Phosphodiesterase-III Inhibitor Prevents Hemorrhagic Transformation Induced by Focal Cerebral Ischemia in Mice Treated with tPA

The purpose of the present study was to investigate whether cilostazol, a phosphodiesterase-III inhibitor and antiplatelet drug, would prevent tPA-associated hemorrhagic transformation. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone at 6 h, with combined tPA plus cilostazol at 6 h, or with vehicle at 6 h. We used multiple imaging (electron microscopy, spectroscopy), histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To further investigate the mechanism of cilostazol to beneficial effect, we also performed an in vitro study with tPA and a phosphodiesterase-III inhibitor in human brain microvascular endothelial cells, pericytes, and astrocytes. Combination therapy with tPA plus cilostazol prevented development of hemorrhagic transformation, reduced brain edema, prevented endothelial injury via reduction MMP-9 activity, and prevented the blood-brain barrier opening by inhibiting decreased claudin-5 expression. These changes significantly reduced the morbidity and mortality at 18 h and 7 days after the reperfusion. Also, the administration of both drugs prevented injury to brain human endothelial cells and human brain pericytes. The present study indicates that a phosphodiesterase-III inhibitor prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA

Revealing the Functions of the Transketolase Enzyme Isoforms in Rhodopseudomonas palustris Using a Systems Biology Approach

BACKGROUND: Rhodopseudomonas palustris (R. palustris) is a purple non-sulfur anoxygenic phototrophic bacterium that belongs to the class of proteobacteria. It is capable of absorbing atmospheric carbon dioxide and converting it to biomass via the process of photosynthesis and the Calvin-Benson-Bassham (CBB) cycle. Transketolase is a key enzyme involved in the CBB cycle. Here, we reveal the functions of transketolase isoforms I and II in R. palustris using a systems biology approach. METHODOLOGY/PRINCIPAL FINDINGS: By measuring growth ability, we found that transketolase could enhance the autotrophic growth and biomass production of R. palustris. Microarray and real-time quantitative PCR revealed that transketolase isoforms I and II were involved in different carbon metabolic pathways. In addition, immunogold staining demonstrated that the two transketolase isoforms had different spatial localizations: transketolase I was primarily associated with the intracytoplasmic membrane (ICM) but transketolase II was mostly distributed in the cytoplasm. Comparative proteomic analysis and network construction of transketolase over-expression and negative control (NC) strains revealed that protein folding, transcriptional regulation, amino acid transport and CBB cycle-associated carbon metabolism were enriched in the transketolase I over-expressed strain. In contrast, ATP synthesis, carbohydrate transport, glycolysis-associated carbon metabolism and CBB cycle-associated carbon metabolism were enriched in the transketolase II over-expressed strain. Furthermore, ATP synthesis assays showed a significant increase in ATP synthesis in the transketolase II over-expressed strain. A PEPCK activity assay showed that PEPCK activity was higher in transketolase over-expressed strains than in the negative control strain. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that the two isoforms of transketolase in R. palustris could affect photoautotrophic growth through both common and divergent metabolic mechanisms

Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells

The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unprecedented miniaturization and parallelization in biological assays. Here, we developed peptide microarrays for the detection of CTL activity. MHC class I-binding peptide epitopes were pipetted onto polymer-coated glass slides. Target cells, loaded with the cell-impermeant dye calcein, were incubated on these arrays, followed by incubation with antigen-expanded CTLs. Cytotoxic activity was detected by release of calcein and detachment of target cells. With only 200,000 cells per microarray, CTLs could be detected at a frequency of 0.5% corresponding to 1,000 antigen-specific T cells. Target cells and CTLs only settled on peptide spots enabling a clear separation of individual epitopes. Even though no physical boundaries were present between the individual spots, peptide loading only occurred locally and cytolytic activity was confined to the spots carrying the specific epitope. The peptide microarrays provide a robust platform that implements the whole process from antigen presentation to the detection of CTL activity in a miniaturized format. The method surpasses all established methods in the minimum numbers of cells required. With antigen uptake occurring on the microarray, further applications are foreseen in the testing of antigen precursors that require uptake and processing prior to presentation

Anti-Tumor Effect against Human Cancer Xenografts by a Fully Human Monoclonal Antibody to a Variant 8-Epitope of CD44R1 Expressed on Cancer Stem Cells

BACKGROUND: CD44 is a major cellular receptor for hyaluronic acids. The stem structure of CD44 encoded by ten normal exons can be enlarged by ten variant exons (v1-v10) by alternative splicing. We have succeeded in preparing MV5 fully human IgM and its class-switched GV5 IgG monoclonal antibody (mAb) recognizing the extracellular domain of a CD44R1 isoform that contains the inserted region coded by variant (v8, v9 and v10) exons and is expressed on the surface of various human epithelial cancer cells. METHODS AND PRINCIPAL FINDINGS: We demonstrated the growth inhibition of human cancer xenografts by a GV5 IgG mAb reshaped from an MV5 IgM. The epitope recognized by MV5 and GV5 was identified to a v8-coding region by the analysis of mAb binding to various recombinant CD44 proteins by enzyme-linked immunosorbent assay. GV5 showed preferential reactivity against various malignant human cells versus normal human cells assessed by flow cytometry and immunohistological analysis. When ME180 human uterine cervix carcinoma cells were subcutaneously inoculated to athymic mice with GV5, significant inhibition of tumor formation was observed. Furthermore, intraperitoneal injections of GV5markedly inhibited the growth of visible established tumors from HSC-3 human larynx carcinoma cells that had been subcutaneously transplanted one week before the first treatment with GV5. From in vitro experiments, antibody-dependent cellular cytotoxicity and internalization of CD44R1 seemed to be possible mechanisms for in vivo anti-tumor activity by GV5. CONCLUSIONS: CD44R1 is an excellent molecular target for mAb therapy of cancer, possibly superior to molecules targeted by existing therapeutic mAb, such as Trastuzumab and Cetuximab recognizing human epidermal growth factor receptor family

