bluuebunny/crossref_metadata_2025 huggingface dataset:
Tissue‐engineered ligament: implant constructs for tooth replacement
Authors: Gault Philippe , Black Annie , Romette Jean‐Louis , Fuente Fabien , Schroeder Klaus , Thillou Fabienne , Brune Thierry , Berdal Ariane , Wurtz Tilmann
Published: June 2010

Gault P, Black A, Romette J‐L, Fuente F, Schroeder K, Thillou F, Brune T, Berdal A, Wurtz T. Tissue‐engineered ligament: implant constructs for tooth replacement. J Clin Periodontol 2010. 37: 750‐758 doi: 10.1111/j.1600-051X.2010.01588.x.

Aim: A tissue‐engineered periodontal ligament (PDL) around implants would represent an important new therapeutic tool to replace lost teeth. The PDL is the key to tooth anchoring; it connects tooth root and alveolar bone, and it sustains bone formation.

Materials and Methods: Cells were isolated from PDL and cultured in a bioreactor on titanium pins. After the formation of multiple cellular layers, pins were implanted in enlarged dental alveolae.

Main Outcome Measures: Cell‐covered implants integrated without adverse effects, and induced bone in their vicinity.

Results: A histological examination of a dog model revealed that cells were arranged in a typical ligament‐like fashion. In human patients, product safety was ascertained for 6–60 months. Probing and motility assessments suggested that the implants were well integrated with mechanical properties similar to those of teeth. Radiographs demonstrated the regeneration of deficient alveolar bone, the development of a lamina dura adjacent to a mineral‐devoid space around the implant and implant migration in an intact bone structure.

Conclusions: New tissue consistent with PDL developed on the surface of dental implants after implantation. This proof‐of‐principal investigation demonstrates the application of ligament‐anchored implants, which have potential advantages over osseointegrated oral implants.
Distinguishing between Net and Global DIF in Polytomous Items
Authors: Penfield Randall D.
Published: June 2010

In this article, I address two competing conceptions of differential item functioning (DIF) in polytomously scored items. The first conception, referred to as net DIF, concerns between‐group differences in the conditional expected value of the polytomous response variable. The second conception, referred to as global DIF, concerns the conditional dependence of group membership and the polytomous response variable. The distinction between net and global DIF is important because different DIF evaluation methods are appropriate for net and global DIF; no currently available method is universally the best for detecting both net and global DIF. Net and global DIF definitions are presented under two different, yet compatible, modeling frameworks: a traditional item response theory (IRT) framework, and a differential step functioning (DSF) framework. The theoretical relationship between the IRT and DSF frameworks is presented. Available methods for evaluating net and global DIF are described, and an applied example of net and global DIF is presented.
Effects of Drying, Packaging, and Temperature on the Quality of Fried Onion Slices
Authors: Asefi Narmela , Mozaffari Mansoure
Published: June 2010

Abstract:  Onion is used widely as a condiment for meat and other types of food in Iran. Because of the high sensitivity of this product to storage conditions and having not enough storage under controlled atmosphere (CA), the effects of drying method, packaging, and temperature during storage on thin slices of fried red Azershahr variety onion were investigated. To prepare this processed food with excellent organoleptic properties and useful in the food service industry onions were sliced (2 mm), deep‐fried with corn frying oil in a batch fryer at 150 °C, and dried to a water content of 3‐4% in a hot‐air dryer at 70 °C for 24 h and a microwave system. Samples were packed in aluminum foil, which was purged by nitrogen gas, and cellophane‐sealed with thermal sewing. Then these samples were kept for one year at laboratory temperature and in a freezer at −18 °C. During the storage time the moisture content, peroxide value, vitamin C content, total microbial count (TMC), and organoleptic characteristics of the samples were analyzed every 2 mo. The results of microbial analyses indicated that after 6 mo all of the samples had higher TMCs than the permission limits so the expiration date was set before that. The moisture contents and peroxide values showed a noticeable increase, probably due to seal failure, whereas vitamin C decreased up to 66% during storage. The loss of sensory properties was parallel to the microbial and chemical results. The findings revealed that the best sample was oven‐dried, packed in aluminum foil under inert gas, and kept in a freezer (OAF) up to 6 mo.
Audiovisual Interventions to Reduce the Use of General Anaesthesia with Paediatric Patients during Radiation Therapy
Authors: Willis D , Barry P
Published: June 2010

Clinical audiovisual interventions were implemented to avoid the use of general anaesthesia with children undergoing radiation therapy treatment. A retrospective audit and case study review was conducted to evaluate the utility of distraction interventions aimed at improving immobilisation and reducing separation anxiety for children aged between 2 and 6 years old who received radiation therapy. A simple, inexpensive audiovisual system was established using commercially available equipment. Paediatric patients could elect to (i) use a closed‐circuit TV system that allowed them to see their carer(s); (ii) watch a DVD movie; or (iii) listen to carer(s) on a microphone during their treatment. Over a 2‐year period (March 2007–May 2009), 37 paediatric patients aged 2–6 years received radiation therapy at the centre. Twenty‐four children participated in audiovisual interventions, and 92% (n = 22) of these children did not require the use of general anaesthesia for some or all of their treatment. Case study review illustrates the utility and limitations of the system. The audit and case studies suggest that the audiovisual interventions provided supportive care and reduced the need to anaesthetise children undergoing radiation therapy treatment.
Use of binaural cues for sound localization in large and small non-echolocating bats: Eidolon helvum and Cynopterus brachyotis
Authors: Heffner Rickye S. , Koay Gimseong , Heffner Henry E.
Published: June 2010

