bluuebunny/crossref_metadata_2025 huggingface dataset:
Analyses of Soft Tissue from
Tyrannosaurus rex
Suggest the Presence of Protein
Authors: Schweitzer Mary Higby , Suo Zhiyong , Avci Recep , Asara John M. , Allen Mark A. , Arce Fernando Teran , Horner John R.
Published: April 2007

We performed multiple analyses of
Tyrannosaurus rex
(specimen MOR 1125) fibrous cortical and medullary tissues remaining after demineralization. The results indicate that collagen I, the main organic component of bone, has been preserved in low concentrations in these tissues. The findings were independently confirmed by mass spectrometry. We propose a possible chemical pathway that may contribute to this preservation. The presence of endogenous protein in dinosaur bone may validate hypotheses about evolutionary relationships, rates, and patterns of molecular change and degradation, as well as the chemical stability of molecules over time.
Optimizing the Laser-Pulse Configuration for Coherent Raman Spectroscopy
Authors: Pestov Dmitry , Murawski Robert K. , Ariunbold Gombojav O. , Wang Xi , Zhi Miaochan , Sokolov Alexei V. , Sautenkov Vladimir A. , Rostovtsev Yuri V. , Dogariu Arthur , Huang Yu , Scully Marlan O.
Published: April 2007

We introduce a hybrid technique that combines the robustness of frequency-resolved coherent anti-Stokes Raman scattering (CARS) with the advantages of time-resolved CARS spectroscopy. Instantaneous coherent broadband excitation of several characteristic molecular vibrations and the subsequent probing of these vibrations by an optimally shaped time-delayed narrowband laser pulse help to suppress the nonresonant background and to retrieve the species-specific signal. We used this technique for coherent Raman spectroscopy of sodium dipicolinate powder, which is similar to calcium dipicolinate (a marker molecule for bacterial endospores, such as
Bacillus subtilis
and
Bacillus anthracis
), and we demonstrated a rapid and highly specific detection scheme that works even in the presence of multiple scattering.
How Best to Look Forward?
Authors: Beck M. Bruce
Published: April 2007

Useless Arithmetic
. Why Environmental Scientists Can't Predict the Future.
By Orrin H. Pilkey and Linda Pilkey-Jarvis
. Columbia University Press, New York, 2007. 246 pp. $29.50, £19. ISBN 9780231132121.

Critiquing a series of examples, the authors argue that many quantitative models only provide a false sense of security and unwarranted confidence in predictions.
Use of Protein-Specific Monoclonal Antibody-Based Latex Agglutination for Rapid Diagnosis of
Burkholderia pseudomallei
Infection in Patients with Community-Acquired Septicemia
Authors: Ekpo Pattama , Rungpanich Utane , Pongsunk Supinya , Naigowit Pimjai , Petkanchanapong Vimon
Published: April 2007

A latex agglutination test employing monoclonal antibody specific to a 30-kDa protein of
Burkholderia pseudomallei
was used to detect the organisms in blood culture specimens from 1,139 patients with community-acquired septicemia. The sensitivity, specificity, and positive and negative predictive values of the test were 96.75%, 99.61%, 96.75%, and 99.61%, respectively.
Transcriptional Activation of Interferon-Stimulated Genes but Not of Cytokine Genes after Primary Infection of Rhesus Macaques with Dengue Virus Type 1
Authors: Sariol Carlos A. , Muñoz-Jordán Jorge L. , Abel Kristina , Rosado Lymarie C. , Pantoja Petraleigh , Giavedoni Luis , Rodriguez Idia Vanessa , White Laura J. , Martínez Melween , Arana Teresa , Kraiselburd Edmundo N.
Published: April 2007

Macaques are the only animal model used to test dengue virus (DENV) vaccine candidates. Nevertheless, the pathogenesis of DENV in macaques is not well understood. In this work, by using Affymetrix oligonucleotide microarrays, we studied the broad transcriptional modifications and cytokine expression profile after infecting rhesus macaques with DENV serotype 1. Five days after infection, these animals produced a potent, innate antiviral immune response by inducing the transcription of signature genes from the interferon (IFN) pathway with demonstrated antiviral activity, such as myxoprotein, 2′,5′-oligoadenylate synthetase, phospholipid scramblase 1, and viperin. Also, IFN regulatory element 7, IFN-stimulated gene 15, and protein ligases linked to the ISGylation process were up-regulated. Unexpectedly, no up-regulation of IFN-α, -β, or -γ genes was detected. Transcription of the genes of interleukin-10 (IL-10), IL-8, IL-6, and tumor necrosis factor alpha was neither up-regulated nor down-regulated. Results were confirmed by real-time PCR and by multiplex cytokine detection in serum samples.
Treatment Failure Related to Intrathecal Immunoglobulin M (IgM) Synthesis, Cerebrospinal Fluid IgM, and Interleukin-10 in Patients with Hemolymphatic-Stage Sleeping Sickness
Authors: Lejon Veerle , Robays Jo , N'Siesi François Xavier , Mumba Dieudonné , Hoogstoel Annemie , Bisser Sylvie , Reiber Hansotto , Boelaert Marleen , Büscher Philippe
Published: April 2007

