bluuebunny/crossref_metadata_2025 huggingface dataset:
Indirect electrochemiluminescence detection of lysine and histidine separated by capillary electrophoresis based on charge displacement
Authors: Yu Huan , Xu Lei , You Tianyan
Published: June 2012

Indirect electrochemiluminescence (ECL) detection was applied for the analysis of lysine (Lys) and histidine (His) separated by capillary electrophoresis (CE). With the most effective electrophoretic buffer system, which contained 15 mM phosphate buffer (pH = 5.8) and 0.5 mM Tripropylamine (TPA), fast separation of the two basic amino acids could be performed within 7 min. The linear ranges were 10–35 μM, 35–150 μM for Lys; and 5–35 μM, 35–150 μM for His. The detection limits (S/N = 3) were 0.3 μM for Lys and 1.0 μM for His, respectively. The proposed method was also successfully used for the determination of Lys in the oral pharmaceutical formulations. Copyright © 2012 John Wiley & Sons, Ltd.
Molecular dynamics simulation of the induced‐fit binding process of DNA aptamer and L‐argininamide
Authors: Lin Po‐Hsun , Tsai Ching‐Wei , Wu Josephine W. , Ruaan Ruoh‐Chyu , Chen Wen‐Yih
Published: June 2012

Aptamers are rare functional nucleic acids with binding affinity to and specificity for target ligands. Recent experiments have lead to the proposal of an induced‐fit binding mechanism for L‐argininamide (Arm) and its binding aptamer. However, at the molecular level, this mechanism between the aptamer and its coupled ligand is still poorly understood. The present study used explicit solvent molecular dynamics (MD) simulations to examine the critical bases involved in aptamer‐Arm binding and the induced‐fit binding process at atomic resolution. The simulation results revealed that the Watson‐Crick pair (G10‐C16), C9, A12, and C17 bases play important roles in aptamer‐Arm binding, and that binding of Arm results in an aptamer conformation optimized through a general induced‐fit process. In an aqueous solution, the mechanism has the following characteristic stages: (a) adsorption stage, the Arm anchors to the binding site of aptamer with strong electrostatic interaction; (b) binding stage, the Arm fits into the binding site of aptamer by hydrogen‐bond formation; and (c) complex stabilization stage, the hydrogen bonding and electrostatic interactions cooperatively stabilize the complex structure. This study provides dynamics information on the aptamer‐ligand induced‐fit binding mechanism. The critical bases in aptamer‐ligand binding may provide a guideline in aptamer design for molecular recognition engineering.
Controlled feeding of hydrogen peroxide as oxygen source improves production of 5‐ketofructose From L‐sorbose using engineered pyranose 2‐oxidase from Peniophora gigantea
Authors: Schneider Konstantin , Dorscheid Susanne , Witte Klaus , Giffhorn Friedrich , Heinzle Elmar
Published: June 2012

5‐Keto‐D‐fructose is a useful starting material for the synthesis of pyrrolidine iminosugars. It can be prepared by regioselective oxidation of L‐sorbose using pyranose 2‐oxidase (P2Ox) and O2 as a cosubstrate. As the solubility of O2 in aqueous solution is low and the affinity of P2Ox for O2 is poor, we developed a new and efficient process for the production of 5‐keto‐D‐fructose based on engineered P2Ox from Peniophora gigantea and in situ generation of O2 from H2O2 with catalase. This kind of oxygen supply required efficient mixing of the bioreactor which was achieved by controlled feeding of H2O2 close to the impeller tip where energy dissipation rate is highest. Thus bubbling, known to affect enzyme stability, was largely avoided, and the process could be run up to 145% oxygen super‐saturation which speeds‐up P2Ox activity. Under these conditions quantitative oxidation of 180 g L−1 L‐sorbose to 5‐keto‐D‐fructose could be achieved within 4 h, resulting in a threefold higher overall productivity of the process compared to a process using gaseous oxygen supply. In addition, in situ generation of O2 from H2O2 lowered the oxygen demand of the process by a factor of 100 compared to gaseous oxygen supply. Biotechnol. Bioeng. 2012; 109: 2941–2945. © 2012 Wiley Periodicals, Inc.
Crosslinked aggregates of Rhizopus oryzae lipase as industrial biocatalysts: Preparation, optimization, characterization, and application for enantioselective resolution reactions
Authors: Kartal Funda , Kilinc Ali
Published: June 2012

Lipase from Rhizopus oryzae (ROL) was immobilized as crosslinked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and simultaneous crosslinking with glutaraldehyde. The optimum conditions of the immobilization process were determined. Lipase CLEAs showed a twofold increase in activity when Tween 80‐pretreated lipase was used for CLEA preparation. CLEAs were shown to have several advantages compared to free lipase. CLEAs were more stable at 50°C and 60°C as well as for a wide range of pH. After incubation at 50°C, CLEA showed 74% of initial activity whereas free enzyme was totally inactivated. Reduction of Schiff bases has been performed for the first time in the CLEA preparation process significantly improving the chemically modified CLEAs' reusability, thus providing an enzyme with high potential for recycling even under aqueous reaction conditions where enzyme leakage is, in general, one of the major problems. The CLEA retained 91% activity after 10 cycles in aqueous medium. The immobilized enzyme was used for kinetic resolution reactions. Results showed that immobilization had an enhancing effect on the conversion (c) as well as on the enantiomeric ratio (E). ROL CLEA displayed five times higher enantioselectivity for the hydrolysis of (R,S)‐1‐phenylethyl acetate and likewise 1.5 times higher enantioselectivity for the transesterification of racemic (R–S)‐1‐phenylethanol with vinylacetate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 937–945, 2012

