bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of revertant fibres in the mdx mouse using antisense oligonucleotides
Authors: Fall Abbie M , Johnsen Russell , Honeyman Kaite , Iversen Pat , Fletcher Susan , Wilton Stephen D
Published: May 2006

Background

Duchenne muscular dystrophy is a fatal genetic disorder caused by dystrophin gene mutations that result in premature termination of translation and the absence of functional protein. Despite the primary dystrophin gene lesion, immunostaining studies have shown that at least 50% of DMD patients, mdx mice and a canine model of DMD have rare dystrophin-positive or 'revertant' fibres. Fine epitope mapping has shown that the majority of transcripts responsible for revertant fibres exclude multiple exons, one of which includes the dystrophin mutation.

Methods

The mdx mouse model of muscular dystrophy has a nonsense mutation in exon 23 of the dystrophin gene. We have shown that antisense oligonucleotides (AOs) can induce the removal of this exon, resulting in an in-frame mRNA transcript encoding a shortened but functional dystrophin protein. To emulate one exonic combination associated with revertant fibres, we target multiple exons for removal by the application of a group of AOs combined as a "cocktail".

Results

Exons 19–25 were consistently excluded from the dystrophin gene transcript using a cocktail of AOs. This corresponds to an alternatively processed gene transcript that has been sporadically detected in untreated dystrophic mouse muscle, and is presumed to give rise to a revertant dystrophin isoform. The transcript and the resultant correctly localised smaller protein were confirmed by RT-PCR, immunohistochemistry and western blot analysis.

Conclusion

This work demonstrates the feasibility of AO cocktails to by-pass dystrophin mutation hotspots through multi-exon skipping. Multi-exon skipping could be important in expediting an exon skipping therapy to treat DMD, so that the same AO formulations may be applied to several different mutations within particular domains of the dystrophin gene.
Induction of antitumor immunity through xenoplacental immunization
Authors: Zhong Zhaohui , Kusznieruk Kornel P , Popov Igor A , Riordan Neil H , Izadi Hamid , Yijian Li , Sher Salman , Szczurko Orest M , Agadjanyan Michael G , Tullis Richard H , Harandi Amir , Reznik Boris N , Mamikonyan Grigor V , Ichim Thomas E
Published: May 2006

Historically cancer vaccines have yielded suboptimal clinical results. We have developed a novel strategy for eliciting antitumor immunity based upon homology between neoplastic tissue and the developing placenta. Placenta formation shares several key processes with neoplasia, namely: angiogenesis, activation of matrix metalloproteases, and active suppression of immune function. Immune responses against xenoantigens are well known to break self-tolerance. Utilizing xenogeneic placental protein extracts as a vaccine, we have successfully induced anti-tumor immunity against B16 melanoma in C57/BL6 mice, whereas control xenogeneic extracts and B16 tumor extracts where ineffective, or actually promoted tumor growth, respectively. Furthermore, dendritic cells were able to prime tumor immunity when pulsed with the placental xenoantigens. While vaccination-induced tumor regression was abolished in mice depleted of CD4 T cells, both CD4 and CD8 cells were needed to adoptively transfer immunity to naïve mice. Supporting the role of CD8 cells in controlling tumor growth are findings that only freshly isolated CD8 cells from immunized mice were capable of inducing tumor cell caspases-3 activation ex vivo. These data suggest feasibility of using xenogeneic placental preparations as a multivalent vaccine potently targeting not just tumor antigens, but processes that are essential for tumor maintenance of malignant potential.
Diesel exhaust particles increase IL-1β-induced human β-defensin expression via NF-κB-mediated pathway in human lung epithelial cells
Authors: Nam Hae Yun , Ahn Eun-Kyung , Kim Hyung Jung , Lim Young , Lee Chun Beoun , Lee Kyo Young , Vallyathan Val
Published: May 2006

Background

Human β-defensin (hBD)-2, antimicrobial peptide primarily induced in epithelial cells, is a key factor in the innate immune response of the respiratory tract. Several studies showed increased defensin levels in both inflammatory lung diseases, such as cystic fibrosis, diffuse panbronchiolitis, idiopathic pulmonary fibrosis and acute respiratory distress syndrome, and infectious diseases. Recently, epidemiologic studies have demonstrated acute and serious adverse effects of particulate air pollution on respiratory health, especially in people with pre-existing inflammatory lung disease. To elucidate the effect of diesel exhaust particles (DEP) on pulmonary innate immune response, we investigated the hBD-2 and interleukin-8 (IL-8) expression to DEP exposure in interleukin-1 beta (IL-1β)-stimulated A549 cells.

Results

IL-1β markedly up-regulated the hBD-2 promoter activity, and the subsequent DEP exposure increased dose-dependently the expression of hBD-2 and inflammatory cytokine IL-8 at the transcriptional level. In addition, DEP further induced the NF-κB activation in IL-1β-stimulated A549 cells more rapidly than in unstimulated control cells, which was showed by nuclear translocation of p65 NF-κB and degradation of IκB-α. The experiment using two NF-κB inhibitors, PDTC and MG132, confirmed that this increase of hBD-2 expression following DEP exposure was regulated through NF-κB-mediated pathway.

