Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix

Adipose tissue forms when basement membrane extract (Matrigel™) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 μL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma

We have previously demonstrated that fibroblasts and invasive human breast carcinoma (HBC) cells specifically activate matrix metalloproteinase- 2 (MMP-2) when cultured on 3-dimensional gels of type I collagen but not a range of other substrates. We show here the constitutive expression of membrane-type 1 (MT1)-MMP in both fibroblasts, and invasive HBC cell lines, that have fibroblastic attributes presumably acquired through an epithelial- to-mesenchymal transition (EMT). Treatment with collagen type I increased the steady-state MT1-MMP mRNA levels in these cells but did not induce either MT1-MMP expression or MMP-2 activation in noninvasive breast carcinoma cell lines, which retain epithelial features. Basal MT3-MMP mRNA expression had a pattern similar to that of MT1-MMP but was not up-regulated by collagen. MT4- MMP mRNA was seen in both invasive and noninvasive HBC cell lines and was also not collagen-regulated, and MT2-MMP mRNA was not detected in any of the HBC cell lines tested. These data support a role for MT1-MMP in the collagen- induced MMP-2-activation seen in these cells. In situ hybridization analysis of archival breast cancer specimens revealed a close parallel in expression of both collagen type I and MT1-MMP mRNA in peritumoral fibroblasts, which was correlated with aggressiveness of the lesion. Relatively high levels of expression of both mRNA species were seen in fibroblasts close to invasive tumor nests and, although only focally, in certain areas close to preinvasive tumors. These foci may represent hot spots for local degradation and invasive progression. Collectively, these results implicate MT1-MMP in collagen- stimulated MMP-2 activation and suggest that this mechanism may be employed in vivo by both tumor-associated fibroblasts and EMT-derived carcinoma cells to facilitate increased invasion and/or metastasis.

Optimizing I/O cost and managing memory for composition vector method based on correlation matrix calculation in bioinformatics

The generation of a correlation matrix for set of genomic sequences is a common requirement in many bioinformatics problems such as phylogenetic analysis. Each sequence may be millions of bases long and there may be thousands of such sequences which we wish to compare, so not all sequences may fit into main memory at the same time. Each sequence needs to be compared with every other sequence, so we will generally need to page some sequences in and out more than once. In order to minimize execution time we need to minimize this I/O. This paper develops an approach for faster and scalable computing of large-size correlation matrices through the maximal exploitation of available memory and reducing the number of I/O operations. The approach is scalable in the sense that the same algorithms can be executed on different computing platforms with different amounts of memory and can be applied to different bioinformatics problems with different correlation matrix sizes. The significant performance improvement of the approach over previous work is demonstrated through benchmark examples.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Accounting for management costs in sensitivity analyses of matrix population models

Traditional sensitivity and elasticity analyses of matrix population models have been used to inform management decisions, but they ignore the economic costs of manipulating vital rates. For example, the growth rate of a population is often most sensitive to changes in adult survival rate, but this does not mean that increasing that rate is the best option for managing the population because it may be much more expensive than other options. To explore how managers should optimize their manipulation of vital rates, we incorporated the cost of changing those rates into matrix population models. We derived analytic expressions for locations in parameter space where managers should shift between management of fecundity and survival, for the balance between fecundity and survival management at those boundaries, and for the allocation of management resources to sustain that optimal balance. For simple matrices, the optimal budget allocation can often be expressed as simple functions of vital rates and the relative costs of changing them. We applied our method to management of the Helmeted Honeyeater (Lichenostomus melanops cassidix; an endangered Australian bird) and the koala (Phascolarctos cinereus) as examples. Our method showed that cost-efficient management of the Helmeted Honeyeater should focus on increasing fecundity via nest protection, whereas optimal koala management should focus on manipulating both fecundity and survival simultaneously. These findings are contrary to the cost-negligent recommendations of elasticity analysis, which would suggest focusing on managing survival in both cases. A further investigation of Helmeted Honeyeater management options, based on an individual-based model incorporating density dependence, spatial structure, and environmental stochasticity, confirmed that fecundity management was the most cost-effective strategy. Our results demonstrate that decisions that ignore economic factors will reduce management efficiency. ©2006 Society for Conservation Biology.