This article was published online on June 26, 2012. An edit was subsequently requested. This notice is included in the online and print versions to indicate that both have been corrected [27 June 2012].
Aqueous Solutions of Mo‐V‐Phosphoric Heteropoly Acids as Bifunctional Catalysts For Preparation of 9,10‐Anthraquinone and its Hydrogenated Derivatives
Authors: Zhizhina Elena G. , Odyakov Viktor F.
Published: June 2012

Acid‐catalyzed condensation of 1,3‐butadiene with para‐quinones and oxidation of resulting adducts can be performed as a one‐pot process in the presence of aqueous solutions of Mo‐V‐phosphoric heteropoly acids (HPA) with the general formula HaPzMoyVxOb. These solutions have bifunctional catalytic properties; they are both strong Brønsted acids and reversible oxidants. Condensation of hydroquinone, 1,4‐benzoquinone, or 1,4‐naphthoquinone with 1,3‐butadiene in the HPA solutions results in 9,10‐anthraquinone (AQ) in a mixture with tetrahydro‐AQ (THA) and dihydro‐AQ (DHA). In this one‐pot process, which proceeds in the absence of organic solvents, the HPA solution is reduced. A slightly soluble mixture of THA, DHA, and AQ is precipitated and filtered off. The non‐Keggin‐type H12P3Mo18V7O85 solution is the most effective in this process. This solution can be rapidly regenerated by dioxygen or air at temperatures of up to 170 °C and then reused. The prepared THA+DHA+AQ mixture can be used as a catalyst for alkaline delignification of wood. Our homogeneous bifunctional HPA catalyst is very stable and promising for use in similar one‐pot processes.
Synthesis and Biological Evaluation of Adenosines with Heterobicyclic and Polycyclic N6‐Substituents as Adenosine A1 Receptor Agonists
Authors: Gosling Joshua I. , Baker Stephen P. , Haynes John M. , Kassiou Michael , Pouton Colin W. , Warfe Lyndon , White Paul J. , Scammells Peter J.
Published: June 2012

A concise synthesis of a series of N6‐substituted adenosines with bicyclo[3.2.1]octan‐6‐yl and polycyclic N6‐substituents has been developed. The adenosine A1 receptor (A1R) affinity and potency of these compounds was initially assessed using competitive binding assays and cyclic adenosine monophosphate (cAMP) accumulation assays in DDT1 MF‐2 cells. The potency and receptor subtype selectivity of selected examples was further evaluated by measuring their effects on cAMP accumulation at all human adenosine receptor subtypes expressed in CHO cells. The results of these assays indicated that all of the synthesised N6‐substituted adenosines are full agonists at A1R and activate this receptor selectively over the other adenosine receptor subtypes. The two standout compounds in terms of potency were N6‐(3‐thiabicyclo[3.2.1]octan‐6‐yl)adenosine and N6‐(cubanylmethyl)adenosine with EC50 values at human A1R of 2.3 nM and 1.1 nM, respectively. The cubanylmethyl derivative in particular proved to be highly receptor subtype selective. These two compounds were further evaluated in a simulated ischaemia model in cultured cardiomyoblasts, where they were found to impart protective effects under hypoxic conditions that resulted in a significant reduction in cell death.
Molecular investigation of lymph nodes in colon cancer patients using one‐step nucleic acid amplification (OSNA)
Authors: Güller Ulrich , Zettl Andreas , Worni Mathias , Langer Igor , Cabalzar‐Wondberg Daniela , Viehl Carsten T. , Demartines Nicolas , Zuber Markus
Published: June 2012

BACKGROUND:

A new diagnostic system, called one‐step nucleic acid amplification (OSNA), has recently been designed to detect cytokeratin 19 mRNA as a surrogate for lymph node metastases. The objective of this prospective investigation was to compare the performance of OSNA with both standard hematoxylin and eosin (H&E) analysis and intensive histopathology in the detection of colon cancer lymph node metastases.

METHODS:

In total, 313 lymph nodes from 22 consecutive patients with stage I, II, and III colon cancer were assessed. Half of each lymph node was analyzed initially by H&E followed by an intensive histologic workup (5 levels of H&E and immunohistochemistry analyses, the gold standard for the assessment of sensitivity/specificity of OSNA), and the other half was analyzed using OSNA.

RESULTS:

OSNA was more sensitive in detecting small lymph node tumor infiltrates compared with H&E (11 results were OSNA positive/H&E negative). Compared with intensive histopathology, OSNA had 94.5% sensitivity, 97.6% specificity, and a concordance rate of 97.1%. OSNA resulted in an upstaging of 2 of 13 patients (15.3%) with lymph node‐negative colon cancer after standard H&E examination.

