bluuebunny/crossref_metadata_2025 huggingface dataset:
Aptasensor for amplified IgE sensing based on fluorescence quenching by graphene oxide
Authors: Hu Kun , Yang Hu , Zhou Jia , Zhao Shulin , Tian Jianniao
Published: September 2012

Water‐soluble graphene oxide (GO) with a two‐dimensional layered nanostructure was synthesized and used as a quencher to construct a highly sensitive and selective fluorescence resonance energy transfer (FRET) aptasensor for sensing Immunoglobulin E (IgE). The fluorescein isothiocyanate (FITC)‐labeled aptamer could be adsorbed stably onto the surface of GO via π → π stacking interaction, which led to the occurrence of FRET from FITC to GO, and the fluorescence of FITC‐labeled aptamer was quenched by GO via energy transfer. In the presence of IgE, the fluorescence was recovered due to a higher affinity between the aptamer and IgE compared with interactions between GO and the aptamer, leading to a high signal‐to‐background ratio. The fluorescence intensity of the aptamer increased in proportion to the amount of IgE in the sample,so that IgE could be detected with a linear range of 60–225 pM and a detection limit of 22 pM. The assay was highly selective because the aptamer was unaffected by the presence of immunoglobulin G (IgG), human serum albumin (HSA) and bovine serum albumin (BSA). The practical application of the proposed aptasensor was successfully carried out for the determination of IgE in human serum samples. Copyright © 2012 John Wiley & Sons, Ltd.
Preparation, characterization and luminescence properties of BiPO4:Eu nanophosphors
Authors: Zhou Xiaochun , Wang Xiaojun
Published: September 2012

A facile chemical method was employed to prepare fine BiPO4:Eu3+ phosphor particles calcined at the same temperature. Introducing lithium greatly affected the morphology of the samples and further affected the luminescence intensity. The samples were characterized by X‐ray diffraction (XRD), scanning electron microscopy (SEM) and photoluminescence (PL) spectroscopy. The XRD patterns of BiPO4:Eu3+ indicated a monoclinic phase. From the fluorescence spectra, the emission transition 5D0 → 7F1 is more prominent than the normal red emission transition 5D0 → 7F2. Based on the intensity ratios of 5D0 → 7F2 to 5D0 → 7F1 in the emission spectra, it can be concluded that introducing Li+ can improve the symmetry of the crystal lattice and modify the emission intensity. Sharp lines at 395 nm are the strongest of the f–f transitions and match well with near‐UV LED chips. Copyright © 2012 John Wiley & Sons, Ltd.
Effect of amylose deposition on potato tuber starch granule architecture and dynamics as studied by lintnerization
Authors: Wikman Jeanette , Blennow Andreas , Bertoft Eric
Published: September 2012

The effect of amylose deposition on the amylopectin crystalline lamellar organization in potato starch granules was studied by mild acid, so‐called lintnerization, of potato tuber starch transgenically engineered to deposit different levels of amylose. The starch granules were subjected to lintnerization at different temperatures (25, 35, and 45°C) and to two levels of solubilization, ∼ 45 and 80%. The rate of the lintnerization increased with temperature but was suppressed by amylose. The molecular size of the lintner dextrins increased with temperature, but this effect was suppressed by the presence of amylose. At high temperatures and low‐amylose content, the degree of branches was high with the concomitant increase in size in the dextrins. A portion of the branches was resistant to debranching enzymes possibly due to specific structural formations. The effects of temperature suggested a unique granular architecture of potato starch, and a model showing the dependence of temperature on the dynamic arrangement of amylopectin and amylose in the crystalline and amorphous lamellae for the potato starch is suggested. © 2012 Wiley Periodicals, Inc.
Synthetic adhesive oligopeptides with rigid polyhydroxylated amino acids
Authors: Deshmukh Manjeet , Singh Shashi , Geyer Armin
Published: September 2012

Synthetic oligopeptides containing polyhydroxylated bicyclic dipeptide (GlcTap) are investigated for their adhesion properties. The non‐natural amino acid building block composed of GlcTap is derived from glucuronic acid and mimics the hydroxyl‐amino acids of the natural proteins. Peptide oligomers of GlcTap flanked by the amino acids Tyr and Lys were synthesized and characterized. Solution structural studies performed by circular dichromism spectroscopy suggests that poly(LysGlcTapTyr) and poly(GlcTapTyr) adopts extended helical structures. Adhesion of these oligomers to the mica surface is shown by atomic force microscopy spectroscopy. Studies indicate that extended polyproline II polyhydroxylated peptide chains, which bear additional phenolic as well as cationic side chains, can mimic some of the adhesion properties of the natural protein models. Furthermore, obtained data suggest that poly(GlcTapTyr) and poly(LysGlcTapTyr) as outstanding adhesive compounds, which combine efficient synthetic accessibility with promising adhesive properties. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 273–281, 2013.
Review peptides and proteins wired into the electrical circuits: An SPM‐based approach
Authors: Sek Slawomir
Published: September 2012