The authors determined the ability of two old-world non-echolocating bats, Eidolon helvum and Cynopterus brachyotis, to use binaural time and intensity difference cues for localization. The bats were trained to localize pure tones throughout most of their hearing range from loudspeakers located 30° to the left and right of midline. Both species easily localized high frequency tones, indicating they could use the interaural intensity difference cue. However, neither was able to localize low frequency tones even when the tones were amplitude modulated thereby indicating that they could not use ongoing phase difference cues. The authors now know of eight mammals that do not use binaural phase cues for localization, and some possible reasons for this inability are explored.
The scattering of the fundamental torsional mode from axi-symmetric defects with varying depth profile in pipes
Authors: Carandente R. , Ma J. , Cawley P.
Published: June 2010

Guided ultrasonic waves have been successfully applied to detect defects and corrosion in plates and pipes; however, defect sizing remains challenging due to the complex profiles of the defects encountered in practice. A study of the reflection of the fundamental torsional mode T(0,1) from an axi-symmetric defect with varying depth profile has been carried out via finite element modeling and experimental validation. Defects with gradually varying depth are represented by a series of tapered steps. It is revealed that, for both tapered up- and down-steps, the wave packet is reflected only at the start and end of the steps. The reflections from the start and end of the step have been predicted, and the frequency dependence of the overall reflection from the step has been studied. The study shows that the reflection coefficient varies as a function of the ratio of the average length of the tapered defect to the wavelength due to the interference between the waves reflected from the two ends of the defect. The reflection coefficient maxima decrease as the slope of the taper becomes more gradual, this effect being more pronounced when the ratio of the average defect length to the wavelength increases.
Measures of tactual detection and temporal order resolution in congenitally deaf and normal-hearing adults
Authors: Moallem Theodore M. , Reed Charlotte M. , Braida Louis D.
Published: June 2010

To guide the development of tactile speech aids, tactual detection and temporal order discrimination by congenitally deaf and normal-hearing adults have been examined. Tactual detection thresholds for sinusoidal vibrations between 2 and 300 Hz were measured at the left thumb and index finger using an adaptive paradigm. Temporal onset- and offset-order discrimination were tested using stimuli of 50 Hz at the thumb and 250 Hz at the index finger, delivered asynchronously and varied independently in amplitude and duration. Mean detection thresholds for the deaf and normal-hearing groups did not differ significantly at any frequency tested. Temporal onset-order discrimination thresholds varied widely, particularly among congenitally deaf individuals, but no statistically significant difference was found between group means. Both experimental groups exhibited a broad range of discrimination thresholds for temporal offset-order, and mean thresholds did not differ significantly. On the whole, tactual offset-order thresholds were substantially higher than onset-order thresholds. Differences in the relative levels of paired stimuli systematically affected sensitivity to both onset- and offset-orders in most subjects. Differences in the relative durations of paired stimuli had little effect on onset-order discrimination, but had a robust effect on offset-order discrimination thresholds, which was consistent across all subjects.
Influence of the noise sources motion on the estimated Green’s functions from ambient noise cross-correlations
Authors: Sabra Karim G.
Published: June 2010

It has been demonstrated theoretically and experimentally that an estimate of the Green’s function between two receivers can be obtained by cross-correlating acoustic (or elastic) ambient noise recorded at these two receivers. Coherent wavefronts emerge from the noise cross-correlation time function due to the accumulated contributions over time from noise sources whose propagation path pass through both receivers. Previous theoretical studies of the performance of this passive imaging technique have assumed that no relative motion between noise sources and receivers occurs. In this article, the influence of noise sources motion (e.g., aircraft or ship) on this passive imaging technique was investigated theoretically in free space, using a stationary phase approximation, for stationary receivers. The theoretical results were extended to more complex environments, in the high-frequency regime, using first-order expansions of the Green’s function. Although sources motion typically degrades the performance of wideband coherent processing schemes, such as time-delay beamforming, it was found that the Green’s function estimated from ambient noise cross-correlations are not expected to be significantly affected by the Doppler effect, even for supersonic sources. Numerical Monte-Carlo simulations were conducted to confirm these theoretical predictions for both cases of subsonic and supersonic moving sources.
An investigation of community noise in high-rise residential environments
Authors: Alam Sheikh Mahbub , Eang Lee Siew , Tan Alan , Tiong Tan Sze
Published: June 2010