CONCLUSIONS:

OSNA appeared to be a powerful and promising molecular tool for the detection of lymph node metastases in patients with colon cancer. OSNA had similar performance in the detection of lymph node metastases compared with intensive histopathologic investigations and appeared to be superior to standard histology with H&E. Most important, the authors concluded that OSNA may lead to a potential upstaging of >15% of patients with colon cancer. Cancer 2012. © 2012 American Cancer Society.
Strain differences in illumination‐dependent structural changes at mouse photoreceptor ribbon synapses
Authors: Fuchs Michaela , Sendelbeck Anna , Atorf Jenny , Kremers Jan , Brandstätter Johann Helmut
Published: June 2012

Photoreceptor cells encode light signals over a wide range of intensities with graded changes in their membrane potential. At their highly specialized ribbon synapses they transmit the signals to the postsynaptic neurons by the tonic release of glutamate, which is continuously adjusted to changes in light intensity. Such a level of performance requires adaptive mechanisms, and it is suggested that illumination‐dependent changes in ribbon shape and size are one of these adaptive processes. In this study we compared structural properties of synaptic ribbons under various illumination conditions between three mouse strains: the pigmented C57BL/6 and the two albino strains Balb/c and B6(Cg)‐Tyrc‐2J/J (coisogenic to C57BL/6). In addition, electroretinograms (ERGs) recorded in the same groups were compared. In the C57BL/6 mouse a change in illumination did not result in structural alterations of the synaptic ribbon. Similarly, in the B6(Cg)‐Tyrc‐2J/J mouse only minor structural changes were detected. In contrast, the state of adaptation had a large influence on the ribbon structure of the Balb/c mouse. The ERG recordings showed only small functional differences between C57BL/6 and B6(Cg)‐Tyrc‐2J/J mice, but the retinal function of Balb/c mice was strongly compromised. We conclude that illumination‐dependent changes of photoreceptor ribbon structure differ between strains and thus cannot be regarded as a general mechanism for light adaptation. J. Comp. Neurol. 521:69–78, 2013. © 2012 Wiley Periodicals, Inc.
Directed glia‐assisted angiogenesis in a mature neurosensory structure: Pericytes mediate an adaptive response in human dental pulp that maintains blood‐barrier function
Authors: Farahani Ramin M. , Sarrafpour Babak , Simonian Mary , Li Qing , Hunter Neil
Published: June 2012

The specialized tightly controlled microcirculation of craniofacial neurosensory organs is an essential evolutionary adaptation and yet a dilemma where angiogenic remodeling occurs. Despite extreme plasticity of neurosensory structures, the capacity to reconcile barrier phenotype of the microcirculation with an angiogenic cascade is not known. Here we provide primary evidence for such a response in an elemental neurosensory structure, human dental pulp, following chronic carious insult. In response to hypoxic challenge neurosensory odontoblasts express hypoxia‐inducible factor‐1α and notch‐1. Associated radial rearrangement of astrocyte‐like telacytes that communicate through a cell‐poor zone with the microvasculature is observed. Activated pericytes characterized by expression of α‐smooth muscle actin are located adjacent to the telacyte attachment to the vasculature. In this location, endothelial expression of sonic hedgehog parallels expression of notch‐1 by pericytes. The angiogenic response is initiated by pericyte contraction and altered endothelial polarity and proliferation leading to intussusception of endothelial cells and extensive remodeling of basement membrane with upregulation of laminin‐8 and laminin‐5. These responses guide intravascular loop formation that maintains both intact basement membrane and tight junctions. This initial phase is followed by formation of anastomoses that enhance the hemodynamic capacity of the intravascular loops. The formation of anastomoses is mediated by extension of cytonemes from pericytes guided by MHC‐II+/CD‐163+ microglia aligned with the telacytes. The cytonemes seek out pericytes on adjacent intravascular loops to initiate migration of endothelial cells. These findings support a new paradigm for understanding angiogenic capacity of neurosensory structures and aberrations of this response manifest as neurovasculopathies. J. Comp. Neurol. 520:3803–3826, 2012. © 2012 Wiley Periodicals, Inc.
Risk assessment in work environments: modeling and simulation
Authors: Fugini Mariagrazia , Raibulet Claudia , Ubezio Luigi
Published: June 2012

The paper proposes a characterization of risks and a service‐oriented prototype to face risky situations in work environments, such as in industrial plants or building construction areas. A risk is the overture of emergencies that produce human and/or material damages. Therefore, it is particularly critical to identify and manage risks to avoid their evolution into emergencies. In this paper, we outline the technological features of a risk environment and propose a risk model and a service‐based simulation prototype aimed to improve safety in work environments. We discuss engineering issues concerning risk modeling and management. Furthermore, we propose a risk management system solution and its related implemented prototype composed of services able to detect and also to prevent the occurrence of risk conditions. Copyright © 2012 John Wiley & Sons, Ltd.
Spray deposition and characterization of undoped and In‐doped tin disulphide thin films
Authors: Kumar K. Santhosh , Manoharan C. , Amalraj L. , Dhanapandian S. , Kiruthigaa G. , Vijayakumar K.
Published: June 2012

Undoped and Indium doped tin disulphide (SnS2) thin films had been deposited onto glass substrates at Ts = 300 °C using spray pyrolysis technique under atmospheric pressure with stannous chloride, indium chloride and thiourea as precursors. The structural, optical and electrical properties of the deposited films were characterized. The XRD pattern revealed that the undoped and doped films had preferred orientation along (002) plane with hexagonal structure. FESEM micrographs had shown that morphologies of the films changed with indium doping. Optical constant such as refractive index (n), extinction coefficient (k), real and imaginary parts of dielectric constants were evaluated from transmittance and reflectance spectra in UV‐Visible spectroscopy. The optical absorption data were used to determine the band gap energy and it was found to be 2.75 eV for undoped and 2.50 eV for indium doped films respectively. The room temperature dark resistivity was found to be 4.545 × 103 Ω‐cm and 5.406 × 103 Ω‐cm for undoped and In‐doped films respectively.
Unusual Catalysts from Molasses: Synthesis, Properties and Application in Obtaining Biofuels from Algae
Authors: Samorì Chiara , Torri Cristian , Fabbri Daniele , Falini Giuseppe , Faraloni Cecilia , Galletti Paola , Spera Silvia , Tagliavini Emilio , Torzillo Giuseppe
Published: June 2012

