bluuebunny/crossref_metadata_2025 huggingface dataset:
Optimization and Application of a Multiplex Bead-Based Assay To Quantify Serotype-Specific IgG againstStreptococcus pneumoniaePolysaccharides: Response to the Booster Vaccine after Immunization with the Pneumococcal 7-Valent Conjugate Vaccine
Authors: Elberse Karin E. M. , Tcherniaeva Irina , Berbers Guy A. M. , Schouls Leo M.
Published: February 2010

We describe the optimization and application of a multiplex bead-based assay (Luminex) to quantify antibodies against polysaccharides of 13 pneumococcal serotypes. In the optimized multiplex immunoassay (MIA), intravenous immune globulin was introduced as an in-house reference serum, and nonspecific reacting antibodies were adsorbed with the commercial product pneumococcal C polysaccharides Multi. The antibody concentrations were assessed in 188 serum samples obtained pre- and post-booster vaccination at 11 months after administration of a primary series of the pneumococcal seven-valent conjugate vaccine (PCV-7) at 2, 3, and 4 months of age. The results of the MIA were compared with those of the ELISA for the serotypes included in the seven-valent conjugated polysaccharide vaccine and for a non-vaccine serotype, serotype 6A. The geometric mean concentrations of the antibodies determined by MIA were slightly higher than those determined by ELISA. The correlations between the assays were good, withR2values ranging from 0.84 to 0.91 for all serotypes except serotype 19F, for whichR2was 0.70. The concentrations of antibody against serotype 6A increased after the administration of PCV-7 due to cross-reactivity with serotype 6B. The differences between the results obtained by ELISA and MIA suggest that the internationally established protective threshold of 0.35 μg/ml should be reevaluated for use in the MIA and may need to be amended separately for each serotype.
Coincorporation of LpxL1 and PagL Mutant Lipopolysaccharides into Liposomes withNeisseria meningitidis Opacity Protein: Influence on Endotoxic and Adjuvant Activity
Authors: Arenas Jesús , van Dijken Harry , Kuipers Betsy , Hamstra Hendrik Jan , Tommassen Jan , van der Ley Peter
Published: January 2010

Wild-type lipopolysaccharide (LPS) ofNeisseria meningitidisnormally contains six acyl chains. Penta-acylated LPS forms were generated through inactivation of thelpxL1gene or through the expression of theBordetella bronchiseptica pagLgene inN. meningitidis. The resulting LPS species, designated LpxL1 LPS and PagL LPS, respectively, display reduced endotoxic activity compared to wild-type LPS. Here, we determined the adjuvant potential of PagL LPS by comparison with the broadly used LpxL1 LPS. We also investigated the potential benefit for adjuvanticity of coincorporating these LPS species, together with the meningococcal opacity-associated protein OpaJ as a model antigen, in a liposomal delivery system. PagL LPS showed a higher endotoxic activity than LpxL1 LPS, and their incorporation into liposomes significantly reduced their endotoxic activity as determined by measuring the induction of interleukin-6 (IL-6) production in a murine macrophage cell line. To determine the adjuvant effect, BALB/c mice were immunized with OpaJ-containing liposomes and either free LPS or LPS coincorporated into the proteoliposomes. OpaJ-containing liposomes adjuvanted with AlPO4or not adjuvanted at all were included as control groups. In the appropriate dose, PagL LPS showed a superior adjuvant effect compared with LpxL1 LPS, and for both LPS types, free LPS showed a higher adjuvant effect than when coincorporated into the liposomes, as evidenced by higher titers of IgG2a and IgG2b antibodies against OpaJ+meningococci and higher bactericidal titers. In conclusion, PagL LPS is a better adjuvant than LpxL1 LPS, but coincorporation of either LPS into proteoliposomes did not improve their adjuvant activity.
IgA Antibody Response of Swine to Foot-and-Mouth Disease Virus Infection and Vaccination
Authors: Pacheco Juan M. , Butler John E. , Jew Jessica , Ferman Geoffrey S. , Zhu James , Golde William T.
Published: January 2010

Foot-and-mouth disease virus (FMDV) continues to be a significant economic problem worldwide. Control of the disease involves the use of killed-virus vaccines, a control measure developed decades ago. After natural infection, the primary site of replication of FMDV is the pharyngeal area, suggesting that a mucosal immune response is the most effective. Humoral immunity to killed-virus vaccination induces antibodies that can prevent the clinical disease but not local infection. Determining whether infection or vaccination stimulates IgA-mediated local immunity depends on the method of analysis. Different assays have been described to analyze the quality of antibody responses of cattle and swine to FMDV, including indirect double-antibody sandwich enzyme-linked immunosorbent assay (IDAS-ELISA) and antibody capture assay-ELISA (ACA-ELISA). We tested these assays on swine and show that vaccinated animals had FMDV-specific IgM and IgG but no IgA in either serum or saliva. After the infection, both assays detected FMDV-specific IgM, IgG, and IgA in serum. Notably, serum IgA was more readily detected using the ACA-ELISA, whereas IgA was not detected in saliva with this assay. FMDV-specific IgA antibodies were detected in saliva samples using the IDAS-ELISA. These data show that parenterally administered, killed-virus vaccine does not induce a mucosal antibody response to FMDV and illuminates limitations and appropriate applications of the two ELISAs used to measure FMDV-specific responses. Further, the presence of the IgA antivirus in serum correlates with the presence of such antibodies in saliva.
Diagnosing Human Anisakiasis: Recombinant Ani s 1 and Ani s 7 Allergens versus the UniCAP 100 Fluorescence Enzyme Immunoassay
Authors: Anadón A. M. , Rodríguez E. , Gárate M. T. , Cuéllar C. , Romarís F. , Chivato T. , Rodero M. , González-Díaz H. , Ubeira F. M.
Published: January 2010

