bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the Association of the Human Cytomegalovirus DNA Polymerase Subunit UL44 with the Viral DNA Replication Factor UL84
Authors: Strang Blair L. , Sinigalia Elisa , Silva Laurie A. , Coen Donald M. , Loregian Arianna 
Published: May 2009

ABSTRACT

The central enzyme responsible for human cytomegalovirus (HCMV) DNA synthesis is a virally encoded DNA polymerase that includes a catalytic subunit, UL54, and a homodimeric accessory subunit, UL44, the presumptive HCMV DNA polymerase processivity factor. The structure of UL44 is similar to that of the eukaryotic processivity factor proliferating cell nuclear antigen (PCNA), which interacts with numerous other proteins required for faithful DNA replication. We sought to determine whether, like PCNA, UL44 is capable of interacting with multiple DNA replication proteins and, if so, whether these proteins bind UL44 at the site corresponding to where multiple proteins bind to PCNA. Initially, several proteins, including the viral DNA replication factors UL84 and UL57, were identified by mass spectrometry in immunoprecipitates of UL44 from infected cell lysate. The association of UL44/UL84, but not UL44/UL57, was confirmed by reciprocal coimmunoprecipitation of these proteins from infected cell lysates and was resistant to nuclease treatment. Yeast two-hybrid analyses demonstrated that the substitution of residues in UL44 that prevent UL44 homodimerization or abrogate the binding of UL54 to UL44 do not abrogate the UL44/UL84 interaction. Reciprocal glutathione-
S
-transferase (GST) pulldown experiments using bacterially expressed UL44 and UL84 confirmed these results and, further, demonstrated that a UL54-derived peptide that competes with UL54 for UL44 binding does not prevent the association of UL84 with UL44. Taken together, our results strongly suggest that UL44 and UL84 interact directly using a region of UL44 different from the UL54 binding site. Thus, UL44 can bind interacting replication proteins using a mechanism different from that of PCNA.
Association of Increased Pathogenicity of Asian H5N1 Highly Pathogenic Avian Influenza Viruses in Chickens with Highly Efficient Viral Replication Accompanied by Early Destruction of Innate Immune Responses
Authors: Suzuki Koutaro , Okada Hironao , Itoh Toshihiro , Tada Tatsuya , Mase Masaji , Nakamura Kikuyasu , Kubo Masanori , Tsukamoto Kenji 
Published: May 2009

ABSTRACT
The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.
Autographa californica
Multiple Nucleopolyhedrovirus
me53
(
ac140
) Is a Nonessential Gene Required for Efficient Budded-Virus Production
Authors: de Jong Jondavid , Arif Basil M. , Theilmann David A. , Krell Peter J. 
Published: May 2009

ABSTRACT

me53
is a highly conserved baculovirus gene found in all lepidopteran baculoviruses that have been fully sequenced to date. The putative ME53 protein contains a zinc finger domain and has been previously described as a major early transcript. We generated an
me53
-null bacmid (AcΔme53GFP), as well as a repair virus (AcRepME53:HA-GFP) carrying
me53
with a C-terminal hemagglutinin (HA) tag, under the control of its native early and late promoter elements. Sf9 and BTI-Tn-
5b1
cells transfected with AcΔme53GFP resulted in a 3-log reduction in budded-virus (BV) production compared to both the parental
Autographa californica
multiple nucleopolyhedrosis virus and the repair bacmids, demonstrating that although
me53
is not essential for replication, replication is compromised in its absence. Our data also suggest that
me53
does not affect DNA replication. Cell fractionation showed that ME53 is found in both the nucleus and the cytoplasm as early as 6 h postinfection. Deletion of the early transcriptional start site resulted in a 10- to 360-fold reduction of BV yield; however, deletion of the late promoter (ATAAG) resulted in a 160- to 1,000-fold reduction, suggesting that, in the context of BV production, ME53 is required both early and late in the infection cycle. Additional Western blot analysis of purified virions from the repair virus revealed that ME53:HA is associated with both BV and occlusion-derived virions. Together, these results indicate that
me53
, although not essential for viral replication, is required for efficient BV production.
Analysis of Modified Human Papillomavirus Type 16 L1 Capsomeres: the Ability To Assemble into Larger Particles Correlates with Higher Immunogenicity
Authors: Schädlich Lysann , Senger Tilo , Gerlach Britta , Mücke Norbert , Klein Corinna , Bravo Ignacio G. , Müller Martin , Gissmann Lutz 
Published: May 2009