Development of molecular scale electronics stimulates the search for new functional materials and compounds. One of the promising directions of further growth within this field is bioelectronics, which assumes the use of electron transfer (ET)‐mediating properties of bio‐related compounds. Because of the structural and functional versatility, peptides and proteins are particularly well suited to perform as single‐molecule‐based elements of circuits or sensing devices. In this review, it is demonstrated that purposely designed systems such as molecular junctions are excellent platforms for the studies of ET properties of peptides and proteins at nanoscale level. Using experimental approach based on scanning probe microscopy, individual molecules can be directly wired between two metallic contacts, and their conductance can be determined. ET behavior of the peptides and proteins can be analyzed in terms of their applicability as molecular wires, switches, diodes and elements of sensing devices. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 71–81, 2013.
Design, expression, and purification of de novo transmembrane “hairpin” peptides
Authors: Tulumello David V. , Johnson Rachel M. , Isupov Inga , Deber Charles M.
Published: September 2012

While our understanding of the folding and structure of water‐soluble proteins has progressed to the point where they can be artificially designed and produced from first principles, there has been only limited work toward the de novo design of membrane proteins. Such studies have been hindered in large part due to the practical challenges in the production and characterization of multispanning transmembrane (TM) proteins that arise from their highly hydrophobic character. In this work, we used molecular biology cloning techniques to produce a library of partially randomized Ala‐ and Ile‐rich de novo helix–loop–helix (hairpin) TM constructs as models for tertiary TM–TM folding. From this plasmid DNA library, we selected sequences corresponding to hairpins with 0, 1, or 2 putative TM segments. While purification protocols could be adapted for application with a broad range of designed protein hairpins, bacterial expression of constructs with multiple predicted TM segments was limited as it is with native membrane proteins. Examples of the peptide hairpins obtained were characterized by circular dichroism spectroscopy, tryptophan fluorescence, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). We found that hairpins composed of two TM segments display characteristic behavior on detergent solubilization, such as an increase in helical structure (vs. that in aqueous buffer), and sequence‐dependent migration rates in SDS‐PAGE analysis—features that may serve as structural hallmarks to verify dual TM topology in hairpin sequences. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 546–556, 2012.
Quantifying the branching frequency of virtual filamentous microbes using fractal analysis
Authors: Barry David J.
Published: September 2012

The productivity of an industrial fermentation process involving a filamentous microbe is heavily dependent on the morphological form adopted by the organism. The development of systems capable of rapidly and accurately characterizing morphology within a given process represents a significant challenge, as the complex phenotypes that are manifested are not easily quantified. Conventional parameters employed in these analyses are often of limited value, as they reveal little about the branching behavior of the organism; an important consideration given the demonstrated link between branching frequency and metabolite production. In this study, the influence of branching behavior on the spatial distribution of mycelia grown in silico is examined through fractal analysis. It is demonstrated that fractal dimension, quantified based on the frequency distribution of parameterized boundary curves, and lacunarity act as robust estimators of branching behavior. The analysis can, in theory, be applied to any morphological form, providing universally applicable process parameters for more complete data acquisition. Biotechnol. Bioeng. 2013; 110: 437–447. © 2012 Wiley Periodicals, Inc.
Dynamic transcription factor activity profiling in 2D and 3D cell cultures
Authors: Bellis Abigail D. , Bernabé Beatriz Peñalver , Weiss Michael S. , Shin Seungjin , Weng Stanley , Broadbelt Linda J. , Shea Lonnie D.
Published: September 2012

Live‐cell assays to measure cellular function performed within 3D cultures have the potential to elucidate the underlying processes behind disease progression and tissue formation. Cells cultured in 3D interact and remodel their microenvironment and can develop into complex structures. We have developed a transcription factor (TF) activity array that uses bioluminescence imaging (BLI) of lentiviral delivered luminescent reporter constructs that allows for the non‐invasive imaging of TF activity in both 2D and 3D culture. Imaging can be applied repeatedly throughout culture to capture dynamic TF activity, though appropriate normalization is necessary. We investigated in‐well normalization using Gaussia or Renilla luciferase, and external well normalization using firefly luciferase. Gaussia and Renilla luciferase were each unable to provide consistent normalization for long‐term measurement of TF activity. However, external well normalization provided low variability and accounted for changes in cellular dynamics. Using external normalization, dynamic TF activities were quantified for five TFs. The array captured expected changes in TF activity to stimuli, however the array also provided dynamic profiles within 2D and 3D that have not been previously characterized. The development of the technology to dynamically track TF activity within cells cultured in both 2D and 3D can provide greater understanding of complex cellular processes. Biotechnol. Bioeng. 2013; 110: 563–572. © 2012 Wiley Periodicals, Inc.
Towards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case
Authors: Roussel Guillaume , Perpète Eric A. , Matagne André , Tinti Emmanuel , Michaux Catherine
Published: September 2012