Commercially available serological methods for serodiagnosis of human anisakiasis either are poorly specific or do not include some of the most relevantAnisakisallergens. The use of selected recombinant allergens may improve serodiagnosis. To compare the diagnostic and clinical values of enzyme-linked immunosorbent assay (ELISA) methods based on Ani s 1 and Ani s 7 recombinant allergens and of the UniCAP 100 fluorescence enzyme immunoassay (CAP FEIA) system, we tested sera from 495 allergic and 25 non-food-related allergic patients. The decay in specific IgE antibodies in serum was also investigated in 15 positive patients over a period of 6 to 38 months. Considering sera that tested positive by either Ani s 1 or Ani s 7 ELISA, the CAP FEIA classified 25% of sera as falsely positive, mainly in the group of patients with the lowest levels of anti-AnisakisIgE antibodies, and 1.28% of positive sera as falsely negative. Considering allergens individually, the overall sensitivities of Ani s 7 ELISA and Ani s 1 ELISA were 94% and 61%, respectively. The results also showed that anti-AnisakisIgE antibodies can be detected in serum for longer with Ani s 1 ELISA than with Ani s 7 ELISA and CAP FEIA (P< 0.01). Our findings suggest that ELISA methods with Ani s 7 and Ani s 1 allergens as targets of IgE antibodies are currently the best option for serodiagnosis of human anisakiasis, combining specificity and sensitivity. The different persistence of anti-Ani s 1 and anti-Ani s 7 antibodies in serum may help clinicians to distinguish between recent and oldAnisakisinfections.
Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of
Mycobacterium tuberculosis
Authors: Eklund Daniel , Welin Amanda , Schön Thomas , Stendahl Olle , Huygen Kris , Lerm Maria
Published: January 2010

Intracellular pathogens such as
Mycobacterium tuberculosis
have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of
M. tuberculosis
. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics.
Analysis of the IgG Immune Response toTreponema phagedenis-Like Spirochetes in Individual Dairy Cattle with Papillomatous Digital Dermatitis
Authors: Moe Kyaw Kyaw , Yano Takahisa , Misumi Kazuhiro , Kubota Chikara , Yamazaki Wataru , Muguruma Michio , Misawa Naoaki
Published: January 2010

Papillomatous digital dermatitis (PDD) is a major infectious disease of the foot skin in dairy cattle.Treponema phagedenis-like spirochetes have been consistently detected in PDD lesions, and antibodies against these organisms have been demonstrated in affected cattle. However, little is known about the dominant antigens recognized by the immune system of affected cattle. Here, we investigated the IgG immune response toT. phagedenis-like isolates by Western blotting with different sera using whole-cell lysates and extracted glycolipid from 18 and 8 isolates, respectively, including those from different cattle on the same or different farms, isolates from different lesions affecting a single cow, and different isolates from the same lesion affecting a single cow. The reactivity of sera in Western blot assays revealed different banding patterns or showed no bands, suggesting that considerable antigenic variations, including glycolipid, may exist among the isolates, even in those from single individuals. With use of a total of 151 serum samples collected from three groups of cattle, i.e., PDD-positive cows on PDD-positive farms (group A), PDD-negative cows on PDD-positive farms (group B), and cows on PDD-free farms (group C), the levels of IgG antibodies against fourT. phagedenis-like isolates were measured by enzyme-linked immunosorbent assay (ELISA). The optical density in groups A and B was significantly higher than that in group C, even though the value varied among the antigens used. Therefore, combinations of multipleTreponemaspecies should be used for serological analysis and the development of a suitable vaccine because of antigenic variations.
Novel Method for Genotyping Hepatitis B Virus on the Basis of TaqMan Real-Time PCR
Authors: Malmström Sebastian , Berglin-Enquist Ida , Lindh Magnus
Published: January 2010

Chronic infection with hepatitis B virus (HBV) is an important cause of cirrhosis and cancer of the liver. HBV is currently classified into eight genotypes, A to H. Accumulated evidence shows that the genotype influences both the clinical course of infection and the response to treatment. We describe a new method for genotyping based on TaqMan real-time PCR, which identifies all HBV genotypes without post-PCR processing. In this assay, each sample is processed in four multiplex real-time PCRs, each targeting two or three genotype-specific segments of HBV. By analyzing 185 samples representing all genotypes and different proportions of genotype mixtures, we could validate high accuracy of the assay. We conclude that this new assay represents a significant advancement for both diagnostics and clinical research because it is accurate, practical, and based on a technique that is well established in many virological laboratories.
Molecular Phylogenetic Diversity of Dermatologic and Other Human Pathogenic Fusarial Isolates from Hospitals in Northern and Central Italy
Authors: Migheli Quirico , Balmas Virgilio , Harak Henry , Sanna Silvana , Scherm Barbara , Aoki Takayuki , O'Donnell Kerry
Published: January 2010