Human African trypanosomiasis treatment is stage dependent, but the tests used for staging are controversial. Central nervous system involvement and its relationship with suramin treatment failure were assessed in 60 patients with parasitologically confirmed hemolymphatic-stage
Trypanosoma brucei gambiense
infection (white blood cell count of ≤5/μl and no trypanosomes in the cerebrospinal fluid [CSF]). The prognostic value of CSF interleukin-10, immunoglobulin M (IgM; as determined by nephelometry and the point-of-care LATEX/IgM test), total protein, and trypanosome-specific antibody was assessed. The IgM and interleukin-10 levels in serum were measured; and the presence of neurological signs, intrathecal IgM synthesis, and blood-CSF barrier dysfunction was determined. After suramin treatment, 14 of 60 patients had relapses (23%). Relapses were significantly correlated with intrathecal IgM synthesis (odds ratio [OR], 46; 95% confidence interval [CI], 8 to 260), a CSF IgM concentration of ≥1.9 mg/liter (OR, 11.7; 95% CI, 2.7 to 50), a CSF end titer by the LATEX/IgM assay of ≥2 (OR, 10.4; 95% CI, 2.5 to 44), and a CSF interleukin-10 concentration of >10 pg/ml (OR, 5; 95% CI, 1.3 to 20). The sensitivities of these markers for treatment failure ranged from 43 to 79%, and the specificities ranged from 74 to 93%. The results show that
T. brucei gambiense
-infected patients who have signs of neuroinflammation in CSF and who are treated with drugs recommended for use at the hemolymphatic stage are at risk of treatment failure. This highlights the need for the development and the evaluation of accurate point-of-care tests for the staging of human African trypanosomiasis.
Identification and Distribution of
Mycobacterium leprae
Genotypes in a Region of High Leprosy Prevalence in China: a 3-Year Molecular Epidemiological Study
Authors: Weng Xiaoman , Wang Zheng , Liu Jian , Kimura Miyako , Black William C. , Brennan Patrick J. , Li Huanying , Vissa Varalakshmi D.
Published: April 2007

Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been proposed as a means of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population are lacking. To this end, a study was initiated to assess the diversity and distribution of prevalent
Mycobacterium leprae
strains in Qiubei County, Yunnan Province, People's Republic of China, where the annual detection rate of leprosy is 10-fold higher than the national average rate. Sixty-eight newly diagnosed leprosy patients were included in the study. MLVA at eight
M. leprae
loci was applied using DNA extracts from skin biopsies. The number of alleles per locus ranged from 4 to 24, providing adequate strain discrimination. MLVA strain typing identified several clusters of patients whose
M. leprae
specimens shared similar VNTR profiles. Two of these clusters were comprised of patients who resided predominantly in the north and northwest parts of Qiubei County. Furthermore, it was found that multicase families are common in this county: 23 of the 68 patients were from 11 families. Intrafamilial VNTR profiles closely matched within six families, although they were different between the families. Moreover, VNTR patterns related to those found in some multicase families were also detected in patients in the same or adjacent townships, indicating the utility of VNTR strain typing to identify and detect short-range transmission events. Social contact through village markets is proposed as a means of transmission.
Bronchoalveolar Lavage Galactomannan in Diagnosis of Invasive Pulmonary Aspergillosis among Solid-Organ Transplant Recipients
Authors: Clancy Cornelius J. , Jaber Reia A. , Leather Helen L. , Wingard John R. , Staley Benjamin , Wheat L. Joseph , Cline Christina L. , Rand Kenneth H. , Schain Denise , Baz Maher , Nguyen M. Hong
Published: April 2007

We review the experience at our institution with galactomannan (GM) testing of bronchoalveolar lavage (BAL) fluid in the diagnosis of invasive pulmonary aspergillosis (IPA) among solid-organ transplant recipients. Among 81 patients for whom BAL GM testing was ordered (heart, 24; kidney, 22; liver, 19; lung, 16), there were five cases of proven or probable IPA. All five patients had BAL GM of ≥2.1 and survived following antifungal therapy. The sensitivity, specificity, and positive and negative predictive values for BAL GM testing at a cutoff of ≥1.0 were 100%, 90.8%, 41.7%, and 100%, respectively. The sensitivity of BAL GM testing was better than that of conventional tests such as serum GM or BAL cytology and culture. Moreover, a positive BAL GM test diagnosed IPA several days to 4 weeks before other methods for three patients. Twelve patients had BAL GM of ≥0.5 but no evidence of IPA. Among these, lung transplant recipients accounted for 41.7% (5/12) of the false-positive results, reflecting frequent colonization of airways in this population. Excluding lung transplants, the specificity and positive predictive value for other solid-organ transplants increased to 92.9% and 62.5%, respectively (cutoff, ≥1.0). In conclusion, BAL GM testing facilitated more-rapid diagnoses of IPA and the institution of antifungal therapy among non-lung solid-organ transplant recipients and helped to rule out IPA.
Analysis of the First Australian Strains of
Bartonella quintana
Reveals Unique Genotypes
Authors: Woolley Mark W. , Gordon David L. , Wetherall Bruce L.
Published: April 2007

Bartonella quintana
is increasingly recognized as a cause of clinical disease in various geographical locations. We characterized three Australian strains associated with endocarditis, using established molecular-typing techniques, the 16S/23S rRNA intergenic spacer (ITS) region, and multispacer typing (MST). All strains examined demonstrated novel ITS and/or MST genotypes. Further characterization of Australian strains is required to determine whether there is an association between genotype and geographical location.
Peptoniphilus gorbachii
sp. nov.,
Peptoniphilus olsenii
sp. nov., and
Anaerococcus murdochii
sp. nov. Isolated from Clinical Specimens of Human Origin
Authors: Song Yuli , Liu Chengxu , Finegold Sydney M.
Published: April 2007