It has recently been reported that 2‐methyl‐2,4‐pentanediol (MPD) can modulate the protein‐binding properties of sodium dodecyl sulfate (SDS), turning it into a non‐denaturing detergent. Indeed both alpha (the lysozyme) and beta (the carbonic anhydrase II) soluble enzymes, as well as a beta membrane protein (PagP) have been successfully refolded into their native form by using this amphiphatic alcohol. In order to support the universal character of our MPD‐based technique, we have extended its transferability to the Omp2a trimeric membrane porin. The far‐UV circular dichroism signature of Omp2a refolded with our original procedure is identical to that obtained by classical techniques, clearly indicating a proper refolding. Moreover, we show that the optimal SDS/MPD ratio for refolding Omp2a is similar to what has been observed for other types of proteins. While the protocol allows refolding at higher protein concentration (up to 4 mg/mL) and ionic strength (up to 1 M NaCl) than other refolding methods, it is also more efficient at basic pH values and medium temperature (20–40°C). Finally, the key role of the cosolvent was highlighted by a thorough study of the efficiency of MPD analogues, and a high variability was observed, as they can be able or unable to induce refolding at low or high salt concentrations. Biotechnol. Bioeng. 2013; 110: 417–423. © 2012 Wiley Periodicals, Inc.
Combination of FASP and fully automated 2D‐LC‐MS/MS allows in‐depth proteomic characterization of mouse zymogen granules
Authors: Sun Xionghua , Jiang Xiaogang
Published: September 2012

Zymogen granule (ZG) constituents play important roles in pancreatic injury and disease. In previous studies, proteomic analyses with rat zymogen granules were separated by two‐dimensional gel electrophoresis or one‐dimensional SDS–PAGE, followed by in‐gel tryptic digestion. In order to overcome the disadvantage of in‐gel digestion and to carry out further in‐depth proteomic analysis of the zymogen granules, in this study, by combining a filter‐aided sample preparation method and fully automated 2D‐LC‐MS/MS technique, 800 ZG proteins were identified with at least two unique peptides for each protein, 75% of which have not been previously reported. The identified proteins revealed broad diversity in protein identity and function. This is the largest dataset of ZG proteome, and also the first dataset of the mouse ZG proteome, which may help elucidate on the molecular architecture of ZGs and their functions. Copyright © 2012 John Wiley & Sons, Ltd.
BDNF Val66Met polymorphism interacts with sex to influence bimanual motor control in healthy humans
Authors: Smolders Ruud , Rijpkema Mark , Franke Barbara , Fernández Guillén
Published: September 2012

Brain‐derived neurotrophic factor (BDNF) plays a critical role in brain development. A common single nucleotide polymorphism in the gene encoding BDNF (rs6265, Val66Met) affects BDNF release and has been associated with altered learning and memory performance, and with structural changes in brain morphology and corpus callosum integrity. BDNF Val66Met has more recently been shown to influence motor learning and performance. Some of the BDNF effects seem to be modulated by an individual's sex, but currently the relationship between BDNF and sex in the motor domain remains elusive. Here, we investigate the relationship between BDNF Val66Met genotype and an individual's sex in the motor system. Seventy‐six healthy, previously genotyped, individuals performed a task in which the participant drew lines at different angles of varying difficulty. Subjects controlled the horizontal and vertical movement of the line on a computer screen by rotating two cylinders. We used this bimanual motor control task to measure contributions from both current motor function and the pre‐existing interhemispheric connectivity. We report that BDNF genotype interacts with sex to influence the motor performance of healthy participants in this bimanual motor control task. We further report that the BDNF genotype by sex interaction was present in the more difficult trials only, which is in line with earlier findings that genetic effects may become apparent only when a system is challenged. Our results emphasize the importance of taking sex into account when investigating the role of BDNF genotype in the motor system.
Cellular basis for singing motor pattern generation in the field cricket (Gryllus bimaculatus DeGeer)
Authors: Schöneich Stefan , Hedwig Berthold
Published: September 2012

The singing behavior of male crickets allows analyzing a central pattern generator (CPG) that was shaped by sexual selection for reliable production of species‐specific communication signals. After localizing the essential ganglia for singing in Gryllus bimaculatus, we now studied the calling song CPG at the cellular level. Fictive singing was initiated by pharmacological brain stimulation. The motor pattern underlying syllables and chirps was recorded as alternating spike bursts of wing‐opener and wing‐closer motoneurons in a truncated wing nerve; it precisely reflected the natural calling song. During fictive singing, we intracellularly recorded and stained interneurons in thoracic and abdominal ganglia and tested their impact on the song pattern by intracellular current injections. We identified three interneurons of the metathoracic and first unfused abdominal ganglion that rhythmically de‐ and hyperpolarized in phase with the syllable pattern and spiked strictly before the wing‐opener motoneurons. Depolarizing current injection in two of these opener interneurons caused additional rhythmic singing activity, which reliably reset the ongoing chirp rhythm. The closely intermeshing arborizations of the singing interneurons revealed the dorsal midline neuropiles of the metathoracic and three most anterior abdominal neuromeres as the anatomical location of singing pattern generation. In the same neuropiles, we also recorded several closer interneurons that rhythmically hyper‐ and depolarized in the syllable rhythm and spiked strictly before the wing‐closer motoneurons. Some of them received pronounced inhibition at the beginning of each chirp. Hyperpolarizing current injection in the dendrite revealed postinhibitory rebound depolarization as one functional mechanism of central pattern generation in singing crickets.
Structural Investigations on the Anti‐HIV G‐Quadruplex‐Forming Oligonucleotide TGGGAG and Its Analogues: Evidence for the Presence of an A‐Tetrad
Authors: Virgilio Antonella , Esposito Veronica , Citarella Giuseppe , Mayol Luciano , Galeone Aldo
Published: September 2012

