An in vitro study showing the three-dimensional microenvironment influence over the behavior of head and neck squamous cell carcinoma

Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-kappa B and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.

A BEM-FEM model for the dynamic analysis of building structures founded on elastic or poroelastic soils.

[EN]This work presents a time-harmonic boundary elementfinite element three-dimensional model for the dynamic analysis of building structures founded on elastic or porelastic soils. The building foundation and soil domains are modelled as homogeneous, isotropic, elastic or poroelastic media using boundary elements.

Ground state properties of neutron-rich Mg isotopes

Studies in regions of the nuclear chart in which the model predictions of properties of nuclei fail can bring a better understanding of the strong interaction in the nuclear medium. To such regions belongs the so called "island of inversion" centered around Ne, Na and Mg isotopes with 20 neutrons in which unexpected ground-state spins, large deformations and dense low-energy spectra appear. This is a strong argument that the magic N = 20 is not a closed shell in this area. In this thesis investigations of isotope shifts of stable 24,25,26Mg, as well as spins and magnetic moments of short-lived 29,31Mg are presented. The successful studies were performed at the ISOLDE facility at CERN using collinear laser and beta-NMR spectroscopy techniques. The isotopes were investigated as single-charged ions in the 280-nm transition from the atomic ground state 2S1/2 to one of the two lowest excited states 2P1/2,3/2 using continuous wave laser beams. The isotope-shift measurements with fluorescence detection for the three stable isotopes show that it is feasible to perform the same studies on radioactive Mg isotopes up to the "island of inversion". This will allow to determine differences in the mean charge square radii and interpret them in terms of deformation. The high detection efficiency for beta particles and optical pumping close to saturation allowed to obtain very good beta-asymmetry signals for 29Mg and 31Mg with half-lives around 1 s and production yields about 10^5 ions/s. For this purpose the ions were implanted into a host crystal lattice. Such detection of the atomic resonances revealed their hyperfine structure, which gives the sign and a first estimate of the value of the magnetic moment. The nuclear magnetic resonance gave also their g-factors with the relative uncertainty smaller than 0.2 %. By combining the two techniques also the nuclear spin of both isotopes could be unambiguously determined. The measured spins and g-factors show that 29Mg with 17 neutrons lies outside the "island of inversion". On the other hand, 31Mg with 19 neutrons has an unexpected ground-state spin which can be explained only by promoting at least two neutrons across the N = 20 shell gap. This places the above nucleus inside the "island". However, modern shell-model approaches cannot predict this level as the ground state but only as one of the low-lying states, even though they reproduce very well the experimental g-factor. This indicates that modifications to the available interactions are required. Future measurements include isotope shift measurements on radioactive Mg isotopes and beta-NMR studies on 33Mg.

Laser systems for collinear spectroscopy and the charge radius of 12 Be

Die kollineare Laserspektroskopie hat sich in den vergangenen drei Jahrzehnten zur Bestimmung der Kernladungsradien mittelschwerer und schwerer kurzlebiger Atomkerne in ausgezeichneter Weise bewährt. Auf die Isotope sehr leichter Elemente konnte sie allerdings erst kürzlich erweitert werden. Dieser Bereich der Nuklidkarte ist von besonderem Interesse, denn die ersten ab-initio Modelle der Kernphysik, die den Aufbau eines Atomkerns basierend auf individuellen Nukleonen und realistischenWechselwirkungspotentialen beschreiben, sind gegenwärtig nur für die leichtesten Elemente anwendbar. Außerdem existiertrnin dieser Region eine besonders exotische Form von Atomkernen, die sogenanntenrnHalokerne. Die Isotopenkette der Berylliumisotope zeichnet sich durch das Auftreten des Ein-Neutronen Halokerns 11Be und des Zwei- oder Vier-Neutronen-Halos 14Be aus. Dem Isotop 12Be kommt durch seine Position zwischen diesen beiden Exoten und den im Schalenmodell erwarteten magischen Schalenabschluss N = 8 eine besondere Bedeutung zu.rnIm Rahmen dieser Arbeit wurden mehrere frequenzstabilisierte Lasersysteme für die kollineare Laserspektroskopie aufgebaut. An TRIGA-SPEC stehen nun unter anderem ein frequenzverdoppeltes Diodenlasersystem mit Trapezverstärker und frequenzkammstabilisierter Titan-Saphirlaser mit Frequenzverdopplungsstufe für die Spektroskopie an refraktären Elementen oberhalb von Molybdän zur Verfügung, die für erste Testexperimente eingesetzt wurden. Außerdem wurde die effiziente Frequenzvervierfachung eines Titan-Saphirlasers demonstriert. An ISOLDE/CERN wurde ein frequenzkammstabilisierter und ein jodstabilisierter Farbstofflaser installiert und für die Laserspektroskopie an 9,10,11,12Be eingesetzt. Durch das verbesserte Lasersystem und den Einsatz eines verzögerten Koinzidenznachweises für Photonen und Ionen gelang es die Empfindlichkeitrnder Berylliumspektroskopie um mehr als zwei Größenordnungen zu steigern und damit die früheren Messungen an 7−11Be erstmals auf das Isotop 12Be auszuweiten. Außerdem wurde die Genauigkeit der absoluten Übergangsfrequenzen und der Isotopieverschiebungen der Isotope 9,10,11Be signifikant verbessert.rnDurch den Vergleich mit Ergebnissen des Fermionic Molecular Dynamics Modells kann der Trend der Ladungsradien der leichteren Isotope durch die ausgeprägte Clusterstruktur der Berylliumkerne erklärt werden. Für 12Be wird ersichtlich, dass der Grundzustand durch eine (sd)2 Konfiguration statt der vom Schalenmodell erwarteten p2 Konfiguration dominiert wird. Dies ist ein klares Indiz für das bereits zuvor beobachtete Verschwinden des N = 8 Schalenabschlusses bei 12Be.