Fifty-eight fusaria isolated from 50 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusarioses in northern and central Italy. Sequence typing revealed that the isolates were nearly evenly divided among theFusarium solanispecies complex (FSSC;n= 18), theF. oxysporumspecies complex (FOSC;n= 20), and theGibberella(Fusarium)fujikuroispecies complex (GFSC;n= 20). The three-locus typing scheme used for members of the FSSC identified 18 novel STs distributed among six phylogenetically distinct species, yielding an index of discrimination of 1.0. Phylogenetic analysis of the FOSC two-locus data set identified nine STs, including four which were novel, and nine isolates of ST 33, the previously described widespread clonal lineage. With the inclusion of eight epidemiologically unrelated ST 33 isolates, the FOSC typing scheme scored a discrimination index of 0.787. The two-locus GFSC typing scheme, which was primarily designed to identify species, received the lowest discrimination index, with a score of 0.492. The GFSC scheme, however, was used to successfully identify 17 isolates asF. verticillioides, 2 asF. sacchari, and 1 asF. guttiforme. This is the first report thatF. guttiformecauses a human mycotic infection, which was supported by detailed morphological analysis. In addition, the results of a pathogenicity experiment revealed that the human isolate ofF. guttiformewas able to induce fusariosis of pineapple, heretofore its only known host.
Hepatitis E Virus (HEV) Strains in Serum Samples Can Replicate Efficiently in Cultured Cells Despite the Coexistence of HEV Antibodies: Characterization of HEV Virions in Blood Circulation
Authors: Takahashi Masaharu , Tanaka Toshinori , Takahashi Hideyuki , Hoshino Yu , Nagashima Shigeo , Jirintai  , Mizuo Hitoshi , Yazaki Yasuyuki , Takagi Tomofumi , Azuma Masahiro , Kusano Eiji , Isoda Norio , Sugano Kentaro , Okamoto Hiroaki
Published: January 2010

We recently developed a cell culture system for hepatitis E virus (HEV) in PLC/PRF/5 and A549 cells, using fecal specimens from HEV-infected patients. Since transfusion-associated hepatitis E has been reported, we examined PLC/PRF/5 and A549 cells for the ability to support replication of HEV in various serum samples obtained from 23 patients with genotype 1, 3, or 4 HEV. HEV progenies emerged in culture media of PLC/PRF/5 cells, regardless of the coexistence of HEV antibodies in serum but dependent on the load of HEV inoculated (31% at 2.0 × 104copies per well and 100% at ≥3.5 × 104copies per well), and were successfully passaged in A549 cells. HEV particles in serum, with or without HEV antibodies, banded at a sucrose density of 1.15 to 1.16 g/ml, which was markedly lower than that for HEV particles in feces, at 1.27 to 1.28 g/ml, and were nonneutralizable by immune sera in this cell culture system. An immuno-capture PCR assay of HEV virions treated with or without detergent indicated that HEV particles in serum are associated with lipids and HEV ORF3 protein, similar to those in culture supernatant. By immunoprecipitation, it was found that >90% of HEV particles in the circulation exist as free virions not complexed with immunoglobulins, despite the coexistence of HEV antibodies. These results suggest that ourin vitrocell culture system can be used for propagation of a wide variety of HEV strains in sera from various infected patients, allowing extended studies on viral replication specific to different HEV strains.
Combined Real-Time PCR and
rpoB
Gene Pyrosequencing for Rapid Identification of
Mycobacterium tuberculosis
and Determination of Rifampin Resistance Directly in Clinical Specimens
Authors: Halse Tanya A. , Edwards Justine , Cunningham Phyllis L. , Wolfgang William J. , Dumas Nellie B. , Escuyer Vincent E. , Musser Kimberlee A.
Published: January 2010

Our laboratory has developed a rapid, sensitive, and specific molecular approach for detection in clinical specimens, within 48 h of receipt, of both
Mycobacterium tuberculosis
complex (MTBC) DNA and mutations within the 81-bp core region of the
rpoB
gene that are associated with rifampin (RIF) resistance. This approach, which combines an initial real-time PCR with internal inhibition assessment and a pyrosequencing assay, was validated for direct use with clinical specimens. To assess the suitability of real-time PCR for use with respiratory, nonrespiratory, acid-fast bacillus (AFB)-positive and AFB-negative specimens, we evaluated specimens received in our laboratory between 11 October 2007 and 30 June 2009. With culture used as the “gold standard,” the sensitivity, specificity, and positive and negative predictive values were determined for 1,316 specimens to be as follows: for respiratory specimens, 94.7%, 99.9%, 99.6%, and 98.6%, respectively; for nonrespiratory specimens, 88.5%, 100.0%, 100.0%, and 96.9%, respectively; for AFB-positive specimens, 99.6%, 100.0%, 100.0%, and 97.7%, respectively; and for AFB-negative specimens, 75.4%, 99.9%, 98.0%, and 98.4%, respectively. PCR inhibition was determined to be minimal in this assay, occurring in 0.2% of tests. The
rpoB
gene pyrosequencing assay was evaluated in a similar prospective study, in which 148 clinical specimens positive for MTBC DNA by real-time PCR were tested. The final results revealed that the results of direct testing of clinical specimens by the pyrosequencing assay were 98.6% concordant with the results of conventional testing for susceptibility to RIF in liquid culture and that our assay displayed adequate sensitivity for 96.6% of the clinical specimens tested. Used together, these assays provide reliable results that aid with the initial management of patients with suspected tuberculosis prior to the availability of the results for cultured material, and they also provide the ability to predict RIF resistance in
Mycobacterium tuberculosis
-positive specimens in as little as 48 h from the time of clinical specimen receipt.
Rapid Detection of
Staphylococcus aureus
Panton-Valentine Leukocidin in Clinical Specimens by Enzyme-Linked Immunosorbent Assay and Immunochromatographic Tests
Authors: Badiou Cedric , Dumitrescu Oana , George Narelle , Forbes Andrea R. N. , Drougka Eleanna , Chan Kian Sing , Ramdani-Bouguessa Nadjia , Meugnier Helene , Bes Michele , Vandenesch Francois , Etienne Jerome , Hsu Li Yang , Tazir Mohamed , Spiliopoulou Iris , Nimmo Graeme R. , Hulten Kristina G. , Lina Gerard
Published: February 2010