Several anti‐HIV aptamers adopt DNA quadruplex structures. Among these, “Hotoda's aptamer” (base sequence TGGGAG) was one of the first to be discovered. Although it has been the topic of some recent research, no detailed structural investigations have been reported. Here we report structural investigations on this aptamer and analogues with related sequences, by using UV, CD, and NMR spectroscopy as well as electrophoretic techniques. The addition of a 3′‐end thymine has allowed us to obtain a single, investigable quadruplex structure. Data clearly point to the presence of an A‐tetrad. Furthermore, the effects of the incorporation of an 8‐methyl‐2′‐deoxyguanosine at the 5′‐end of the G‐run were investigated.
Porcine model of intracoronary pulverization of stent struts by rotablation atherectomy
Authors: Quang Thuy Tran , Hatem Raja , Rousseau Guy , Dube Bruno , Samson Caroline , Schampaert Erick , Charron Thierry
Published: September 2012

Objective

To evaluate the feasibility and safety of rotablation atherectomy in a suboptimally expanded stent.

Methods

Seven pigs underwent suboptimal stent expansion in the left anterior descending coronary. Pulverization of the stent struts was performed by rotablation atherectomy with two different burr sizes. Two types of control porcine models were used: pigs with fully expanded stents and pigs without stents. Continuous electrocardiogram readings as well as microscopic and radiologic analysis of cardiac tissue were performed.

Results

Rotablation atherectomy reduces the suboptimally expanded stent by (26.95 ± 5.03)%. Ninety‐five percent of the metal microparticles, imbedded in the suboptimally expanded stent group cardiac tissue, are less than 15 μm. Transient vasospasm and ST segments elevations were observed during rotablation atherectomy, which returned to basal conditions at the end of the intervention.

Conclusion

Our study demonstrated the feasibility and safety of using rotablation atherectomy to pulverize stent struts in a suboptimally expanded stent. © 2012 Wiley Periodicals, Inc.
Investigation of Promoted Cu/ZnO/Al2O3 Methanol Steam Reforming Nanocatalysts by Full Factorial Design
Authors: Sharifi Pajaie H. , Taghizadeh M.
Published: September 2012

A Cu/ZnO/Al2O3 nanocatalyst was applied for hydrogen production via steam reforming of methanol in a fixed‐bed reactor. Modified forms of the catalyst were prepared by adding small amounts of Ba, Zr, and Ce oxides. The catalysts were characterized by means of N2 adsorption‐desorption, X‐ray diffraction, and scanning electron microscope techniques. Full factorial design was used to optimize the required number of experiments and evaluate the catalytic activity in a fixed‐bed reactor. The oxide additives reduced the production of carbon monoxide and increased the selectivity of carbon dioxide as well as the yield of hydrogen production. Among the studied catalysts, the Cu/ZnO/Al2O3/CeO2/ZrO2 catalyst presented the best performance.
Independent Association Between Obstructive Sleep Apnea and Noncalcified Coronary Plaque Demonstrated by Noninvasive Coronary Computed Tomography Angiography
Authors: Sharma Sunil , Gebregziabher Mulugeta , Parker Adrian T. , Abro Joseph A. , Armstrong Andrew M. , Schoepf U. Joseph
Published: September 2012

Background:

Coronary artery atherosclerosis has been associated with obstructive sleep apnea (OSA). However, the type and severity of plaque formation have not been characterized. This study evaluated the association of coronary noncalcified plaques and severity of stenosis in patients with OSA.

Hypothesis:

Methods:

This study was a retrospective analysis of 81 patients, 49 with OSA and 32 without OSA, who had undergone multidetector‐row helical computed tomography scanning. The board‐certified radiologist was blinded to the diagnosis of OSA and reviewed the scans for plaque characterization, severity of stenosis, and number of vessels involved.

Results:

Of the 81 patients reviewed, the mean apnea‐hypopnea index in the OSA group was 42.2 vs 7.5 in the non‐OSA group. The groups did not significantly differ in the distribution of comorbid conditions. We found that among the patients with OSA, 63% had noncalcified/mixed plaques, as opposed to 16% in the non‐OSA group (P < 0.0001), with unadjusted odds ratio of 9.3 (3.0, 28.4). After adjustment for other risk factors such as age, sex, race, hypercholesterolemia, and history of smoking, the association remained strong, with an odds ratio of 7.0 (1.9, 26.5; P < 0.05).

Conclusions:

Our study finds that the frequency of noncalcified/mixed plaques is much higher in patients with OSA than in non‐OSA patients. Patients with OSA also have more severe stenosis and a higher number of vessels involved. This study adds to a growing body of data regarding our understanding of the association of OSA and atherosclerosis.

Dr. Sharma, Dr. Schoepf, Mr. Parker, Mr. Abro, and Mr. Armstrong contributed to data collection and analysis, drafting of the manuscript, and final approval. Dr. Gebregziabher contributed to data analysis, drafting of the manuscript, and final approval. Dr. Sharma had access to and takes responsibility for the integrity of the data and the accuracy of the data analysis.

The authors of this manuscript have certified that they comply with the principles of ethical publishing (Shewan LG, Coats AJ. Ethics in the authorship and publishing of scientific articles. Int J Cardiol. 2010;144:1‐2.).