Acid catalysts were prepared by sulfonation of carbon materials obtained from the pyrolysis of sugar beet molasses, a cheap, viscous byproduct in the processing of sugar beets into sugar. Conditions for the pyrolysis of molasses (temperature and time) influenced catalyst performance; the best combination came from pyrolysis at low temperature (420 °C) for a relatively long time (8–15 h), which ensured better stability of the final material. The most effective molasses catalyst was highly active in the esterification of fatty acids with methanol (100 % yield after 3 h) and more active than common solid acidic catalysts in the transesterification of vegetable oils with 25–75 wt % of acid content (55–96 % yield after 8 h). A tandem process using a solid acid molasses catalyst and potassium hydroxide in methanol was developed to de‐acidificate and transesterificate algal oils from Chlamydomonas reinhardtii, Nannochloropsis gaditana, and Phaeodactylum tricornutum, which contain high amounts of free fatty acids. The amount of catalyst required for the de‐acidification step was influenced by the chemical composition of the algal oil, thus operational conditions were determined not only in relation to free fatty acids content in the oil, but according to the composition of the lipid extract of each algal species.
Evolution of a sexually dimorphic trait in a broadly distributed topminnow (Fundulus olivaceus)
Authors: Schaefer Jacob F. , Duvernell David D. , Kreiser Brian R. , Champagne Charles , Clark Scott R. , Gutierrez Melissa , Stewart Laura K. , Coleman Chazz
Published: June 2012

Understanding the interaction between sexual and natural selection within variable environments is crucial to our understanding of evolutionary processes. The handicap principle predicts females will prefer males with exaggerated traits provided those traits are indicators of male quality to ensure direct or indirect female benefits. Spatial variability in ecological factors is expected to alter the balance between sexual and natural selection that defines the evolution of such traits. Male and female blackspotted topminnows (Fundulidae: Fundulus olivaceus) display prominent black dorsolateral spots that are variable in number across its broad range. We investigated variability in spot phenotypes at 117 sites across 13 river systems and asked if the trait was sexually dimorphic and positively correlated with measures of fitness (condition and gonadosomatic index [GSI]). Laboratory and mesocosm experiments assessed female mate choice and predation pressure on spot phenotypes. Environmental and community data collected at sampling locations were used to assess predictive models of spot density at the individual, site, and river system level. Greater number of spots was positively correlated with measures of fitness in males. Males with more spots were preferred by females and suffered greater mortality due to predation. Water clarity (turbidity) was the best predictor of spot density on the drainage scale, indicating that sexual and natural selection for the trait may be mediated by local light environments.
Sex‐specific effects of developmental environment on reproductive trait expression in Drosophila melanogaster
Authors: Edward Dominic A. , Chapman Tracey
Published: June 2012

Variation in the expression of reproductive traits provides the raw material upon which sexual selection can act. It is therefore important to understand how key factors such as environmental variation influence the expression of reproductive traits, as these will have a fundamental effect on the evolution of mating systems. It is also important to consider the effects of environmental variation upon reproductive traits in both sexes and to make comparisons with the environment to which the organism is adapted. In this study, we addressed these issues in a systematic study of the effect of a key environmental factor, variation in larval density, on reproductive trait expression in male and female Drosophila melanogaster. To do this, we compared reproductive trait expression when flies were reared under controlled conditions at eight different larval densities that covered a 20‐fold range. Then, to place these results in a relevant context, we compared the results to those from flies sourced directly from stock cages. Many reproductive traits were surprisingly insensitive to variation in larval density. A notable exception was nonlinear variation in female fecundity. In contrast, we found much bigger differences in comparisons with flies from stock cages—including differences in body size, latency to mate, copulation duration, fecundity, and male share of paternity in a competitive environment. For a number of traits, even densities of 1000 larvae per vial (125 larvae per mL of food) did not phenocopy stock cage individuals. This study reveals novel patterns of sex‐specific sensitivity to environmental variation that will influence the strength of sexual selection. It also illustrates the importance of comparisons with the environment to which individuals are adapted.
Understanding of the impact of chemicals on amphibians: a meta‐analytic review
Authors: Egea‐Serrano Andrés , Relyea Rick A. , Tejedo Miguel , Torralva Mar
Published: June 2012

Many studies have assessed the impact of different pollutants on amphibians across a variety of experimental venues (laboratory, mesocosm, and enclosure conditions). Past reviews, using vote‐counting methods, have described pollution as one of the major threats faced by amphibians. However, vote‐counting methods lack strong statistical power, do not permit one to determine the magnitudes of effects, and do not compare responses among predefined groups. To address these challenges, we conducted a meta‐analysis of experimental studies that measured the effects of different chemical pollutants (nitrogenous and phosphorous compounds, pesticides, road deicers, heavy metals, and other wastewater contaminants) at environmentally relevant concentrations on amphibian survival, mass, time to hatching, time to metamorphosis, and frequency of abnormalities. The overall effect size of pollutant exposure was a medium decrease in amphibian survival and mass and a large increase in abnormality frequency. This translates to a 14.3% decrease in survival, a 7.5% decrease in mass, and a 535% increase in abnormality frequency across all studies. In contrast, we found no overall effect of pollutants on time to hatching and time to metamorphosis. We also found that effect sizes differed among experimental venues and among types of pollutants, but we only detected weak differences among amphibian families. These results suggest that variation in sensitivity to contaminants is generally independent of phylogeny. Some publication bias (i.e., selective reporting) was detected, but only for mass and the interaction effect size among stressors. We conclude that the overall impact of pollution on amphibians is moderately to largely negative. This implies that pollutants at environmentally relevant concentrations pose an important threat to amphibians and may play a role in their present global decline.
Irf5‐deficient mice are protected from pristane‐induced lupus via increased Th2 cytokines and altered IgG class switching
Authors: Feng Di , Yang Lisong , Bi Xiaohui , Stone Rivka C. , Patel Priya , Barnes Betsy J.
Published: June 2012

