonstrated that a six-nucleotide sequence, GGGCAG, that is complementary to the 3′ region of the 18S rRNA and a minimal length of 180 nucleotides are required for the enhancement of translation induced by the 3′ UTR.
Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis
Authors: Salvatore Mirella , Basler Christopher F. , Parisien Jean-Patrick , Horvath Curt M. , Bourmakina Svetlana , Zheng Hongyong , Muster Thomas , Palese Peter , García-Sastre Adolfo
Published: October 2010

The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-α/β) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein—lacking approximately half of its carboxy-terminal end—showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.
Mutations of the Woodchuck Hepatitis Virus Polymerase Gene That Confer Resistance to Lamivudine and 2′-Fluoro-5-Methyl-β-
l
-Arabinofuranosyluracil
Authors: Yamamoto Toshiki , Litwin Samuel , Zhou Tianlun , Zhu Yuao , Condreay Lynn , Furman Phillip , Mason William S.
Published: October 2010

Administration of either lamivudine (2′-deoxy-3′-thiacytidine) or
l
-FMAU (2′-fluoro-5-methyl-β-
l
-arabinofuranosyluracil) to woodchucks chronically infected with woodchuck hepatitis virus (WHV) induces a transient decline in virus titers. However, within 6 to 12 months, virus titers begin to increase towards pretreatment levels. This is associated with the emergence of virus strains with mutations of the B and C regions of the viral DNA polymerase (T. Zhou et al., Antimicrob. Agents Chemother. 43:1947–1954, 1999; Y. Zhu et al., J. Virol. 75:311–322, 2001). The present study was carried out to determine which of the mutants that we have identified conferred resistance to lamivudine and/or to
l
-FMAU. When inserted into a laboratory strain of WHV, each of the mutations, or combinations of mutations, of regions B and C produced a DNA replication-competent virus and typically conferred resistance to both nucleoside analogs in cell culture. Sequencing of the polymerase active site also occasionally revealed other mutations, but these did not appear to contribute to drug resistance. Moreover, in transfected cells, most of the mutants synthesized viral DNA nearly as efficiently as wild-type WHV. Computational models suggested that persistence of several of the WHV mutants as prevalent species in the serum and, by inference, liver for up to 6 months following drug withdrawal required a replication efficiency of at least 10 to 30% of that of the wild type. However, their delayed emergence during therapy suggested replication efficiency in the presence of the drug that was still well below that of wild-type WHV in the absence of the drug.
Proteolytic Processing of Human Cytomegalovirus Glycoprotein B Is Dispensable for Viral Growth in Culture
Authors: Strive Tanja , Borst Eva , Messerle Martin , Radsak Klaus
Published: October 2010

Glycoprotein B (gB) of human cytomegalovirus (HCMV), which is considered essential for the viral life cycle, is proteolytically processed during maturation. Since gB homologues of several other herpesviruses remain uncleaved, the relevance of this property of HCMV gB for viral infectivity is unclear. Here we report on the construction of a viral mutant in which the recognition site of gB for the cellular endoprotease furin was destroyed. Because mutagenesis of essential proteins may result in a lethal phenotype, a replication-deficient HCMV gB-null genome encoding enhanced green fluorescent protein was constructed, and complementation by mutant gBs was initially evaluated in transient-cotransfection assays. Cotransfection of plasmids expressing authentic gB or gB with a mutated cleavage site (gB-ΔFur) led to the formation of green fluorescent miniplaques which were considered to result from one cycle of phenotypic complementation of the gB-null genome. To verify these results, two recombinant HCMV genomes were constructed: HCMV-BAC-ΔMhdI, with a deletion of hydrophobic domain 1 of gB that appeared to be essential for viral growth in the cotransfection experiments, and HCMV-BACΔFur, in which the gB cleavage site was mutated by amino acid substitution. Consistent with the results of the cotransfection assays, only the ΔFur mutant replicated in human fibroblasts, showing growth kinetics comparable to that of wild-type virus. gB in mutant-infected cells was uncleaved, whereas glycosylation and transport to the cell surface were not impaired. Extracellular mutant virus contained exclusively uncleaved gB, indicating that proteolytic processing of gB is dispensable for viral replication in cell culture.
Detection of a Novel Unglycosylated Form of Hepatitis C Virus E2 Envelope Protein That Is Located in the Cytosol and Interacts with PKR
Authors: Pavio Nicole , Taylor Deborah R. , Lai Michael M. C.
Published: October 2010

