Rapid fabrication of collagen bundles mimicking tumor-associated collagen architectures.

Stromal collagen is upregulated surrounding a solid tumor and presents as dense, thick, linearized, and aligned bundles. The collagen bundles are continually remodeled during tumor progression, and their orientation with respect to the tumor boundary has been correlated with invasive state. Currently, reconstituted-collagen gels are the standard in vitro tumor cell-extracellular matrix interaction model. The reticular, dense, and isotropic nanofiber (~900 nm-diameter, on average) gels do not, however, recapitulate the in vivo structural features of collagen bundling and alignment. Here, we present a rapid and simple method to fabricate bundles of collagen type I, whose average thickness may be varied between about 4 µm and 9 µm dependent upon diluent temperature and ionic strength. The durability and versatility of the collagen bundles was demonstrated with their incorporation into two in vitro models where the thickness and alignment of the collagen bundles resembled various in vivo arrangements. First, collagen bundles aligned by a microfluidic device elicited cancer cell contact guidance and enhanced their directional migration. Second, the presence of the collagen bundles in a bio-inert agarose hydrogel was shown to provide a route for cancer cell outgrowth. The unique structural features of the collagen bundles advance the physiological relevance of in vitro collagen-based tumor models for accurately capturing tumor cell-extracellular matrix interactions. STATEMENT OF SIGNIFICANCE: Collagen in the tumor microenvironment is upregulated and remodeled into dense, thick, and aligned bundles that are associated with invasive state. Current collagen-based in vitro models are based on reticular, isotropic nanofiber gels that do not fully recapitulate in vivo tumor stromal collagen. We present a simple and robust method of rapidly fabricating cell-scale collagen bundles that better mimic the remodeled collagen surrounding a tumor. Interacting with the bundles, cancer cells exhibited drastically different phenotypic behaviors, compared to nanofiber scaffolds. This work reveals the importance of microscale architecture of in vitro tumor models. The collagen bundles provide physiologically relevant collagen morphologies that may be easily incorporated into existing models of tumor cell-extracellular matrix interactions.

Toward Morphologically Relevant Extracellular Matrix in Vitro Models: 3D Fiber Reinforced Hydrogels.

The extracellular matrix (ECM) is known to play an important role in the health of cells and tissues. Not only are chemical signals transmitted via bonds and tightly controlled diffusion, but the structure of the ECM also provides important physical signaling for the cells attached to it. The structure is composed of a mesh of fibrous proteins, such as collagen, embedded in a hydrated gel matrix of glycosaminoglycans. To study cell behavior with respect to the combined morphology and mechanics of such matrices is not currently possible with the types of 3D cell culture matrices available. Most of the cell culture matrices are single-phase bio- or polymeric hydrogels. Therefore, here we developed a continuous hybrid manufacturing process to make fiber-reinforced composite hydrogels. A far field electrospinning process was used to deposit the fibrous component with the aid of guiding electrodes; and a gravity-assisted, droplet-based system controlled the rate of addition of the cell-laden hydrogel component. The addition of the fibrous component slightly increased the elastic modulus of the pure hydrogel. The cells that were embedded into the fiber-reinforced hydrogels were viable for 8 days. The cells were randomly placed in the matrix such that some had no contact to the fibers and others were initially in proximity to fibers. The cells with no contact to fibers grew into spheroidal clusters within the hydrogel, and those in proximity to the fibers spread out and grew along the fibers showing that the fiber-reinforced hydrogels are able to control cell behavior with morphological cues.

Determining Relative Importance and Effective Settings for Genetic Algorithm Control Parameters.

Setting the control parameters of a genetic algorithm to obtain good results is a long-standing problem. We define an experiment design and analysis method to determine relative importance and effective settings for control parameters of any evolutionary algorithm, and we apply this method to a classic binary-encoded genetic algorithm (GA). Subsequently, as reported elsewhere, we applied the GA, with the control parameter settings determined here, to steer a population of cloud-computing simulators toward behaviors that reveal degraded performance and system collapse. GA-steered simulators could serve as a design tool, empowering system engineers to identify and mitigate low-probability, costly failure scenarios. In the existing GA literature, we uncovered conflicting opinions and evidence regarding key GA control parameters and effective settings to adopt. Consequently, we designed and executed an experiment to determine relative importance and effective settings for seven GA control parameters, when applied across a set of numerical optimization problems drawn from the literature. This paper describes our experiment design, analysis, and results. We found that crossover most significantly influenced GA success, followed by mutation rate and population size and then by rerandomization point and elite selection. Selection method and the precision used within the chromosome to represent numerical values had least influence. Our findings are robust over 60 numerical optimization problems.

Elastic free energy drives the shape of prevascular solid tumors.