Conclusion

These results demonstrated that DEP exposure increases the expression of antimicrobial peptide and inflammatory cytokine at the transcriptional level in IL-1β-primed A549 epithelial cells and suggested that the increase is mediated at least partially through NF-κB activation. Therefore, DEP exposure may contribute to enhance the airway-responsiveness especially on the patients suffering from chronic respiratory disease.
Three minimum tile paths from bacterial artificial chromosome libraries of the soybean (Glycine max cv. 'Forrest'): tools for structural and functional genomics
Authors: Shultz JL , Yesudas C , Yaegashi S , Afzal AJ , Kazi S , Lightfoot DA
Published: May 2006

Background

The creation of minimally redundant tile paths (hereafter MTP) from contiguous sets of overlapping clones (hereafter contigs) in physical maps is a critical step for structural and functional genomics. Build 4 of the physical map of soybean (Glycine max L. Merr. cv. 'Forrest') showed the 1 Gbp haploid genome was composed of 0.7 Gbp diploid, 0.1 Gbp tetraploid and 0.2 Gbp octoploid regions. Therefore, the size of the unique genome was about 0.8 Gbp. The aim here was to create MTP sub-libraries from the soybean cv. Forrest physical map builds 2 to 4.

Results

The first MTP, named MTP2, was 14,208 clones (of mean insert size 140 kbp) picked from the 5,597 contigs of build 2. MTP2 was constructed from three BAC libraries (Bam HI (B), Hin dIII (H) and Eco RI (E) inserts). MTP2 encompassed the contigs of build 3 that derived from build 2 by a series of contig merges. MTP2 encompassed 2 Gbp compared to the soybean haploid genome of 1 Gbp and does not distinguish regions by ploidy. The second and third MTPs, called MTP4BH and MTP4E, were each based on build 4. Each was semi-automatically selected from 2,854 contigs. MTP4BH was 4,608 B and H insert clones of mean size 173 kbp in the large (27.6 kbp) T-DNA vector pCLD04541. MTP4BH was suitable for plant transformation and functional genomics. MTP4E was 4,608 BAC clones with large inserts (mean 175 kbp) in the small (7.5 kbp) pECBAC1 vector. MTP4E was suitable for DNA sequencing. MTP4BH and MTP4E clones each encompassed about 0.8 Gbp, the 0.7 Gbp diploid regions and 0.05 Gbp each from the tetraploid and octoploid regions. MTP2 and MTP4BH were used for BAC-end sequencing, EST integration, micro-satellite integration into the physical map and high information content fingerprinting. MTP4E will be used for genome sequence by pooled genomic clone index.

Conclusion

Each MTP and associated BES will be useful to deconvolute and ultimately finish the whole genome shotgun sequence of soybean.
The short coiled-coil domain-containing protein UNC-69 cooperates with UNC-76 to regulate axonal outgrowth and normal presynaptic organization in Caenorhabditis elegans
Authors: Su Cheng-Wen , Tharin Suzanne , Jin Yishi , Wightman Bruce , Spector Mona , Meili David , Tsung Nancy , Rhiner Christa , Bourikas Dimitris , Stoeckli Esther , Garriga Gian , Horvitz H Robert , Hengartner Michael O
Published: May 2006

Background

The nematode Caenorhabditis elegans has been used extensively to identify the genetic requirements for proper nervous system development and function. Key to this process is the direction of vesicles to the growing axons and dendrites, which is required for growth-cone extension and synapse formation in the developing neurons. The contribution and mechanism of membrane traffic in neuronal development are not fully understood, however.

Results

We show that the C. elegans gene unc-69 is required for axon outgrowth, guidance, fasciculation and normal presynaptic organization. We identify UNC-69 as an evolutionarily conserved 108-amino-acid protein with a short coiled-coil domain. UNC-69 interacts physically with UNC-76, mutations in which produce similar defects to loss of unc-69 function. In addition, a weak reduction-of-function allele, unc-69(ju69), preferentially causes mislocalization of the synaptic vesicle marker synaptobrevin. UNC-69 and UNC-76 colocalize as puncta in neuronal processes and cooperate to regulate axon extension and synapse formation. The chicken UNC-69 homolog is highly expressed in the developing central nervous system, and its inactivation by RNA interference leads to axon guidance defects.

Conclusion

We have identified a novel protein complex, composed of UNC-69 and UNC-76, which promotes axonal growth and normal presynaptic organization in C. elegans. As both proteins are conserved through evolution, we suggest that the mammalian homologs of UNC-69 and UNC-76 (SCOCO and FEZ, respectively) may function similarly.
HIV and AIDS Stigma Violates Human Rights in Five African Countries
Authors: Kohi Thecla W , Makoae Lucy , Chirwa Maureen , Holzemer William L , RenéPhetlhu Deliwe , Uys Leana , Naidoo Joanne , Dlamini Priscilla S , Greeff Minrie
Published: May 2006

The situation and human rights of people living with HIV and AIDS were explored through focus groups in five African countries (Lesotho, Malawi, South Africa, Swaziland and Tanzania). A descriptive qualitative research design was used. The 251 informants were people living with HIV and AIDS, and nurse managers and nurse clinicians from urban and rural settings. NVivo™ software was used to identify specific incidents related to human rights, which were compared with the Universal Declaration of Human Rights. The findings revealed that the human rights of people living with HIV and AIDS were violated in a variety of ways, including denial of access to adequate or no health care/services, and denial of home care, termination or refusal of employment, and denial of the right to earn an income, produce food or obtain loans. The informants living with HIV and AIDS were also abused verbally and physically. Country governments and health professionals need to address these issues to ensure the human rights of all people.
Whistleblowing and Organizational Ethics
Authors: Ray Susan L
Published: May 2006