Staphylococcus aureus
strains producing Panton-Valentine leukocidin (PVL) have been epidemiologically linked to specific human infections. To evaluate immunological tests that may be used to diagnose infections with PVL-producing strains, we prospectively collected pus, respiratory tract specimens, and joint fluid specimens from which
S. aureus
had been isolated in clinical laboratories in six countries. An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT) targeting LukS-PV were performed directly with clinical samples for the detection of PVL. The same tests were applied to
S. aureus
culture supernatants. The corresponding
S. aureus
isolates were characterized by PCR for the presence of the PVL locus (
lukS-PV
and
lukF-PV
) and the
mec
A
gene. A total of 185 samples from 144 skin infections, 23 bone and joint infections, and 18 lower respiratory tract infections were analyzed. By PCR, 72/185
S. aureus
isolates were PVL locus positive (PVL
+
); 28 of these were also
mecA
positive. PVL was detected in the supernatants of all PVL
+
strains by both ELISA and an ICT, while no signal was observed with PVL-negative strains. The PVL concentrations in human clinical samples that grew PVL
+
strains ranged from 0 to 399 μg/ml by ELISA. By the use of 0.015 μg/ml of PVL as a cutoff value, PVL was detected in 65/72 (90%) of the clinical samples by ELISA. The sensitivity and specificity of the ELISA test were 90% and 100%, respectively. By the ICT, PVL was detected in 57/72 (79%) of the samples, and the sensitivity and specificity of ICT were 79% and 100%, respectively. PVL is expressed by
S. aureus
during human infection, and a PVL-specific ELISA and ICT could be reliable tests for the diagnosis of infections caused by PVL-producing strains.
Prevalence of Non-Penicillin-Susceptible Group B Streptococcus in Philadelphia and Specificity of Penicillin Resistance Screening Methods
Authors: Kasahara Kei , Baltus Andrew J. , Lee Sun-Hwa , Edelstein Martha A. , Edelstein Paul H.
Published: January 2010

A total of 1,991 group B streptococcus (GBS) isolates, collected in 2008 and 2009, were tested for non-penicillin susceptibility by broth microdilution, disk testing, and oxacillin screening agar. No GBS isolates were non-penicillin susceptible. Oxacillin and ceftizoxime disk testing results showed that proposed screening criteria are nonspecific. The oxacillin screening agar was specific but of unknown sensitivity.
Stable Formation of Compositionally Unique Stress Granules in Virus-Infected Cells
Authors: Piotrowska Joanna , Hansen Spencer J. , Park Nogi , Jamka Katarzyna , Sarnow Peter , Gustin Kurt E.
Published: January 2010

Stress granules are sites of mRNA storage formed in response to a variety of stresses, including viral infections. Here, the mechanisms and consequences of stress granule formation during poliovirus infection were examined. The results indicate that stress granules containing T-cell-restricted intracellular antigen 1 (TIA-1) and mRNA are stably constituted in infected cells despite lacking intact RasGAP SH3-domain binding protein 1 (G3BP) and eukaryotic initiation factor 4G. Fluorescent
in situ
hybridization revealed that stress granules in infected cells do not contain significant amounts of viral positive-strand RNA. Infection does not prevent stress granule formation in response to heat shock, indicating that poliovirus does not block
de novo
stress granule formation. A mutant TIA-1 protein that prevents stress granule formation during oxidative stress also prevents formation in infected cells. However, stress granule formation during infection is more dependent upon ongoing transcription than is formation during oxidative stress or heat shock. Furthermore, Sam68 is recruited to stress granules in infected cells but not to stress granules formed in response to oxidative stress or heat shock. These results demonstrate that stress granule formation in poliovirus-infected cells utilizes a transcription-dependent pathway that results in the appearance of stable, compositionally unique stress granules.
Characterization of the Varicella-Zoster Virus ORF50 Gene, Which Encodes Glycoprotein M
Authors: Sadaoka Tomohiko , Yanagi Tatsuya , Yamanishi Koichi , Mori Yasuko
Published: January 2010

