bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial genetic features of eastern oysters (Crassostrea virginica Gmelin) in the Gulf of Mexico: northward movement of a secondary contact zone
Authors: Anderson Joel D. , Karel William J. , Mace Christopher E. , Bartram Brian L. , Hare Matthew P.
Published: April 2014

The eastern oyster (Crassostrea virginica Gmelin) is an economically and ecologically valuable marine bivalve occurring in the Gulf of Mexico. This study builds upon previous research that identified two divergent populations of eastern oysters in the western Gulf of Mexico. Allelic and genotypic patterns from 11 microsatellite markers were used to assess genetic structure and migration between the previously described oyster populations in Texas. The main findings are as follows: (1) there are two distinct populations (FST = 0.392, P < 0.001) of oysters that overlap in the Corpus Christi/Aransas Bay estuarine complex in Texas, (2) the distribution of genotypes among individuals in the contact zone suggests limited hybridization between populations, (3) the variables of salinity, temperature, dissolved oxygen, turbidity and depth are not correlated with allele frequencies on reefs in the contact zone or when analyzed across Texas, and (4) there is little evidence of directional selection acting on the loci assayed here, although patterns at four markers suggested the influence of balancing selection based on outlier analyses. These results are consistent with long‐term historical isolation between populations, followed by secondary contact. Recent hydrological changes in the area of secondary contact may be promoting migration in areas that were previously inhospitable to eastern oysters, and observed differences in the timing of spawning may limit hybridization between populations. Comparison of these findings with the results of an earlier study of oysters in Texas suggests that the secondary contact zone has shifted approximately 27 km north, in as little as a 23‐year span.
DNA vaccines encoding membrane‐bound or secreted forms of heat shock protein 70 exhibit improved potency
Authors: Garrod Tamsin J. , Grubor‐Bauk Branka , Gargett Tessa , Li Yanrui , Miller Darren S. , Yu Wenbo , Major Lee , Burrell Christopher J. , Wesselingh Steven , Suhrbier Andreas , Gowans Eric J.
Published: April 2014

Traditional vaccine strategies are inefficient against challenge with complex pathogens including HIV; therefore, novel vaccine technologies are required. DNA vaccines are attractive as they are relatively cheap and easy to manufacture, but a major limitation has been their lack of immunogenicity in humans, which may be overcome with the incorporation of an adjuvant. HSP70 is a recognised damage‐associated molecular pattern, which is a potential adjuvant. We investigated the immunogenicity of a DNA vaccine encoding HIV gag and HSP70; the latter was genetically modified to produce cytoplasmic, secreted or membrane‐bound HSP70, the expression of which was controlled by an independent promoter. The DNA was administered to C57BL/6 mice to evaluate gag‐specific T‐cell responses. Our results demonstrated the ability of membrane‐bound and secreted HSP70 to significantly enhance gag‐specific T‐cell responses and increase the breadth of T‐cell responses to include subdominant epitopes. Membrane‐bound or secreted HSP70 also significantly improved the multifunctionality of HIV‐specific T cells and T‐cell proliferation, which is important for maintaining T‐cell integrity. Most importantly, the inclusion of membrane‐bound HSP70, secreted HSP70 or a combination significantly increased protection in mice challenged with EcoHIV, a chimeric virus that replicates in mouse leukocytes in vivo.
Tetraspanins CD9 and CD151 at the immune synapse support T‐cell integrin signaling
Authors: Rocha‐Perugini Vera , González‐Granado José Maria , Tejera Emilio , López‐Martín Soraya , Yañez‐Mó Maria , Sánchez‐Madrid Francisco
Published: April 2014

Understanding how the immune response is activated and amplified requires detailed knowledge of the stages in the formation of the immunological synapse (IS) between T lymphocytes and antigen‐presenting cells (APCs). We show that tetraspanins CD9 and CD151 congregate at the T‐cell side of the IS. Silencing of CD9 or CD151 blunts the IL‐2 secretion and expression of the activation marker CD69 by APC‐conjugated T lymphocytes, but does not affect the accumulation of CD3 or actin to the IS, or the translocation of the microtubule‐organizing center toward the T‐B contact area. CD9 or CD151 silencing diminishes the relocalization of α4β1 integrin to the IS and reduces the accumulation of high‐affinity β1 integrins at the cell–cell contact. These changes are accompanied by diminished phosphorylation of the integrin downstream targets FAK and ERK1/2. Our results suggest that CD9 and CD151 support integrin‐mediated signaling at the IS.
Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL‐10 and suppress T‐cell proliferation in sarcoidosis
Authors: Crawshaw Anjali , Kendrick Yvonne R. , McMichael Andrew J. , Ho Ling‐Pei
Published: April 2014

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T‐cell expansion of T helper 1 (Th1) cells. The cause of T‐cell overactivity is unknown. We hypothesized that interleukin‐10 (IL‐10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T‐cell activity. Focusing on IL‐10‐producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid‐naïve sarcoidosis patients (n = 51) produced less IL‐10 compared to controls, and were less able to suppress T‐cell proliferation. In addition, monocytic IL‐10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT‐specific defects might be responsible for this reduced IL‐10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL‐10 production. Moreover, iNKT cells enhanced monocytic IL‐10 production in vitro. Defective IL‐10 production and T‐cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d‐ and cell contact‐dependent process. We suggest that reduced iNKT‐cell numbers in sarcoidosis may lead to impaired monocytic IL‐10 production and unchecked T‐cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes.
One‐Pot Transformation of Methylarenes into Aromatic Aldehydes under Metal‐Free Conditions
Authors: Tabata Masayuki , Moriyama Katsuhiko , Togo Hideo
Published: April 2014