High-rise dwellers in Singapore are often subjected to several community noise sources in close proximity. These include food center, children playground, soccer playground, basketball playground, waste disposal truck, etc. A scientific and reliable approach is required for evaluation of the community noise and its impact on high-rise dwellers. A comprehensive noise survey by a cluster sampling technique, conducted among 522 households in five residential towns in Singapore, showed that community noise was one of the prime sources of noise in a high-rise residential environment. From a subjective noise survey, undertaken concurrent with objective noise measurements, a mean outdoor noise level of 59 dBA was established as an acceptable noise level in the indoor environment. To investigate the level of noise exposure from different community noise sources, software modeling and simulations were carried out. The predicted results were validated with field measured data at five 16 story residential buildings. Analysis of noise exposure data showed that except for waste disposal truck, noise exposure due to other community noise sources (building distance of 15 m) were within the established acceptable noise level. A factor analysis of the survey data identified the key factors related to the disturbance due to community noise sources.
Perception of articulatory dynamics from acoustic signatures
Authors: Iskarous Khalil , Nam Hosung , Whalen D. H.
Published: June 2010

This study investigated the degree to which the articulatory trajectory of the tongue dorsum in the production of a vowel-vowel sequence is perceptually relevant. Previous research has shown that the tongue dorsum takes a path that leads to a pattern of area function change, termed the pivot pattern. In this study, articulatory synthesis was used to generate paths of tongue motion for the production of the vowel sequence /ai/. These paths differed in their curvature, leading to stimuli that conform to the pivot pattern and stimuli that violate it. Participants gave naturalness ratings and discriminated the stimuli. The acoustic properties were also compared to acoustic measurements made on productions of /ai/ by 34 speakers. The curvature of the tongue path and the curvature of the F1-F2 trajectory correlate highly with the naturalness-rating task results, but not the discrimination results. However, the particular way in which constriction location changes, particularly whether the change is discrete or continuous, and the maximal velocity of F2 through the transition, explain the perceptual patterns evident in both perception tasks, as well as the patterns in the observed acoustic data. Consequences of these results for the links between production and perception and the segmentation problem are discussed.
Predicting transmission of shaped sonic booms into a residential house structure
Authors: Sizov Natalia V. , Plotkin Kenneth J. , Hobbs Christopher M.
Published: June 2010

Human perception of sonic booms is a major impediment to commercial supersonic flight. Shaping, which reduces the audible shock waves of a boom, can make outdoor perception of booms acceptable. Perception of sonic booms experienced indoors is of concern, and it is not yet established whether shaped booms offer benefit to indoor listeners. A better understanding of the transmission of shaped booms into building structures is needed. In the authors’ earlier work the vibration response of house elements subjected to different sonic boom wave shapes was evaluated using a single degree of freedom model. This paper expands that approach with a modal analysis model. The acceleration of building elements and the resulting sound pressure inside a room are computed in the time and frequency domains. Analytical results are compared with experimental data measured by NASA during sonic boom tests conducted at Edwards Air Force Base in 2007. The effects of wave signature parameters on transmission are studied to evaluate the advantages of various kinds of minimized boom shapes.
Vocal tract adjustments in the high soprano range
Authors: Garnier Maëva , Henrich Nathalie , Smith John , Wolfe Joe
Published: June 2010

Twelve sopranos with different levels of expertise (4 nonexperts, 4 advanced, 4 professionals) sustained pitches from A4 (∼440 Hz) to their highest pitch (ranging from C6 to D7, i.e., from ∼1000 to 2300 Hz). The frequencies of their first two vocal tract resonances (R1 and R2) were measured by broadband excitation at the mouth and compared with the voice harmonics (f0, 2f0, etc). Lip articulation was measured from simultaneous video recordings. Adjustment of R1 near to f0 (R1:f0 tuning) was observed below C6 to D6 (∼1000–1200 Hz) for both expert and non-expert singers. Experts began this tuning at lower pitches. Some singers combine R2:2f0 adjustment with R1:f0 tuning. Some singers increased mouth area with increasing pitch over the whole R1:f0 tuning range. Other singers showed this strategy on the higher part of the R1:f0 range only, and used another, as yet unidentified, articulatory strategy on the lower part. To achieve very high pitches, some singers extended the range of R1:f0 tuning as far as E6 to F#6 (∼1300–1500 Hz) while others adjusted R2 near f0 over the highest pitch range.
Meteorological influence on sound propagation between adjacent city canyons: A real-life experiment
Authors: Van Renterghem Timothy , Botteldooren Dick
Published: June 2010

Sound propagation between a courtyard and an adjacent street canyon, as influenced by a wide range of meteorological conditions, was investigated by means of a real-life experiment in a dense urban setting. During several months, test signals were emitted on a regular base by an outdoor loudspeaker in the courtyard and recorded by wall-mounted microphones in the courtyard and the street canyon. Detailed meteorological observations were made at nearby buildings with sensors at roof level. A thorough quality check of the recorded signals was performed, given the large amount of shielding and the presence of background noise. With increasing wind speed and sound frequency, a strong increase in coherence loss was observed. The wide variety of measured vertical temperature lapses was shown to have no effect given the short propagation distance. With increasing downwind wind speed, refraction into the shielded canyon was observed to a limited degree only. The rather small effect of building-induced refraction of sound by wind could be qualitatively explained by the geometry of the city canyons under study.
Metamorphic constraints on the evolution of the gneisses from the parautochthonous and allochthonous polycyclic belts, Grenville Province, western Quebec
Authors: Indares A. , Martignole J.
Published: June 2010