Characteristics and alteration origins of matrix minerals in volcaniclastic kimberlite of the Muskox pipe (Nunavut, Canada)

The matrix of volcaniclastic kimberlite (VK) from the Muskox pipe (Northern Slave Province, Nunavut, Canada) is interpreted to represent an overprint of an original clastic matrix. Muskox VK is subdivided into three different matrix mineral assemblages that reflect differences in the proportions of original primary matrix constituents, temperature of formation and nature of the altering fluids. Using whole rock X-ray fluorescence (XRF), whole rock X-ray diffraction (XRD), microprobe analyses, back-scatter electron (BSE) imaging, petrography and core logging, we find that most matrix minerals (serpentine, phlogopite, chlorite, saponite, monticellite, Fe-Ti oxides and calcite) lack either primary igneous or primary clastic textures. The mineralogy and textures are most consistent with formation through alteration overprinting of an original clastic matrix that form by retrograde reactions as the deposit cools, or, in the case of calcite, by precipitation from Ca-bearing fluids into a secondary porosity. The first mineral assemblage consists largely of serpentine, phlogopite, calcite, Fe-Ti oxides and monticellite and occurs in VK with relatively fresh framework clasts. Alteration reactions, driven by deuteric fluids derived from the juvenile constituents, promote the crystallisation of minerals that indicate relatively high temperatures of formation (> 400 °C). Lower-temperature minerals are not present because permeability was occluded before the deposit cooled to low temperatures, thus shielding the facies from further interaction with fluids. The other two matrix mineral assemblages consist largely of serpentine, phlogopite, calcite, +/- diopside, and +/- chlorite. They form in VK that contains more country rock, which may have caused the deposit to be cooler upon emplacement. Most framework components are completely altered, suggesting that larger volumes of fluids drove the alteration reactions. These fluids were likely of meteoric provenance and became heated by the volcaniclastic debris when they percolated into the VK infill. Most alteration reactions ceased at temperatures > 200 °C, as indicated by the absence or paucity of lower-temperature phases in most samples, such as saponite. Recognition that Muskox VK contains an original clastic matrix is a necessary first step for evaluating the textural configuration, which is important for reconstructing the physical processes responsible for the formation of the deposit.

Hierarchical clustering of the genetic connectivity matrix reveals the network topology of gene action on brain microstructure: An N=531 twin study

Genetic correlation (rg) analysis determines how much of the correlation between two measures is due to common genetic influences. In an analysis of 4 Tesla diffusion tensor images (DTI) from 531 healthy young adult twins and their siblings, we generalized the concept of genetic correlation to determine common genetic influences on white matter integrity, measured by fractional anisotropy (FA), at all points of the brain, yielding an NxN genetic correlation matrix rg(x,y) between FA values at all pairs of voxels in the brain. With hierarchical clustering, we identified brain regions with relatively homogeneous genetic determinants, to boost the power to identify causal single nucleotide polymorphisms (SNP). We applied genome-wide association (GWA) to assess associations between 529,497 SNPs and FA in clusters defined by hubs of the clustered genetic correlation matrix. We identified a network of genes, with a scale-free topology, that influences white matter integrity over multiple brain regions.

Discussion of “Generalized estimating equations: Notes on the choice of the working correlation matrix”

Objective To discuss generalized estimating equations as an extension of generalized linear models by commenting on the paper of Ziegler and Vens "Generalized Estimating Equations. Notes on the Choice of the Working Correlation Matrix". Methods Inviting an international group of experts to comment on this paper. Results Several perspectives have been taken by the discussants. Econometricians have established parallels to the generalized method of moments (GMM). Statisticians discussed model assumptions and the aspect of missing data Applied statisticians; commented on practical aspects in data analysis. Conclusions In general, careful modeling correlation is encouraged when considering estimation efficiency and other implications, and a comparison of choosing instruments in GMM and generalized estimating equations, (GEE) would be worthwhile. Some theoretical drawbacks of GEE need to be further addressed and require careful analysis of data This particularly applies to the situation when data are missing at random.

Fast Public Key Cryptosystem Based On Matrix Rings

A public key cryptosystem is proposed, which is based on the assumption that finding the square root of an element in a large finite ring is computationally infeasible in the absence of a knowledge of the ring structure. The encryption and decryption operations are very fast, and the data expansion is 1:2.

Estimation in a multiplicative mixed model involving a genetic relationship matrix.

Genetic models partitioning additive and non-additive genetic effects for populations tested in replicated multi-environment trials (METs) in a plant breeding program have recently been presented in the literature. For these data, the variance model involves the direct product of a large numerator relationship matrix A, and a complex structure for the genotype by environment interaction effects, generally of a factor analytic (FA) form. With MET data, we expect a high correlation in genotype rankings between environments, leading to non-positive definite covariance matrices. Estimation methods for reduced rank models have been derived for the FA formulation with independent genotypes, and we employ these estimation methods for the more complex case involving the numerator relationship matrix. We examine the performance of differing genetic models for MET data with an embedded pedigree structure, and consider the magnitude of the non-additive variance. The capacity of existing software packages to fit these complex models is largely due to the use of the sparse matrix methodology and the average information algorithm. Here, we present an extension to the standard formulation necessary for estimation with a factor analytic structure across multiple environments.