The ORF50 gene of the varicella-zoster virus (VZV) encodes glycoprotein M (gM), which is conserved among all herpesviruses and is important for the cell-to-cell spread of VZV. However, few analyses of ORF50 gene expression or its posttranscriptional and translational modifications have been published. Here we found that in VZV-infected cells, ORF50 encoded four transcripts: a full-size transcript, which was translated into the gM, and three alternatively spliced transcripts, which were not translated. Using a splicing-negative mutant virus, we showed that the alternative transcripts were nonessential for viral growth in cell culture. In addition, we found that two amino acid mutations of gM, V42P and G301M, blocked gM's maturation and transport to the
trans
-Golgi network, which is generally recognized as the viral assembly complex. We also found that the mutations disrupted gM's interaction with glycoprotein N (gN), revealing their interaction through a bond that is otherwise unreported for herpesviruses. Using this gM maturation-negative virus, we found that immature gM and gN were incorporated into intracellularly isolated virus particles and that mature gM was required for efficient viral growth via cell-to-cell spread but not for virion morphogenesis. The virus particles were more abundant at the abnormally enlarged perinuclear cisternae than those of the parental virus, but they were also found at the cell surface and in the culture medium. Additionally, in the gM maturation-negative mutant virus-infected melanoma cells, typical syncytium formation was rarely seen, again indicating that mature gM functions in cell-to-cell spread via enhancement of syncytium formation.
The ORF3 Protein of Hepatitis E Virus Delays Degradation of Activated Growth Factor Receptors by Interacting with CIN85 and Blocking Formation of the Cbl-CIN85 Complex
Authors: Chandra Vivek , Kalia Manjula , Hajela Krishnan , Jameel Shahid
Published: February 2010

Hepatitis E virus (HEV) causes an acute self-limiting disease that is endemic in developing countries. Previous studies suggested that the ORF3 protein (pORF3) of HEV is required for infection
in vivo
and is likely to modulate the host response. Our previous work showed that pORF3 localizes to early and recycling endosomes and causes a delay in the postinternalization trafficking of epidermal growth factor receptor (EGFR) to late endosomes/lysosomes. Here we report that pORF3 also delays the trafficking and degradation of activated hepatocyte growth factor receptor (c-Met) and delineate the mechanistic details of these effects. A mutant ORF3 protein, which does not localize to endosomes, also showed similar effects on growth factor receptor trafficking, making this effect independent of the endosomal localization of pORF3. The ORF3 protein was found to interact with CIN85, a multidomain adaptor protein implicated in the Cbl-mediated downregulation of receptor tyrosine kinases. This interaction competed with the formation of the growth factor receptor-Cbl-CIN85 complex, resulting in the reduced ubiquitination of CIN85 and trafficking of the growth factor receptor complex toward late endosomes/lysosomes. We propose that through its effects on growth factor receptor trafficking, pORF3 prolongs endomembrane growth factor signaling and promotes cell survival to contribute positively to viral replication and pathogenesis.
Expression of Varicella-Zoster Virus Immediate-Early Regulatory Protein IE63 in Neurons of Latently Infected Human Sensory Ganglia
Authors: Zerboni Leigh , Sobel Raymond A. , Ramachandran Vasavi , Rajamani Jaya , Ruyechan William , Abendroth Allison , Arvin Ann
Published: January 2010

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediate-early 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained.
Electrochemical behavior of poly(3,4-ethylenedioxy)thiophene doped with hexacyanoferrate anions in different electrolyte media
Authors: Melato Ana I. , Abrantes Luisa M.
Published: February 2010

This paper describes in detail the electrochemical properties of potentiodynamically synthesized poly(3,4-ethylenedioxy)thiophene (PEDOTh) modified by the entrapment of hexacyanoferrate anions (Fe(CN)63–). Cyclic voltammetric data clearly shows that during polymer redox conversion/immobilized anion reduction and re-oxidation, charge compensation occurs at the expenses of the insertion–deinsertion of appropriate cations from solution. In aqueous electrolyte mainly protons are involved whereas in organic media the process requires small highly mobile ions such as lithium. PEDOTh/Pt and PEDOTh/Fe(CN)63–/Pt have been used for the detection of ascorbic acid (AA) in mixed medium (0.1 M TBAPF6 in acetonitrile + 8% H2O). Compared to that of bare platinum electrode, the analyte oxidation peak potential occurs at significant less positive values at both modified electrodes, revealing their good electrocatalytic activity. For the polymer incorporating ferricyanide ion, the observed decrease in the AA oxidation onset potential along with the fading away of Fe(CN)63– reduction signal, supports a mechanism taking into account the mediator character of the embedded anion.
Effects of annual crown pruning and serial propagation on rooting of stem cuttings from Douglas-fir
Authors: Copes Donald L.
Published: January 2010

Success of rooting from stem cuttings of 14 Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco) clones increased significantly from 42% in 1974 to 67% in 1981. The increase was slightly more than 3% each year despite ortet age increasing from 10 and 13 years in 1974 to 17 and 20 years in 1981. The greatest increase occurred from the 3rd through the 6th year of annual rooting trials. Yearly pruning of ortets and rooted ramets of the same clones is thought to have reinvigorated shoots from the pruned crowns of trees. Several annual prunings seemed to be required before average rooting potential increased markedly. No translocation of the reinvigoration stimulus from the pruned to the adjacent unpruned areas of the same trees was detected. Rooting of cuttings from ortets from pruned areas was at least 9% more than cuttings from unpruned areas. No additional invigoration from serial propagation of cuttings was noted for rooting potential, but such cuttings grew a little taller and slightly less plagiotropic 1 year after rooting than were ramets propagated from pruned ortets. Significant clonal variation was found with all treatments. Evidence of a change in physiological vigor of the ortets and rooted ramets was externally visible in the formation of abundant lammas shoots on pruned areas of the 17- and 20-year-old ortets and rooted ramets. Abundant lammas growth is normally found only on Douglas-fir trees less than 10 years old.
Conifer fine root and mycorrhizal root biomass within the forest floors of Douglas-fir stands of different ages and site productivities
Authors: Vogt Kristiina A. , Moore Erin E. , Vogt Daniel J. , Redlin Mark J. , Edmonds Robert L.
Published: January 2010