On the basis of studies of the transformation of benzylic bromides into the corresponding aromatic aldehydes by treatment with N‐methylmorpholine N‐oxide, various methylarenes were treated either with DBDMH in the presence of AIBN in acetonitrile at reflux (Method A) or with NBS in CCl4 under irradiation with a tungsten lamp at 30 °C (Method B), followed by treatment with N‐methylmorpholine N‐oxide to provide aromatic aldehydes in good yields. These methods could be adopted in one‐pot transformations of methylarenes into aromatic aldehydes under conditions free of less toxic reagents and transition metals.
Neighboring ortho‐Hydroxy Group Directed Catalytic Asymmetric Triple Domino Reactions of Acetaldehyde with (E)‐2‐(2‐Nitrovinyl)phenols
Authors: Ramachary Dhevalapally B. , Reddy P. Srinivasa , Prasad M. Shiva
Published: April 2014

Herein we demonstrate the utilization of neighboring ortho‐hydroxy group participation in the pretransition state of enamine‐ and iminium‐based triple domino reactions for high reactions rates and asymmetric induction. Enantiomerically pure, drug‐like chromanes and tetrahydro‐6H‐benzo[c]chromenes having three to four contiguous stereocenters are synthesized through triple domino Michael/aldol/oxa‐Michael reactions catalyzed by (R)‐2‐{diphenyl[(trimethylsilyl)oxy]methyl}pyrrolidine followed by Wittig and Michael/Wittig–Horner reactions from simple precursors under mild conditions.
Mortality salience and evaluations of in‐group versus out‐group critics: The role of criticism legitimacy and perceived threat
Authors: Khoo Bernice L. Z. , See Ya Hui Michelle
Published: April 2014

Significant terror management research has examined the impact of mortality salience on evaluations toward in‐group versus out‐group and attitudinally similar versus dissimilar others. However, relatively little research has examined evaluations when group membership is disentangled from attitude similarity. The current research examined the impact of mortality salience on evaluations toward in‐group and out‐group critics when people are less likely to rely on group membership as a heuristic. In Experiment 1, the results showed that in the control condition, participants rated an in‐group member who provided unjustified criticism more positively than an out‐group member who provided the same criticism. Under mortality salience, the reverse occurred: An in‐group member who provided unjustified criticism was rated more negatively than an out‐group member. Experiment 2 showed that under mortality salience, the derogation of an in‐group critic who provided unjustified criticism was mediated by perceptions of threat. Implications for reactions to group‐directed criticism as well as mortality salience effects are discussed. Copyright © 2014 John Wiley & Sons, Ltd.
The ultra‐social animal
Authors: Tomasello Michael
Published: April 2014

In evolutionary perspective, what is most remarkable about human sociality is its many and diverse forms of cooperation. Here, I provide an overview of some recent research, mostly from our laboratory, comparing human children with their nearest living relatives, the great apes, in various tests of collaboration, prosocial behavior, conformity, and group‐mindedness (e.g., following and enforcing social norms). This is done in the context of a hypothetical evolutionary scenario comprising two ordered steps: a first step in which early humans began collaborating with others in unique ways in their everyday foraging and a second step in which modern humans began forming cultural groups. Humans' unique forms of sociality help to explain their unique forms of cognition and morality. © 2014. The Authors. European Journal of Social Psychology published by John Wiley & Sons, Ltd.
Surface modification of PDMS microchips with poly(ethylene glycol) derivatives for μTAS applications
Authors: de Campos Richard Piffer Soares , Yoshida Inez Valeria Pagotto , da Silva José Alberto Fracassi
Published: April 2014

In this work is presented a method for the modification of native PDMS surface in order to improve its applicability as a substrate for microfluidic devices, especially in the analysis of nonpolar analytes. Therefore, poly(ethylene glycol) divinyl ether modified PDMS substrate was obtained by surface modification of native PDMS. The modified substrate was characterized by attenuated total reflectance infrared spectroscopy, water contact angle measurements, and by evaluating the adsorption of rhodamine B and the magnitude of the EOF mobility. The reaction was confirmed by the spectroscopic evaluation. The formation of a well‐spread water film over the surface immediately after the modification was an indicative of the modified surface hydrophilicity. This characteristic was maintained for approximately ten days, with a gradual return to a hydrophobic state. Fluorescence assays showed that the nonpolar adsorption property of PDMS was significantly decreased. The EOF mobility obtained was 3.6 × 10−4 cm2 V−1s−1, higher than the typical values found for native PDMS. Due to the better wettability promoted by the modification, the filling of the microchannels with aqueous solutions was facilitated and trapping of air bubbles was not observed.
Highly sensitive microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer for the detection of neuron‐specific enolase
Authors: Yang Tingzhen , Vdovenko Marina , Jin Xue , Sakharov Ivan Yu. , Zhao Shulin
Published: April 2014

A microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer (CRET) was developed for highly sensitive detection of neuron‐specific enolase (NSE). The CRET system consisted of horseradish peroxidase (HRP)/luminol as a light donor and fluorescein isothiocyanate as an acceptor. When fluorescein isothiocyanate‐labeled antibody binds with HRP‐labeled antigen to form immunocomplex, the donor and acceptor are brought close each other and CRET occurs in the immunocomplex. In the MCE, the immunocomplex and excess HRP–NSE were separated, and the chemiluminescense intensity of immunocomplex was used to estimate NSE concentration. The calibration curve showed a linearity in the range of NSE concentrations from 9.0 to 950 pM with a correlation coefficient of 0.9964. Based on a S/N of 3, the detection limit for NSE determination was estimated to be 4.5 pM, which is two‐order magnitude lower than that of without CRET detection. This assay was applied for NSE quantification in human serum. The obtained results demonstrated that the proposed immunoassay may serve as an alternative tool for clinical analysis of NSE.
Cationic isotachophoresis separation of the biomarker cardiac troponin I from a high‐abundance contaminant, serum albumin
Authors: Jacroux Thomas , Bottenus Danny , Rieck Bennett , Ivory Cornelius F. , Dong Wen‐ji
Published: April 2014

Cationic ITP was used to separate and concentrate fluorescently tagged cardiac troponin I (cTnI) from two proteins with similar isoelectric properties in a PMMA straight‐channel microfluidic chip. In an initial set of experiments, cTnI was effectively separated from R‐Phycoerythrin using cationic ITP in a pH 8 buffer system. Then, a second set of experiments was conducted in which cTnI was separated from a serum contaminant, albumin. Each experiment took ∼10 min or less at low electric field strengths (34 V/cm) and demonstrated that cationic ITP could be used as an on‐chip removal technique to isolate cTnI from albumin. In addition to the experimental work, a 1D numerical simulation of our cationic ITP experiments has been included to qualitatively validate experimental observations.
Biomarker discovery for neuroendocrine cervical cancer
Authors: Lin Li‐Hsun , Chang Shing‐Jyh , Hu Ren‐Yu , Lin Meng‐Wei , Lin Szu‐Ting , Huang Shun‐Hong , Lyu Ping‐Chiang , Chou Hsiu‐Chuan , Lai Zih‐Yin , Chuang Yung‐Jen , Chan Hong‐Lin
Published: April 2014

Neuroendocrine cervical cancer is an aggressive but rare form of cervical cancer. The majority of neuroendocrine cervical cancer patients present with advanced‐stage diseases. However, the limited numbers of neuroendocrine tumor markers are insufficient for clinical purposes. Thus, we used a proteomic approach combining lysine labeling 2D‐DIGE and MALDI‐TOF MS to investigate the biomarkers for neuroendocrine cervical cancer. By analyzing the global proteome alteration between the neuroendocrine cervical cancer line (HM‐1) and non‐neuroendocrine cervical cancer lines (CaSki cells, ME‐180 cells, and Hela cells), we identified 82 proteins exhibiting marked changes between HM‐1 and CaSki cells, and between ME‐180 and Hela cells. Several proteins involved in protein folding, cytoskeleton, transcription control, signal transduction, glycolysis, and redox regulation exhibited significant changes in abundance. Proteomic and immunoblot analyses indicated respective 49.88‐fold and 25‐fold increased levels of transgelin in HM‐1 cells compared with that in other non‐neuroendocrine cervical cancer cell lines, implying that transgelin is a biomarker for neuroendocrine cervical cancer. In summary, we used a comprehensive neuroendocrine/non‐neuroendocrine cervical cancer model based proteomic approach for identifying neuroendocrine cervical cancer markers, which might contribute to the prognosis and diagnosis of neuroendocrine cervical cancer.
Tissue phosphoproteomics with PolyMAC identifies potential therapeutic targets in a transgenic mouse model of HER2 positive breast cancer
Authors: Searleman Adam C. , Iliuk Anton B. , Collier Timothy S. , Chodosh Lewis A. , Tao W. Andy , Bose Ron
Published: April 2014

Altered protein phosphorylation is a feature of many human cancers that can be targeted therapeutically. Phosphopeptide enrichment is a critical step for maximizing the depth of phosphoproteome coverage by MS, but remains challenging for tissue specimens because of their high complexity. We describe the first analysis of a tissue phosphoproteome using polymer‐based metal ion affinity capture (PolyMAC), a nanopolymer that has excellent yield and specificity for phosphopeptide enrichment, on a transgenic mouse model of HER2‐driven breast cancer. By combining phosphotyrosine immunoprecipitation with PolyMAC, 411 unique peptides with 139 phosphotyrosine, 45 phosphoserine, and 29 phosphothreonine sites were identified from five LC‐MS/MS runs. Combining reverse phase liquid chromatography fractionation at pH 8.0 with PolyMAC identified 1571 unique peptides with 1279 phosphoserine, 213 phosphothreonine, and 21 phosphotyrosine sites from eight LC‐MS/MS runs. Linear motif analysis indicated that many of the phosphosites correspond to well‐known phosphorylation motifs. Analysis of the tyrosine phosphoproteome with the Drug Gene Interaction database uncovered a network of potential therapeutic targets centered on Src family kinases with inhibitors that are either FDA‐approved or in clinical development. These results demonstrate that PolyMAC is well suited for phosphoproteomic analysis of tissue specimens.
Chiral separation of 12 cathinone analogs by cyclodextrin‐assisted capillary electrophoresis with UV and mass spectrometry detection
Authors: Merola Gustavo , Fu Hanzhuo , Tagliaro Franco , Macchia Teodora , McCord Bruce R.
Published: April 2014