The tectono-metamorphic history of polycyclic "grey gneisses" located in the central Grenville Province of western Quebec has been constrained along a transect perpendicular to the length of the Grenville Orogen. Two terranes, the Réservoir Dozois terrane (RDT) and the Réservoir Baskatong terrane (RBT), were recognized from their structural, lithological, and geochronological characteristics. This subdivision has been confirmed by application of geothermobarometric techniques to appropriate mineral assemblages.The RDT is the southern extension of the parautochthonous belt of the Grenville Province, which in this area is composed of Archean rocks of upper-amphibolite grade. During the Grenvillian Orogeny, northwest-directed thrusting resulted in the tectonic burial of this terrane as a single tectonic unit, in contrast with the northern part of the parautochthonous belt, where several slices were imbricated against the Grenville Front. Maximum P–T conditions in the RDT (850 MPa, 720 °C) were likely Grenvillian and were followed by pervasive retrogression down to the hornblende–epidote subfacies. Locally, the RDT is overlain by remnants of thrust slices composed of monocyclic metasedimentary rocks that were deformed and metamorphosed in the granulite facies during the Grenvillian Orogeny.To the southeast, the RBT is an allochthonous or exotic terrane probably of Proterozoic age. It also experienced tectonic burial by thrusting (1030 MPa, 710 °C) during the Grenvillian Orogeny, whose thermal climax (790 °C) coincided with charnockite emplacement during decompression to 850 MPa.These two terranes are separated by a narrow strip of sheared rocks, the Renzy shear belt (RSB), which comprises mafic and ultramafic rocks subjected to high P and T (975 MPa, 745 °C). In view of the significant discrepancy between the metamorphic histories of the two terranes separated by the RSB, major tectonic transport has to be envisaged along this zone.
Arctodus simus from the Alaskan Arctic Slope
Authors: Churcher C. S. , Morgan A. V. , Carter L. D.
Published: June 2010

Arctodus simus yukonensis, the extinct northern North American short-faced bear, is represented by an immature left humerus lacking its unfused proximal epiphysis, which was recovered from a point bar on the Ikpikpuk River, Alaska (69°41′N, 154°54′W). This is the northernmost record of this bear. The specimen is dated at 27 190 ± 280 BP (Iso Trace TO-2539) on 14C analysis, which lies within the observed age distribtution of Arctodus. The individual is larger than average for the species based on the dimensions of the distal articulation, despite its immaturity, and may have been male.
EFFECTS OF THE GENERALIZED UNCERTAINTY PRINCIPLE ON THE INFLATION PARAMETERS
Authors: NOZARI KOUROSH , AKHSHABI SIAMAK
Published: June 2010

We investigate the effects of the generalized uncertainty principle on the inflationary dynamics of the early universe from both the standard and the braneworld viewpoint. We choose the Randall–Sundrum II model as our underlying braneworld scenario. We find that the quantum-gravitational effects lead to a spectral index which is not scale-invariant. Also, the amplitude of density fluctuations is reduced by increasing the strength of quantum-gravitational corrections. However, the tensor-to-scalar ratio increases by incorporation of these quantum gravity effects. We outline possible manifestations of these quantum gravity effects in the recent and future observations.
The Discovery of Zinc Fingers and Their Applications in Gene Regulation and Genome Manipulation
Authors: Klug Aaron
Published: June 2010

An account is given of the discovery of the classical Cys2His2 zinc finger, arising from the interpretation of biochemical studies on the interaction of the Xenopus protein transcription factor IIIA with 5S RNA, and of structural studies on its structure and its interaction with DNA. The finger is a self-contained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines, and by an inner hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA binding proteins generally make use of the two-fold symmetry of the double helix, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome.

The zinc finger design is ideally suited for engineering proteins to target specific genes. In the first example of their application in 1994, a three-finger protein was constructed to block the expression of an oncogene transformed into a mouse cell line. In addition, a reporter gene was activated by targeting an inserted zinc finger promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that by combining zinc fingers with other effector domains, e.g., from nucleases or integrases, to form chimeric proteins, genomes could be manipulated or modified. Several applications of such engineered zinc finger proteins are described here, including some of therapeutic importance.
Origins of Specificity in Protein-DNA Recognition
Authors: Rohs Remo , Jin Xiangshu , West Sean M. , Joshi Rohit , Honig Barry , Mann Richard S.
Published: June 2010

Specific interactions between proteins and DNA are fundamental to many biological processes. In this review, we provide a revised view of protein-DNA interactions that emphasizes the importance of the three-dimensional structures of both macromolecules. We divide protein-DNA interactions into two categories: those when the protein recognizes the unique chemical signatures of the DNA bases (base readout) and those when the protein recognizes a sequence-dependent DNA shape (shape readout). We further divide base readout into those interactions that occur in the major groove from those that occur in the minor groove. Analogously, the readout of the DNA shape is subdivided into global shape recognition (for example, when the DNA helix exhibits an overall bend) and local shape recognition (for example, when a base pair step is kinked or a region of the minor groove is narrow). Based on the >1500 structures of protein-DNA complexes now available in the Protein Data Bank, we argue that individual DNA-binding proteins combine multiple readout mechanisms to achieve DNA-binding specificity. Specificity that distinguishes between families frequently involves base readout in the major groove, whereas shape readout is often exploited for higher resolution specificity, to distinguish between members within the same DNA-binding protein family.
Transcript Elongation by RNA Polymerase II
Authors: Selth Luke A. , Sigurdsson Stefan , Svejstrup Jesper Q.
Published: June 2010