The hepatitis C virus (HCV) envelope protein E2 has been shown to accumulate in the lumen of the endoplasmic reticulum (ER) as a properly folded glycoprotein as well as large aggregates of misfolded proteins. In the present study, we have identified an additional unglycosylated species, with an apparent molecular mass of 38 kDa (E2-p38). In contrast to the glycosylated E2, E2-p38 is significantly less stable and is degraded through the proteasome pathway. Correspondingly, E2-p38 is found to be ubiquitinated. E2-p38 is localized mostly in the cytosol, in contrast to the glycosylated form, which is exclusively membrane associated. Alpha interferon (IFN-α) treatment or overexpression of the double-stranded RNA-activated protein kinase (PKR) significantly increased the stability of E2-p38, consistent with a previous report (D. R. Taylor, S. T. Shi, P. R. Romano, G. N. Barber, and M. M. Lai, Science 285:107–110, 1999) that E2 interacts with PKR and inhibits its kinase activity. Direct interaction between PKR and E2-p38, but not the glycosylated form of E2, was also observed. These results show that E2-p38 is the form of E2 that interacts with PKR in the cytosol and may contribute to the resistance of HCV to IFN-α. Thus, an ER protein can exist in the cytosol as an unglycosylated species and impair cellular functions.
Complement Component C1q Enhances the Biological Activity of Influenza Virus Hemagglutinin-Specific Antibodies Depending on Their Fine Antigen Specificity and Heavy-Chain Isotype
Authors: Feng Jing Qi , Mozdzanowska Krystyna , Gerhard Walter
Published: October 2010

We have previously observed that selected influenza virus hemagglutinin (HA)-specific monoclonal antibodies (MAbs) with poor virus-neutralizing (VN) activity in vitro exhibited greatly enhanced VN activity in vivo after administration to SCID mice. The same Abs displayed improved VN activity also when tested in vitro in the presence of noninactivated serum from SCID mice. To identify Ab-dependent properties and serum components that contributed to enhancement of Ab activity, we screened a large panel of HA-specific MAbs for hemagglutination inhibition (HI) in the presence of noninactivated serum from naive mice (NMS). We found that HI activity was enhanced by NMS depending on the Ab’s fine specificity (antigenic region Cb/E > Ca/A,D > Sa,Sb/B), its heavy-chain isotype (immunoglobulin G2 [IgG2] > IgG3; IgG1 and IgM negative), and to some extent also on its derivation (primary response > memory response). On average, the HI activity of Cb/E-specific MAbs of the IgG2 isotype isolated from the primary response was enhanced by 20-fold. VN activity was enhanced significantly but less strongly than HI activity. Enhancement (i) was destroyed by heat inactivation (30 min, 56°C); (ii) did not require C3, the central complement component; (iii) was abolished by treatment of serum with anti-C1q; and (iv) could be reproduced with purified C1q, the binding moiety of C1, the first complement component. We believe that this is the first description of a direct C1q-mediated enhancement of antiviral Ab activities.
Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus
Authors: Spaderna S. , Blessing H. , Bogner E. , Britt W. , Mach M.
Published: October 2010

Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.
In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors
Authors: Kuhn Ulrich , Terunuma Atsushi , Pfutzner Wolfgang , Foster Ruth Ann , Vogel Jonathan C.
Published: October 2010

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.
Comparison of Gene Transfer Efficiencies and Gene Expression Levels Achieved with Equine Infectious Anemia Virus- and Human Immunodeficiency Virus Type 1-Derived Lentivirus Vectors
Authors: O’Rourke J. P. , Newbound G. C. , Kohn D. B. , Olsen J. C. , Bunnell B. A.
Published: October 2010