In this study, a rapid chiral separation of 12 cathinones analogs has been developed and validated using cyclodextrin‐assisted CE with UV and TOF‐MS detection. Optimum separation was obtained on a 57.5 cm × 50 μm capillary using a buffer system consisting of 10 mM β‐cyclodextrin (β‐CD) in a 100 mM phosphate buffer for CE‐UV, and 0.6% v/v highly sulfated‐γ‐cyclodextrin (HS‐γ‐CD) in a 50 mM phosphate buffer for CE‐MS. In the CE‐MS experiment, a partial filling technique was employed to ensure that a minimum amount of cyclodextrin entered the mass spectrometer. All analytes were separated within 18 min in the CE‐UV separation and identified by TOF‐MS. Ten compounds were enantiomerically separated using β‐CD in the UV mode and an additional two more were enantiomerically separated using HS‐γ‐CD in the MS mode. Detection limits down to 1.0 ng/mL were obtained. The method was then applied to examine seized drugs.
Separation and characterization of phlorotannins from brown algae Cystoseira abies‐marina by comprehensive two‐dimensional liquid chromatography
Authors: Montero Lidia , Herrero Miguel , Ibáñez Elena , Cifuentes Alejandro
Published: April 2014

Phlorotannins are an important class of polyphenolic compounds only found in brown algae. The chemical analysis of these bioactive polyphenols is rather difficult due to the great chemical variability and complexity of the natural composition of these components in algae, forming large phloroglucinol polymers. In the present work, a new approach based on the use of comprehensive 2D LC (LC × LC) is shown to analyze this complex family of compounds. The developed LC × LC methodology is based on the coupling of a hydrophilic‐interaction LC (HILIC)‐based separation in the first dimension and an RP‐based separation in the second dimension. The employment of this online coupling together with DAD and MS/MS allowed the separation and identification of more than 50 compounds in a Cystoseira abies‐marina brown alga extract. Phlorotannins containing from 5 to 17 phloroglucinol units were identified in this sample by HILIC × RP‐DAD‐MS/MS. Besides, using the 2,4‐dimethoxybenzaldehyde assay, it was possible to determine that the total amount of phlorotannins present in the extract was 40.2 mg phloroglucinol equivalents per gram of extract. To our knowledge, this work is the first demonstration of the usefulness of HILIC × RP‐DAD‐MS/MS for the determination of phlorotannins.
Genome‐Wide Family‐Based Linkage Analysis of Exome Chip Variants and Cardiometabolic Risk
Authors: Hellwege Jacklyn N. , Palmer Nicholette D. , Raffield Laura M. , Ng Maggie C.Y. , Hawkins Gregory A. , Long Jirong , Lorenzo Carlos , Norris Jill M. , Ida Chen Y.‐D. , Speliotes Elizabeth K. , Rotter Jerome I. , Langefeld Carl D. , Wagenknecht Lynne E. , Bowden Donald W.
Published: April 2014

Linkage analysis of complex traits has had limited success in identifying trait‐influencing loci. Recently, coding variants have been implicated as the basis for some biomedical associations. We tested whether coding variants are the basis for linkage peaks of complex traits in 42 African‐American (n = 596) and 90 Hispanic (n = 1,414) families in the Insulin Resistance Atherosclerosis Family Study (IRASFS) using Illumina HumanExome Beadchips. A total of 92,157 variants in African Americans (34%) and 81,559 (31%) in Hispanics were polymorphic and tested using two‐point linkage and association analyses with 37 cardiometabolic phenotypes. In African Americans 77 LOD scores greater than 3 were observed. The highest LOD score was 4.91 with the APOE SNP rs7412 (MAF = 0.13) with plasma apolipoprotein B (ApoB). This SNP was associated with ApoB (P‐value = 4 × 10−19) and accounted for 16.2% of the variance in African Americans. In Hispanic families, 104 LOD scores were greater than 3. The strongest evidence of linkage (LOD = 4.29) was with rs5882 (MAF = 0.46) in CETP with HDL. CETP variants were strongly associated with HDL (0.00049 < P‐value <4.6 × 10−12), accounting for up to 4.5% of the variance. These loci have previously been shown to have effects on the biomedical traits evaluated here. Thus, evidence of strong linkage in this genome wide survey of primarily coding variants was uncommon. Loci with strong evidence of linkage was characterized by large contributions to the variance, and, in these cases, are common variants. Less compelling evidence of linkage and association was observed with additional loci that may require larger family sets to confirm.
Bayesian Latent Variable Collapsing Model for Detecting Rare Variant Interaction Effect in Twin Study
Authors: He Liang , Sillanpää Mikko J. , Ripatti Samuli , Pitkäniemi Janne
Published: April 2014

By analyzing more next‐generation sequencing data, researchers have affirmed that rare genetic variants are widespread among populations and likely play an important role in complex phenotypes. Recently, a handful of statistical models have been developed to analyze rare variant (RV) association in different study designs. However, due to the scarce occurrence of minor alleles in data, appropriate statistical methods for detecting RV interaction effects are still difficult to develop. We propose a hierarchical Bayesian latent variable collapsing method (BLVCM), which circumvents the obstacles by parameterizing the signals of RVs with latent variables in a Bayesian framework and is parameterized for twin data. The BLVCM can tackle nonassociated variants, allow both protective and deleterious effects, capture SNP‐SNP synergistic effect, provide estimates for the gene level and individual SNP contributions, and can be applied to both independent and various twin designs. We assessed the statistical properties of the BLVCM using simulated data, and found that it achieved better performance in terms of power for interaction effect detection compared to the Granvil and the SKAT. As proof of practical application, the BLVCM was then applied to a twin study analysis of more than 20,000 gene regions to identify significant RVs associated with low‐density lipoprotein cholesterol level. The results show that some of the findings are consistent with previous studies, and we identified some novel gene regions with significant SNP–SNP synergistic effects.
Spreading depression requires microglia and is decreased by their M2a polarization from environmental enrichment
Authors: Pusic Kae M. , Pusic Aya D. , Kemme Jordan , Kraig Richard P.
Published: April 2014