Three groups of previously unknown gram-positive, anaerobic, coccus-shaped bacteria were characterized using phenotypic and molecular taxonomic methods. Phenotypic and genotypic data demonstrate that these organisms are distinct, and each group represents a previously unknown subline within
Clostridium
cluster XIII. Two groups are most closely related to
Peptoniphilus harei
in the genus
Peptoniphilus
, and the other group is most closely related to
Anaerococcus lactolyticus
in the genus
Anaerococcus
. Based on the findings, three novel species,
Peptoniphilus gorbachii
sp. nov.,
Peptoniphilus olsenii
sp. nov., and
Anaerococcus murdochii
sp. nov., are proposed. The type strains of
Peptoniphilus gorbachii
sp. nov.,
Peptoniphilus olsenii
sp. nov., and
Anaerococcus murdochii
sp. nov. are WAL 10418
T
(= CCUG 53341
T
= ATCC BAA-1383
T
), WAL 12922
T
(= CCUG 53342
T
= ATCC BAA-1384
T
), and WAL 17230
T
(= CCUG 53340
T
= ATCC BAA-1385
T
), respectively.
Outbreak of Fungemia among Neonates Caused by
Candida haemulonii
Resistant to Amphotericin B, Itraconazole, and Fluconazole
Authors: Khan Zia U. , Al-Sweih Noura A. , Ahmad Suhail , Al-Kazemi Nawal , Khan Seema , Joseph Leena , Chandy Rachel
Published: April 2007

The first outbreak of
Candida haemulonii
fungemia is described. The seven isolates from the blood of four neonates were identified by DNA sequencing of the ribosomal DNA. They were all resistant to amphotericin B, fluconazole, and itraconazole. This report highlights the emergence of
C. haemulonii
as an opportunistic pathogen in immunocompromised patients.
Performance of a Rapid Assay (Binax NOW) for Detection of Respiratory Syncytial Virus at a Children's Hospital over a 3-Year Period
Authors: Cruz Andrea T. , Cazacu Andreea C. , Greer Jewel M. , Demmler Gail J.
Published: April 2007

A rapid assay, Binax NOW RSV, was compared to viral culture for 14,756 pediatric respiratory specimens obtained from 2003 to 2006. There were 794 viral culture-confirmed respiratory syncytial virus infections. Sensitivity was 81%, and specificity was 93.2%. Sensitivity was greatest for neonates (91.1% versus 80.7% [
P
< 0.01]).
Clinical Evaluation of the Sensititre YeastOne Plate for Testing Susceptibility of Filamentous Fungi to Posaconazole
Authors: Patel Reena , Mendrick Cara , Knapp Cindy C. , Grist Roger , McNicholas Paul M.
Published: April 2007

Sensititre YeastOne colorimetric antifungal panels were compared with the CLSI (formerly NCCLS) M38-A reference method for testing the susceptibility of filamentous fungi to posaconazole; agreement (±2 log
2
dilutions) between the two methods was 97%. These data confirm the utility of YeastOne panels for measuring the susceptibility of filamentous fungi to posaconazole.
PCR-Restriction Fragment Length Polymorphism for Rapid, Low-Cost Identification of Isoniazid-Resistant
Mycobacterium tuberculosis
Authors: Caws Maxine , Tho Dau Quang , Duy Phan Minh , Lan Nguyen Thi Ngoc , Hoa Dai Viet , Torok Mili Estee , Chau Tran Thi Hong , Van Vinh Chau Nguyen , Chinh Nguyen Tran , Farrar Jeremy
Published: April 2007

PCR-restriction fragment length poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant
Mycobacterium tuberculosis
. One hundred consecutive isolates from a Vietnamese tuberculosis hospital were tested by MspA1I PCR-RFLP for the detection of isoniazid-resistant
katG
_315 mutants. The test had a sensitivity of 80% and a specificity of 100% against conventional phenotypic drug susceptibility testing. The positive and negative predictive values were 1 and 0.86, respectively. None of the discrepant isolates had mutant
katG
_315 codons by sequencing. The test is cheap (less than $1.50 per test), specific, and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of
katG
_315 mutants among isoniazid-resistant
M. tuberculosis
isolates.
Analysis of Murine Hepatitis Virus Strain A59 Temperature-Sensitive Mutant TS-LA6 Suggests that nsp10 Plays a Critical Role in Polyprotein Processing
Authors: Donaldson Eric F. , Graham Rachel L. , Sims Amy C. , Denison Mark R. , Baric Ralph S.
Published: April 2007

Coronaviruses are the largest RNA viruses, and their genomes encode replication machinery capable of efficient replication of both positive- and negative-strand viral RNAs as well as enzymes capable of processing large viral polyproteins into putative replication intermediates and mature proteins. A model described recently by Sawicki et al. (S. G. Sawicki, D. L. Sawicki, D. Younker, Y. Meyer, V. Thiel, H. Stokes, and S. G. Siddell, PLoS Pathog. 1:e39, 2005), based upon complementation studies of known temperature-sensitive (TS) mutants of murine hepatitis virus (MHV) strain A59, proposes that an intermediate comprised of nsp4 to nsp10/11 (∼150 kDa) is involved in negative-strand synthesis. Furthermore, the mature forms of nsp4 to nsp10 are thought to serve as cofactors with other replicase proteins to assemble a larger replication complex specifically formed to transcribe positive-strand RNAs. In this study, we introduced a single-amino-acid change (nsp10:Q65E) associated with the TS-LA6 phenotype into nsp10 of the infectious clone of MHV. Growth kinetic studies demonstrated that this mutation was sufficient to generate the TS phenotype at permissive and nonpermissive temperatures. Our results demonstrate that the TS mutant variant of nsp10 inhibits the main protease, 3CLpro, blocking its function completely at the nonpermissive temperature. These results implicate nsp10 as being a critical factor in the activation of 3CLpro function. We discuss how these findings challenge the current hypothesis that nsp4 to nsp10/11 functions as a single cistron in negative-strand RNA synthesis and analyze recent complementation data in light of these new findings.
Epstein-Barr Virus Thymidine Kinase Is a Centrosomal Resident Precisely Localized to the Periphery of Centrioles
Authors: Gill Michael B. , Kutok Jeffery L. , Fingeroth Joyce D.
Published: April 2007