ABSTRACTL1 capsomeres purified fromEscherichia colirepresent an economic alternative to the recently launched virus-like particle (VLP)-based prophylactic vaccines against infection with human papillomavirus types 16 and 18 (HPV-16 and HPV-18), which are causative agents of cervical cancer. It was recently reported that capsomeres are much less immunogenic than VLPs. Numerous modifications of the L1 protein leading to the formation of capsomeres but preventing capsid assembly have been described, such as the replacement of the cysteine residues that form capsid-stabilizing disulfide bonds or the deletion of helix 4. So far, the influence of these modifications on immunogenicity has not been thoroughly investigated. Here, we describe the purification of eight different HPV-16 L1 proteins as capsomeres fromEscherichia coli. We compared them for yield, structure, and immunogenicity in mice. All L1 proteins formed almost identical pentameric structures yet differed strongly in their immunogenicity, especially regarding the humoral immune responses. Immunization of TLR4−/−mice and DNA immunization by the same constructs confirmed that immunogenicity was independent of different degrees of contamination with copurifying immune-stimulatory molecules fromE. coli. We hypothesize that immunogenicity correlates with the intrinsic ability of the capsomeres to assemble into larger particles, as only assembly-competent L1 proteins induced high antibody responses. One of the proteins (L1ΔN10) proved to be the most immunogenic, inducing antibody titers equivalent to those generated in response to VLPs. However, preassembly prior to injection did not increase immunogenicity. Our data suggest that certain L1 constructs can be used to produce highly immunogenic capsomeres in bacteria as economic alternatives to VLP-based formulations.
Do physiotherapy staff record treatment time accurately? An observational study
Authors: Bagley Pam , Hudson Mary , Green John , Forster Anne , Young John 
Published: May 2009

Objective: To assess the reliability of duration of treatment time measured by physiotherapy staff in early-stage stroke patients. Design: Comparison of physiotherapy staff’s recording of treatment sessions and video recording. Setting: Rehabilitation stroke unit in a general hospital. Subjects: Thirty-nine stroke patients without trunk control or who were unable to stand with an erect trunk without the support of two therapists recruited to a randomized trial evaluating the Oswestry Standing Frame. Twenty-six physiotherapy staff who were involved in patient treatment. Main measures: Contemporaneous recording by physiotherapy staff of treatment time (in minutes) compared with video recording. Statistical analysis: Intraclass correlation with 95% confidence interval and the Bland and Altman method for assessing agreement by calculating the mean difference (standard deviation; 95% confidence interval), reliability coefficient and 95% limits of agreement for the differences between the measurements. Results: The mean duration (standard deviation, SD) of treatment time recorded by physiotherapy staff was 32 (11) minutes compared with 25 (9) minutes as evidenced in the video recording. The mean difference (SD) was —6 (9) minutes (95% confidence interval (CI) —9 to —3). The reliability coefficient was 18 minutes and the 95% limits of agreement were —24 to 12 minutes. Intraclass correlation coefficient for agreement between the two methods was 0.50 (95% CI 0.12 to 0.73). Conclusions: Physiotherapy staff’s recording of duration of treatment time was not reliable and was systematically greater than the video recording.
Polo-like kinase 1 is overexpressed in acute myeloid leukemia and its inhibition preferentially targets the proliferation of leukemic cells
Authors: Renner Annelies G. , Dos Santos Cédric , Recher Christian , Bailly Christian , Créancier Laurent , Kruczynski Anna , Payrastre Bernard , Manenti Stéphane 
Published: May 2009

Abstract
Polo-like kinase 1 (Plk1) is a major mitotic regulator overexpressed in many solid tumors. Its role in hematopoietic malignancies is still poorly characterized. In this study, we demonstrate that Plk1 is highly expressed in leukemic cell lines, and overexpressed in a majority of samples from patients with acute myeloid leukemia compared with normal progenitors. A pharmacologic inhibitor, BI2536, blocks proliferation in established cell lines, and dramatically inhibits the clonogenic potential of leukemic cells from patients. Plk1 knockdown by small interfering RNA also blocked proliferation of leukemic cell lines and the clonogenic potential of primary cells from patients. Interestingly, normal primary hematopoietic progenitors are less sensitive to Plk1 inhibition than leukemic cells, whose proliferation is dramatically decreased by the inhibitor. These results highlight Plk1 as a potentially interesting therapeutic target for the treatment of acute myeloid leukemia.
A murine model for induction of long-term immunologic tolerance to factor VIII does not require persistent detectable levels of plasma factor VIII and involves contributions from Foxp3+ T regulatory cells
Authors: Matsui Hideto , Shibata Masaru , Brown Brian , Labelle Andrea , Hegadorn Carol , Andrews Chandler , Chuah Marinee , VandenDriessche Thierry , Miao Carol H. , Hough Christine , Lillicrap David 
Published: May 2009