Microglia play an important role in fine‐tuning neuronal activity. In part, this involves their production of tumor necrosis factor‐alpha (TNFα), which increases neuronal excitability. Excessive synaptic activity is necessary to initiate spreading depression (SD). Increased microglial production of proinflammatory cytokines promotes initiation of SD, which, when recurrent, may play a role in conversion of episodic to high frequency and chronic migraine. Previous work shows that this potentiation of SD occurs through increased microglial production of TNFα and reactive oxygen species, both of which are associated with an M1‐skewed microglial population. Hence, we explored the role of microglia and their M1 polarization in SD initiation. Selective ablation of microglia from rat hippocampal slice cultures confirmed that microglia are essential for initiation of SD. Application of minocycline to dampen M1 signaling led to increased SD threshold. In addition, we found that SD threshold was increased in rats exposed to environmental enrichment. These rats had increased neocortical levels of interleukin‐11 (IL‐11), which decreases TNFα signaling and polarized microglia to an M2a‐dominant phenotype. M2a microglia reduce proinflammatory signaling and increase production of anti‐inflammatory cytokines, and therefore may protect against SD. Nasal administration of IL‐11 to mimic effects of environmental enrichment likewise increased M2a polarization and increased SD threshold, an effect also seen in vitro. Similarly, application of conditioned medium from M2a polarized primary microglia to slice cultures also increased SD threshold. Thus, microglia and their polarization state play an essential role in SD initiation, and perhaps by extension migraine with aura and migraine. GLIA 2014;62:1176–1194
Changing hydrologic connectivity due to permafrost thaw in the lower Liard River valley, NWT, Canada
Authors: Connon Ryan F. , Quinton William L. , Craig James R. , Hayashi Masaki
Published: April 2014

Flows from river basins in northwestern Canada have been rising in the last two decades as a result of climate warming. In the wetland‐dominated basins that characterise the southern margin of permafrost, permafrost thaw and disappearance, and resulting land‐cover change, is occurring at an unprecedented rate. The impact of this thaw on runoff generation in headwater basins is poorly understood. Permafrost thaw has the potential to fundamentally alter the cycling and storage of moisture inputs in this region by altering the type and relative proportions of the major land‐cover types, such as peat plateaus, channel fens and flat bogs. This paper examines streamflow changes in the four Water Survey of Canada gauged river basins (152–2050 km2) in the lower Liard River valley, Northwest Territories, Canada, a region where permafrost thaw has produced widespread loss of forest and concomitant expansion of permafrost‐free wetlands. Annual runoff in the lower Liard Valley increased by between 112 and 160 mm over the period of 1996–2012. The Mann‐Kendall non‐parametric statistical test and the Kendall‐Theil robust line were used to ascertain changes in streamflow. Historical aerial photographs from 1977 and high‐resolution satellite imagery (WorldView 2) from 2010 were used to measure the rate and pattern of permafrost thaw in a representative 6 km2 area of Scotty Creek. Permafrost thaw‐induced land‐cover change is both increasing the adjacency between runoff producing and transmitting land cover types and transforming certain land covers that store water into ones that produce runoff. This land‐cover change was found to be the single most important factor (37–61 mm) contributing to the observed increase in river discharge. Other contributing factors include increases in plateau runoff contributing areas (20–32 mm), increases in annual effective precipitation depth (18–30 mm), contribution of water from the melt of ice within permafrost (9 mm) and increases in baseflow (0.9–6.8 mm). Although runoff has significantly (p < 0.05) increased in all four basins, the largest increases are in basins with a relatively high cover of flat bogs. Copyright © 2014 John Wiley & Sons, Ltd.
Novel biomimetic tripolymer scaffolds consisting of chitosan, collagen type 1, and hyaluronic acid for bone marrow‐derived human mesenchymal stem cells‐based bone tissue engineering
Authors: Mathews Smitha , Bhonde Ramesh , Gupta Pawan Kumar , Totey Satish
Published: April 2014

Human bone marrow‐derived mesenchymal stem cells (hMSCs) are an ideal osteogenic cell source for bone tissue engineering (BTE). A scaffold, in the context of BTE, is the extracellular matrix (ECM) that provides the unique microenvironment and play significant role in regulating cell behavior, differentiation, and development in an in vitro culture system. In this study, we have developed novel biomimetic tripolymer scaffolds for BTE using an ECM protein, collagen type 1; an ECM glycosaminoglycan, hyaluronic acid; and a natural osteoconductive polymer, chitosan. The scaffolds were characterized by scanning electron microscopy (SEM) and swelling ratio. The scaffolds were seeded with hMSCs and tested for cytocompatibility and osteogenic potential. The scaffolds supported cell adhesion, enhanced cell proliferation, promoted cell migration, showed good cell viability, and osteogenic potential. The cells were able to migrate out from the scaffolds in favorable conditions. SEM, alkaline phosphatase assay, and immunofluorescent staining confirmed the differentiation of hMSCs to osteogenic lineage in the scaffolds. In conclusion, we have successfully developed biomimetic scaffolds that supported the proliferation and differentiation of hMSCs. These scaffolds hold great promise as a cell‐delivery vehicle for regenerative therapies and as a support system for enhancing bone regeneration. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 1825–1834, 2014.
Fluorescent in situ hybridization of synaptic proteins imaged with super‐resolution STED microscopy
Authors: Zhang William I. , Röhse Heiko , Rizzoli Silvio O. , Opazo Felipe
Published: April 2014