The authors have no funding, financial relationships, or conflicts of interest to disclose.
Photoluminescent hierarchical Zno micro‐flower and its surface wettability
Authors: Ji X. B. , Lu W. C. , Ma H. P.
Published: September 2012

The flower‐like ZnO with micro‐nano hierarchical structure is successfully obtained by a simple hydrothermal synthesis, using sodium dodecyl benzene sulfonate (SDBS) as a structure direct agent. The resulted ZnO micro‐flowers are very uniform in morphology with particle sizes around 1 µm. A number of techniques, including X‐ray diffraction (XRD), field emission scan electron microscopy (FESEM), energy‐dispersive spectroscopy (EDS), fourier transform infrared (FTIR) spectra and thermogravimetry analysis (TGA), are used to characterize the obtained ZnO. The self‐assemble of ZnO nano‐sheets under the direction of SDBS leads to the formation of ZnO micro‐flowers. The room temperature photoluminescence property of the obtained flower‐like ZnO exhibits a broad visible light emission. The surface of as‐made ZnO shows a very hydrophilic property, while the special micro‐nano hierarchical structure enables the ZnO micro‐flower a superhydrophobic surface after modification of fluoroalkylsilane.
Merging Sustainability with Organocatalysis in the Formation of Organic Carbonates by Using CO2 as a Feedstock
Authors: Whiteoak Christopher J. , Nova Ainara , Maseras Feliu , Kleij Arjan W.
Published: September 2012

The use of phenolic compounds as organocatalysts is discussed in the context of the atom‐efficient cycloaddition of carbon dioxide to epoxides, forming useful cyclic organic carbonate products. The presence and cooperative nature of adjacent phenolic groups in the catalyst structure results in significantly enhanced catalytic efficiencies, allowing these CO2 fixation reactions to operate efficiently under virtually ambient conditions. The cooperative effect has also been studied by computational methods. Furthermore, when the cycloaddition reactions are carried out on a larger scale and under solvent‐free conditions, further enhancements in activity are observed, combined with the advantageous requirement of reduced loadings of the binary organocatalyst system. The reported system is among one of the mildest and most effective metal‐free catalysts for this conversion and contributes to a much more sustainable development of organic carbonate production; this feature has not been the main focus of previous contributions in this area.
Ferritin as a novel reporter gene for photoacoustic molecular imaging
Authors: Ha Seung Han , Carson Andrew R. , Kim Kang
Published: September 2012

Reporter genes may serve as endogenous contrast agents in the field of photoacoustic (PA) molecular imaging (PMI), enabling greater characterization of detailed cellular processes and disease progression. To demonstrate the feasibility of using ferritin as a reporter gene, human melanoma SK‐24 (SK‐MEL‐24) cells were co‐transfected with plasmid expressing human heavy chain ferritin (H‐FT) and plasmid expressing enhanced green fluorescent protein (pEGFP‐C1) using lipofectamine™ 2000. Nontransfected SK‐MEL‐24 cells served as a negative control. Fluorescent imaging of GFP confirmed transfection and transgene expression in co‐transfected cells. To detect iron accumulation due to ferritin overexpression in SK‐MEL‐24 cells, a focused high‐frequency ultrasonic transducer (60 MHz, f/1.5), synchronized to a pulsed laser (fluence < 5 mJ/cm2) was used to scan the PA signal at a wide range NIR wavelengths (850–950 nm). PA signal intensity from H‐FT transfected SK‐MEL‐24 cells was about 5–9 dB higher than nontransfected SK‐MEL‐24 cells at 850–950 nm. Immunofluorescence and RT‐PCR analysis both indicate high levels of ferritin expression in H‐FT transfected SK‐MEL24 cells, with little ferritin expression in nontransfected SK‐MEL‐24 cells. In this study, the feasibility of using ferritin as a reporter gene for PMI has been demonstrated in vitro. The use of ferritin as a reporter gene represents a novel concept for PMI using an endogenous contrast agent and may provide various opportunities for molecular imaging and basic science research. © 2012 International Society for Advancement of Cytometry
Integrins regulate centrosome integrity and astrocyte polarization following a wound
Authors: Peng Huashan , Ong Yen May , Shah Waris Ali , Holland Paul C. , Carbonetto Salvatore
Published: September 2012

In response to a wound, astrocytes in culture extend microtubule‐rich processes and polarize, orienting their centrosomes and Golgi apparatus woundside. β1 Integrin null astrocytes fail to extend processes toward the wound, and are disoriented, and often migrate away orthogonal, to the wound. The centrosome is unusually fragmented in β1 integrin null astrocytes. Expression of a β1 integrin cDNA in the null background yields cells with intact centrosomes that polarize and extend processes normally. Fragmented centrosomes rapidly assemble following integrin ligation and cell attachment. However, several experiments indicated that cell adhesion is not necessary. For example, astrocytes in suspension expressing a chimeric β1 subunit that can be activated by an antibody assemble centrosomes suggesting that β1 activation is sufficient to cause centrosome assembly in the absence of cell adhesion. siRNA knockdown of PCM1, a major centrosomal protein, inhibits cell polarization, consistent with the notion that centrosomes are necessary for polarity and that integrins regulate polarity via centrosome integrity. Screening inhibitors of molecules downstream of integrins indicate that neither FAK nor ILK is involved in regulation of centrosome integrity. In contrast, blebbistatin, a specific inhibitor of non‐muscle myosin II (NMII), mimics the response of β1 integrin null astrocytes by disrupting centrosome integrity and cell polarization. Blebbistatin also inhibits integrin‐mediated centrosome assembly in astrocytes attaching to fibronectin, consistent with the hypothesis that NMII functions downstream of integrins in regulating centrosome integrity. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 333–353, 2013.
Jelly belly trans‐synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture
Authors: Rohrbough Jeffrey , Kent Karla S. , Broadie Kendal , Weiss Joseph B.
Published: September 2012