It is well established that the mechanical environment influences cell functions in health and disease. Here, we address how the mechanical environment influences tumor growth, in particular, the shape of solid tumors. In an in vitro tumor model, which isolates mechanical interactions between cancer tumor cells and a hydrogel, we find that tumors grow as ellipsoids, resembling the same, oft-reported observation of in vivo tumors. Specifically, an oblate ellipsoidal tumor shape robustly occurs when the tumors grow in hydrogels that are stiffer than the tumors, but when they grow in more compliant hydrogels they remain closer to spherical in shape. Using large scale, nonlinear elasticity computations we show that the oblate ellipsoidal shape minimizes the elastic free energy of the tumor-hydrogel system. Having eliminated a number of other candidate explanations, we hypothesize that minimization of the elastic free energy is the reason for predominance of the experimentally observed ellipsoidal shape. This result may hold significance for explaining the shape progression of early solid tumors in vivo and is an important step in understanding the processes underlying solid tumor growth.

Instantaneous fabrication of arrays of normally closed, adjustable, and reversible nanochannels by tunnel cracking.

A direct fabrication method capable of producing fully-reversible, tunable nanochannel arrays, without the use of a molding step, is described. It is based on tunnel cracking of a readily-prepared brittle layer constrained between elastomeric substrates. The resulting nanochannels have adjustable cross-sections that can be reversibly opened, closed, widened and narrowed merely by applying and removing tensile strains to the substrate. This permits reversible trapping and release of nanoparticles, and easy priming or unclogging of the nanochannels for user-friendly and robust operations. The ease of fabrication and operation required to open and close the nanochannels is superior to previous approaches.

In silico estimates of the free energy rates in growing tumor spheroids.

The physics of solid tumor growth can be considered at three distinct size scales: the tumor scale, the cell-extracellular matrix (ECM) scale and the sub-cellular scale. In this paper we consider the tumor scale in the interest of eventually developing a system-level understanding of the progression of cancer. At this scale, cell populations and chemical species are best treated as concentration fields that vary with time and space. The cells have chemo-mechanical interactions with each other and with the ECM, consume glucose and oxygen that are transported through the tumor, and create chemical by-products. We present a continuum mathematical model for the biochemical dynamics and mechanics that govern tumor growth. The biochemical dynamics and mechanics also engender free energy changes that serve as universal measures for comparison of these processes. Within our mathematical framework we therefore consider the free energy inequality, which arises from the first and second laws of thermodynamics. With the model we compute preliminary estimates of the free energy rates of a growing tumor in its pre-vascular stage by using currently available data from single cells and multicellular tumor spheroids.

External compression-induced fracture patterning on the surface of poly(dimethylsiloxane) cubes and microspheres.

This article describes a method for creating submicrometer surface patterns on cubes and microspheres. In this method, PDMS cubes and microspheres are exposed to oxygen plasma, which creates a very thin, hard, surface-modified layer on a compliant substrate. These are then compressed, causing the layer to crack in patterns dictated by the distribution of tensile stresses in the surface layer. Cracks with submicrometer widths were generated on 1 cm3 cubes and 800-microm-diameter microspheres, and the resulting crack patterns were observed. Finite-element simulations of the tensile stress distributions reveal that the fracture patterns arise from different mechanisms in the cubes and spheres. In particular, pattern formation is associated with frictional contact in the cubes but not in the microspheres, where geometrical effects associated with changes in the cross-sectional area along the axis lead to the generation of tensile stress. These observations and analyses provide a foundation on which to predict and guide crack pattern formation on a wide variety of small 3D objects. In anticipation of future applications in materials science and biology, we demonstrate the selective deposition of compounds into the cracks to make them functionally differentiable from the rest of the surface.

The mechanical properties of a surface-modified layer on poly(dimethylsiloxane).

Surface-modification of the elastomer poly(dimethylsiloxane) by exposure to oxygen plasma for four minutes creates a thin, stiff film. In this study, the thickness and mechanical properties of this surface-modified layer were determined. Using the phase image capabilities of a tapping-mode atomic-force microscope, the surface-modified region was distinguished from the bulk PDMS; specifically, it suggested a graded surface layer to a depth of about 200 nm. Load-displacement data for elastic indentation using a compliant AFM cantilever was analyzed as a plate bending on an elastic foundation to determine the elastic modulus of the surface (37 MPa). An applied uniaxial strain generated a series of parallel nano-cracks with spacing on the order of a few microns. Numerical analyses of this cracking phenomenon showed that the depth of these cracks was in the range of 300-600 nm and that the surface layer was extremely brittle, with its toughness in the range of 0.1-0.3 J/m(2).

Distinctive immunoglobulin E anti-house dust allergen-binding specificities in a tropical Australian Aboriginal community.