The purpose of this article is to discuss an external whistleblowing event that occurred after all internal whistleblowing through the hierarchy of the organization had failed. It is argued that an organization that does not support those that whistle blow because of violation of professional standards is indicative of a failure of organizational ethics. Several ways to build an ethics infrastructure that could reduce the need to resort to external whistleblowing are discussed. A relational ethics approach is presented as a way to eliminate the negative consequences of whistleblowing by fostering an interdependent moral community to address ethical concerns.
Chairperson and Faculty Gender in Academic Emergency Medicine Departments
Authors: Cheng David , Promes Susan , Clem Kathleen , Shah Anand , Pietrobon Ricardo
Published: May 2006

Objectives:
Despite the influx of female physicians in academic medicine departments, there are a small number of women in faculty and departmental leadership positions in emergency medicine (EM). The objective of this study was to determine if the gender of the chairperson of an academic EM department is associated with the gender of the residency program director (RPD) and gender proportion of its faculty.

Methods:
This was a retrospective analysis of 133 academic EM departments using the Society for Academic Emergency Medicine online residency catalog, program Web site, or e‐mail. Main outcome measures were proportion of female EM faculty and gender of the RPD.

Results:
Data were available for 133 academic departments. Women chaired 7.5% (n= 10) of departments and comprised 22.3% of all faculty and 15.0% (n= 20) of RPD positions. EM departments that were chaired by women had a significantly higher percentage of female faculty compared with those led by men (31% vs. 22%; p = 0.01). Similarly, departments that were chaired by women had a significantly higher proportion of female RPDs compared with those chaired by men (50% vs. 12%; p < 0.01). Compared with departments chaired by men, the RPD was 5.0 times (95% confidence interval = 1.9 to 27.8; p < 0.01) more likely to be a woman if the chairperson was also a woman.

Conclusions:
An academic EM department was more likely to have a higher proportion of female faculty and a female RPD when the department chairperson was female.
Myosin light chain kinase plays a role in the regulation of epithelial cell survival
Authors: Connell Laureen E. , Helfman David M.
Published: May 2006

Myosin II activation is essential for stress fiber and focal adhesion formation, and is implicated in integrin-mediated signaling events. In this study we investigated the role of acto-myosin contractility, and its main regulators, i.e. myosin light chain kinase (MLCK) and Rho-kinase (ROCK) in cell survival in normal and Ras-transformed MCF-10A epithelial cells. Treatment of cells with pharmacological inhibitors of MLCK (ML-7 and ML-9), or expression of dominant-negative MLCK, led to apoptosis in normal and transformed MCF-10A cells. By contrast, treatment of cells with a ROCK inhibitor (Y-27632) did not induce apoptosis in these cells. Apoptosis following inhibition of myosin II activation by MLCK is probably meditated through the death receptor pathway because expression of dominant-negative FADD blocked apoptosis. The apoptosis observed after MLCK inhibition is rescued by pre-treatment of cells with integrin-activating antibodies. In addition, this rescue of apoptosis is dependent on FAK activity, suggesting the participation of an integrin-dependent signaling pathway. These studies demonstrate a newly discovered role for MLCK in the generation of pro-survival signals in both untransformed and transformed epithelial cells and supports previous work suggesting distinct cellular roles for Rho-kinase- and MLCK-dependent regulation of myosin II.
Ryanodine receptor interaction with the SNARE-associated protein snapin
Authors: Zissimopoulos Spyros , West Duncan J. , Williams Alan J. , Lai F. Anthony
Published: May 2006

The ryanodine receptor (RyR) is a widely expressed intracellular calcium (Ca2+)-release channel regulating processes such as muscle contraction and neurotransmission. Snapin, a ubiquitously expressed SNARE-associated protein, has been implicated in neurotransmission. Here, we report the identification of snapin as a novel RyR2-interacting protein. Snapin binds to a 170-residue predicted ryanodine receptor cytosolic loop (RyR2 residues 4596-4765), containing a hydrophobic segment required for snapin interaction. Ryanodine receptor binding of snapin is not isoform specific and is conserved in homologous RyR1 and RyR3 fragments. Consistent with peptide fragment studies, snapin interacts with the native ryanodine receptor from skeletal muscle, heart and brain. The snapin-RyR1 association appears to sensitise the channel to Ca2+ activation in [3H]ryanodine-binding studies. Deletion analysis indicates that the ryanodine receptor interacts with the snapin C-terminus, the same region as the SNAP25-binding site. Competition experiments with native ryanodine receptor and SNAP25 suggest that these two proteins share an overlapping binding site on snapin. Thus, regulation of the association between ryanodine receptor and snapin might constitute part of the elusive molecular mechanism by which ryanodine-sensitive Ca2+ stores modulate neurosecretion.
Caveolin-1 is required for signaling and membrane targeting of EphB1 receptor tyrosine kinase
Authors: Vihanto Meri M. , Vindis Cecile , Djonov Valentin , Cerretti Douglas P. , Huynh-Do Uyen
Published: May 2006