Laser scanning microscopy combined with image restoration to analyse a 3D model of the human epithelial airway barrier

A laser scanning microscope collects information from a thin, focal plane and ignores out of focus information. During the past few years it has become the standard imaging method to characterise cellular morphology and structures in static as well as in living samples. Laser scanning microscopy combined with digital image restoration is an excellent tool for analysing the cellular cytoarchitecture, expression of specific proteins and interactions of various cell types, thus defining valid criteria for the optimisation of cell culture models. We have used this tool to establish and evaluate a three dimensional model of the human epithelial airway wall.

Finite Element Modeling of Shell Shape in the Freshwater Turtle Pseudemys concinna Reveals a Trade-Off between Mechanical Strength and Hydrodynamic Efficiency

Aquatic species can experience different selective pressures on morphology in different flow regimes. Species inhabiting lotic regimes often adapt to these conditions by evolving low-drag (i.e., streamlined) morphologies that reduce the likelihood of dislodgment or displacement. However, hydrodynamic factors are not the only selective pressures influencing organismal morphology and shapes well suited to flow conditions may compromise performance in other roles. We investigated the possibility of morphological trade-offs in the turtle Pseudemys concinna. Individuals living in lotic environments have flatter, more streamlined shells than those living in lentic environments; however, this flatter shape may also make the shells less capable of resisting predator-induced loads. We tested the idea that ‘‘lotic’’ shell shapes are weaker than ‘‘lentic’’ shell shapes, concomitantly examining effects of sex. Geometric morphometric data were used to transform an existing finite element shell model into a series of models corresponding to the shapes of individual turtles. Models were assigned identical material properties and loaded under identical conditions, and the stresses produced by a series of eight loads were extracted to describe the strength of the shells. ‘‘Lotic’’ shell shapes produced significantly higher stresses than ‘‘lentic’’ shell shapes, indicating that the former is weaker than the latter. Females had significantly stronger shell shapes than males, although these differences were less consistent than differences between flow regimes. We conclude that, despite the potential for many-to-one mapping of shell shape onto strength, P. concinna experiences a trade-off in shell shape between hydrodynamic and mechanical performance. This trade-off may be evident in many other turtle species or any other aquatic species that also depend on a shell for defense. However, evolution of body size may provide an avenue of escape from this trade-off in some cases, as changes in size can drastically affect mechanical performance while having little effect on hydrodynamic performance.

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

Early and Middle Holocene Hunter-Gatherer Occupations in Western Amazonia: The Hidden Shell Middens

We report on previously unknown early archaeological sites in the Bolivian lowlands, demonstrating for the first time early and middle Holocene human presence in western Amazonia. Multidisciplinary research in forest islands situated in seasonally-inundated savannahs has revealed stratified shell middens produced by human foragers as early as 10,000 years ago, making them the oldest archaeological sites in the region. The absence of stone resources and partial burial by recent alluvial sediments has meant that these kinds of deposits have, until now, remained unidentified. We conducted core sampling, archaeological excavations and an interdisciplinary study of the stratigraphy and recovered materials from three shell midden mounds. Based on multiple lines of evidence, including radiocarbon dating, sedimentary proxies (elements, steroids and black carbon), micromorphology and faunal analysis, we demonstrate the anthropogenic origin and antiquity of these sites. In a tropical and geomorphologically active landscape often considered challenging both for early human occupation and for the preservation of hunter-gatherer sites, the newly discovered shell middens provide evidence for early to middle Holocene occupation and illustrate the potential for identifying and interpreting early open-air archaeological sites in western Amazonia. The existence of early hunter-gatherer sites in the Bolivian lowlands sheds new light on the region’s past and offers a new context within which the late Holocene “Earthmovers” of the Llanos de Moxos could have emerged.