BACKGROUND: There is evidence that the specificity of the IgE binding in allergy tests can vary for different populations. OBJECTIVE: We aimed to examine the allergenic specificity of IgE binding in sera from house dust mite (HDM)-atopic subjects in a tropical Australian Aboriginal community. METHODS: Sera shown to contain IgE antibodies to an HDM extract of Dermatophagoides pteronyssinus were examined for IgE binding to a panel of nine purified HDM allergens from this mite species by quantitative microtitre assays. IgG antibody binding (IgG1 and IgG4) was also measured. RESULTS: The IgE-binding activity in the sera from the Aboriginal community was not directed to the expected major groups 1 and 2 HDM allergens but instead to the group 4 amylase allergen. There was also little IgE binding to the potentially cross-reactive tropomyosin (Der p 10) or arginine kinase (Der p 20) allergens. The IgG4 antibody was rarely detected and limited to the Der p 4 allergen. IgG1 antibody binding was frequently measured to all the allergens regardless of an individual's atopic status, whereas in urban communities it is restricted to the major allergens and to atopic subjects. CONCLUSION: The high IgE anti-HDM response of Australian Aboriginals predominantly bound Der p 4 and not the Der p 1 and 2 allergens, showing a distinctive allergy that could affect the disease outcome and diagnosis.

Tuneable elastomeric nanochannels for nanofluidic manipulation.

Fluidic transport through nanochannels offers new opportunities to probe fundamental nanoscale transport phenomena and to develop tools for manipulating DNA, proteins, small molecules and nanoparticles. The small size of nanofabricated devices and the accompanying increase in the effect of surface forces, however, pose challenges in designing and fabricating flexible nanofluidic systems that can dynamically adjust their transport characteristics according to the handling needs of various molecules and nanoparticles. Here, we describe the use of nanoscale fracturing of oxidized poly(dimethylsiloxane) to conveniently fabricate nanofluidic systems with arrays of nanochannels that can actively manipulate nanofluidic transport through dynamic modulation of the channel cross-section. We present the design parameters for engineering material properties and channel geometry to achieve reversible nanochannel deformation using remarkably small forces. We demonstrate the versatility of the elastomeric nanochannels through tuneable sieving and trapping of nanoparticles, dynamic manipulation of the conformation of single DNA molecules and in situ photofabrication of movable polymeric nanostructures.

Effect of seating, vision and direction of horizontal oscillation on motion sickness.

BACKGROUND: Low frequency horizontal oscillation can cause motion sickness in some forms of transport, but the influence of the characteristics of the motion and the visual and postural conditions of the body on sickness are not known. HYPOTHESES: It was hypothesised that body position, vision and direction of motion will have an effect on motion sickness. METHOD: There were 72 seated subjects who were exposed to horizontal oscillation at 0.25 Hz, 0.7 ms(-2) r.m.s. (peak-to-peak displacement of 0.8 m) for up to 30 min while in 1 of 6 conditions. Three conditions involved fore-and-aft motion and three involved lateral motion. For motion in each axis, subjects sat within a closed cabin with either: a) a high backrest with their eyes open; b) a low backrest with their eyes open; or c) a low backrest with their eyes closed and blindfolded. Subjects provided ratings of their motion sickness symptoms at 1-min intervals during the 30-min exposures. RESULTS: The most nauseogenic stimulus was fore-and-aft motion with a low backrest and the eyes open. Self-ratings of motion sickness susceptibility provided by subjects before participating in the experiment were positively correlated with their illness ratings during the experiment. CONCLUSIONS: Restraint to the upper body during exposure to horizontal acceleration may reduce the susceptibility to motion sickness caused by horizontal oscillation. The relative nauseogenicity of fore-and-aft and lateral oscillation depends on the support given to the upper body. In the conditions of the experiment the effects of the postural support given to the subjects and their prior susceptibility to motion sickness were greater than any effect of the visual conditions.

Molecular characterization of the group 4 house dust mite allergen from Dermatophagoides pteronyssinus and its amylase homologue from Euroglyphus maynei.

BACKGROUND: Of the ten recognised groups of Dermatophagoides pteronyssinus allergens, the group 4 is the only group that has not been characterised at the molecular level. METHODS: Primers were designed to PCR amplify Der p 4 (D. pteronyssinus) and Eur m 4 (Euroglyphus maynei) cDNA. These fragments were used to screen the corresponding cDNA libraries and the cDNA clones obtained were subsequently sequenced. The coding regions of Der p 4 and Eur m 4 were cloned into the pET expression vector and recombinant histidine-tagged proteins expressed in Escherichia coli. RESULTS: cDNA clones which included the mature protein coding sequence for Der p 4 and Eur m 4 were sequenced. The Der p 4 and Eur m 4 genes were found to code for 496 amino acid mature proteins with residues important for the function of alpha-amylase highly conserved. Der p 4 and Eur m 4 were calculated to be 90% identical and a BLAST search of the GenBank database found these sequences to be approximately 50% identical to insect and mammalian alpha-amylases. The calculated molecular weights of Der p 4 and Eur m 4 were approximately 57,000, although recombinant Der p 4 and Eur m 4 migrate on SDS-PAGE at about 60,000. Der p 4 recombinant protein was found to bind specific IgE in 3 of the 10 house dust mite allergic patients tested. CONCLUSIONS: This paper describes the first cDNA sequence of Der p 4 and Eur m 4 confirming that this allergen is house dust mite alpha-amylase.