The thymidine kinase (TK) encoded by Epstein-Barr virus (EBV) differs not only from that of the alphaherpesviruses but also from that of the gamma-2 herpesvirus subfamily. Because cellular location is frequently a determinant of regulatory function, to gain insight into additional role(s) of EBV TK and to uncover how the lymphocryptovirus and rhadinovirus enzymes differ, the subcellular localizations of EBV TK and the related cercopithecine herpesvirus-15 TK were investigated. We show that in contrast to those of the other family members, the gamma-1 herpesvirus TKs localize to the centrosome and even more precisely to the periphery of the centriole, tightly encircling the tubulin-rich centrioles in a microtubule-independent fashion. Centrosomal localization is observed in diverse cell types and occurs whether the protein is expressed independently or in the context of lytic EBV infection. Surprisingly, analysis of mutants revealed that the unique N-terminal domain was not critical for targeting to the centrosome, but rather, peptide sequences located C terminal to this domain were key. This is the first herpesvirus protein documented to reside in the centrosome, or microtubule-organizing center, an amembranous organelle that regulates the structural biology of the cell cycle through control of chromosome separation and cytokinesis. More recently, proteasome-mediated degradation of cell cycle regulatory proteins, production and loading of antigenic peptides onto HLA molecules, and transient homing of diverse virion proteins required for entry and/or egress have been shown to be coordinated at the centrosome. Potential implications of centrosomal localization for EBV TK function are discussed.
Epidermal Growth Factor Receptor Is Not Required for Human Cytomegalovirus Entry or Signaling
Authors: Isaacson Marisa K. , Feire Adam L. , Compton Teresa
Published: April 2007

Human cytomegalovirus (HCMV) can bind, fuse, and initiate gene expression in a diverse range of vertebrate cell types. This broad cellular tropism suggests that multiple receptors and/or universally distributed receptors mediate HCMV entry. Our laboratory has recently discovered that certain β1 and β3 integrin heterodimers are critical mediators of HCMV entry into permissive fibroblasts (A. L. Feire, H. Koss, and T. Compton, Proc. Natl. Acad. Sci. USA 101:15470-15475, 2004). It has also been reported that epidermal growth factor receptor (EGFR) is necessary for HCMV-mediated signaling and entry (X. Wang, S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. E. Huang, Nature 424:456-461, 2003). Integrins are known to signal synergistically with growth factor receptors, and this coordination was recently reported for EGFR and β3 integrins in the context of HCMV entry (X. Wang, D. Y. Huang, S. M. Huong, and E. S. Huang, Nat. Med. 11:515-521, 2005). However, EGFR-negative cell lines, such as hematopoietic cells, are known to be infected by HCMV. Therefore, we wished to confirm a role for EGFR in HCMV entry and then examine any interaction between β1 integrins and EGFR during the entry process. Surprisingly, we were unable to detect any role for EGFR in the process of HCMV entry into fibroblast, epithelial, or endothelial cell lines. Additionally, HCMV did not activate the EGFR kinase in fibroblast cell lines. We first examined HCMV entry into two EGFR-positive or -negative cell lines but observed no increase in entry when EGFR was expressed to high levels. Physically blocking EGFR with a neutralizing antibody in fibroblast, epithelial, or endothelial cell lines or blocking EGFR kinase signaling with a chemical inhibitor in fibroblast cells did not inhibit virus entry. Lastly, we were unable to detect phosphorylation of EGFR in fibroblasts cells in response to HCMV stimulation. Our findings demonstrate that EGFR does not play a significant role in HCMV entry or signaling. These results suggest that specific integrin heterodimers either act alone as the primary entry receptors or interact in conjunction with an additional receptor(s), other than EGFR, to facilitate virus entry.
Hepatitis B Virus X Protein Stimulates the Mitochondrial Translocation of Raf-1 via Oxidative Stress
Authors: Chen Jun , Siddiqui Aleem
Published: April 2007

The human hepatitis B virus (HBV) X protein (HBx) plays a crucial role(s) in the viral life cycle and contributes to the onset of hepatocellular carcinoma (HCC). HBx caused the mitochondrial translocation of Raf-1 kinase either alone or in the context of whole-viral-genome transfections. Mitochondrial translocation of Raf-1 is mediated by HBx-induced oxidative stress and was dependent upon the phosphorylation of Raf-1 at the serine
338/339
and Y
340/341
residues by p21-activated protein kinase 1 and Src kinase, respectively. These studies provide an insight into the mechanisms by which HBV induces intracellular events relevant to liver disease pathogenesis, including HCC.
Characterization and Experimental Transmission of an Oncogenic Papillomavirus in Female Macaques
Authors: Wood Charles E. , Chen Zigui , Cline J. Mark , Miller Brigitte E. , Burk Robert D.
Published: April 2007