Super‐resolution fluorescence microscopy is still a developing field. One of the limitations has been that standard labeling assays, which had been developed for conventional imaging, must be adjusted and optimized for each super‐resolution method. These methods are more sensitive to noise, and require more intense labeling than conventional microscopy, which is not always trivial to achieve. Here, we describe the use of stimulation‐emission depletion (STED) microscopy to locate messenger RNAs (mRNAs) in single neurons with high spatial precision. We address several technical difficulties we encountered in using fluorescent in situ hybridization (FISH) for STED imaging. We optimized the experimental protocol to detect mRNAs and proteins simultaneously, by performing FISH and immunostaining on the same samples. We tested our imaging approach in primary hippocampal neurons, studying the mRNAs of three important presynaptic proteins (synaptobrevin, synaptotagmin, and synaptophysin). Our approach allowed us to relate changes in mRNA levels and localization to neuronal physiology, under different activity regimes and also during neuronal development. We conclude that FISH can be performed efficiently using super‐resolution techniques. This should contribute significantly to the clarification of the molecular mechanisms that govern mRNA distribution and dynamics within cells. Microsc. Res. Tech. 77:517–527, 2014. © 2014 Wiley Periodicals, Inc.
Practical methods for improving homogeneity in 3 tesla breast imaging
Authors: Winkler Simone A. , Rutt Brian K.
Published: April 2014

Purpose

To improve image contrast and
field homogeneity in 3 Tesla (T) breast MR.

Materials and Methods

Two practical
shimming methods for 3T breast MR are presented; low‐cost passive shimming using local pads of high dielectric permittivity (εr from 0 to 100), and two‐channel radiofrequency (RF) shimming (adjusting Q‐I amplitude ratios and phase differences of 0 to −4 dB and 90 to 45 degrees), as well as a combination of both methods. The technique has been studied both in simulation using a numerical body model with added mammary tissue and in vivo in six subjects.

Results

Large improvements are observed with both methods, leading to a decrease in left–right
asymmetry ratio of 1.24 to 1.00 (simulation) and from 1.26 to 1.01 (in vivo). RF safety was not adversely affected.

Conclusion

Both RF shimming and dielectric shimming were shown to improve inhomogeneity in the
field in 3T breast MR. J. Magn. Reson. Imaging 2015;41:992–999. © 2014 Wiley Periodicals, Inc.
Measurement of tumor blood flow in head and neck squamous cell carcinoma by pseudo‐continuous arterial spin labeling: Comparison with dynamic contrast‐enhanced MRI
Authors: Fujima Noriyuki , Kudo Kohsuke , Tsukahara Akiko , Yoshida Daisuke , Sakashita Tomohiro , Homma Akihiro , Tha Khin Khin , Shirato Hiroki
Published: April 2014

Purpose

To investigate the feasibility of tumor blood flow (TBF) measurement in head and neck squamous cell carcinoma (HNSCC) using pseudo‐continuous arterial spin labeling (pCASL) in a comparison with dynamic contrast‐enhanced (DCE) perfusion.

Materials and Methods

We prospectively scanned 18 patients with HNSCC using 3T magnetic resonance imaging (MRI) with both pCASL and DCE perfusion. Quantitative TBF value in the whole‐tumor region of interest (ROI), and regional TBF in the ROIs of the central and peripheral areas in the tumor were respectively measured. Relative TBF value in the whole‐tumor ROI was also calculated. We determined the correlation and agreement between each measured TBF by pCASL and DCE perfusion using Pearson's correlation coefficients, intraclass correlation coefficients (ICC), and Bland–Altman analysis.

Results

In the whole‐tumor ROIs, significant correlation was observed between the absolute TBF values (r = 0.72, P < 0.01), with an ICC of 0.72; moreover, higher correlation was observed in the relative TBF (r = 0.79). The correlation was higher in the peripheral ROI (r = 0.70) than the central ROI (r = 0.65), with an ICC of 0.62 and 0.54, respectively. Bland–Altman plots revealed the underestimation of TBF by pCASL in central ROIs.

Conclusion

TBF measurement by pCASL was feasible in patients with HNSCC. J. Magn. Reson. Imaging 2015;41:983–991. © 2014 Wiley Periodicals, Inc.
GABA‐induced uncoupling of GABA/benzodiazepine site interactions is associated with increased phosphorylation of the GABAA receptor
Authors: Gutiérrez María L. , Ferreri María C. , Farb David H. , Gravielle María C.
Published: April 2014