The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway.

We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB. AtzB catalyzed the transformation of hydroxyatrazine to N-isopropylammelide. The product was identified by use of high-performance liquid chromatography, mass spectrometery, and nuclear magnetic resonance spectroscopy. Tn5 mutagenesis of pMD1 was used to determine that atzB was located 8 kb downstream of atzA. Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce plasmid pATZB-2. The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2. ORF1 was identified as the coding region of atzB by demonstrating that (i) only ORF1 was transcribed in Pseudomonas sp. strain ADP, (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) codon usage was consistent with ORF1 being translated. AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamination of the s-triazine substrate melamine. The atzA and atzB genes catalyze the first two steps of the metabolic pathway in a bacterium that rapidly metabolizes atrazine to carbon dioxide, ammonia, and chloride.

Cloning, characterization, and expression of a gene region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine.

We previously identified a Pseudomonas sp. strain, ADP, which rapidly metabolized atrazine in liquid culture, agar plates, and soils (R. T. Mandelbaum, D. L. Allan, L. P. Wackett, Appl. Environ. Microbiol. 61:1451-1457, 1995). In this study, we report the cloning and partial characterization of a gene region from Pseudomonas sp. strain ADP that encodes atrazine degradation activity. A 22-kb EcoRI genomic DNA fragment, designated pMD1, was shown to encode atrazine dechlorination activity in Escherichia coli DH5 alpha. Atrazine degradation was demonstrated by a zone-clearing assay on agar medium containing crystalline atrazine and by chromatographic methods. A gene conferring the atrazine-clearing phenotype was subsequently subcloned as a 1.9-kb AvaI fragment in pACYC184, designated pMD4, and was expressed in E. coli. This result and random Tn5 mutagenesis established that the 1.9-kb AvaI fragment was essential for atrazine dechlorination. High-pressure liquid and thin-layer chromatographic analyses were used to rigorously establish that E. coli containing pMD4 degraded atrazine and accumulated hydroxyatrazine. Hydroxyatrazine was detected only transiently in E. coli containing pMD1. This is consistent with the idea that hydroxyatrazine is the first metabolite in atrazine degradation by Pseudomonas sp. strain ADP. A 0.6-kb ApaI-PstI fragment from pMD4, containing the putative atrazine chlorohydrolase gene, hybridized to DNA from atrazine-degrading bacteria isolated in Switzerland and Louisiana.(ABSTRACT TRUNCATED AT 250 WORDS)

A Saccharomyces cerevisiae RAD52 allele expressing a C-terminal truncation protein: activities and intragenic complementation of missense mutations.

A nonsense allele of the yeast RAD52 gene, rad52-327, which expresses the N-terminal 65% of the protein was compared to two missense alleles, rad52-1 and rad52-2, and to a deletion allele. While the rad52-1 and the deletion mutants have severe defects in DNA repair, recombination and sporulation, the rad52-327 and rad52-2 mutants retain either partial or complete capabilities in repair and recombination. These two mutants behave similarly in most tests of repair and recombination during mitotic growth. One difference between these two alleles is that a homozygous rad52-2 diploid fails to sporulate, whereas the homozygous rad52-327 diploid sporulates weakly. The low level of sporulation by the rad52-327 diploid is accompanied by a low percentage of spore viability. Among these viable spores the frequency of crossing over for markers along chromosome VII is the same as that found in wild-type spores. rad52-327 complements rad52-2 for repair and sporulation. Weaker intragenic complementation occurs between rad52-327 and rad52-1.

Bilateral carpal tunnel syndrome associated with interleukin 2 therapy.

We report the development of synchronous bilateral carpal tunnel syndrome in a woman with metastatic colorectal cancer, undergoing treatment with recombinant interleukin 2. A carpal tunnel decompression was carried out on the hand which was more severely affected, with a gradual recovery in median nerve function. To the best of our knowledge, this is the first reported case of carpal tunnel syndrome in association with recombinant interleukin 2.

Pycnodysostosis.

The first Scottish family with pycnodysostosis is reported. The clinical and radiological findings in the two affected men are recorded.