Live and dead conifer fine root (≤2 mm) and mycorrhizal root biomasses were determined in the forest floors of 16 stands of Douglas-fir (Pseudotsugamenziesii (Dougl.) Forbes) of low (IV) or high (II) productivity. The maximum biomass of both live conifer and mycorrhizal root biomass occurred at canopy closure in site II and IV stands. After crown closure this biomass decreased significantly in site II but not in site IV stands. During and following crown closure, site IV stands generally had a significantly higher live conifer root biomass than site II stands. Only in the 65- to 75- and 150- to 163-year age groupings of stands was a significantly higher live mycorrhizal root biomass measured in site IV than in site II stands. The proportion of dead to total conifer fine root biomass fluctuated from 13 to 56% in site II stands and from 26 to 76% in site IV stands. The percentage of total mycorrhizal roots that were dead fluctuated from 25 to 57% in site II stands and from 19 to 76% in site IV stands. Generally the highest percentage of live root tips infected by mycorrhizal fungi occurred in the 45- and 46-year-old site II stands and in the 33-, 49-, 67-, and 69-year-old site IV stands.
Compétition pour le rayonnement solaire en début de succession secondaire dans une érablière à bouleau jaune et hêtre
Authors: Bellefleur P. , LaRocque G.
Published: January 2010

We demonstrate that sugar maple (Acersaccharum Marsh.), yellow birch (Betulaalleghaniensis Britton), and beech (Fagusgrandifolia Ehrh.) compete for solar radiation against other woody species (noncommercial) and herbaceous species, following clear-cutting. The experiment was carried out at the Duchesnay Forest Station (Quebec, Canada) in a sugar maple – yellow birch – beech community and consisted of one control and two treatments: (1) elimination of noncommercial woody species and (2) elimination of noncommercial woody species and herbaceous species. The graph of solar radiation interception versus cover indicates that crown overlapping does not contribute significantly to any increase in solar radiation interception. Surface soil temperature is strongly linked to the decrease in solar radiation due to its interception by plants.
CODING PARTITIONS OF REGULAR SETS
Authors: BÉAL MARIE-PIERRE , BURDERI FABIO , RESTIVO ANTONIO
Published: January 2010

A coding partition of a set of words partitions this set into classes such that whenever a sequence, of minimal length, has two distinct factorizations, the words of these factorizations belong to the same class. The canonical coding partition is the finest coding partition that partitions the set of words in at most one unambiguous class and other classes that localize the ambiguities in the factorizations of finite sequences.

We prove that the canonical coding partition of a regular set contains a finite number of regular classes and we give an algorithm for computing this partition. From this we derive a canonical decomposition of a regular monoid into a free product of finitely many regular monoids.
A HUMERAL COORDINATE SYSTEM FOR IN VIVO 3-D KINEMATICS OF THE GLENOHUMERAL JOINT
Authors: Amadi Hippolite O. , Majed Addie , Emery Roger J. H. , Bull Anthony M. J.
Published: January 2010

The aim of this study was to define axes from clearly identifiable landmarks on the proximal aspect of the humerus and to compare these for reasonable best alternatives to the use of the humeral canal and elbow epicondylar axes to define a humeral coordinate frame (HCF). The elbow epicondylar axis (EC) and six different humeral canal axes (HC) based on varying lengths of humerus were quantified from 21 computed tomography (CT) scans of humeri. Six additional axes were defined using the proximal humerus only. These included a line from the center of a sphere fit on the humeral head to the 3D surface area centroid of the greater tubercle region, (GT). The inclinations of these axes relative to EC were calculated. GT was found to be the most closely aligned to EC (13.4° ± 6.8°). The inclinations of the other axes ranged from 36.3° to 86.8°. The HC axis orientation was found to be insensitive to humeral shaft lengths (variability, within average: 0.6°). This was chosen as one of two axes for the HCF. It was also the most inter-subject related axis to EC with inclination standard deviation of ±1.8°. EC was therefore predicted from this such that if the superior axis [1 0 0] of an image scan is maintained and the humerus rotated to make its quantified HC align superiorly in the direction [0.98 0.01 0.01], then its EC axis lies laterally in the direction [0 0 1]. This study demonstrates that it is possible with confidence to apply an orthogonal coordinate frame to the humerus based on proximal imaging data only.
TIEG1-NULL OSTEOCYTES DISPLAY DEFECTS IN THEIR MORPHOLOGY, DENSITY AND SURROUNDING BONE MATRIX
Authors: Haddad Oualid , Hawse John R. , Subramaniam Malayannan , Spelsberg Thomas C. , Bensamoun Sabine F.
Published: January 2010

Through the development of TGFβ-inducible early gene-1 (TIEG1) knockout (KO) mice, we have demonstrated that TIEG1 plays an important role in osteoblast-mediated bone mineralization, and in bone resistance to mechanical strain. To further investigate the influence of TIEG1 in skeletal maintenance, osteocytes were analyzed by transmission electron microscopy using TIEG1 KO and wild-type mouse femurs at one, three and eight months of age. The results revealed an age-dependent change in osteocyte surface and density, suggesting a role for TIEG1 in osteocyte development. Moreover, there was a decrease in the amount of hypomineralized bone matrix surrounding the osteocytes in TIEG1 KO mice relative to wild-type controls. While little is known about the function or importance of this hypomineralized bone matrix immediately adjacent to osteocytes, this study reveals significant differences in this bone microenvironment and suggests that osteocyte function may be compromised in the absence of TIEG1 expression.
INVESTIGATION INTO CHANGES IN COLLAGEN STRUCTURE OF ARTICULAR CARTILAGE AND WEAR PARTICLES OF KNEE JOINTS FOR OSTEOARTHRITIC WEAR ASSESSMENT
Authors: Peng Zhongxiao , Fasiolo Mark , Hart Kane
Published: January 2010