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans‐synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb–Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild‐type postsynaptic Alk expression in Alk partial loss‐of‐function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb–Alk signaling triggers the Ras‐MAP kinase cascade in both pre‐ and postsynaptic compartments. These novel roles for Jeb–Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013
ZNF385B is characteristically expressed in germinal center B cells and involved in B‐cell apoptosis
Authors: Iijima Kazutoshi , Yamada Hiroyuki , Miharu Masashi , Imadome Ken‐Ichi , Miyagawa Yoshitaka , Akimoto Shingo , Kobayashi Kenichiro , Okita Hajime , Nakazawa Atsuko , Fujiwara Shigeyoshi , Fujimoto Junichiro , Kiyokawa Nobutaka
Published: September 2012

We previously identified zinc finger (ZF) protein ZNF385B as a molecule specifically expressed in Burkitt's lymphoma (BL) among hematologic malignancies. Here, we investigated ZNF385B expression in healthy B cells in a variety of hematological tissues by RT‐PCR and immunohistochemistry. ZNF385B expression was found to be limited to a subset of GC B cells, the healthy counterpart to BL B cells. To elucidate the function of ZNF385B in healthy B cells, we established a tetracycline‐controlled protein‐inducible system in B‐cell lines and observed that ectopic expression of the longest transcript variant of ZNF385B, possessing four ZF domains, induced upregulation of PERP and FAS/CD95, a downstream target of p53, and activation of caspase, resulting in apoptosis induction. However, a ZNF385B deletion mutant with three ZF domains corresponding to shorter isoforms, did not induce upregulation; rather it inhibited apoptosis induced by CD20 cross‐linking and BCR stimulation. The direct binding of ZNF385B with p53 has suggested the involvement of ZNF385B in B‐cell apoptosis via modulation of p53 transactivation; our data indicate that ZNF385B characteristically expressed in GC B cells has both proapoptotic and antiapoptotic activities depending on the type of isoform and should be a novel player in GC B‐cell selection.
Mature proteins derived from Epstein–Barr virus fail to feed into the MHC class I antigenic pool
Authors: Fiebiger Benjamin M. , Moosmann Andreas , Behrends Uta , Mautner Josef
Published: September 2012

The immediate presentation of peptide epitopes on MHC class I (MHC I) after antigen expression has led to the concept that MHC I ligands are mostly derived from defective ribosomal products (DRiPs), a subset of newly synthesized proteins that are rapidly degraded by the proteasome. Whether and to what extent mature proteins contribute to the antigenic pool, however, has remained elusive. Here, we developed a conditional antigen expression system that allows studying antigen presentation from mature proteins by inducing their rapid proteasomal degradation in the absence of further antigen synthesis. Target cells in which expression of two Epstein–Barr virus (EBV) antigens was induced were rapidly recognized by antigen‐specific CD8+ T cells in a time‐ and dosage‐dependent manner, demonstrating that antigen presentation was linked to antigen synthesis. By contrast, T cells failed to recognize target cells containing large amounts of mature protein even after induction of their rapid proteasomal degradation. Thus, the presentation of these antigens proved to be strictly dependent on protein synthesis whereas mature proteins failed to furnish the antigenic pool. These results have implications for the design of immunotherapeutic strategies that aim at targeting proteins with increased half‐lives and are hence overexpressed in tumors.
Normal phase LC coupled with direct analysis in real time MS for the chiral analysis of 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol and jasmonic acid
Authors: Chang Cuilan , Zhou Zhigui , Yang Youyou , Han Yehua , Bai Yu , Zhao Meiping , Liu Huwei
Published: September 2012

Normal phase chiral LC (NPLC) has been proved to be powerful and efficient for chiral separation. However, the combination of NPLC with ESI or atmospheric pressure chemical ionization MS is restricted by the poor ionization efficiency and thermal fragmentations of analytes to some extent. Direct analysis in real time MS (DART‐MS) is an ambient ionization technique that shows high ionization efficiency of the analytes in the normal phase mobile phase. In this work, we coupled chiral NPLC to DART‐MS for the chiral qualitative and quantitative analysis of 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol and jasmonic acid enantiomers. Satisfactory results for the enantiomers of 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol operating in the positive mode were obtained in terms of linearity (2.5–250 μg/mL, R2, 0.999–1.000) and repeatability (25 μg/mL, RSDs, 4.7–5.6%). Moreover, chiral NPLC‐DART‐MS resulted in the simultaneous chiral separation and detection of jasmonic acid enantiomers, which are very difficult to be analyzed by NPLC‐ESI‐MS and NPLC‐APCI‐MS. Compared with the coupled techniques of NPLC‐ESI‐MS and NPLC‐APCI‐MS, NPLC‐DART‐MS showed advantages in increasing the ionization efficiency and reducing the in‐source thermal fragmentation of analytes.
Sample preconcentration in open microchannels combined with MALDI‐MS
Authors: Mikkonen Saara , Rokhas Maria Khihon , Jacksén Johan , Emmer Åsa
Published: September 2012