Abstract
Under certain instances, factor VIII (FVIII) stimulates an immune response, and the resulting neutralizing antibodies present a significant clinical challenge. Immunotherapies to re-establish or induce long-term tolerance would be beneficial, and an in-depth knowledge of mechanisms involved in tolerance induction is essential to develop immune-modulating strategies. We have developed a murine model system for studying mechanisms involved in induction of immunologic tolerance to FVIII in hemophilia A mice. We used lentiviral vectors to deliver the canine FVIII transgene to neonatal hemophilic mice and demonstrated that induction of long-term FVIII tolerance could be achieved. Hemophilia A mice are capable of mounting a robust immune response to FVIII after neonatal gene transfer, and tolerance induction is dependent on the route of delivery and type of promoter used. High-level expression of FVIII was not required for tolerance induction and, indeed, tolerance developed in some animals without evidence of detectable plasma FVIII. Tolerance to FVIII could be adoptively transferred to naive hemophilia recipient mice, and FVIII-stimulated splenocytes isolated from tolerized mice expressed increased levels of interleukin-10 and decreased levels of interleukin-6 and interferon-γ. Finally, induction of FVIII tolerance mediated by this protocol is associated with a FVIII-expandable population of CD4+CD25+Foxp3+ regulatory T cells.
Psychotropic medication use among nursing home residents in Austria: a cross-sectional study
Authors: Mann Eva , Köpke Sascha , Haastert Burkhard , Pitkälä Kaisu , Meyer Gabriele 
Published: May 2009

Abstract

Background
The use of psychotropic medications and their adverse effects in frail elderly has been debated extensively. However, recent data from European studies show that these drugs are still frequently prescribed in nursing home residents. In Austria, prevalence data are lacking. We aimed to determine the prevalence of psychotropic medication prescription in Austrian nursing homes and to explore characteristics associated with their prescription.

Methods
Cross-sectional study and association analysis in forty-eight out of 50 nursing homes with 1844 out of a total of 2005 residents in a defined urban-rural region in Austria. Prescribed medication was retrieved from residents' charts. Psychotropic medications were coded according to the Anatomical Therapeutic Chemical Classification 2005. Cluster-adjusted multiple logistic regression analysis was performed to investigate institutional and residents' characteristics associated with prescription.

Results
Residents' mean age was 81; 73% of residents were female. Mean cluster-adjusted prevalence of residents with at least one psychotropic medication was 74.6% (95% confidence interval, CI, 72.0–77.2). A total of 45.9% (95% CI 42.7–49.1) had at least one prescription of an antipsychotic medication. Two third of all antipsychotic medications were prescribed for bedtime use only. Anxiolytics were prescribed in 22.2% (95% CI 20.0–24.5), hypnotics in 13.3% (95% CI 11.3–15.4), and antidepressants in 36.8% (95% CI 34.1–39.6) of residents. None of the institutional characteristics and only few residents' characteristics were significantly associated with psychotropic medication prescription. Permanent restlessness was positively associated with psychotropic medication prescription (AOR 1.54, 95% CI 1.32–1.79) whereas cognitive impairment was inversely associated (AOR 0.70, 95% CI 0.56–0.88).

Conclusion
Frequency of psychotropic medication prescription is high in Austrian nursing homes compared to recent published data from other countries. Interventions should aim at reduction and optimisation of prescriptions.
Groucho binds two conserved regions of LEF-1 for HDAC-dependent repression
Authors: Arce Laura , Pate Kira T , Waterman Marian L 
Published: May 2009

AbstractBackgroundDrosophilaGroucho and its human Transducin-like-Enhancer of Split orthologs (TLEs) function as transcription co-repressors within the context of Wnt signaling, a pathway with strong links to cancer. The current model for how Groucho/TLE's modify Wnt signaling is by direct competition with β-catenin for LEF/TCF binding. The molecular events involved in this competitive interaction are not defined and the actions of Groucho/TLEs within the context of Wnt-linked cancer are unknown.MethodsWe usedin vitroprotein interaction assays with the LEF/TCF family member LEF-1, andin vivoassays with Wnt reporter plasmids to define Groucho/TLE interaction and repressor function.ResultsMapping studies reveal that Groucho/TLE binds two regions in LEF-1. The primary site of recognition is a 20 amino acid region in the Context Dependent Regulatory domain. An auxiliary site is in the High Mobility Group DNA binding domain. Mutation of an eight amino acid sequence within the primary region (RFSHHMIP) results in a loss of Groucho action in a transient reporter assay.DrosophilaGroucho, human TLE-1, and a truncated human TLE isoform Amino-enhancer-of-split (AES), work equivalently to repress LEF-1•β-catenin transcription in transient reporter assays, and these actions are sensitive to the HDAC inhibitor Trichostatin A. A survey of Groucho/TLE action in a panel of six colon cancer cell lines with elevated β-catenin shows that Groucho is not able to repress transcription in a subset of these cell lines.ConclusionOur data shows that Groucho/TLE repression requires two sites of interaction in LEF-1 and that a central, conserved amino acid sequence within the primary region (F S/T/P/xx y I/L/V) is critical. Our data also reveals that AES opposes LEF-1 transcription activation and that both Groucho and AES repression require histone deacetylase activity suggesting multiple steps in Groucho competition with β-catenin. The variable ability of Groucho/TLE to oppose Wnt signaling in colon cancer cells suggests there may be defects in one or more of these steps.
Mung Bean nuclease mapping of RNAs 3' end
Authors: Bellavia Daniele , Sisino Giorgia , Papadopoulos Giorgio L , Forte Giusi I , Barbieri Rainer 
Published: May 2009