Eph receptor tyrosine kinases are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Caveolae are flask-shaped invaginations of the cell membrane; their major structural protein, caveolin-1, has been shown to regulate signaling molecules localized in these micro-domains. The interaction of caveolin-1 with several of these proteins is mediated by the binding of its scaffolding domain to a region containing hydrophobic amino acids within these proteins. The presence of such a motif within the EphB1 kinase domain prompted us to investigate the caveolar localization and regulation of EphB1 by caveolin-1. We report that EphB1 receptors are localized in caveolae, and directly interact with caveolin-1 upon ligand stimulation. This interaction, as well as EphB1-mediated activation of extracellular-signal-regulated kinase (ERK), was abrogated by overexpression of a caveolin-1 mutant lacking a functional scaffolding domain. Interaction between Ephs and caveolin-1 is not restricted to the B-subclass of receptors, since we show that EphA2 also interacts with caveolin-1. Furthermore, we demonstrate that the caveolin-binding motif within the kinase domain of EphB1 is primordial for its correct membrane targeting. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of EphB1.
Basal body and flagellum mutants reveal a rotational constraint of the central pair microtubules in the axonemes of trypanosomes
Authors: Gadelha C. , Wickstead B. , McKean P. G. , Gull K.
Published: May 2006

Productive beating of eukaryotic flagella and cilia requires a strict regulation of axonemal dynein activation. Fundamental to any description of axonemal beating is an understanding of the significance of the central pair microtubules and the degree to which central pair rotation has a role. However, for the majority of organisms, it is unclear whether the central pair actually rotates. Using an extra-axonemal structure as a fixed reference, we analysed the orientation of the central pair in African trypanosomes and other kinetoplastid protozoa. A geometric correction allowed the superposition of data from many cross-sections, demonstrating that the axis of the central pair is invariant and that there is no central pair rotation in these organisms. Analysis of mutants depleted in particular flagellar and basal body proteins [γ-tubulin, δ-tubulin, Parkin co-regulated gene product (PACRG) or the paraflagellar rod protein PFR2] allowed a dissection of the mechanisms for central pair constraint. This demonstrated that orientation is independent of flagellum attachment and beating, but is influenced by constraints along its length and is entirely dependent on correct positioning at the basal plate.
Syndecan-1 regulates αvβ5 integrin activity in B82L fibroblasts
Authors: McQuade K. J. , Beauvais D. M. , Burbach B. J. , Rapraeger A. C.
Published: May 2006

B82L mouse fibroblasts respond to fibronectin or vitronectin via a syndecan-1-mediated activation of the αvβ5 integrin. Cells attached to syndecan-1-specific antibody display only filopodial extension. However, the syndecan-anchored cells extend lamellipodia when the antibody-substratum is supplemented with serum, or low concentrations of adsorbed vitronectin or fibronectin, that are not sufficient to activate the integrin when plated alone. Integrin activation is blocked by treatment with (Arg-Gly-Asp)-containing peptides and function-blocking antibodies that target αv integrins, as well as by siRNA-mediated silencing of β5 integrin expression. In addition, αvβ5-mediated cell attachment and spreading on high concentrations of vitronectin is blocked by competition with recombinant syndecan-1 ectodomain core protein and by downregulation of mouse syndecan-1 expression by mouse-specific siRNA. Taking advantage of the species-specificity of the siRNA, rescue experiments in which human syndecan-1 constructs are expressed trace the activation site to the syndecan-1 ectodomain. Moreover, both full-length mouse and human syndecan-1 co-immunoprecipitate with the β5 integrin subunit, but fail to do so if the syndecan is displaced by competition with soluble, recombinant syndecan-1 ectodomain. These results suggest that the ectodomain of the syndecan-1 core protein contains an active site that assembles into a complex with the αvβ5 integrin and regulates αvβ5 integrin activity.
Wingless signaling modulates cadherin-mediated cell adhesion inDrosophilaimaginal disc cells
Authors: Wodarz A. , Stewart D. B. , Nelson W. J. , Nusse R.
Published: May 2006

Armadillo, the Drosophila homolog of β-catenin, plays a crucial role in both the Wingless signal transduction pathway and cadherin-mediated cell-cell adhesion, raising the possibility that Wg signaling affects cell adhesion. Here, we use a tissue culture system that allows conditional activation of the Wingless signaling pathway and modulation of E-cadherin expression levels. We show that activation of the Wingless signaling pathway leads to the accumulation of hypophosphorylated Armadillo in the cytoplasm and in cellular processes, and to a concomitant reduction of membrane-associated Armadillo. Activation of the Wingless pathway causes a loss of E-cadherin from the cell surface, reduced cell adhesion and increased spreading of the cells on the substratum. After the initial loss of E-cadherin from the cell surface, E-cadherin gene expression is increased by Wingless. We suggest that Wingless signaling causes changes in Armadillo levels and subcellular localization that result in a transient reduction of cadherin-mediated cell adhesion, thus facilitating cell shape changes, division and movement of cells in epithelial tissues.
Flat clathrin coats on endosomes mediate degradative protein sorting by scaffolding Hrs in dynamic microdomains
Authors: Raiborg C. , Wesche J. , Malerød L. , Stenmark H.
Published: May 2006