Until recently, it was generally assumed that essentially all regulation of transcription takes place via regions adjacent to the coding region of a gene—namely promoters and enhancers—and that, after recruitment to the promoter, the polymerase simply behaves like a machine, quickly “reading the gene.” However, over the past decade a revolution in this thinking has occurred, culminating in the idea that transcript elongation is extremely complex and highly regulated and, moreover, that this process significantly affects both the organization and integrity of the genome. This review addresses basic aspects of transcript elongation by RNA polymerase II (RNAPII) and how it relates to other DNA-related processes.
From Virus Structure to Chromatin: X-ray Diffraction to Three-Dimensional Electron Microscopy
Authors: Klug Aaron
Published: June 2010

Early influences led me first to medical school with a view to microbiology, but I felt the lack of a deeper foundation and changed to chemistry, which in turn led me to physics and mathematics. I moved to the University of Cape Town to work on the X-ray crystallography of some small organic compounds. I developed a new method of using molecular structure factors to solve the crystal structure, which won me a research studentship to Trinity College Cambridge and the Cavendish Laboratory. There I worked on the austenite-pearlite transition in steel. This is governed by the dissipation of latent heat, and I ended up numerically solving partial differential equations. I used the idea of nucleation and growth during the phase change, which had its echo when I later tackled the assembly of Tobacco mosaic virus (TMV) from its constituent RNA and protein subunits.

I wanted to move on to X-ray structure analysis of large biological molecules and obtained a Nuffield Fellowship to work in J.D. Bernal's department at Birkbeck College, London. There, I met Rosalind Franklin, who had taken up the study of TMV. I was able to interpret some of Franklin's beautiful X-ray diffraction patterns of the virus particle. From then on, my fate was sealed.

After Franklin's untimely death in 1958, I moved in 1962 to the newly built MRC Laboratory of Molecular Biology in Cambridge, which, under Max Perutz, housed the original MRC unit from the Cavendish Laboratory. I was thus privileged to join the Laboratory at an early stage in its expansion and consequently able to take advantage of, and to help build up, its then unique environment of intellectual and technological sophistication. There I have remained ever since.
The mechanism of ascorbic acid-induced differentiation of ATDC5 chondrogenic cells
Authors: Temu Tecla M. , Wu Ke-Ying , Gruppuso Philip A. , Phornphutkul Chanika
Published: June 2010

The ATDC5 cell line exhibits a multistep process of chondrogenic differentiation analogous to that observed during endochondral bone formation. Previous investigators have induced ATDC5 cells to differentiate by exposing them to insulin at high concentrations. We have observed spontaneous differentiation of ATDC5 cells maintained in ascorbic acid-containing α-MEM. A comparison of the differentiation events in response to high-dose insulin vs. ascorbic acid showed similar expression patterns of key genes, including collagen II, Runx2, Sox9, Indian hedgehog, and collagen X. We took advantage of the action of ascorbic acid to examine signaling events associated with differentiation. In contrast to high-dose insulin, which downregulates both IGF-I and insulin receptors, there were only minimal changes in the abundance of these receptors during ascorbic acid-induced differentiation. Furthermore, ascorbic acid exposure was associated with ERK activation, and ERK inhibition attenuated ascorbic acid-induced differentiation. This was in contrast to the inhibitory effect of ERK activation during IGF-I-induced differentiation. Inhibition of collagen formation with a proline analog markedly attenuated the differentiating effect of ascorbic acid on ATDC5 cells. When plates were conditioned with ATDC5 cells exposed to ascorbic acid, ATDC5 cells were able to differentiate in the absence of ascorbic acid. Our results indicate that matrix formation early in the differentiation process is essential for ascorbic acid-induced ATDC5 differentiation. We conclude that ascorbic acid can promote the differentiation of ATDC5 cells by promoting the formation of collagenous matrix and that matrix formation mediates activation of the ERK signaling pathway, which promotes the differentiation program.
Cellular depletion of atypical PKCλ is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells
Authors: Stretton Clare , Evans Ashleigh , Hundal Harinder S.
Published: June 2010