Cervical cancer is one of the leading causes of cancer mortality in women worldwide, yet few suitable animal models currently exist for study of this disease. Virtually all cases of cervical cancer in women are caused by specific types of genital human papillomavirus (HPV). In this study, we investigated naturally occurring genital PVs in female cynomolgus macaques (
Macaca fascicularis
) without breeding contact for at least 3.5 years. Exfoliated cervicovaginal cells from 19 of 54 animals tested positive for at least one PV. Seven different PVs were identified, including four novel genotypes and two genotypes (RhPV-d and RhPV-a) previously identified in rhesus macaques (
Macaca mulatta
). Four PV types were associated with cervical intraepithelial neoplasia (CIN), which resembled human CIN by endoscopy, cervical cytology, histology, and immunohistochemistry. The presence of CIN was highly associated with PV infection (
P
< 0.0001). The most prevalent virus type was RhPV-d, which was identified in 60% of animals with CIN. An RhPV-d genome sequenced from a high-grade CIN lesion was found to be phylogenetically related to the highly oncogenic HPV16. Transfer of cervical cytobrush samples from donor animals naturally carrying RhPV-d resulted in new infections in 4 of 12 previously virus-free animals and abnormal cytology and histology in 1 of 4 infected animals after 18 weeks of infection. Experimental transmission was confirmed by E1^E4 reverse transcription-PCR products and RhPV-d sequence identity with the donor variant. These findings identify key similarities between macaque and human oncogenic PVs which should prove useful in the study of viral persistence, carcinogenesis, and therapeutic development.
Diminished Potential for B-Lymphoid Differentiation after Murine Leukemia Virus Infection In Vivo and in EML Hematopoietic Progenitor Cells
Authors: Finstad Samantha L. , Rosenberg Naomi , Levy Laura S.
Published: April 2007

Infection with a recombinant murine-feline gammaretrovirus, MoFe2, or with the parent virus, Moloney murine leukemia virus, caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective, in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state.
Human Immunodeficiency Virus Type 1 cDNAs Produced in the Presence of APOBEC3G Exhibit Defects in Plus-Strand DNA Transfer and Integration
Authors: Mbisa Jean L. , Barr Rebekah , Thomas James A. , Vandegraaff Nick , Dorweiler Irene J. , Svarovskaia Evguenia S. , Brown William L. , Mansky Louis M. , Gorelick Robert J. , Harris Reuben S. , Engelman Alan , Pathak Vinay K.
Published: April 2007

Encapsidation of host restriction factor APOBEC3G (A3G) into
vif
-deficient human immunodeficiency virus type 1 (HIV-1) blocks virus replication at least partly by C-to-U deamination of viral minus-strand DNA, resulting in G-to-A hypermutation. A3G may also inhibit HIV-1 replication by reducing viral DNA synthesis and inducing viral DNA degradation. To gain further insight into the mechanisms of viral inhibition, we examined the metabolism of A3G-exposed viral DNA. We observed that an overall 35-fold decrease in viral infectivity was accompanied by a five- to sevenfold reduction in viral DNA synthesis. Wild-type A3G induced an additional fivefold decrease in the amount of viral DNA that was integrated into the host cell genome and similarly reduced the efficiency with which HIV-1 preintegration complexes (PICs) integrated into a target DNA in vitro. The A3G C-terminal catalytic domain was required for both of these antiviral activities. Southern blotting analysis of PICs showed that A3G reduced the efficiency and specificity of primer tRNA processing and removal, resulting in viral DNA ends that are inefficient substrates for integration and plus-strand DNA transfer. However, the decrease in plus-strand DNA transfer did not account for all of the observed decrease in viral DNA synthesis associated with A3G. These novel observations suggest that HIV-1 cDNA produced in the presence of A3G exhibits defects in primer tRNA processing, plus-strand DNA transfer, and integration.
The Host Protein Staufen1 Participates in Human Immunodeficiency Virus Type 1 Assembly in Live Cells by Influencing pr55
Gag
Multimerization
Authors: Chatel-Chaix Laurent , Abrahamyan Levon , Fréchina Céline , Mouland Andrew J. , DesGroseillers Luc
Published: April 2007

Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55
Gag
. In this report, we tested whether Stau1 modulates pr55
Gag
assembly using a new and specific pr55
Gag
oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55
Gag
-pr55
Gag
BRET levels, suggesting a role for Stau1 in regulating pr55
Gag
oligomerization during assembly. This effect of Stau1 on pr55
Gag
oligomerization was observed only in membranes, a cellular compartment in which pr55
Gag
assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55
Gag
-pr55
Gag
BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55
Gag
complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55
Gag
-pr55
Gag
interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.
U
S
3 of Herpes Simplex Virus Type 1 Encodes a Promiscuous Protein Kinase That Phosphorylates and Alters Localization of Lamin A/C in Infected Cells
Authors: Mou Fan , Forest Tom , Baines Joel D.
Published: April 2007