Our bodies deteriorate from wear and tear processes, which give rise to ache and pain. This degenerative process is medically referred to as osteoarthritis (OA) and it is estimated to affect a large portion of the population at some stage in their life. There is a need for research into new and improved techniques that might be developed into a schema to aid in the early diagnosis and prognosis of a patient's condition. This can be achieved by studying the morphology of the collagen fibers in the wear particles generated to gain an insight into the osteoarthritic condition exhibited by the joint. The study has been conducted in three phases. Firstly, an animal model has been used to generate samples of cartilage and wear particles for the study. A suitable staining technique has then been developed that allows the three-dimensional visualization and quantitative analysis of the structure of the collagen matrix of sheep cartilage and in wear particles. Finally, correlation of the changes in the collagen matrix as per OA severity has been studied. The study has identified key numerical parameters to characterize distinctive wear features of the cartilage and wear debris. A good correlation of the wear features of the cartilage and wear particle samples has been found. The positive results attained by this study suggest that with the aid of further research and development, it is distinctly possible to develop improved diagnostic procedures for clinical osteoarthritic assessment.
COMPARISON OF KINEMATIC AND ELECTROMYOGRAPHIC PATTERNS DURING CYCLIC NECK MOTIONS IN ASYMPTOMATIC YOUNG AND MIDDLE-AGE ADULTS
Authors: Wang Shwu-Fen , Tang Pei-Fang , Lin Jiu-Jenq , Lin Kwan-Hwa
Published: January 2010

The purposes of this study were to investigate the activation of neck muscles during movement of the head from the neutral position to the maximum points and back to the neutral position. The experiment was designed to compare between groups. There were in total 26 subjects: 11 in the young group (mean age 24.3 years old) and 15 in the middle-age group (mean age 55.9 years old) were recruited. The active ROM, velocity, and surface EMG activities of the left and right sternocleidomastoid (SCM) muscles were recorded during repetitive cervical movements in the six directions of flexion/extension, right/left rotation and side-bending. The data were evaluated with repeated-measures analysis of variance (ANOVA). The active ROM and maximum force in both flexion and extension were significantly lower for the middle-age group than for the young group. However, the integrated EMG values for the left and right SCM muscles during movement of the head to and from the maximum points of the ROM in three cardinal planes were significantly greater in the middle-age group than in the young group. Whether these over-activation of SCM is related to degeneration of the cervical spines requires further investigation.
Essential role for STIM1/Orai1-mediated calcium influx in PDGF-induced smooth muscle migration
Authors: Bisaillon Jonathan M. , Motiani Rajender K. , Gonzalez-Cobos José C. , Potier Marie , Halligan Katharine E. , Alzawahra Wael F. , Barroso Margarida , Singer Harold A. , Jourd'heuil David , Trebak Mohamed
Published: January 2010

We recently demonstrated that thapsigargin-induced passive store depletion activates Ca2+ entry in vascular smooth muscle cells (VSMC) through stromal interaction molecule 1 (STIM1)/Orai1, independently of transient receptor potential canonical (TRPC) channels. However, under physiological stimulations, despite the ubiquitous depletion of inositol 1,4,5-trisphosphate-sensitive stores, many VSMC PLC-coupled agonists (e.g., vasopressin and endothelin) activate various store-independent Ca2+ entry channels. Platelet-derived growth factor (PDGF) is an important VSMC promigratory agonist with an established role in vascular disease. Nevertheless, the molecular identity of the Ca2+ channels activated by PDGF in VSMC remains unknown. Here we show that inhibitors of store-operated Ca2+ entry (Gd3+ and 2-aminoethoxydiphenyl borate at concentrations as low as 5 μM) prevent PDGF-mediated Ca2+ entry in cultured rat aortic VSMC. Protein knockdown of STIM1, Orai1, and PDGF receptor-β (PDGFRβ) impaired PDGF-mediated Ca2+ influx, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Scratch wound assay showed that knockdown of STIM1, Orai1, or PDGFRβ inhibited PDGF-mediated VSMC migration, but knockdown of STIM2, Orai2, and Orai3 was without effect. STIM1, Orai1, and PDGFRβ mRNA levels were upregulated in vivo in VSMC from balloon-injured rat carotid arteries compared with noninjured control vessels. Protein levels of STIM1 and Orai1 were also upregulated in medial and neointimal VSMC from injured carotid arteries compared with noninjured vessels, as assessed by immunofluorescence microscopy. These results establish that STIM1 and Orai1 are important components for PDGF-mediated Ca2+ entry and migration in VSMC and are upregulated in vivo during vascular injury and provide insights linking PDGF to STIM1/Orai1 during neointima formation.
Early transcription from the maternal genome controlling blastomere integrity in mouse two-cell-stage embryos
Authors: Han Zhiming , Mtango Namdori R. , Zhong Zhisheng , Vassena Rita , Latham Keith E.
Published: January 2010