Measurement of the cross-section of high transverse momentum vector bosons reconstructed as single jets and studies of jet substructure in pp collisions at √s = 7 TeV with the ATLAS detector

This paper presents a measurement of the cross-section for high transverse momentum W and Z bosons produced in pp collisions and decaying to all-hadronic final states. The data used in the analysis were recorded by the ATLAS detector at the CERN Large Hadron Collider at a centre-of-mass energy of √s = 7 TeV;{\rm Te}{\rm V}$and correspond to an integrated luminosity of$4.6\;{\rm f}{{{\rm b}}^{-1}}$. The measurement is performed by reconstructing the boosted W or Z bosons in single jets. The reconstructed jet mass is used to identify the W and Z bosons, and a jet substructure method based on energy cluster information in the jet centre-of-mass frame is used to suppress the large multi-jet background. The cross-section for events with a hadronically decaying W or Z boson, with transverse momentum${{p}_{{\rm T}}}\gt 320\;{\rm Ge}{\rm V}$and pseudorapidity$|\eta |\lt 1.9$, is measured to be${{\sigma }_{W+Z}}=8.5\pm 1.7$ pb and is compared to next-to-leading-order calculations. The selected events are further used to study jet grooming techniques

Search for direct pair production of the top squark in all-hadronic final states in proton-proton collisions at s√=8 TeV with the ATLAS detector

The results of a search for direct pair production of the scalar partner to the top quark using an integrated luminosity of 20.1fb−1 of proton–proton collision data at √s = 8 TeV recorded with the ATLAS detector at the LHC are reported. The top squark is assumed to decay via t˜→tχ˜01 or t˜→ bχ˜±1 →bW(∗)χ˜01 , where χ˜01 (χ˜±1 ) denotes the lightest neutralino (chargino) in supersymmetric models. The search targets a fully-hadronic final state in events with four or more jets and large missing transverse momentum. No significant excess over the Standard Model background prediction is observed, and exclusion limits are reported in terms of the top squark and neutralino masses and as a function of the branching fraction of t˜ → tχ˜01 . For a branching fraction of 100%, top squark masses in the range 270–645 GeV are excluded for χ˜01 masses below 30 GeV. For a branching fraction of 50% to either t˜ → tχ˜01 or t˜ → bχ˜±1 , and assuming the χ˜±1 mass to be twice the χ˜01 mass, top squark masses in the range 250–550 GeV are excluded for χ˜01 masses below 60 GeV

Search for pair-produced long-lived neutral particles decaying to jets in the ATLAS hadronic calorimeter in ppcollisions at √s=8TeV

The ATLAS detector at the Large Hadron Collider at CERN is used to search for the decay of a scalar boson to a pair of long-lived particles, neutral under the Standard Model gauge group, in 20.3fb−1of data collected in proton–proton collisions at √s=8TeV. This search is sensitive to long-lived particles that decay to Standard Model particles producing jets at the outer edge of the ATLAS electromagnetic calorimeter or inside the hadronic calorimeter. No significant excess of events is observed. Limits are reported on the product of the scalar boson production cross section times branching ratio into long-lived neutral particles as a function of the proper lifetime of the particles. Limits are reported for boson masses from 100 GeVto 900 GeV, and a long-lived neutral particle mass from 10 GeVto 150 GeV

Hagfish and lamprey Hox genes reveal conservation of temporal colinearity in vertebrates

Hox genes exert fundamental roles for proper regional specification along the main rostro-caudal axis of animal embryos. They are generally expressed in restricted spatial domains according to their position in the cluster (spatial colinearity)—a feature that is conserved across bilaterians. In jawed vertebrates (gnathostomes), the position in the cluster also determines the onset of expression of Hox genes (a feature known as whole-cluster temporal colinearity (WTC)), while in invertebrates this phenomenon is displayed as a subcluster-level temporal colinearity. However, little is known about the expression profile of Hox genes in jawless vertebrates (cyclostomes); therefore, the evolutionary origin of WTC, as seen in gnathostomes, remains a mystery. Here, we show that Hox genes in cyclostomes are expressed according to WTC during development. We investigated the Hox repertoire and Hox gene expression profiles in three different species—a hagfish, a lamprey and a shark—encompassing the two major groups of vertebrates, and found that these are expressed following a whole-cluster, temporally staggered pattern, indicating that WTC has been conserved during the past 500 million years despite drastically different genome evolution and morphological outputs between jawless and jawed vertebrates

Specific Evolution of F1-Like ATPases in Mycoplasmas

F1F0 ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F1F0 ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the α, β, γ and ε subunits of F1 ATPases and could form an F1-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F1-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F1-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F1-like structure is associated with a hypothetical X0 sector located in the membrane of mycoplasma cells