Polymorphisms in the transcription factor interferon (IFN) regulatory factor 5 (IRF5) have been identified that show a strong association with an increased risk of developing the autoimmune disease systemic lupus erythematosus (SLE). A potential pathological role for IRF5 in SLE development is supported by the fact that increased IRF5 mRNA and protein are observed in primary blood cells of SLE patients and this correlates with an increased risk of developing the disease. Here, we demonstrate that IRF5 is required for pristane‐induced SLE via its ability to control multiple facets of autoimmunity. We show that IRF5 is required for pathological hypergammaglobulinemia and, in the absence of IRF5, IgG class switching is reduced. Examination of in vivo cytokine expression (and autoantibody production) identified an increase in Irf5−/− mice of Th2 cytokines. In addition, we provide clear evidence that loss of Irf5 significantly weakens the in vivo type I IFN signature critical for disease pathogenesis in this model of murine lupus. Together, these findings demonstrate the importance of IRF5 for autoimmunity and provide a significant new insight into how overexpression of IRF5 in blood cells of SLE patients may contribute to disease pathogenesis.
HLA‐G1 and HLA‐G5 active dimers are present in malignant cells and effusions: The influence of the tumor microenvironment
Authors: Zilberman Sonia , Schenowitz Chantal , Agaugué Sophie , Favier Benoît , Riteau Béatrice , Rouzier Roman , Carosella Edgardo D. , Rouas‐Freiss Nathalie , Menier Catherine
Published: June 2012

Dimers of the nonclassical HLA‐G class I molecule have recently been shown to be active structures that mediate inhibition of NK‐cell cytotoxic activity through interaction with the immunoglobulin‐like transcript (ILT)‐2 inhibitory receptor. However, this has only been proven in trophoblasts and HLA‐G transfectants. Here, we document for the first time the existence of HLA‐G dimers in cancer. Indeed, we identified both surface and soluble HLA‐G dimers in tumor cells and malignant ascites respectively. Interestingly, factors from the tumor microenvironment, such as interferons, enhanced the formation of HLA‐G dimers and increased the protection of tumors from NK cell‐mediated lysis. These data emphasize the impact of HLA‐G conformation on its efficiency at inhibiting the antitumor response and thus favoring tumor progression. In view of these results, the effect of the tumor microenvironment on upregulation of HLA‐G function deserves particular attention when designing cancer immunotherapy protocols.
Peptide‐MHC class I stability is a better predictor than peptide affinity of CTL immunogenicity
Authors: Harndahl Mikkel , Rasmussen Michael , Roder Gustav , Dalgaard Pedersen Ida , Sørensen Mikael , Nielsen Morten , Buus Søren
Published: June 2012

Efficient presentation of peptide‐MHC class I (pMHC‐I) complexes to immune T cells should benefit from a stable peptide‐MHC‐I interaction. However, it has been difficult to distinguish stability from other requirements for MHC‐I binding, for example, affinity. We have recently established a high‐throughput assay for pMHC‐I stability. Here, we have generated a large database containing stability measurements of pMHC‐I complexes, and re‐examined a previously reported unbiased analysis of the relative contributions of antigen processing and presentation in defining cytotoxic T lymphocyte (CTL) immunogenicity [Assarsson et al., J. Immunol. 2007. 178: 7890–7901]. Using an affinity‐balanced approach, we demonstrated that immunogenic peptides tend to be more stably bound to MHC‐I molecules compared with nonimmunogenic peptides. We also developed a bioinformatics method to predict pMHC‐I stability, which suggested that 30% of the nonimmunogenic binders hitherto classified as “holes in the T‐cell repertoire” can be explained as being unstably bound to MHC‐I. Finally, we suggest that nonoptimal anchor residues in position 2 of the peptide are particularly prone to cause unstable interactions with MHC‐I. We conclude that the availability of accurate predictors of pMHC‐I stability might be helpful in the elucidation of MHC‐I restricted antigen presentation, and might be instrumental in future search strategies for MHC‐I epitopes.
Subcellular localization of FOXP3 in human regulatory and nonregulatory T cells
Authors: Magg Thomas , Mannert Joachim , Ellwart Joachim W. , Schmid Irene , Albert Michael H.
Published: June 2012