The use‐dependent regulation of the GABAA receptor occurs under physiological, pathological, and pharmacological conditions. Tolerance induced by prolonged administration of benzodiazepines is associated with changes in GABAA receptor function. Chronic exposure of neurons to GABA for 48 hr induces a downregulation of the GABAA receptor number and an uncoupling of the GABA/benzodiazepine site interactions. A single brief exposure (t1/2 = 3 min) of rat neocortical neurons to the neurotransmitter initiates a process that results in uncoupling hours later (t1/2 = 12 hr) without alterations in the number of GABAA receptors and provides a paradigm to study the uncoupling mechanism selectively. Here we report that uncoupling induced by a brief GABAA receptor activation is blocked by the coincubation with inhibitors of protein kinases A and C, indicating that the uncoupling is mediated by the activation of a phosphorylation cascade. GABA‐induced uncoupling is accompanied by subunit‐selective changes in the GABAA receptor mRNA levels. However, the GABA‐induced downregulation of the α3 subunit mRNA level is not altered by the kinase inhibitors, suggesting that the uncoupling is the result of a posttranscriptional regulatory process. GABA exposure also produces an increase in the serine phosphorylation on the GABAA receptor γ2 subunit. Taken together, our results suggest that the GABA‐induced uncoupling is mediated by a posttranscriptional mechanism involving an increase in the phosphorylation of GABAA receptors. The uncoupling of the GABAA receptor may represent a compensatory mechanism to control GABAergic neurotransmission under conditions in which receptors are persistently activated. © 2014 Wiley Periodicals, Inc.
Determination of polar organic compounds in atmospheric aerosols by gas chromatography with ion trap tandem mass spectrometry
Authors: Visentin Marco , Pietrogrande Maria Chiara
Published: April 2014

A gas chromatography with ion trap mass spectrometry method has been developed and validated for the analysis of 27 polar organic compounds in atmospheric aerosols. The target analytes were low‐molecular‐weight carboxylic acids and methoxyphenols, as relevant markers of source emissions and photochemical processes of organic aerosols. The operative parameters were optimized in order to achieve the best sensitivity and selectivity for the analysis. In comparison with the previous gas chromatography with mass spectrometry procedure based on single ion monitoring detection, the tandem mass spectrometry technique increased the analytical sensitivity by reducing detection limits for standard solutions from 1–2.6 to 0.1–0.4 ng/μL ranges (concentrations in the injected solution). In addition, it enhanced selectivity by reducing matrix interferences and chemical noise in the chromatogram. The applicability of the developed method in air quality monitoring campaigns was effectively checked by analyzing environmental samples collected in the Po Valley (Northern Italy) in different seasons. The obtained results indicate that the ion trap mass spectrometer may be an ideal alternative to high‐resolution mass spectrometers for the user‐friendly and cost‐effective determination of a wide range of molecular tracers in airborne particulate matter.
Identification and analysis of alkaloids in cortex Phellodendron amurense by high‐performance liquid chromatography with electrospray ionization mass spectrometry coupled with photodiode array detection
Authors: Xian Xiaoyan , Sun Bohang , Ye Xueting , Zhang Guanying , Hou Pengyi , Gao Huiyuan
Published: April 2014

Alkaloids from Cortex Phellodendron amurense Rupr. were identified to determine the material basis for the bioactivity of this herb. HPLC–ESI‐MS with photodiode array detection coupled to XCharge C18 column was applied to analyze the alkaloids qualitatively and quantitatively. A total of 37 alkaloids were identified and tentatively characterized from the ethanol extract by online ESI‐MSn fragmentation and UV spectral analysis. A total of ten alkaloids, including four novel natural products, were tentatively identified for the first time in P. amurense. The fragmentation pathways for certain compounds were analyzed. The contents of a pair of isomers (columbamine and jatrorrhizine) and four main alkaloids (phellodendrine, magnoflorine, berberine, and palmatine) were simultaneously quantified using the aforementioned method. Results showed that the newly discovered and known components of P. amurense were helpful in determining the material basis for the bioactivity of the herb. The application of the XCharge C18 column is a suitable and practical method for the isolation of alkaloids in plants.
Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography
Authors: Chen Miao , Liu Liangliang , Chen Xiaoqing
Published: April 2014

A simple, rapid, and effective assay based on ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target‐guidance of ultrafiltration combined with the high‐performance liquid chromatography experiment, liquiritin (1), isoliquiritin (2), and liquiritigenin (3) were separated by high‐speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC50 of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high‐performance liquid chromatography and high‐speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.
Minimization of carryover for high‐throughput liquid chromatography with tandem mass spectrometry analysis of 14 mycotoxins in corn grits
Authors: Tamura Masayoshi , Matsumoto Keiko , Watanabe Jun , Iida Junko , Nagatomi Yasushi , Mochizuki Naoki
Published: April 2014

A method for the simultaneous analysis of 14 mycotoxins with the minimization of carryover was developed. Our verification experiments suggested that the carryover occurred due to the chelation of fumonisins with the metal. To wash the fumonisins from the metal, the inner surface of the injection needle was rinsed with 10 mM trisodium citrate and 1% formic acid in water/methanol/acetonitrile/isopropanol after each injection, and the analysis was performed on a metal‐free Mastro C18 column. This approach remarkably minimized the carryover of fumonisins. Fourteen mycotoxins in samples were extracted with 2% acetic acid in water/acetonitrile and a quick, easy, cheap, effective, rugged, and safe extraction kit, purified on a MultiSep 229 Ochra, and then quantified by liquid chromatography with tandem mass spectrometry. Determinations performed using this method produced a linearity greater than 0.99 and recoveries ranging from 72.6 to 117.4%, with good intraday precision from 4.0 to 12.4%, and interday precision from 6.5 to 17.0%. The limits of detection ranged from 0.01 to 0.71 μg/kg, demonstrating that a highly sensitive method for the simultaneous analysis of mycotoxins over a wide range of concentrations was achieved with minimal carryover. When 12 samples of commercially available corn grits were analyzed with this method, deoxynivalenol, fumonisin B1, fumonisin B2, fumonisin B3, and zearalenone were present most frequently.
Determination of Sudan dyes in chili pepper powder by online solid‐phase extraction with a butyl methacrylate monolithic column coupled to liquid chromatography with tandem mass spectrometry
Authors: Liu Yao , Wang Man‐Man , Ai Lian‐Feng , Zhang Chang‐Kun , Li Xin , Wang Xue‐Sheng
Published: April 2014