Atypical protein kinase C (aPKC) isoforms (λ and ζ) have been implicated in the control of insulin-stimulated glucose uptake in adipose and skeletal muscle, but their precise role in this process remains unclear, especially in light of accumulating evidence showing that, in response to numerous stimuli, including insulin and lipids such as ceramide, activation of aPKCs acts to negatively regulate key insulin-signaling molecules, such as insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB)/cAMP-dependent PKC (Akt). In this study, we have depleted PKCλ in L6 skeletal muscle cells using RNA interference and assessed the effect this has upon insulin action. Muscle cells did not express detectable amounts of PKCζ. Depletion of PKCλ (>95%) had no significant effect on the expression of proteins participating in insulin signaling [i.e., insulin receptor, IRS-1, phosphatidylinositol 3-kinase (PI 3-kinase), PKB, or phosphate and tensin homolog deleted on chromosome 10] or those involved in glucose transport [Akt substrate of 160 kDa, glucose transporter (GLUT)1, or GLUT4]. However, PKCλ-depleted muscle cells exhibited greater activation of PKB/Akt and phosphorylation of its downstream target glycogen synthase kinase 3, in the basal state and displayed greater responsiveness to submaximal doses of insulin with respect to p85-PI 3-kinase/IRS-1 association and PKB activation. The increase in basal and insulin-induced signaling resulted in an associated enhancement of basal and insulin-stimulated glucose transport, both of which were inhibited by the PI 3-kinase inhibitor wortmannin. Additionally, like RNAi-mediated depletion of PKCλ, overexpression of a dominant-negative mutant of PKCζ induced a similar insulin-sensitizing effect on PKB activation. Our findings indicate that aPKCs are likely to play an important role in restraining proximal insulin signaling events but appear dispensable with respect to insulin-stimulated glucose uptake in cultured L6 muscle cells.
Low Level ofHer-2Locus Amplification by Fluorescent In Situ Hybridization Does Not Correlate with Her-2 Protein Overexpression by Immunohistochemistry in Barrett's Esophagus
Authors: Rygiel Agnieszka M. , Milano Francesca , ten Kate Fiebo J. , Bergman Jacques J. G. H. M. , Krishnadath Kausillia K.
Published: June 2010

An accurate evaluation of theHer-2status has important prognostic and therapeutic implications in many carcinomas. The aim of the study was to correlateHer-2locus (17q11.2) amplification and chromosome 17 gains as assessed by fluorescent in situ hybridization (FISH) with Her-2 protein overexpression by immunohistochemistry (IHC) in patients with Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). We analyzed 34 patients withHer-2amplification and/or chromosome 17gains using FISH on brush cytology specimens. Seven patients (21%) showed highHer-2locus amplification (Her-2: Cep17≥5 : 1), 5 (15%) showed lowHer-2locus amplification (Her-2: Cep17≥2<5 : 1), and 22 (65%) displayed gains of chromosome 17 only. Further, we confirmedHer-2amplification on corresponding biopsies that were taken at the same occasion as the cytologybrushings. Then, we compared the FISH results with IHC data obtained from the corresponding biopsies and showed that low level of Her-2 amplification does not correlate with Her-2 protein overexpression (score+3/+2;P=1), in contrast to the high amplification level (P=.001). Thus, in our population of BE and EAC patients, low level ofHer-2amplification does not result in detectable level of Her-2 protein as assessed by IHC.
Direct Inhibition of Elastase Activity by Indole-3-Carbinol Triggers a CD40-TRAF Regulatory Cascade That Disrupts NF-κB Transcriptional Activity in Human Breast Cancer Cells
Authors: Aronchik Ida , Bjeldanes Leonard F. , Firestone Gary L.
Published: June 2010

Treatment of highly tumorigenic MDA-MB-231 human breast cancer cells with indole-3-carbinol (I3C) directly inhibited the extracellular elastase-dependent cleavage of membrane-associated CD40, a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 signaling has been implicated in regulating cell survival, apoptosis, and proliferation, as well as in sensitizing breast cancer cells to chemotherapy, and is therefore an important potential target of novel breast cancer treatments. The I3C-dependent accumulation of full-length unprocessed CD40 protein caused a shift in CD40 signaling through TNF receptor–associated factors (TRAF), including the TRAF1/TRAF2 positive regulators and TRAF3 negative regulator of NF-κB transcription factor activity. Because TRAF1 is a transcriptional target gene of NF-κB, I3C disrupted a positive feedback loop involving these critical cell survival components. siRNA ablation of elastase expression mimicked the I3C inhibition of CD40 protein processing and G1 cell cycle arrest, whereas siRNA knockdown of TRAF3 and the NF-κB inhibitor IκB prevented the I3C-induced cell cycle arrest. In contrast, siRNA knockdown of PTEN had no effect on the I3C control of NF-κB activity, showing the importance of CD40 signaling in regulating this transcription factor. Our study provides the first direct in vitro evidence that I3C directly inhibits the elastase-mediated proteolytic processing of CD40, which alters downstream signaling to disrupt NF-κB–induced cell survival and proliferative responses. Furthermore, we have established a new I3C-mediated antiproliferative cascade that has significant therapeutic potential for treatment of human cancers associated with high levels of elastase and its CD40 membrane substrate. Cancer Res; 70(12); 4961–71. ©2010 AACR.
Increased Immunogenicity of Tumor-Associated Antigen, Mucin 1, Engineered to Express α-Gal Epitopes: A Novel Approach to Immunotherapy in Pancreatic Cancer
Authors: Deguchi Takashi , Tanemura Masahiro , Miyoshi Eiji , Nagano Hiroaki , Machida Tomohiko , Ohmura Yoshiaki , Kobayashi Shogo , Marubashi Shigeru , Eguchi Hidetoshi , Takeda Yutaka , Ito Toshinori , Mori Masaki , Doki Yuichiro , Sawa Yoshiki
Published: June 2010