The transcriptional regulator FOXP3 is an important determinant of regulatory T (Treg) cell development and function and is frequently used to quantitate Treg cells. However, FOXP3 is also expressed in recently activated conventional human T cells. Here, we investigated the FOXP3 expression patterns in Treg and activated T cells at a cellular level. Upon activation, human CD4+CD25− T cells expressed FOXP3 mainly in the cytoplasm, in sharp contrast to human CD4+CD25+ Treg cells, where we found FOXP3 to be predominantly expressed in the nucleus. A GFP‐FOXP3‐fusion protein shuttled from the nucleus to the cytoplasm in transfected primary human T cells. We identified two novel leucine‐rich nuclear export signals in FOXP3. Site‐directed mutagenesis of both sequences completely abolished nuclear export of FOXP3 in human T cells. Both export sequences localized to exons affected by alternative splicing. The three isoforms FOXP3Δ2, FOXP3Δ7, and FOXP3Δ2Δ7 localized preferentially to the nucleus. Additionally, forced expression of nucleus‐directed FOXP3 induced a Treg‐cell‐associated gene expression pattern and induced regulatory capacity. These findings should aid in the interpretation of future studies utilizing FOXP3 expression as a Treg‐cell marker and shed some light on the molecular mechanisms controlling subcellular FOXP3 localization in human T cells.
Combinatorial roles for histamine H1‐H2 and H3‐H4 receptors in autoimmune inflammatory disease of the central nervous system
Authors: Saligrama Naresha , Noubade Rajkumar , Case Laure K , del Rio Roxana , Teuscher Cory
Published: June 2012

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system in which histamine (HA) and its receptors have been implicated in disease pathogenesis. HA exerts its effects through four different G protein‐coupled receptors designated H1‐H4. We previously examined the effects of traditional single HA receptor (HR) knockouts (KOs) in experimental allergic encephalomyelitis (EAE), the autoimmune model of MS. Our results revealed that H1R and H2R are propathogenic, while H3R and H4R are antipathogenic. This suggests that combinatorial targeting of HRs may be an effective disease‐modifying therapy (DMT) in MS. To test this hypothesis, we generated H1H2RKO and H3H4RKO mice and studied them for susceptibility to EAE. Compared with wild‐type (WT) mice, H1H2RKO mice developed a less severe clinical disease course, whereas the disease course of H3H4RKO mice was more severe. H1H2RKO mice also developed less neuropathology and disrupted blood brain barrier permeability compared with WT and H3H4RKO mice. Additionally, splenocytes from immunized H1H2RKO mice produced less interferon(IFN)‐γ and interleukin(IL)‐17. These findings support the concept that combined pharmacological targeting of HRs may be an appropriate ancillary DMT in MS and other immunopathologic diseases.
Sin1 regulates Treg‐cell development but is not required for T‐cell growth and proliferation
Authors: Chang Xing , Lazorchak Adam S. , Liu Dou , Su Bing
Published: June 2012

Mammalian Sin1 plays key roles in the regulation of mitogen‐activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling. Sin1 is an essential component of mTOR complex 2 (mTORC2). The functions of Sin1 and mTORC2 remain largely unknown in T cells. Here, we investigate Sin1 function in T cells using mice that lack Sin1 in the hematopoietic system. Sin1 deficiency blocks the mTORC2‐dependent Akt phosphorylation in T cells during development and activation. Sin1‐deficient T cells exhibit normal thymic cellularity and percentages of double‐negative, double‐positive, and single‐positive CD4+ and CD8+ thymocytes. Sin1 deficiency does not impair T‐cell receptor (TCR) induced growth and proliferation. Sin1 appears dispensable for in vitro CD4+ helper cell differentiation. However, Sin1 deficiency results in an increased proportion of Foxp3+ natural T‐regulatory (nTreg) cells in the thymus. The TGF‐β‐dependent differen‐tiation of CD4+ T cells in vitro is enhanced by the inhibition of mTOR but not by loss of Sin1 function. Our results reveal that Sin1 and mTORC2 are dispensable for the development and activation of T cells but play a role in nTreg‐cell differentiation.
Apoptotic tumor cells induce IL‐27 release from human DCs to activate Treg cells that express CD69 and attenuate cytotoxicity
Authors: Sekar Divya , Hahn Christina , Brüne Bernhard , Roberts Edward , Weigert Andreas
Published: June 2012

Intrinsic immunosuppression is a major obstacle for successful cancer therapy. The mechanisms for the induction and regulation of immunosuppression in humans are ill defined. A microenvironmental component that might prevent antitumor immunity is the presence of dying tumor cells, which are abundant following conventional cancer ablation methods such as chemo‐ or radiotherapy. Shedding of apoptotic debris and/or secretion of factors to the tumor bed or draining lymph nodes thus might have a profound impact on professional phagocytes, such as DCs, and subsequent priming of lymphocytes. Here, we exposed human DCs to supernatants of live, apoptotic, or necrotic human breast cancer cells and cocultured them with autologous T cells. Priming with apoptotic debris prevented DCs from establishing cytotoxicity toward live human tumor cells by inducing a Treg‐cell population, defined by coexpression of CD39 and CD69. Immunosuppression via Treg cells was transferable and required the release of sphingosine‐1‐phosphate (S1P) from apoptotic cells, acting via S1P receptor 4 on DCs to induce IL‐27 secretion. We propose that CD69 expression on CD39+ Treg cells enables them to interact with CD73‐expressing CD8+ T cells to generate adenosine, thereby suppressing cytotoxicity. These findings aid the understanding of how dying tumor cells limit antitumor immunity.
Human CD1c (BDCA‐1)+ myeloid dendritic cells secrete IL‐10 and display an immuno‐regulatory phenotype and function in response to Escherichia coli
Authors: Kassianos Andrew J. , Hardy Melinda Y. , Ju Xinsheng , Vijayan Dipti , Ding Yitian , Vulink Annelie J. E. , McDonald Kylie J. , Jongbloed Sarah L. , Wadley Robert B. , Wells Christine , Hart Derek N. J. , Radford Kristen J.
Published: June 2012