A poly(butyl methacrylate‐co‐ethylene dimethacrylate) monolithic column was fabricated and used as a novel sorbent for online solid‐phase extraction coupled to liquid chromatography with tandem mass spectrometry for the simultaneous determination of Sudan I–IV in chili pepper powder. The prepared columns were characterized by scanning electron microscopy, nitrogen adsorption‐desorption, and pressure drop measurements. Online solid‐phase extraction was performed on the synthesized monolithic column using 10 mM ammonium acetate solution as the loading solution with the aid of an online cleanup chromatography system. The desorption of Sudan I–IV was achieved with acetonitrile as the eluting solution at the flow rate of 0.5 mL/min. The extracted analytes were subsequently eluted into a C18 analytical column for chromatographic separation using a mixture of 10% acetonitrile/90% formic acid (0.5%) solution as the mobile phase. Under the optimized conditions, the developed method had linear range of 1.0–50 μg/kg, a detection limit of 0.3 μg/kg, and a quantification limit of 1.0 μg/kg for each analyte. The intraday and interday recoveries of Sudan I–IV in chili pepper powder samples ranged from 94.8 to 100.9% and 94.9 to 99.4%, respectively. The intraday and interday precision were between 3.37–7.01% and 5.01–7.68%, respectively.
COL6A3 expression in adipocytes associates with insulin resistance and depends on PPARγ and adipocyte size
Authors: Dankel Simon N. , Svärd Jessica , Matthä Simone , Claussnitzer Melina , Klöting Nora , Glunk Viktoria , Fandalyuk Zinayida , Grytten Elise , Solsvik Margit H. , Nielsen Hans‐Jørgen , Busch Christian , Hauner Hans , Blüher Matthias , Skurk Thomas , Sagen Jørn V. , Mellgren Gunnar
Published: April 2014

Objective

COL6A3 may modulate adipose tissue function in obesity and insulin resistance. A role for human adipocytes linking COL6A3 with insulin resistance warrants exploration.

Methods

COL6A3 mRNA in abdominal subcutaneous adipose samples was compared between (1) BMI‐matched obese subjects resistant or sensitive to insulin (surgical whole tissue biopsies, n = 30/group), (2) lean/overweight and obese subjects (isolated adipocytes from collagenase‐treated surgical biopsies, n = 11/group), (3) developing primary human adipocytes with/without knockdown of the insulin‐sensitizing adipogenic gene PPARG (collagenase‐treated lipoaspirate, n = 5), and (4) small and large adipocytes from lean/overweight subjects (collagenase‐treated surgical biopsies or lipoaspirate, n = 10). Insulin resistance and sensitivity were assessed by euglycemic‐hyperinsulinemic clamp (glucose infusion rate <60 and >70 μmol kg−1 min−1, respectively) (1), or by HOMA‐IR and TG/HDL ratio (2).

Results

Whole tissue COL6A3 mRNA was 2.6‐fold higher in insulin resistant compared to sensitive subjects (P < 0.001). In isolated adipocytes, COL6A3 mRNA correlated positively with BMI (P = 0.007), HOMA‐IR (P = 0.039), and TG/HDL (P = 0.004). PPARG knockdown in developing adipocytes increased COL6A3 mRNA 1.5‐fold (P = 0.043). The inverse relationship with adipocyte development was further supported by 2.8‐fold higher COL6A3 mRNA in small compared to large adipocytes (P = 0.004).

Conclusion

Increased adipocyte COL6A3 expression associates with insulin resistance in humans, which may involve impaired PPARγ‐mediated adipocyte development.
Hydrophilic polydopamine‐coated magnetic graphene nanocomposites for highly efficient tryptic immobilization
Authors: Shi Chenyi , Deng Chunhui , Li Yan , Zhang Xiangmin , Yang Pengyuan
Published: April 2014

In this work, polydopamine‐coated magnetic graphene (MG@PDA) nanocomposites were synthesized by a facile method. Trypsin was then directly immobilized on the surface of the nanocomposites through simple PDA chemistry with no need for introducing any other coupling groups. The as‐made MG@PDA nanocomposites inherit not only the large surface area of graphene which makes them capable of immobilizing high amount of trypsin (up to 0.175 mg/mg), but also the good hydrophilicity of PDA which greatly improves their biocompatibility. Moreover, the strong magnetic responsibility makes them easy to be separated from the digested peptide solution when applying a magnetic field. The feasibility of the trypsin‐immobilized MG@PDA (MG@PDA‐trypsin) nanocomposites for protein digestion was investigated and the results indicated their high digestion efficiency in a short digestion time (10 min). In addition, the reusability and stability of the MG@PDA‐trypsin nanocomposites were also tested in our work. To further confirm the efficiency of MG@PDA‐trypsin nanocomposites for proteome analysis, they were applied to digest proteins extracted from skimmed milk, followed by nano RPLC‐ESI‐MS/MS analysis, and a total of 321 proteins were identified, much more than those obtained by 16‐h in‐solution digestion (264 proteins), indicating the great potential of MG@PDA‐trypsin nanocomposites as the supports for high‐throughput proteome study.