Endocytosed membrane proteins that are destined for degradation in lysosomes are ubiquitylated and recognised by sorting complexes on endosome membranes. The ubiquitin-binding sorting component Hrs as well as ubiquitylated cargo are enriched in a characteristic flat clathrin coat on the endosome membrane. The function of clathrin within this coat has not been investigated. Here, we show that both clathrin and the clathrin-box motif of Hrs are required for the clustering of Hrs into restricted microdomains. The C-terminus of Hrs, which contains the clathrin-box, is sufficient to redirect a phosphatidylinositol(3)-phosphate-binding protein into the Hrs- and clathrin-containing microdomains. Although these microdomains show little lateral diffusion in the membrane, they are dynamic structures that exchange Hrs and clathrin with similar kinetics, and acquire the downstream sorting component Tsg101. The clathrin-mediated clustering is essential for the function of Hrs in degradative protein sorting. We conclude that clathrin is responsible for concentrating Hrs in endosomal microdomains specialised for recognition of ubiquitylated membrane proteins, thus enabling efficient sorting of cargo into the degradative pathway.
Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion molecular systems
Authors: Sonnet C. , Lafuste P. , Arnold L. , Brigitte M. , Poron F. , Authier F. , Chrétien F. , Gherardi R. K. , Chazaud B.
Published: May 2006

The mechanisms underlying stromal cell supportive functions are incompletely understood but probably implicate a mixture of cytokines, matrix components and cell adhesion molecules. Skeletal muscle uses recruited macrophages to support post-injury regeneration. We and others have previously shown that macrophages secrete mitogenic factors for myogenic cells. Here, we focused on macrophage-elicited survival signals. We demonstrated that: (1) macrophage influx is temporally correlated with the disappearance of TUNEL-positive apoptotic myogenic cells during post-injury muscle regeneration in mice; (2) direct cell-cell contacts between human macrophages and myogenic cells rescue myogenic cells from apoptosis, as assessed by decreased annexin V labelling and caspase-3 activity, and by increased DIOC-6 staining, Bcl-2 expression and phosphorylation of Akt and ERK1/2 survival pathways; (3) four pro-survival cell-cell adhesion molecular systems detected by DNA macroarray are expressed by macrophages and myogenic cells in vitro and in vivo - VCAM-1-VLA-4, ICAM-1-LFA-1, PECAM-1-PECAM-1 and CX3CL1-CX3CR1; (4) macrophages deliver anti-apoptotic signals through all four adhesion systems, as assessed by functional analyses with blocking antibodies; and (5) macrophages more strongly rescue differentiated myotubes, which must achieve adhesion-induced stabilisation of their structure to survive. Macrophages could secure these cells until they establish final association with the matrix.
Pyrene-modified DNA Aptamer as a Fluorescent Biosensor with High Affinity and Specificity for ATP Sensing
Authors: Kamekawa Nobuyuki , Shimomura Yukinori , Nakamura Mitsunobu , Yamana Kazushige
Published: May 2006

The fluorescence properties of anti-ATP apatmers labeled with a pyrenylmethyl group at the 2′-positin were investigated. The labeling of the nucleoside residue at the stem region adjacent to the ligand-binding pocket gave a fluorescent biosensor having high affinity yet retaining its specificity for ATP.
Oxygen Atom Transfer Catalytic Property of Oxorhenium(V) Complex with 2-Methylquinolin-8-ylamide and Tetrachlorocatecholate
Authors: Ohashi Tetuya , Miyashita Yoshitaro , Fujisawa Kiyoshi , Okamoto Ken-ichi
Published: May 2006

New oxorhenium(V) complex was prepared and characterized by an X-ray crystal analysis. The complex showed characteristic releasing property of monodentate ligand and high oxygen atom transfer catalytic activity from 2,6-lutidine-N-oxide to PPh3.
Dye Sensitization of ZnO by Unsymmetrical Squaraine Dyes Suppressing Aggregation
Authors: Otsuka Atsuhiro , Funabiki Kazumasa , Sugiyama Naoyuki , Yoshida Tsukasa , Minoura Hideki , Matsui Masaki
Published: May 2006

We have developed an efficient, cyan-colored, and novel squaraine-dye-sensitized ZnO solar cell, producing a maximum incident photon-to-electron conversion efficiency (IPCE) of 59% at 630 nm. The solar cell sensitized by Sq 3 having bulky groups to prevent aggregation of dyes has achieved a 1.5% solar-to-electricity conversion efficiency (η) with a short-circuit current density of 4.04 mA cm−2, an open-circuit photovoltage of 0.61 V, and a fill factor of 0.61 under standard AM 1.5 irradiation (100 mW cm−2).
Determination of N-Desmethyl- and N-Bisdesmethyl Metabolites of Sibutramine in Doping Control Analysis Using Liquid Chromatography-Tandem Mass Spectrometry
Authors: Thevis Mario , Sigmund Gerd , Schiffer Anna-Katharina , Schänzer Wilhelm
Published: May 2006

Since January 2006, the list of prohibited substances established by the World Anti-Doping Agency includes the antidepressant/anti-obesity drug Sibutramine. Due to its rapid degradation to its active metabolites N-desmethyl and N-bisdesmethyl sibutramine, reference compounds were synthesized and included into an existing screening assay to allow the unambiguous determination of these metabolic products in human urine using liquid–liquid extraction followed by liquid chromatography/tandem mass spectrometry. Characteristic product ions, obtained after electrospray ionization and collision-induced dissociation, were elucidated using high resolution/high accuracy mass measurements with a hybrid linear ion trap/orbitrap mass analyzer. Based on diagnostic product ions, the extended screening procedure was validated for both sibutramine metabolites using a triple quadrupole mass spectrometer. Items such as lower limits of detection (6–40 ng mL−1), recoveries (39–42%), intraday precision (low: 5.5–10.6%, medium: 4.9–5.9%), high: 12.8–16.4%) and interday precision (low: 15.0–22.8%, medium: 17.7–18.6%), high: 16.5–25.6%) were evaluated and a clinical spot urine sample was analyzed to demonstrate the applicability of the developed assay in sports drug testing.
Differential association of protein subunits with the human RNase MRP and RNase P complexes
Authors: Welting Tim J.M. , Kikkert Bastiaan J. , van Venrooij Walther J. , Pruijn Ger J.M.
Published: May 2006

RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60–80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60–80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.
'Dual operating' – an old innovation
Authors: Petri GJ , Banerjee TS
Published: May 2006

Surgical waiting lists have been a feature of the NHS landscape for so long that British patients and health professionals have grown accustomed to their existence and often accept them as inevitable. They are usually explained by 'lack of resources' or, more specifically, by 'lack of surgeons'. It is well known that for many years the UK has lagged behind other rich European nations in the proportion of the gross domestic product spent on public health and it is often assumed that underfunding alone is responsible for waiting lists. This may not be entirely correct.
The 12th annual conference of Women in Surgical Training
Authors: Wynn-Mackenzie Clare
Published: May 2006

Getting to the Top and Staying There was the theme of the 12th annual conference of Women in Surgical Training (WIST) on 27 March 2006 and this topic was certainly reflected in the experiences of the 160 delegates, who ranged from medical students to retired consultant surgeons.
The eagle project takes off
Authors: Thomas Bill
Published: May 2006

Surgical education is absolutely crucial for the future of surgery as a profession and for the advancement of patient safety and care. There are multiple challenges currently facing surgical training and education, such as the European Working Time Directive, Modernising Medical Careers and service and activity targets, but just as important is the need for training and education to keep pace with the enormous strides in technology and medical innovation.
Increased Integrity of Free Circulating DNA in Sera of Patients with Colorectal or Periampullary Cancer: Direct Quantitative PCR for ALU Repeats
Authors: Umetani Naoyuki , Kim Joseph , Hiramatsu Suzanne , Reber Howard A , Hines Oscar J , Bilchik Anton J , Hoon Dave S B
Published: May 2006

Background: Cell-free DNA circulating in blood is a candidate biomarker for malignant tumors. Unlike uniformly truncated DNA released from apoptotic nondiseased cells, DNA released from dead cancer cells varies in size. We developed a novel method to measure the ratio of longer to shorter DNA fragments (DNA integrity) in serum as a potential biomarker for patients with colorectal cancer (CRC) or periampullary cancers (PACs).

Methods: Sera from 32 patients with CRC (3 stage I, 14 stage II, 6 stage III, and 9 stage IV patients), 19 patients with PACs (2 stage I, 9 stage II, 1 stage III, and 7 stage IV patients), and 51 healthy volunteers were assessed by quantitative real-time PCR of ALU repeats (ALU-qPCR) with 2 sets of primers (115 and 247 bp) amplifying different lengths of DNA. We used serum directly as a template for ALU-qPCR without DNA purification. DNA integrity was determined as ratio of qPCR results of 247-bp ALU over 115-bp ALU.

Results: ALU-qPCR had a detection limit of 0.01 pg of DNA. Eliminating DNA purification reduced technical artifacts and reagent/labor costs. Serum DNA integrity was significantly increased for stage I/II and III/IV CRC and stage I/II and III/IV PACs (P = 0.002, P = 0.006, P = 0.022, and P <0.0001, respectively). ROC curves for detecting CRC and PACs had areas under the curves of 0.78 and 0.80, respectively.

Conclusions: Direct ALU-qPCR is a robust, highly sensitive, and high-throughput method to measure serum DNA integrity. DNA integrity is a potential serum biomarker for detection and evaluation of CRC and PACs.
The Medial Temporal Lobe Distinguishes Old from New Independently of Consciousness
Authors: Daselaar Sander M. , Fleck Mathias S. , Prince Steven E. , Cabeza Roberto
Published: May 2006

Although it is widely accepted that the medial temporal lobes (MTLs) are critical for becoming aware that something happened in the past, there is virtually no evidence whether MTL sensitivity to event oldness also depends on conscious awareness. Using event-related functional magnetic resonance imaging, we show that activity in posterior MTL tracks whether an item is actually old (true oldness), regardless of participants' awareness of oldness (perceived oldness). Confirming its sensitivity to the objective nature of the stimulus, activity in this region was strongly correlated with individual memory performance (r= 0.74). At the same time, we found that memory errors (misses) were associated with activity in an anterior MTL region, which signaled whether an item was consciously experienced as new (perceived novelty). Logistic regression analyses based on individual trial activity indicated that the two MTL regions showed opposing relationships with behavior, and that memory performance was determined by their joint activity. Furthermore, functional connectivity analyses showed that perceived novelty activity in the posterior MTL inhibited true oldness activity in the anterior MTL. These findings indicate that participants' behavior reflected the combined effects of multiple MTL regions. More generally, our results show that parts of MTL can distinguish old from new independently of consciousness.
Precise Long-Range Synchronization of Activity and Silence in Neocortical Neurons during Slow-Wave Sleep
Authors: Volgushev Maxim , Chauvette Sylvain , Mukovski Mikhail , Timofeev Igor
Published: May 2006