Mucin 1 (MUC1), a bound mucin glycoprotein, is overexpressed and aberrantly glycosylated in >80% of human ductal pancreatic carcinoma. Evidence suggests that MUC1 can be used as a tumor marker and is a potential target for immunotherapy of pancreatic cancer. However, vaccination with MUC1 peptides fails to stimulate the immune response against cancer cells because immunity toward tumor-associated antigens (TAA), including MUC1, in cancer patients is relatively weak, and the presentation of these TAAs to the immune system is poor due to their low immunogenicity. We investigated whether vaccination with immunogenetically enhanced MUC1 (by expressing α-gal epitopes; Galα1-3Galβ1-4GlcNAc-R) can elicit effective antibody production for MUC1 itself as well as certain TAAs derived from pancreatic cancer cells and induced tumor-specific T-cell responses. We also used α1,3galactosyltransferase (α1,3GT) knockout mice that were preimmunized with pig kidney and transplanted with B16F10 melanoma cells transfected with MUC1 expression vector. Vaccination of these mice with α-gal MUC1 resulted in marked inhibition of tumor growth and significant improvement of overall survival time compared with mice vaccinated with MUC1 alone (P = 0.003). Furthermore, vaccination with pancreatic cancer cells expressing α-gal epitopes induced immune responses against not only differentiated cancer cells but also cancer stem cells. The results suggested that vaccination using cells engineered to express α-gal epitopes is a novel strategy for treatment of pancreatic cancer. Cancer Res; 70(13); 5259–69. ©2010 AACR.
High Wnt Signaling Represses the Proapoptotic Proteoglycan syndecan-2 in Osteosarcoma Cells
Authors: Dieudonné François-Xavier , Marion Allison , Haÿ Eric , Marie Pierre Jacques , Modrowski Dominique
Published: June 2010

Osteosarcoma is characterized by frequent relapse and metastatic disease associated with resistance to chemotherapy. We previously showed that syndecan-2 is a mediator of the antioncogenic effect of chemotherapeutic drugs. The purpose of this work was to elucidate molecular mechanisms responsible for the low expression of syndecan-2 in osteosarcoma. We compared the regulatory activity of cis-acting DNA sequences of the syndecan-2 gene in osteosarcoma and osteoblastic cell lines. We identified a DNA region that negatively regulates syndecan-2 transcription in the osteosarcoma cells. T-cell factors (TCF) bind to this sequence in vivo. Wnt3a stimulation, β-catenin activation, and TCF overexpression resulted in syndecan-2 repression, whereas Wnt inhibition using sFRP-1 increased syndecan-2 expression in U2OS cells. RhoA activation blunted the stimulatory effect of sFRP-1 on syndecan-2 transcription, whereas RhoA inhibition enhanced syndecan-2 expression. These results indicate that Wnt/β-catenin and Wnt/RhoA signaling contribute to syndecan-2 repression. The alteration of syndecan-2 expression in osteosarcoma cell lines also seemed to be related to a higher shedding, controlled by Wnt/RhoA. Conversely, syndecan-2 was found to activate its own expression in U2OS cells through RhoA inhibition. These data identify a molecular network that may contribute to the low expression of the proapoptotic proteoglycan syndecan-2 in osteosarcoma cells. The high activity of the canonical Wnt pathway in the different osteosarcoma cells induces a constitutive repression of syndecan-2 transcription, whereas Wnt/RhoA signaling blocks the amplification loop of syndecan-2 expression. Our results identify syndecan-2 as a Wnt target and bring new insights into a possible pathologic role of Wnt signaling in osteosarcoma. Cancer Res; 70(13); 5399–408. ©2010 AACR.
Development of Human Single-Chain Antibodies to the Transferrin Receptor that Effectively Antagonize the Growth of Leukemias and Lymphomas
Authors: Crépin Ronan , Goenaga Anne-Laure , Jullienne Betsy , Bougherara Houcine , Legay Christine , Benihoud Karim , Marks James D. , Poul Marie-Alix
Published: June 2010

The major route of iron uptake by cells occurs through transferrin receptor (TfR)–mediated endocytosis of diferric-charged plasma transferrin (holo-Tf). In this work, we pursued TfR antibodies as potential cancer therapeutics, characterizing human single-chain variable antibody fragments (scFv) specific for the human TfR isolated from a phage display library. We hypothesized that many of these antibodies would function as ligand mimetics because scFvs from the library were selected for binding and internalization into living cells. In support of this hypothesis, the anti-TfR scFvs identified were antagonists of TfR binding to holo-Tf, particularly two of the most potent antibodies, 3TF12 and 3GH7, which blocked the in vitro proliferation of a number of hematopoietic cancer cell lines. We optimized this activity of 3TF12 and 3GH7 by engineering 55-kDa bivalent antibody formats, namely, F12CH and H7CH, which could block cell proliferation with an IC50 of 0.1 μg/mL. We found that the mechanism of the scFv antibody cytotoxicity was unique compared with cytotoxic anti-TfR monoclonal antibodies that have been described, causing cell surface upregulation of TfR along with the inhibition of holo-Tf cell uptake and induction of cell death. In a nude mouse model of erythroleukemia, administration of F12CH reduced tumor growth. Together, our findings define a new class of fully human anti-TfR antibodies suitable for immunotherapy against tumors whose proliferation relies on high levels of TfR and iron uptake, such as acute lymphoid and myeloid leukemias. Cancer Res; 70(13); 5497–506. ©2010 AACR.
Drug-Induced Vessel Remodeling in Bone Metastases as Assessed by Dynamic Contrast Enhanced Magnetic Resonance Imaging and Vessel Size Imaging: A LongitudinalIn vivoStudy
Authors: Bäuerle Tobias , Merz Maximilian , Komljenovic Dorde , Zwick Stefan , Semmler Wolfhard
Published: June 2010

Purpose: The aim of this study was to assess the antiangiogenic treatment effects of zoledronic acid (ZA) and sunitinib malate (SM) noninvasively in experimental breast cancer bone metastases by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and vessel size imaging.