Blastomere cytofragmentation in mammalian embryos poses a significant problem in applied and clinical embryology. Mouse two-cell-stage embryos display strain-dependent differences in the rate of cytofragmentation, with a high rate observed in C3H/HeJ embryos and a lower rate observed in C57BL/6 embryos. The maternally inherited genome exerts the strongest effect on the process, with lesser effects mediated by the paternally inherited genome and the ooplasm. The effect of the maternal genome is transcription dependent and independent of the mitochondrial strain of origin. To identify molecular mechanisms that underlie cytofragmentation, we evaluated transcriptional activities of embryos possessing maternal pronuclei (mPN) of different origins. The mPN from C57BL/6 and C3H/HeJ strains directed specific transcription at the two-cell stage of mRNAs corresponding to 935 and 864 Affymetrix probe set IDs, respectively. Comparing transcriptomes of two-cell-stage embryos with different mPN revealed 64 transcribed genes with differential expression (1.4-fold or greater). Some of these genes occupy molecular pathways that may regulate cytofragmentation via a combination of effects related to apoptosis and effects on the cytoskeleton. These results implicate specific molecular mechanisms that may regulate cytofragmentation in early mammalian embryos. The most striking effect of mPN strain of origin on gene expression was on adenylate cyclase 2 ( Adcy2). Treatment with dibutyryl cAMP (dbcAMP) elicits a high rate and severe form of cytofragmentation, and the effective dbcAMP concentration varies with maternal genotype. An activator of exchange proteins directly activated by cAMP (EPACs, or RAPGEF 3 and 4) 8-pCPT-2′- O-methyl-cAMP, elicits a high level of fragmentation while the PKA-specific activator N6-benzoyl-cAMP does not. Inhibition of A kinase anchor protein activities with st-Ht31 induces fragmentation. Inhibition of phosphatidylinositol 3-kinase signaling also induces fragmentation. These results reveal novel mechanisms by which maternal genotype affects cytofragmentation, including a system of opposing signaling pathways that most likely operate by controlling cytoskeletal function.
ADP-ribosylation factor 6 modulates adrenergic stimulated lipolysis in adipocytes
Authors: Liu Yingqiu , Zhou Dequan , Abumrad Nada A. , Su Xiong
Published: January 2010

ADP-ribosylation factor 6 (Arf6) is a small GTPase that influences membrane receptor trafficking and the actin cytoskeleton. In adipocytes, Arf6 regulates the trafficking of the glucose transporter type 4 (GLUT4) and consequently insulin-stimulated glucose transport. Previous studies also indicated a role of Arf6 in adrenergic receptor trafficking, but whether this contributes to the control of lipolysis in adipocytes remains unknown. This was examined in the present study by using RNA interference (RNAi) and pharmaceutical inhibition in murine cultured 3T3-L1 adipocytes. Downregulation of Arf6 by RNAi impairs isoproterenol-stimulated lipolysis specifically but does not alter triacylglycerol (TAG) synthesis or the insulin signaling pathway. Neither total TAG amounts nor TAG fatty acid compositions are altered. The inhibitory effect on lipolysis is mimicked by dynasore, a specific inhibitor for dynamin, which is required for endocytosis. In contrast, lipolysis triggered by reagents that bypass events at the plasma membrane (e.g., forskolin, isobutylmethylxanthine or 8-bromo-cAMP) is not affected. Moreover, Arf6 protein levels in white adipose tissues are markedly increased in ob/ob mice, whereas they are decreased in obesity-resistant CD36 null mice. These changes reflect at least in part alterations in Arf6 mRNA levels. Collectively, these results suggest a role of the endocytic pathway and its regulation by Arf6 in adrenergic stimulation of lipolysis in adipocytes and potentially in the development of obesity.
Biogenesis of the mitochondrial Tom40 channel in skeletal muscle from aged animals and its adaptability to chronic contractile activity
Authors: Joseph Anna-Maria , Ljubicic Vladimir , Adhihetty Peter J. , Hood David A.
Published: January 2010

Evidence exists that mitochondrial content and/or function is reduced in muscle of aging individuals. The purposes of this study were to investigate the contribution of outer membrane protein import and assembly processes to this decline and to determine whether the assembly process could adapt to chronic contractile activity (CCA). Tom40 assembly into the translocases of the outer membrane (TOM complex) was measured in subsarcolemmal mitochondria obtained from young (6 mo old) and aged (36 mo old) Fischer 344 × Brown Norway animals. While the initial import of Tom40 did not differ between young and aged animals, its subsequent assembly into the final ∼380 kDa complex was 2.2-fold higher ( P < 0.05) in mitochondria from aged compared with young animals. This was associated with a higher abundance of Tom22, a protein vital for the assembly process. CCA induced a greater initial import and subsequent assembly of Tom40 in mitochondria from young animals, resulting in a CCA-induced 75% increase ( P < 0.05) in Tom40 within mitochondria. This effect of CCA was attenuated in mitochondria from old animals. These data suggest that the import and assembly of proteins into the outer membrane do not contribute to reduced mitochondrial content or function in aged animals. Indeed, the greater assembly rate in mitochondria from aged animals may be a compensatory mechanism attempting to offset any decrements in mitochondrial content or function within aged muscle. Our data also indicate the potential of CCA to contribute to increased mitochondrial biogenesis in muscle through changes in the outer membrane import and assembly pathway.