In this work, a method for preconcentrating samples in 1 cm long, 50–150 μm wide open microchannels is presented. Platinum electrodes were positioned at the channel ends, voltage was applied, and charged analyte was preconcentrated at the oppositely charged side during continuous supply of sample. The preconcentration was initially studied in a closed system, where an influence on the analyte position from a pH gradient, generated by water electrolysis, was observed. In the open channel, the analyte distribution after preconcentration was evaluated using MALDI‐MS with the channel as MALDI target. MALDI matrix was applied with an airbrush or by electrospray matrix deposition and by using the latter technique higher degrees of crystallization in the channels were obtained. After preconcentrating a 1 nM cytochrome c solution for 5 min, corresponding to a supplied amount of 1.25 fmol, a signal on the cathodic channel end could be detected. When a solution of cytochrome c trypsin digest was supplied, the peptides were preconcentrated at different positions along the channel depending on their charge.
K+, Na+, and Mg2+ on DNA translocation in silicon nitride nanopores
Authors: Uplinger James , Thomas Brian , Rollings Ryan , Fologea Daniel , McNabb David , Li Jiali
Published: September 2012

In this work, we report on how salt concentration and cation species affect DNA translocation in voltage‐biased silicon nitride nanopores. The translocation of dsDNA in linear, circular, and supercoiled forms was measured in salt solutions containing KCl, NaCl, and MgCl2. As the KCl concentrations were decreased from 1 to 0.1 M, the time taken by a DNA molecule to pass through a nanopore was shorter and the frequency of the translocation in a folded configuration was reduced, suggesting an increase in DNA electrophoretic mobility and DNA persistence length. When the salt concentration was kept at 1 M, but replacing K+ with Na+, longer DNA translocation times (td) were observed. The addition of low concentrations of MgCl2 with 1.6 M KCl resulted in longer td and an increased frequency of supercoiled DNA molecules in a branched form. These observations were consistent with the greater counterion charge screening ability of Na+ and Mg2+ as compared to K+. In addition, we demonstrated that dsDNA molecules indeed translocated through a ∼10 nm nanopore by PCR amplification and gel electrophoresis. We also compared the dependence of DNA mobility and conformation on KCl concentration and cation species measured at single molecule level by silicon nitride nanopores with existing bulk‐based experimental results and theoretical predictions.
Effect of electrolyte pH on CIEF with narrow pH range ampholytes
Authors: Páger Csilla , Vargová Andrea , Takácsi‐Nagy Anna , Dörnyei Ágnes , Kilár Ferenc
Published: September 2012

CIEF of components following sequential injection of ampholytes and the sample zone offers unique advantages for analysis. The most important one of these is the efficient separation of amphoteric compounds having pIs outside the pH range of the ampholytes applied, but the resolution of the components can be increased by an adequate setup in the injection protocol. In this study, the effect of the pH of the anolyte and catholyte on the selectivity and speed of the isoelectric focusing was investigated. Changes in the pH values significantly influenced the resolution and the length of the pH gradient, while changes in the charge state of components were also observed. Three ampholyte solutions (from different suppliers) covering only two pH units were used for the analyses of substituted nitrophenol dyes in uncoated capillary. With appropriate setup, the components, with pIs not covered by the ampholyte pH range, migrated in charged state outside the pH gradient. This phenomenon is preferable for coupling isoelectric focusing to MS detection, by evading the undesirable ion suppression effect of ampholytes.
Chemical and toxicological assessment of a full-scale biosolid compost
Authors: Patureau Dominique , Delgenes Nadine , Muller Mathieu , Dagnino Sonia , Lhoutellier Claire , Delgenes Jean-Philippe , Balaguer Patrick , Hernandez-Raquet Guillermina
Published: September 2012

The impact of a full-scale biosolid composting plant on the fate of a broad range of priority organic pollutants was investigated. Chemical analysis was performed at different steps of the process during two seasons. Simultaneously, the toxicological quality was assessed using estrogen α-, dioxin-, and pregnane X-receptor reporter cell lines. Mass-balance calculation highlighted the removal of easily degradable pollutants during composting. The important variations observed for each compound and for the two seasons might be explained by pollutant-fate dependency on process parameters like temperature. The final compost displayed low pregnane X activity but high estrogenic activity. The dioxin-like activity stayed constant through the process. The chemical and toxicological results highlight the importance of combining both approaches to accurately assess the compost quality. Such compilation of data on full-scale processes may be also very helpful for the environmental risk assessment of new organic waste disposal practices. Environ. Toxicol. Chem. 2012; 31: 2748–2756. © 2012 SETAC
Polyploidy road to therapy‐induced cellular senescence and escape
Authors: Wang Qin , Wu Peter C. , Dong David Z. , Ivanova Iana , Chu Elizabeth , Zeliadt Steven , Vesselle Hubert , Wu Daniel Y.
Published: September 2012