Statistical model-based segmentation of the proximal femur in digital antero-posterior (AP) pelvic radiographs.

PURPOSE    Segmentation of the proximal femur in digital antero-posterior (AP) pelvic radiographs is required to create a three-dimensional model of the hip joint for use in planning and treatment. However, manually extracting the femoral contour is tedious and prone to subjective bias, while automatic segmentation must accommodate poor image quality, anatomical structure overlap, and femur deformity. A new method was developed for femur segmentation in AP pelvic radiographs. METHODS    Using manual annotations on 100 AP pelvic radiographs, a statistical shape model (SSM) and a statistical appearance model (SAM) of the femur contour were constructed. The SSM and SAM were used to segment new AP pelvic radiographs with a three-stage approach. At initialization, the mean SSM model is coarsely registered to the femur in the AP radiograph through a scaled rigid registration. Mahalanobis distance defined on the SAM is employed as the search criteria for each annotated suggested landmark location. Dynamic programming was used to eliminate ambiguities. After all landmarks are assigned, a regularized non-rigid registration method deforms the current mean shape of SSM to produce a new segmentation of proximal femur. The second and third stages are iteratively executed to convergence. RESULTS    A set of 100 clinical AP pelvic radiographs (not used for training) were evaluated. The mean segmentation error was [Formula: see text], requiring [Formula: see text] s per case when implemented with Matlab. The influence of the initialization on segmentation results was tested by six clinicians, demonstrating no significance difference. CONCLUSIONS    A fast, robust and accurate method for femur segmentation in digital AP pelvic radiographs was developed by combining SSM and SAM with dynamic programming. This method can be extended to segmentation of other bony structures such as the pelvis.

A convolution model for energy transport in a therapeutic fast neutron beam

A three-dimensional model has been proposed that uses Monte Carlo and fast Fourier transform convolution techniques to calculate the dose distribution from a fast neutron beam. This method transports scattered neutrons and photons in the forward, lateral, and backward directions and protons, electrons, and positrons in the forward and lateral directions by convolving energy spread kernels with initial interaction available energy distributions. The primary neutron and photon spectrums have been derived from narrow beam attenuation measurements. The positions and strengths of the effective primary neutron, scattered neutron, and photon sources have been derived from dual ion chamber measurements. The size of the effective primary neutron source has been measured using a copper activation technique. Heterogeneous tissue calculations require a weighted sum of two convolutions for each component since the kernels must be invariant for FFT convolution. Comparisons between calculations and measurements were performed for several water and heterogeneous phantom geometries. ^

Three-dimensional printing for perioperative planning of complex aortic arch surgery

Purpose In this study, we show the use of three-dimensional printing models for preoperative planning of surgery for patients with complex aortic arch anomalies. Description A 70-year-old man with an extensively arteriosclerotic aneurysm reaching from the ascending aorta to the descending aorta was referred to our center for complete aortic arch replacement. We visualized and reconstructed computed tomography data of the patient and fabricated a flexible three-dimensional model of the aortic arch including the aneurysm. Evaluation This model was very helpful for the preoperative decision making and planning of the frozen elephant trunk procedure owing to the exact and lifelike illustration of the native aortic arch. Conclusions Three-dimensional models are helpful in preoperative planning and postoperative evaluation of frozen elephant trunk procedures in patients with complex aortic anatomy.

A functionally defined model for the M2 proton channel of influenza A virus suggests a mechanism for its ion selectivity

The M2 protein from influenza A virus forms proton-selective channels that are essential to viral function and are the target of the drug amantadine. Cys scanning was used to generate a series of mutants with successive substitutions in the transmembrane segment of the protein, and the mutants were expressed in Xenopus laevis oocytes. The effect of the mutations on reversal potential, ion currents, and amantadine resistance were measured. Fourier analysis revealed a periodicity consistent with a four-stranded coiled coil or helical bundle. A three-dimensional model of this structure suggests a possible mechanism for the proton selectivity of the M2 channel of influenza virus.

RNA replication by Q beta replicase: a working model.

Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Q beta replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I- and site II-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Q beta replicase.

Assessing advertising in a hierarchical decision model

Many destination marketing organizations in the United States and elsewhere are facing budget retrenchment for tourism marketing, especially for advertising. This study evaluates a three-stage model using Random Coefficient Logit (RCL) approach which controls for correlations between different non-independent alternatives and considers heterogeneity within individual’s responses to advertising. The results of this study indicate that the proposed RCL model results in a significantly better fit as compared to traditional logit models, and indicates that tourism advertising significantly influences tourist decisions with several variables (age, income, distance and Internet access) moderating these decisions differently depending on decision stage and product type. These findings suggest that this approach provides a better foundation for assessing, and in turn, designing more effective advertising campaigns.