This report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) lentiviral vectors in a variety of human cell types in vitro. EIAV and HIV vectors transduced equivalent numbers of proliferating and G
1
/S- and G
2
/M-arrested cells, and both had very low efficiencies of transduction into G
0
-arrested cells. Analysis of the levels of both the enhanced green fluorescent protein (EGFP) and mRNA demonstrated that the HIV-transduced cells expressed greater levels of EGFP protein and RNA than the EIAV-transduced cells. Measurements of vector-derived EGFP RNA half-lives were fourfold higher with the HIV vector than with the EIAV vector. Long-term culture of EIAV-transduced human cells showed a significant decrease in the number of cells expressing the transgene; however, no corresponding loss was found in EIAV-transduced equine cells. In contrast, only a moderate decrease in the number of transgene-expressing cells was seen with the HIV vectors. Taken together, these results demonstrate that the EIAV vectors transduced human cells with efficiencies similar to those of the HIV vectors. However, our data indicate that transgene expression from EIAV vectors is limited by the instability of vector-derived RNA transcripts and silencing of the EIAV vectors over time.
Analysis of the Disease Potential of a Recombinant Retrovirus Containing Friend Murine Leukemia Virus Sequences and a Unique Long Terminal Repeat from Feline Leukemia Virus
Authors: Nishigaki Kazuo , Hanson Charlotte , Thompson Delores , Yugawa Takashi , Hisasue Masaharu , Tsujimoto Hajime , Ruscetti Sandra
Published: October 2010

We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage.
Affinity Purification of an Archaeal DNA Replication Protein Network
Authors: Li Zhuo , Santangelo Thomas J. , Čuboňová Ľubomíra , Reeve John N. , Kelman Zvi
Published: October 2010

Nineteen
Thermococcus kodakarensis
strains have been constructed, each of which synthesizes a different His
6
-tagged protein known or predicted to be a component of the archaeal DNA replication machinery. Using the His
6
-tagged proteins, stable complexes assembled
in vivo
have been isolated directly from clarified cell lysates and the
T. kodakarensis
proteins present have been identified by mass spectrometry. Based on the results obtained, a network of interactions among the archaeal replication proteins has been established that confirms previously documented and predicted interactions, provides experimental evidence for previously unrecognized interactions between proteins with known functions and with unknown functions, and establishes a firm experimental foundation for archaeal replication research. The proteins identified and their participation in archaeal DNA replication are discussed and related to their bacterial and eukaryotic counterparts.

IMPORTANCE
DNA replication is a central and essential event in all cell cycles. Historically, the biological world was divided into prokaryotes and eukaryotes, based on the absence or presence of a nuclear membrane, and many components of the DNA replication machinery have been identified and characterized as conserved or nonconserved in prokaryotic versus eukaryotic organisms. However, it is now known that there are two evolutionarily distinct prokaryotic domains,
Bacteria
and
Archaea
, and to date, most prokaryotic replication research has investigated bacterial replication. Here, we have taken advantage of recently developed genetic techniques to isolate and identify many proteins likely to be components of the archaeal DNA replication machinery. The results confirm and extend predictions from genome sequencing that the archaeal replication system is less complex but more closely related to a eukaryotic than to a bacterial replication system.
Optimising ankle sprain prevention: a critical review and practical appraisal of the literature
Authors: Verhagen E A L M , Bay K
Published: November 2010

Objectives

To establish the effect of preventive measures and assert the optimal prevention strategy for acute lateral ligament injury to the ankle.

Methods

An electronic literature search was employed to look for published randomised controlled trials, a controlled trials or time interventions containing research questions regarding the prevention of lateral ankle ligament injuries. Two reviewers reviewed relevant studies for strengths and weaknesses in design and methodology, according to a standardised set of predefined criteria. A total of 24 relevant studies met the criteria for inclusion and were analysed.

Results

Overall taping, bracing and neuromuscular training were all effective for the prevention of ankle-sprain recurrences. The RRs of these prophylactic measures are of similar magnitude, ranging from 0.2 to 0.5 when compared with control groups. Although preventive effects have been reported in a general athletic population, evidence suggests this overall effect is due to a strong preventive effect in previously injured athletes and that any effect on fresh ankle sprains is either non-existent or very low.

Conclusions

Based on these outcomes, a combination of an external prophylactic measure (tape or brace) with neuromuscular training will achieve the best preventive outcomes with minimal burden for the athlete.