Human blood myeloid DCs can be subdivided into CD1c (BDCA‐1)+ and CD141 (BDCA‐3)+ subsets that display unique gene expression profiles, suggesting specialized functions. CD1c+ DCs express TLR4 while CD141+ DCs do not, thus predicting that these two subsets have differential capacities to respond to Escherichia coli. We isolated highly purified CD1c+ and CD141+ DCs and compared them to in vitro generated monocyte‐derived DCs (MoDCs) following stimulation with whole E. coli. As expected, MoDCs produced high levels of the proinflammatory cytokines TNF, IL‐6, and IL‐12, were potent inducers of Th1 responses, and processed E. coli‐derived Ag. In contrast, CD1c+ DCs produced only low levels of TNF, IL‐6, and IL‐12 and instead produced high levels of the anti‐inflammatory cytokine IL‐10 and regulatory molecules IDO and soluble CD25. Moreover, E. coli‐activated CD1c+ DCs suppressed T‐cell proliferation in an IL‐10‐dependent manner. Contrary to their mouse CD8+ DC counterparts, human CD141+ DCs did not phagocytose or process E. coli‐derived Ag and failed to secrete cytokines in response to E. coli. These data demonstrate substantial differences in the nature of the response of human blood DC subsets to E. coli.
Phox activity of differentiated PLB‐985 cells is enhanced, in an agonist specific manner, by the PLA2 activity of Prdx6‐PLA2
Authors: Ellison Michael A. , Thurman Gail W. , Ambruso Daniel R.
Published: June 2012

Peroxiredoxin 6‐phospholipase A2 (Prdx6‐PLA2) is a bi‐functional enzyme with peroxi‐redoxin (Prdx) and phospholipase A2 (PLA2) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB‐985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged. Reintroduction of shRNA‐resistant Prdx6‐PLA2 into the knockdown cells by stable transfection with a Prdx6‐PLA2 expression plasmid restored the fMLF response, as did reintroduction of Prdx6‐PLA2 mutated in the Prdx active site; reintroduction of PLA2 active site mutants, however, failed to restore the response. Thus, the PLA2 activity of Prdx6‐PLA2 in intact cells mediates its ability to enhance phox activity in response to fMLF. In combination with previous publications by other groups, our work indicates that various PLA2 isoforms can enhance oxidase activity but they are differentially important in different cell types and in the response to different agonists.
Enhanced GITR/GITRL interactions augment IL‐27 expression and induce IL‐10‐producing Tr‐1 like cells
Authors: Carrier Yijun , Whitters Matthew J. , Miyashiro Joy S. , LaBranche Timothy P. , Ramon Hilda E. , Benoit Stephen E. , Ryan Mark S. , Keegan Sean P. , Guay Heath , Douhan John , Collins Mary , Dunussi‐Joannopoulos Kyri , Medley Quintus G.
Published: June 2012

The glucocorticoid‐induced TNFR‐related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under‐stand the role of long‐term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five‐fold expansion of the Treg‐cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T‐cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3− IL‐10‐producing type 1 regulatory T (Tr‐1)‐like cells that suppress naïve T‐cell proliferation in an IL‐10‐dependent fashion. Increased IL‐27 production from Tg APCs and activation of c‐Maf in the Tr1‐like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T‐cell responses, which includes promoting the differentiation of Tr‐1‐like cells, which contribute to the maintenance of peripheral T‐cell tolerance.
M‐CSF induces the expression of a membrane‐bound form of IL‐18 in a subset of human monocytes differentiating in vitro toward macrophages
Authors: Bellora Francesca , Castriconi Roberta , Doni Andrea , Cantoni Claudia , Moretta Lorenzo , Mantovani Alberto , Moretta Alessandro , Bottino Cristina
Published: June 2012

IL‐18 is a proinflammatory cytokine belonging to the “IL‐1 family” that has been shown to play a prominent role in the induction of type 1 immune responses. Here, we show that M‐CSFinduces the expression of a membrane‐bound form of IL‐18 (mIL‐18) in a subset of human blood monocytes differentiating toward macrophages. While monocytes, DC, and GM‐CSF‐treated monocytes did not express mIL‐18, its expression was detected in approximately 30–40% of M‐CSF‐primed macrophages differentiating from both CD16− and CD16+ monocytes. Treatment with the caspase‐1 inhibitor significantly reduced mIL‐18 expression suggesting the requirement of an assembled inflammasome for IL‐18 surface expression. Polarization toward M2 did not modify mIL‐18 expression. On the contrary, LPS stimulation of both M0 and M2 (mIL‐18+) macrophages induced shedding of mIL‐18, which was likely mediated by the activation of cellular protease(s). Importantly, the soluble form IL‐18 (sIL‐18) induced in autologous resting NK cells both the expression of CCR7 and the production of high amounts of IFN‐γ, which was virtually abrogated by Ab‐mediated neutralization of sIL‐18. Overall our data shed new light on the cells and mechanisms leading to the release of sIL‐18, the major IFN‐γ‐inducing factor in both physiological and pathological immune responses.
Human TCRγδ+T cells represent a novel target for IL‐27 activity
Authors: Morandi Fabio , Prigione Ignazia , Airoldi Irma
Published: June 2012