The herpes simplex virus type 1 (HSV-1) U
S
3 gene encodes a serine/threonine kinase that, when inactivated, causes capsids to aggregate aberrantly between the inner and outer nuclear membranes (INM and ONM, respectively) within evaginations/extensions of the perinuclear space. In both Hep2 cells and an engineered cell line derived from Hep2 cells expressing lamin A/C fused to enhanced green fluorescent protein (eGFP-lamin A/C), lamin A/C localized mostly in a reticular pattern with small regions of the INM devoid of eGFP-lamin A/C when they were either mock infected or infected with wild-type HSV-1(F). Cells infected with HSV-1(F) also contained some larger diffuse regions lacking lamin A/C. Proteins U
L
31 and U
L
34, markers of potential envelopment sites at the INM and perinuclear virions, localized within the regions devoid of lamin A/C and also in regions containing lamin A/C. Similar to previous observations with Vero cells (S. L. Bjerke and R. J. Roller, Virology 347:261-276, 2006), the proteins U
L
34 and U
L
31 localized exclusively in very discrete regions of the nuclear lamina lacking lamin A/C in the absence of U
S
3 kinase activity. To determine how U
S
3 alters lamin A/C distribution, U
S
3 was purified and shown to phosphorylate lamin A/C at multiple sites in vitro, despite the presence of only one putative U
S
3 kinase consensus site in the lamin A/C sequence. U
S
3 kinase activity was also sufficient to invoke partial solubilization of lamin A/C from permeabilized Hep2 cell nuclei in an ATP-dependent manner. Two-dimensional electrophoretic analyses of lamin A/C revealed that lamin A/C is phosphorylated in HSV-infected cells, and the full spectrum of phosphorylation requires U
S
3 kinase activity. These data suggest that U
S
3 kinase activity regulates HSV-1 capsid nuclear egress at least in part by phosphorylation of lamin A/C.
Comparative Study of Methods for Detecting Sequence Compartmentalization in Human Immunodeficiency Virus Type 1
Authors: Zárate Selene , Pond Sergei L. Kosakovsky , Shapshak Paul , Frost Simon D. W.
Published: April 2007

Human immunodeficiency virus (HIV) infects different organs and tissues. During these infection events, subpopulations of HIV type 1 (HIV-1) develop and, if viral trafficking is restricted between subpopulations, the viruses can follow independent evolutionary histories, i.e., become compartmentalized. This phenomenon is usually detected via comparative sequence analysis and has been reported for viruses isolated from the central nervous system (CNS) and the genital tract. Several approaches have been proposed to study the compartmentalization of HIV sequences, but to date, no rigorous comparison of the most commonly employed methods has been made. In this study, we systematically compared inferences made by six different methods for detecting compartmentalization based on three data sets: (i) a sample of 45 patients with sequences gathered from the CNS, (ii) sequences from the female genital tract of 18 patients, and (iii) a set of simulated sequences. We found that different methods often reached contradictory conclusions. Methods based on the topology of a phylogenetic tree derived from clonal sequences were generally more sensitive in detecting compartmentalization than those that relied solely upon pairwise genetic distances between sequences. However, as the branching structure in a phylogenetic tree is often uncertain, especially for short, low-diversity, or recombinant sequences, tree-based approaches may need to be modified to take phylogenetic uncertainty into account. Given the frequently discordant predictions of different methods and the strengths and weaknesses of each particular methodology, we recommend that a suite of several approaches be used for reliable inference of compartmentalized population structure.
A Borna Disease Virus Vector for Expression of Foreign Genes in Neurons of Rodents
Authors: Schneider Urs , Ackermann Andreas , Staeheli Peter
Published: April 2007

An expression cassette for green fluorescent protein was successfully inserted at a site near the 5′ end of the genome of Borna disease virus (BDV). When introduced into a mutant virus with highly active polymerase, the foreign gene was strongly expressed in neurons of infected rats. Since BDV can establish long-term persistence in the central nervous system of rodents, it may be used to engineer efficient vectors for specific delivery of foreign genes into highly differentiated neurons.
The X Protein of Borna Disease Virus Serves Essential Functions in the Viral Multiplication Cycle
Authors: Poenisch Marion , Wille Sandra , Ackermann Andreas , Staeheli Peter , Schneider Urs
Published: April 2007

The X gene of Borna disease virus (BDV) encodes a nonstructural 10-kDa protein that can interact with viral polymerase cofactor P, thus regulating polymerase activity. It remained unknown whether X is essential for virus multiplication. All our attempts to generate mutant BDV with a nonfunctional X gene proved unsuccessful. However, a mutant virus with an inactive X gene was able to replicate in Vero cells if an artificial gene cassette encoding X was inserted at a site near the 5′ end of the viral genome. These results indicate that X performs essential viral functions.
Human Immunodeficiency Virus Type 1 Variants Resistant to First- and Second-Version Fusion Inhibitors and Cytopathic in Ex Vivo Human Lymphoid Tissue
Authors: Chinnadurai Raghavan , Rajan Devi , Münch Jan , Kirchhoff Frank
Published: April 2007

Human immunodeficiency virus type 1 (HIV-1) fusion inhibitors blocking viral entry by binding the gp41 heptad repeat 1 (HR1) region offer great promise for antiretroviral therapy, and the first of these inhibitors, T20 (Fuzeon; enfuvirtide), is successfully used in the clinic. It has been reported previously that changes in the 3-amino-acid GIV motif at positions 36 to 38 of gp41 HR1 mediate resistance to T20 but usually not to second-version fusion inhibitors, such as T1249, which target an overlapping but distinct region in HR1 including a conserved hydrophobic pocket (HP). Based on the common lack of cross-resistance and the difficulty of selecting T1249-resistant HIV-1 variants, it has been suggested that the determinants of resistance to first- and second-version fusion inhibitors may be different. To further assess HIV-1 resistance to fusion inhibitors and to analyze where changes in HR1 are tolerated, we randomized 16 codons in the HR1 region, including those making contact with HR2 codons and/or encoding residues in the GIV motif and the HP. We found that changes only at positions 37I, 38V, and 40Q near the N terminus of HR1 were tolerated. The propagation of randomly gp41-mutated HIV-1 variants in the presence of T1249 allowed the effective selection of highly resistant forms, all containing changes in the IV residues. Overall, the extent of T1249 resistance was inversely correlated to viral fitness and cytopathicity. Notably, one HIV-1 mutant showing ∼10-fold-reduced susceptibility to T1249 inhibition replicated with wild type-like kinetics and caused substantial CD4
+
-T-cell depletion in ex vivo-infected human lymphoid tissue in the presence and absence of an inhibitor. Taken together, our results show that the GIV motif also plays a key role in resistance to second-version fusion inhibitors and suggest that some resistant HIV-1 variants may be pathogenic in vivo.
Cytidine Deaminases APOBEC3G and APOBEC3F Interact with Human Immunodeficiency Virus Type 1 Integrase and Inhibit Proviral DNA Formation
Authors: Luo Kun , Wang Tao , Liu Bindong , Tian Chunjuan , Xiao Zuoxiang , Kappes John , Yu Xiao-Fang
Published: April 2007