Experimental Design: Nude rats bearing bone metastases after inoculation of MDA-MB-231 breast cancer cells were treated with ZA (40 μg/kg weekly; n = 8 rats), SM (20 mg/kg daily; n = 8 rats), or their combination (ZA and SM; n = 8 rats) and compared with sham-treated controls (n = 10 rats). Vascular changes in bone metastases were longitudinally imaged in vivo using DCE-MRI [amplitude (A) and exchange rate coefficient (kep)] and vessel size imaging [blood volume (BV) and vessel size index (VI)]. In addition, antiresorptive and antitumor changes were assessed in these lesions by flat-panel volumetric computed tomography as well as morphologic MRI and diffusion-weighted imaging.

Results: In bone metastases, significant changes in A, kep, BV, and VI in accordance with decreased blood volume and vessel permeability as well as with increased mean vessel diameters were observed after application of ZA and SM as compared with controls. In this longitudinal study, antiangiogenic changes preceded the inhibition of osteolysis and antitumor effects after treatment.

Conclusions: These results indicate vessel remodeling in breast cancer bone metastases on ZA and SM treatment and implicate substantial effects on imaging and treatment of malignant bone lesions. Clin Cancer Res; 16(12); 3215–25. ©2010 AACR.
Bioengineering a Unique Deimmunized Bispecific Targeted Toxin That Simultaneously Recognizes Human CD22 and CD19 Receptors in a Mouse Model of B-Cell Metastases
Authors: Vallera Daniel A. , Oh Seunguk , Chen Hua , Shu Yanqun , Frankel Arthur E.
Published: June 2010

A drug of high potency and reduced immunogenicity is needed to develop a targeted biological drug that when injected systemically can penetrate to malignant B cells. Therefore, a novel deimmunized bispecific ligand-directed toxin targeted by dual high-affinity single-chain Fvs (scFv) spliced to PE38 with a KDEL COOH-terminus was genetically engineered. The aims were to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit antitoxin antibody responses, and show that mutated drug was effective against systemic B-cell lymphoma in vivo. Both human anti-CD22 scFv and anti-CD19 scFv were cloned onto the same single-chain molecule with truncated pseudomonas exotoxin (PE38) to create the drug. Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISAs were done to determine whether antitoxin antibodies were reduced. Finally, a powerful genetically altered luciferase xenograft model was used that could be imaged in real time to determine the effect on systemic malignant human B-cell lymphoma, Raji-luc. Patient B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B lymphoma were high in CD22 and CD19 expression. 2219KDEL7mut was significantly effective against systemic Raji-luc in mice and prevented metastatic spread. Mutagenesis reduced neutralizing antitoxin antibodies by ∼80% with no apparent loss in in vitro or in vivo activity. Because 2219KDEL7mut immunogenicity was significantly reduced and the drug was highly effective in vivo, we can now give multiple drug treatments with targeted toxins in future clinical trials. Mol Cancer Ther; 9(6); 1872–83. ©2010 AACR.
Ethno-Religious Communities: To the Problem of Identity Markers
Authors: Arakelova Victoria
Published: June 2010

The paper focuses on the phenomenon of ethno-religiousness and, particularly, on the process of the formation of ethno-religious communities. In the spotlight of the research is the Yezidi identity—the stages of its formation from the new syncretic mentality, initially exclusively with the religious vector, and later having acquired the drive to ethnicity. The similar processes can be traced in other cases of ethno-religious identities, e.g., the Mandaeans and the Druzes, both cases being used as comparative material.
A Remnant Remaining: Armenians amid Northern Iraq's Christian Minority
Authors: Logan Darren
Published: June 2010

Northern Mesopotamia is the source for numerous ethnicities and religions. Each has played a role in forming a fragile, beautiful, yet sometimes painful cultural, religious and social montage in the region. Over the course of millennia, under numberless rulers and regimes, and through successive conflicts, many of these once historic majorities gradually faded into the ascending Arab and Islamic milieu. Within this ethnic and religious mosaic comprising Iraq, one finds a relatively large Christian community. Digging deeper still one discovers a small remaining remnant of Armenians. Long ago this ancient Christian people dominated the Armenian Highland, territory now located in Eastern Turkey. Historical evidence indicates an Armenian presence in Mesopotamia for well over several thousand years. With this as background, the paper is a brief consideration of the Christian minorities—chiefly the small Armenian community—located in Iraqi territory officially under the control of the Kurdistan Regional Government (KRG), generally based on the author's field-work in Iraqi Kurdistan from December 2009 to March 2010.