Slow-wave sleep is characterized by alternating periods of activity and silence in corticothalamic networks. Both activity and silence are stable network states, but the mechanisms of their alternation remain unknown. We show, using simultaneous multisite intracellular recordings in cats, that slow rhythm involves all neocortical neurons and that both activity and silence started almost synchronously in cells located up to 12 mm apart. Activity appeared predominantly at the area 5/7 border and spread in both anterior and posterior directions. The activity started earlier in fast-spiking cells and intrinsically bursting cells than in regular-spiking neurons. These results provide direct evidence for two mechanisms of active state generation: spread of activity from a local focus and synchronization of weaker activity, originating at multiple locations. Surprisingly, onsets of silent states were synchronized even more precisely than the onsets of activity, showing no latency bias for location or cell type. This most intriguing finding exposes a major gap in understanding the nature of state alternation. We suggest that it is the synchronous termination of activity and occurrence of silent states of the neuronal network that makes the EEG picture during slow-wave sleep so characteristic. Synchronous onset of silence in distant neurons cannot rely exclusively on properties of individual cells and synapses, such as adaptation of neuronal firing or synaptic depression; instead, it implies the existence of a network mechanism. Revealing this yet unknown large-scale mechanism, which switches network activity to silence, will aid our understanding of the origin of brain rhythms in normal function and pathology.
Silencing Acid-Sensing Ion Channel 1a Alters Cone-Mediated Retinal Function
Authors: Ettaiche Mohammed , Deval Emmanuel , Cougnon Marc , Lazdunski Michel , Voilley Nicolas
Published: May 2006

The action of extracellular protons on retinal activity and phototransduction occurs through pH-sensitive elements, mainly membrane conductances present on the different cell types of the outer and inner nuclear layers and of the ganglion cell layer. Acid-sensing ion channels (ASICs) are depolarizing conductances that are directly activated by protons. We investigated the participation of ASIC1a, a particular isoform of ASICs, in retinal physiologyin vivousing electroretinogram measurements.In situhybridization and immunohistochemistry localized ASIC1a in the outer and inner nuclear layers (cone photoreceptors, horizontal cells, some amacrine and bipolar cells) and in the ganglion cell layer. Both thein vivoknockdown of ASIC1a by antisense oligonucleotides and thein vivoblocking of its activity by PcTx1, a specific venom peptide, were able to decrease significantly and reversibly the photopic a- and b-waves and oscillatory potentials. Our study indicates that ASIC1a is an important channel in normal retinal activity. Being present in the inner segments of cones and inner nuclear layer cells, and mainly at synaptic cleft levels, it could participate in gain adaptation to ambient light of the cone pathway, facilitating cone hyperpolarization in brightness and modulating synaptic transmission of the light-induced visual signal.
Cannabinoid Receptor Type 1 Located on Presynaptic Terminals of Principal Neurons in the Forebrain Controls Glutamatergic Synaptic Transmission
Authors: Domenici Maria R. , Azad Shahnaz C. , Marsicano Giovanni , Schierloh Anja , Wotjak Carsten T. , Dodt Hans-Ulrich , Zieglgänsberger Walter , Lutz Beat , Rammes Gerhard
Published: May 2006

It is widely accepted that cannabinoids regulate GABA release by activation of cannabinoid receptor type 1 (CB1). Results obtained from a variety of brain regions consistently indicate that cannabinoid agonists can also reduce glutamatergic synaptic transmission. However, there are still conflicting data concerning the role of CB1 in cannabinoid-induced inhibition of glutamatergic transmission in cortical areas. Here, we provide direct evidence that activation of CB1 on terminals of principal neurons controls excitatory synaptic responses in the forebrain. In slices of the basolateral amygdala, the CA1 region of the hippocampus, and the primary somatosensory cortex of wild-type mice, application of the CB1 agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2; WIN) (5 μm) reduced evoked excitatory postsynaptic responses. In contrast, in slices obtained from conditional mouse mutants lacking CB1 in all principal forebrain neurons but not in GABAergic interneurons (CB1f/f;CaMKIIαCre), WIN no longer affected glutamatergic synaptic transmission in any of the brain regions tested. Compatible with a presynaptic mechanism, WIN did not change the sensitivity to focally uncagedl-glutamate. WIN reduced glutamatergic responses in slices obtained from mice lacking CB1 exclusively in GABAergic neurons (CB1f/f;Dlx5/6-Cre), thus excluding the involvement of CB1 expressed on GABAergic neurons in this effect of the drug. The present data strongly indicate that excitatory synaptic transmission in forebrain areas is directly modulated by CB1 expressed on presynaptic axon terminals originating from glutamatergic neurons.
Ca2+Influx through Mechanosensitive Channels Inhibits Neurite Outgrowth in Opposition to Other Influx Pathways and Release from Intracellular Stores
Authors: Jacques-Fricke Bridget T. , Seow Yiqi , Gottlieb Philip A. , Sachs Frederick , Gomez Timothy M.
Published: May 2006