APOBEC3G (A3G) is a single-stranded DNA cytidine deaminase that targets retroviral minus-strand DNA and has potent antiviral activity against diverse retroviruses. However, the mechanisms of A3G antiviral functions are incompletely understood. Here we demonstrate that A3G, A3F, and, to a lesser extent, the noncatalytic A3GC291S block human immunodeficiency virus type 1 (HIV-1) replication by interfering with proviral DNA formation. In HIV-1 virions, A3G interacted with HIV-1 integrase and nucleocapsid, key viral factors for reverse transcription and integration. Unlike A3G, the weak antiviral A3C cytidine deaminase did not interact with either of these factors and did not affect viral reverse transcription or proviral DNA formation. Thus, multiple steps of the HIV-1 replication cycle, most noticeably the formation of proviral DNA, are inhibited by both cytidine deamination-dependent and -independent mechanisms.
Mutations in Human Immunodeficiency Virus Type 1 RNase H Primer Grip Enhance 3′-Azido-3′-Deoxythymidine Resistance
Authors: Delviks-Frankenberry Krista A. , Nikolenko Galina N. , Barr Rebekah , Pathak Vinay K.
Published: April 2007

We recently observed that mutations in the human immunodeficiency type 1 (HIV-1) reverse transcriptase (RT) connection domain significantly increase 3′-azido-3′-deoxythymidine (AZT) resistance up to 536 times over wild-type (WT) RT in the presence of thymidine analog resistance mutations (TAMs). These mutations also decreased RT template switching, suggesting that they altered the balance between nucleotide excision and template RNA degradation, which in turn increased AZT resistance. Several residues in the HIV-1 connection domain contact the primer strand and form an RNase H primer grip structure that helps to position the primer-template at the RNase H and polymerase active sites. To test the hypothesis that connection domain mutations enhanced AZT resistance by influencing the RNase H primer grip, we determined the effects of alanine substitutions in RNase H primer grip residues on nucleoside RT inhibitor resistance in the context of a WT, TAM-containing, or K65R-containing polymerase domain. Ten of the 11 RNase H primer grip mutations increased AZT resistance 20 to 243 times above WT levels in the context of a TAM-containing polymerase domain. Furthermore, all mutations in the RNase H primer grip decreased template switching, suggesting that they reduced RNase H activity. These results demonstrate that mutations in the RNase H primer grip region can significantly enhance AZT resistance and support the hypothesis that mutations in the connection and RNase H domains can increase resistance by altering the RNase H primer grip region, changing interactions between RT and the template-primer complex and/or shifting the balance between the polymerase and RNase H activities.
VP3, a Structural Protein of Infectious Pancreatic Necrosis Virus, Interacts with RNA-Dependent RNA Polymerase VP1 and with Double-Stranded RNA
Authors: Pedersen Torunn , Skjesol Astrid , Jørgensen Jorunn B.
Published: April 2007

Infectious pancreatic necrosis virus
(IPNV) is a bisegmented, double-stranded RNA (dsRNA) virus of the
Birnaviridae
family that causes widespread disease in salmonids. Its two genomic segments are encapsulated together with the viral RNA-dependent RNA polymerase, VP1, and the assumed internal protein, VP3, in a single-shell capsid composed of VP2. Major aspects of the molecular biology of IPNV, such as particle assembly and interference with host macromolecules, are as yet poorly understood. To understand the infection process, analysis of viral protein interactions is of crucial importance. In this study, we focus on the interaction properties of VP3, the suggested key organizer of particle assembly in birnaviruses. By applying the yeast two-hybrid system in combination with coimmunoprecipitation, VP3 was proven to bind to VP1 and to self-associate strongly. In addition, VP3 was shown to specifically bind to dsRNA in a sequence-independent manner by in vitro pull-down experiments. The binding between VP3 and VP1 was not dependent on the presence of dsRNA. Deletion analyses mapped the VP3 self-interaction domain within the 101 N-terminal amino acids and the VP1 interaction domain within the 62 C-terminal amino acids of VP3. The C-terminal end was also crucial but not sufficient for the dsRNA binding capacity of VP3. For VP1, the 90 C-terminal amino acids constituted the only dispensable part for maintaining VP3-binding ability. Kinetic analysis revealed the presence of VP1-VP3 complexes prior to the formation of mature virions in IPNV-infected CHSE-214 cells, which indicates a role in promoting the assembly process.
Effects of Use of Alternative Therapies on Quality of Life and Healthcare Spending
Authors: Zhang James X. , Woo Jean , Lau Wallace C. S. , Lee Polly , Chiu Peter , Lam Daniel
Published: April 2007