IL‐27 and TCRγδ+ T lymphocytes play critical roles in both innate and adaptive immune responses in health and disease, including infection and tumors. Although the activity of IL‐27 is well characterized in different human immune cells, no information is available on the role of IL‐27 in human TCRγδ+ T lymphocytes. Here, we provide the first evidence that TCRγδ+ T lymphocytes express both gp130 and WSX‐1 chains of IL‐27R, and that IL‐27 may function in TCRγδ+ T cells by (i) inducing STAT1 and STAT3 phosphorylation, (ii) stimulating cytotoxicity against tumor cells through upregulation of cytotoxic granules production, (iii) reducing the release of Th2‐related cytokines, such as IL‐5 and IL‐13, and inducing IFN‐γ production, and (iv) upregulating the expression of CD62L. These results highlighted a novel immunoregulatory property of human IL‐27 that may be relevant in the immune response against tumors. Our results may offer new perspectives for the development of future clinical trials using IL‐27 and TCRγδ+ cells for cancer immunotherapy.
Site‐specific accumulation of recently activated CD4+Foxp3+ regulatory T cells following adoptive transfer
Authors: Lieberman Scott M. , Kim Jiyeon S. , Corbo‐Rodgers Evann , Kambayashi Taku , Maltzman Jonathan S. , Behrens Edward M. , Turka Laurence A.
Published: June 2012

CD4+Foxp3+ regulatory T (Treg) cells are required for the maintenance of self‐tolerance, as demonstrated by profound autoimmunity in mice and humans with inactivating Foxp3 mutations. Recent studies demonstrate that Treg cells are anatomically compartmentalized within secondary lymphoid organs based on their TCR repertoire and specific organ‐protective function; however, whether this reflects differential homing or in situ selection is not known. Here, using Foxp3‐GFP reporter mice, we have examined the ability of polyclonal Treg cells from cervical LNs to return to their site‐of‐origin following adoptive transfer to nonlymphopenic congenic recipients. We find that bulk cervical LN Treg cells do not home directly to cervical LNs but rather accumulate site specifically over time following transfer. Site‐specific enrichment is both more rapid and more pronounced among a population of recently activated (CD69+) Treg cells. These data suggest that compartmentalization of Treg cells within secondary lymphoid organs may be governed by antigen recognition and implicate CD69 as a potential marker of recently activated Treg cells recognizing locally expressed antigens.
p53 suppresses type II endometrial carcinomas in mice and governs endometrial tumour aggressiveness in humans
Authors: Wild Peter J. , Ikenberg Kristian , Fuchs Thomas J. , Rechsteiner Markus , Georgiev Strahil , Fankhauser Niklaus , Noske Aurelia , Roessle Matthias , Caduff Rosmarie , Dellas Athanassios , Fink Daniel , Moch Holger , Krek Wilhelm , Frew Ian J.
Published: June 2012

Type II endometrial carcinomas are a highly aggressive group of tumour subtypes that are frequently associated with inactivation of the TP53 tumour suppressor gene. We show that mice with endometrium‐specific deletion of Trp53 initially exhibited histological changes that are identical to known precursor lesions of type II endometrial carcinomas in humans and later developed carcinomas representing all type II subtypes. The mTORC1 signalling pathway was frequently activated in these precursor lesions and tumours, suggesting a genetic cooperation between this pathway and Trp53 deficiency in tumour initiation. Consistent with this idea, analyses of 521 human endometrial carcinomas identified frequent mTORC1 pathway activation in type I as well as type II endometrial carcinoma subtypes. mTORC1 pathway activation and p53 expression or mutation status each independently predicted poor patient survival. We suggest that molecular alterations in p53 and the mTORC1 pathway play different roles in the initiation of the different endometrial cancer subtypes, but that combined p53 inactivation and mTORC1 pathway activation are unifying pathogenic features among histologically diverse subtypes of late stage aggressive endometrial tumours.
Eating Disorders in the General Practice: A Case–Control Study on the Utilization of Primary Care
Authors: Van Son Gabriëlle E. , Hoek Hans W. , Van Hoeken Daphne , Schellevis François G. , Van Furth Eric F.
Published: June 2012

Objective

To investigate primary care utilization between patients with an eating disorder (ED) and other patient groups, and between the ED subgroups anorexia nervosa (AN) and bulimia nervosa (BN).

Method

The present study was an observational case–control study. In total, 167 patients with ED were matched with two control groups (with and without mental disorders). General practitioners (GPs) kept electronic records and provided all patient contacts, prescriptions and referrals with a diagnostic code.

Results

Although patients with BN have the highest number of face‐to‐face contacts compared with all other groups, these patients less often seek help for eating problems compared with patients with AN, even when the ED diagnosis is known to the GP. Overall, patients with mental disorders showed a comparable rate of GP care, which was elevated compared with patients without mental disorders.

Discussion

Patients with BN might need more active encouragement by the GP to talk about their eating problems because there are indications that point at an unmet need for GP care. Copyright © 2012 John Wiley & Sons, Ltd and Eating Disorders Association.
Evaluation in Latin America: Paradigms and practices
Authors: Neirotti Nerio
Published: June 2012

This chapter addresses the development of evaluation, as a practice and as a field of knowledge, throughout Latin America in relation to the sociopolitical context, including characteristics of the state and its relations with civil society. It comes from the perspectives of evaluation as a learning tool, as a means to improve accountability and transparency in public administration, and as a dialogic device. The political context is described in terms of three historical moments: (a) the existence of the welfare state, (b) the withdrawal of the state in the framework of the Washington consensus and the emergence of neoconservative policies, and (c) the return of the state and the rise of civil society and active politics. Finally, there is an analysis of opportunities and challenges facing contemporary policy evaluators and decision makers. ©Wiley Periodicals, Inc., and the American Evaluation Association.