Abstract
A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA) and non polyA RNAs (sea urchin 18S and 26S rRNAs). This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases.
Role of biomechanics in the understanding of normal, injured, and healing ligaments and tendons
Authors: Jung Ho-Joong , Fisher Matthew B , Woo Savio L-Y 
Published: May 2009

Abstract
Ligaments and tendons are soft connective tissues which serve essential roles for biomechanical function of the musculoskeletal system by stabilizing and guiding the motion of diarthrodial joints. Nevertheless, these tissues are frequently injured due to repetition and overuse as well as quick cutting motions that involve acceleration and deceleration. These injuries often upset this balance between mobility and stability of the joint which causes damage to other soft tissues manifested as pain and other morbidity, such as osteoarthritis.
The healing of ligament and tendon injuries varies from tissue to tissue. Tendinopathies are ubiquitous and can take up to 12 months for the pain to subside before one could return to normal activity. A ruptured medial collateral ligament (MCL) can generally heal spontaneously; however, its remodeling process takes years and its biomechanical properties remain inferior when compared to the normal MCL. It is also known that a midsubstance anterior cruciate ligament (ACL) tear has limited healing capability, and reconstruction by soft tissue grafts has been regularly performed to regain knee function. However, long term follow-up studies have revealed that 20–25% of patients experience unsatisfactory results. Thus, a better understanding of the function of ligaments and tendons, together with knowledge on their healing potential, may help investigators to develop novel strategies to accelerate and improve the healing process of ligaments and tendons.
With thousands of new papers published in the last ten years that involve biomechanics of ligaments and tendons, there is an increasing appreciation of this subject area. Such attention has positively impacted clinical practice. On the other hand, biomechanical data are complex in nature, and there is a danger of misinterpreting them. Thus, in these review, we will provide the readers with a brief overview of ligaments and tendons and refer them to appropriate methodologies used to obtain their biomechanical properties. Specifically, we hope the reader will pay attention to how the properties of these tissues can be altered due to various experimental and biologic factors. Following this background material, we will present how biomechanics can be applied to gain an understanding of the mechanisms as well as clinical management of various ligament and tendon ailments. To conclude, new technology, including imaging and robotics as well as functional tissue engineering, that could form novel treatment strategies to enhance healing of ligament and tendon are presented.
Neutrophils exhibit distinct phenotypes toward chitosans with different degrees of deacetylation: implications for cartilage repair
Authors: Simard Pascale , Galarneau Hugo , Marois Sébastien , Rusu Daniel , Hoemann Caroline D , Poubelle Patrice E , El-Gabalawy Hani , Fernandes Maria JG 
Published: May 2009

Abstract
Introduction
Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan.

Methods
Human blood neutrophils from healthy donors were isolated by centrifugation on Ficoll-Paque. Chemotaxis was performed using the chemoTX system. Production of superoxide anions was evaluated using the cytochrome c reduction assay. Degranulation was determined by evaluating the release of myeloperoxidase and lactoferrin. The internalization of fluorescently labelled 80% deacetylated chitosan by neutrophils was studied by confocal microscopy.

Results
Neutrophils were dose dependently attracted to 80% deacetylated chitosan. In contrast, 95% deacetylated chitosan was not chemotactic for neutrophils. Moreover, the majority of the chemotactic effect of 80% deacetylated chitosan was mediated by phospholipase-A2-derived bioactive lipids. Contrary to the induction of chemotaxis, neither 80% nor 95% deacetylated chitosan activated the release of granule enzymes or the generation of active oxygen species. Despite the distinct response of neutrophils toward 80% and 95% deacetylated chitosan, both chitosans were internalized by neutrophils.

Conclusions
Eighty per cent deacetylated chitosan induces a phenotype in neutrophils that is distinct from the classical phenotype induced by pro-inflammatory agents. Our observations also indicate that the degree of deacetylation is an important factor to consider in the use of chitosan as an accelerator of repair because neutrophils do not respond to 95% deacetylated chitosan.