Osteoarthritis (OA) is a costly disease that causes much morbidity and mortality in the world, and it was the sixth leading cause of disability in developed countries. We aim to study the utilization pattern of alternative therapies and their effects on quality of life and personal health spending in Chinese OA patients in Hong Kong. Five-hundred forty-seven patients with OA from four regional hospitals in Hong Kong were recruited, and we measured various types of alternative therapies, SF-36 scales, an overall Health Utility Index derived from a pre-scored multi-attribute classification system based upon SF-36 health surveys, health spending per person and out-of-pocket payments and side-effects. The study shows that out of the 547 OA patients, the patients have used a wide spectrum of alternative therapies and often used a multiplicity of them. Payment for alternative therapies constitutes 5% of the overall personal healthcare spending, and 29% of the out-of-pocket payments. The use of alternative therapies was significantly associated with higher personal healthcare spending ( p = 0.01), after adjusting for socioeconomic variables, years of OA and severity of OA. The use of alternative therapies was not significantly associated with an improvement in the quality of life in the regression analysis ( p = 0.64). The use of alternative therapies was statistically significant associated with the side effects, including gastric discomfort and gastric ulcer/bleeding ( p = 0.04, 0.02, respectively). Alternative therapies were used extensively by OA patients in Hong Kong. Clinicians, health policy makers, and insurance carriers should be aware of the potential health and economic effects in practice and policy formulation.
Xiong-gui-tiao-xue-yin (Kyuki-chouketsu-in), a Traditional Herbal Medicine, Stimulates Lactation with Increase in Secretion of Prolactin but not Oxytocin in the Postpartum Period
Authors: Ushiroyama Takahisa , Sakuma Kou , Souen Hekiko , Nakai Gou , Morishima Shouko , Yamashita Yoshiki , Kamegai Hideki
Published: April 2007

The aim of the present investigation was to evaluate the clinical efficacy of Xiong-gui-tiao-xue-yin, a traditional Japanese herbal medicine, in stimulating lactation in the postpartum period. We enrolled 82 women who had a normal delivery in Osaka Medical College Hospital, and randomly assigned them to the following two groups: a group of 41 women who received Xiong-gui-tiao-xue-yin at a dose of 6.0 g/day (Group X), and a group of 41 women who received ergometrine (methylergometrine maleate) at a dose of 0.375 mg/day (Group E). Volume of lactation was determined daily until Day 6 postpartum. Plasma prolactin and oxytocin concentration were measured at Days 1 and 6 postpartum. The results showed that volume of lactation was significantly higher in Group X than in Group E at Days 4 ( p = 0.042), 5 ( p = 0.038), and 6 ( p = 0.046). Significant differences between Groups X and E were noted in plasma prolactin concentration at Days 1 (157.9 ± 78.2 ng/ml and 129.1 ± 64.8 ng/ml ; p = 0.037) and 6 (167.5 ± 95.4 ng/ml and 117.1 ± 53.6 ng/ml ; p = 0.0042) postpartum. On the other hand, at Day 1, oxytocin concentration was significantly higher in Group E than in Group X ( p = 0.0024). No adverse effects were observed in this study. The results of our study demonstrate the beneficial effects of Xiong-gui-tiao-xue-yin on lactation, with increase in prolactin level without increase in oxytocin level in the postpartum period. Therefore, Xiong-gui-tiao-xue-yin can be expected to improve lactation in women in the postpartum period. Further detailed bio-pharmacological studies and clinical trials to investigate the properties of this drug are warranted.
Crossover Clinical Trial to Determine the Effect of Manual Acupuncture at Siguan Points (Bilateral LI4 and LR3) on Intestinal Motility in Healthy Subjects
Authors: Yim Y. K. , Kang W. C. , Cho J. H. , Shin J. W. , Lee N. H. , Choi S. M. , Koo S. T. , Park K. S. , Son C. G.
Published: April 2007

This study examined whether manual acupuncture at the Siguan points (bilateral points LI4 and LR3) affects intestinal motility in healthy human subjects. Twenty healthy male subjects were randomly assigned either to real acupuncture (RA) at Siguan points or sham acupuncture (SA) groups in a crossover manner. All subjects underwent two experimental sessions; the RA group in the first session was treated with SA in the second session after a 2-week washout period, and vice versa. Each subject took 20 radio-markers and was treated with acupuncture 0, 12, 24, and 36 hours after radio-marker intake. Radiographs were taken at 6, 12.5, 24.5, and 48 hours, and the effect of acupuncture on intestinal motility was evaluated based on the distribution of the radio-markers in the ileum, ascending colon, transverse colon, descending colon, sigmoid/ rectum, and outside the body. Defecating habit was monitored during the trial, and complete blood counts were checked before and after the two acupuncture sessions. The RA and SA results showed extremely similar distributions of the radio-markers in these five regions of the alimentary canal and outside the body in radiographs taken at four different times, verifying that there was no effect of manual acupuncture at the Siguan points on intestinal motility, at least in healthy human subjects.
Appearance of Human Meridian-Like Structure and Acupoints and Its Time Correlation by Infrared Thermal Imaging
Authors: Yang Hong-Qin , Xie Shu-Sen , Hu Xiang-Long , Chen Li , Li Hui
Published: April 2007

The meridians and acupoints of human bodies at natural condition are investigated among 30 healthy volunteers by infrared thermal imaging technique. The results give clear evidence of the existence of infrared radiant tracks along human meridian courses. The time dependent evolution of the infrared radiant track is observed for the first time. The time rhythm of acupoints is also studied. Our findings not only support the view that infrared radiant tracks along human meridian courses is a normal vital and physiological phenomenon appearing in human beings, but also offer a potential method for noninvasive diagnostic by studying the physiological function and pathological change of meridians or acupoints by means of thermography.