Therapy‐induced cellular senescence (TCS), characterized by prolonged cell cycle arrest, is an in vivo response of human cancers to chemotherapy and radiation. Unfortunately, TCS is reversible for a subset of senescent cells, leading to cellular reproliferation and ultimately tumor progression. This invariable consequence of TCS recapitulates the clinical treatment experience of patients with advanced cancer. We report the findings of a clinicopathological study in patients with locally advanced non‐small cell lung cancer demonstrating that marker of in vivo TCS following neoadjuvant therapy prognosticate adverse clinical outcome. In our efforts to elucidate key molecular pathways underlying TCS and cell cycle escape, we have previously shown that the deregulation of mitotic kinase Cdk1 and its downstream effectors are important mediators of survival and cell cycle reentry. We now report that aberrant expression of Cdk1 interferes with apoptosis and promotes the formation of polyploid senescent cells during TCS. These polyploid senescent cells represent important transition states through which escape preferentially occurs. The Cdk1 pathway is in part modulated differentially by p21 and p27 two members of the KIP cyclin‐dependent kinase inhibitor family during TCS. Altogether, these studies underscore the importance of TCS in cancer therapeutics.
Inhibitors of vacuolar ATPase proton pumps inhibit human prostate cancer cell invasion and prostate‐specific antigen expression and secretion
Authors: Michel Vera , Licon‐Munoz Yamhilette , Trujillo Kristina , Bisoffi Marco , Parra Karlett J.
Published: September 2012

Vacuolar ATPases (V‐ATPases) comprise specialized and ubiquitously distributed pumps that acidify intracellular compartments and energize membranes. To gain new insights into the roles of V‐ATPases in prostate cancer (PCa), we studied the effects of inhibiting V‐ATPase pumps in androgen‐dependent (LNCaP) and androgen‐independent (C4‐2B) cells of a human PCa progression model. Treatment with nanomolar concentrations of the V‐ATPase inhibitors bafilomycin A or concanamycin A reduced the in vitro invasion in both cell types by 80%, regardless that V‐ATPase was prominent at the plasma membrane of C4‐2B cells and only traces were detected in the low‐metastatic LNCaP parental cells. In both cell types, intracellular V‐ATPase was excessive and co‐localized with prostate‐specific antigen (PSA) in the Golgi compartment. V‐ATPase inhibitors reversibly excluded PSA from the Golgi and led to the accumulation of largely dispersed PSA‐loaded vesicles of lysosomal composition. Inhibition of acridine orange staining and transferrin receptor recycling suggested defective endosomal and lysosomal acidification. The inhibitors, additionally, interfered with the AR‐PSA axis under conditions that reduced invasion. Bafilomycin A significantly reduced steady‐state and R1881‐induced PSA mRNA expression and secretion in the LNCaP cells which are androgen‐dependent, but not in the C4‐2B cells which are androgen ablation‐resistant. In the C4‐2B cells, an increased susceptibility to V‐ATPase inhibitors was detected after longer treatments, as proliferation was reduced and reversibility of bafilomycin‐induced responses impaired. These findings make V‐ATPases attractive targets against early and advanced PCa tumors.
Smoke‐induced microRNA and related proteome alterations. Modulation by chemopreventive agents
Authors: De Flora Silvio , Balansky Roumen , D'Agostini Francesco , Cartiglia Cristina , Longobardi Mariagrazia , Steele Vernon E. , Izzotti Alberto
Published: September 2012

Dysregulation of microRNAs (miRNAs) has important consequences on gene and protein expression since a single miRNA targets a number of genes simultaneously. This article provides a review of published data and ongoing studies regarding the effects of cigarette smoke (CS), either mainstream (MCS) or environmental (ECS), on the expression of miRNAs and related proteins. The results generated in mice, rats, and humans provided evidence that exposure to CS results in an intense dysregulation of miRNA expression in the respiratory tract, which is mainly oriented in the sense of downregulation. In parallel, there was an upregulation of proteins targeted by the downregulated miRNAs. These trends reflect an attempt to defend the respiratory tract by means of antioxidant mechanisms, detoxification of carcinogens, DNA repair, anti‐inflammatory pathways, apoptosis, etc. However, a long‐lasting exposure to CS causes irreversible miRNA alterations that activate carcinogenic mechanisms, such as modulation of oncogenes and oncosuppressor genes, cell proliferation, recruitment of undifferentiated stem cells, inflammation, inhibition of intercellular communications, angiogenesis, invasion, and metastasis. The miRNA alterations induced by CS in the lung of mice and rats are similar to those observed in the human respiratory tract. Since a number of miRNAs that are modulated by CS and/or chemopreventive agents are subjected to single nucleotide polymorphisms in humans, they can be evaluated according to toxicogenomic/pharmacogenomics approaches. A variety of cancer chemopreventive agents tested in our laboratory modulated both baseline and CS‐related miRNA and proteome alterations, thus contributing to evaluate both safety and efficacy of dietary and pharmacological agents.
An Informed Look at Doctoral Vivas (Oral Examinations) in the Preparation of Counselor Educators
Authors: McAdams Charles R. , Robertson Derek L.
Published: September 2012

Viva voce is the Latin expression traditionally used to refer to doctoral oral examinations. Viva voce, or simply viva, examinations are a standard requirement for doctoral candidacy and degree completion in many counselor education programs. Despite the prevalent use of vivas as an assessment and learning tool, the counselor education literature is scant on information about their rationale, method, and impact. Current trends toward transparency and accountability in higher education now require that counselor educators and researchers take steps to ensure that the use of vivas is justified beyond tradition alone.