Ca2+signals are known to be important regulators of neurite outgrowth and steering. Here we show that inhibiting Ca2+influx through stretch-activated channels using various compounds, including a highly specific peptide isolated fromGrammostola spatulataspider venom (GsMTx4), strongly accelerates the rate of neurite extension on diverse substrata and within the intact spinal cord. Consistent with the presence of stretch-activated channels, we show that Ca2+influx is triggered by hypotonic solutions, which can be partially blocked by GsMTx4. Finally, chelating local, but not global, Ca2+signals prevents the acceleration that is normally produced by GsMTx4. Blocking Ca2+influx through other channel types has little or opposite effects, but release from intracellular stores is required for maximal acceleration. Together, our data suggest that Ca2+functions at distinct microdomains in growth cones, with influx through mechanosensitive channels acting to inhibit outgrowth in opposition to influx through other plasma membrane channels and release from stores.
Representation of Head-Centric Flow in the Human Motion Complex
Authors: Goossens Jeroen , Dukelow Sean P. , Menon Ravi S. , Vilis Tutis , van den Berg Albert V.
Published: May 2006

Recent neuroimaging studies have identified putative homologs of macaque middle temporal area (area MT) and medial superior temporal area (area MST) in humans. Little is known about the integration of visual and nonvisual signals in human motion areas compared with monkeys. Through extra-retinal signals, the brain can factor out the components of visual flow on the retina that are induced by eye-in-head and head-in-space rotations and achieve a representation of flow relative to the head (head-centric flow) or body (body-centric flow).

Here, we used functional magnetic resonance imaging to test whether extra-retinal eye-movement signals modulate responses to visual flow in the human MT+ complex. We distinguished between MT and MST and tested whether subdivisions of these areas may transform the retinal flow into head-centric flow.

We report that interactions between eye-movement signals and visual flow are not evenly distributed across MT+. Pursuit hardly influenced the response of MT to flow, whereas the responses in MST to the same retinal stimuli were stronger during pursuit than during fixation. We also identified two subregions in which the flow-related responses were boosted significantly by pursuit, one overlapping part of MST. In addition, we found evidence of a metric relation between rotational flow relative to the head and fMRI signals in a subregion of MST. The latter findings provide an important advance over published single-cell recordings in monkey MST. A visual representation of the rotation of the head in the world derived from head-centric flow may supplement semicircular canals signals and is appropriate for cross-calibrating vestibular and visual signals.
Motor Axon Guidance of the Mammalian Trochlear and Phrenic Nerves: Dependence on the Netrin ReceptorUnc5cand Modifier Loci
Authors: Burgess Robert W. , Jucius Thomas J. , Ackerman Susan L.
Published: May 2006

Netrin signaling is important to guide migrating neurons and axons in many systems. Experiments with vertebrate CNS explants suggested netrin is bifunctional, attracting some axons and repelling others. Netrin1-expressing cells attracted spinal commissural axons and repelled trochlear cranial nerve axons in these experiments. Subsequent genetic studies demonstrated that multiple axon types, including those of the spinal commissural neurons, are attracted to netrinin vivo; however, anin vivorole for netrin signaling in trochlear nerve repulsion has not been observed. Here, we demonstrate that mice with a null mutation in the netrin receptorUnc5con the inbred C57BL/6J (B6) genetic background have ventral/ipsilateral trochlear nerve misprojections. These misprojections are attenuated on a hybrid B6 × SJL background. In addition, B6.Unc5c−/−mice die as neonates of apparent respiratory distress and have incomplete phrenic nerve innervation of the diaphragm muscle. Neither the trochlear nerve misprojections nor the phrenic nerve phenotype was observed in B6 embryos lacking the netrin receptors DCC or Neogenin1, or the ligand netrin1, indicating these signaling molecules are dispensable for guidance of these axons. Like the trochlear nerve, the phrenic nerve phenotype is modified in a B6 × SJL hybrid background. To identify these modifier loci, we performed genome scans of the hybridUnc5c−/−mice and found a major SJL-derived suppressor locus on Chromosome 17. Our results provide the first evidence that genes involved in netrin signaling are necessary for proper mammalian spinal motor axon development and trochlear axon guidance. In addition, they demonstrate the importance of modifier genes in vertebrate axonal guidance.
The Wnt Receptor Ryk Is Required for Wnt5a-Mediated Axon Guidance on the Contralateral Side of the Corpus Callosum
Authors: Keeble Thomas R. , Halford Michael M. , Seaman Clare , Kee Nigel , Macheda Maria , Anderson Richard B. , Stacker Steven A. , Cooper Helen M.
Published: May 2006

Ryk (receptor related to tyrosine kinase) has been shown to be a novel Wnt receptor in bothCaenorhabditis elegansandDrosophila melanogaster. Recently, Ryk–Wnt interactions were shown to guide corticospinal axons down the embryonic mouse spinal cord. Here we show that, inRyk-deficient mice, cortical axons project aberrantly across the major forebrain commissure, the corpus callosum. Many mouse mutants have been described in which loss-of-function mutations result in the inability of callosal axons to cross the midline, thereby forming Probst bundles on the ipsilateral side. In contrast, loss of Ryk does not interfere with the ability of callosal axons to cross the midline but impedes their escape from the midline into the contralateral side. Therefore,Ryk−/−mice display a novel callosal guidance phenotype. We also show that Wnt5a acts as a chemorepulsive ligand for Ryk, driving callosal axons toward the contralateral hemisphere after crossing the midline. In addition, whereas callosal axons do cross the midline inRyk−/−embryos, they are defasciculated on the ipsilateral side, indicating that Ryk also promotes fasciculation of axons before midline crossing. In summary, this study expands the emerging role for Wnts in axon guidance and identifies Ryk as a key guidance receptor in the establishment of the corpus callosum. Our analysis of Ryk function further advances our understanding of the molecular mechanisms underlying the formation of this important commissure.