Latent TGF-beta Binding Proteins : Adhesive functions and matrix association of LTBP-2 and potential functions of LTBP-1 and LTBP-3 in mesothelioma

Latent transforming growth factor-beta (TGF-beta) binding proteins (LTBPs) -1, -3 and -4 are ECM components whose major function is to augment the secretion and matrix targeting of TGF-beta, a multipotent cytokine. LTBP-2 does not bind small latent TGF-beta but has suggested functions as a structural protein in ECM microfibrils. In the current work we focused on analyzing possible adhesive functions of LTBP-2 as well as on characterizing the kinetics and regulation of LTBP-2 secretion and ECM deposition. We also explored the role of TGF-beta binding LTBPs in endothelial cells activated to mimic angiogenesis as well as in malignant mesothelioma. We found that, unlike most adherent cells, several melanoma cell lines efficiently adhered to purified recombinant LTBP-2. Further characterization revealed that the adhesion was mediated by alpha3beta1 and alpha6beta1 integrins. Heparin also inhibited the melanoma cell adhesion suggesting a role for heparan sulphate proteoglycans. LTBP-2 was also identified as a haptotactic substrate for melanoma cell migration. We used cultured human embryonic lung fibroblasts to analyze the temporal and spatial association of LTBP-2 into ECM. By We found that LTBP-2 was efficiently assembled to the ECM only in confluent cultures following the deposition of fibronectin (FN) and fibrillin-1. In early, subconfluent cultures it remained primarily in soluble form after secretion. LTBP-2 colocalized transiently with FN and fibrillin-1. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2 indicating that the ECM association of LTBP-2 depends on a pre-formed fibrillin-1 network. Considering the established role of TGF-beta as a regulator of angiogenesis we induced morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) and followed the fate of LTBP-1 in the endothelial ECM. This resulted in profound proteolytic processing of LTBP-1 and release of latent TGF-beta complexes from the ECM. The processing was coupled with increased activation of MT-MMPs and specific upregulation of MT1-MMP. The major role of MT1-MMP in the proteolysis of LTBP-1 was confirmed by suppressing the expression with lentivirally induced short-hairpin RNAs as well as by various metalloproteinases inhibitors. TGF-beta can promote tumorigenesis of malignant mesothelioma (MM), which is an aggressive tumor of the pleura with poor prognosis. TGF-beta activity was analyzed in a panel of MM tumors by immunohistochemical staining of phosphorylated Smad-2 (P-Smad2). The tumor cells were strongly positive for P-Smad2 whereas LTBP-1 immunoreactivity was abundant in the stroma, and there was a negative correlation between LTBP-1 and P-Smad2 staining. In addition, the high P-Smad2 immunoreactivity correlated with shorter survival of patients. mRNA analysis revealed that TGF-beta1 was the most highly expressed isoform in both normal human pleura and MM tissue. LTBP-1 and LTBP-3 were both abundantly expressed. LTBP-1 was the predominant isoform in established MM cell lines whereas the expression of LTBP-3 was high in control cells. Suppression of LTBP-3 expression by siRNAs resulted in increased TGF-beta activity in MM cell lines accompanied by decreased proliferation. Our results suggest that decreased expression of LTBP-3 in MM could alter the targeting of TGF-beta to the ECM and lead to its increased activation. The current work emphasizes the coordinated process of the assembly and appropriate targeting of LTBPs with distinct adhesive or cytokine harboring properties into the ECM. The hierarchical assembly may have implications in the modulation of signaling events during morphogenesis and tissue remodeling.

An NAD(P)(+)-dependent secondary-alcohol dehydrogenase of Alcaligenes eutrophus: Purification, characterization and its application for the production of chiral alcohols

A soil micro-organism identified as Alcaligenes eutrophus capable of utilizing nerolidol, a sesquiterpene alcohol as the sole source of carbon, contains an inducible NAD(P)(+)-linked secondary-alcohol dehydrogenase (SADH), The enzyme was purified 252-fold from crude cell-free extract by a combination of salt precipitation, ion-exchange and affinity-matrix chromatography, Native and SDS/PAGE PAGE of the purified enzyme showed a single protein band and the enzyme appears to be a homotetramer having an apparent molecular mass of 139 kDa comprising four identical subunits of 38.5 kDa, The isoelectric point (pi) of SADH was determined to be 6.2, Depending on pH of the reaction media, the enzyme carried out both oxidation and reductions of various terpenoids and steroids, At pH 5.5, the enzyme catalysed the stereospecific reduction of prochiral ketones to optically active (S)-alcohols and the oxidation reaction was predominated over the former at pH 9.5, NADP(+) and NADPH were respectively preferred over NAD(+) and NADH for oxidation and reduction reactions, The K-m values for testosterone, NADP(+) and NAD(+) were 11.8, 55.6, and 122 mu M respectively, Neither enzyme was significantly inhibited by metal-binding agents, but some thiol-blocking compounds inhibited it, SADH tolerates moderate concentrations of water-miscible organic solvents such as ethanol, methanol, acetone and dioxan, Some of the properties of this enzyme were found to be significantly different from those thus far described.

Regulation of extracellular matrix genes by arecoline in primary gingival fibroblasts requires epithelial factors

Background and Objective: Oral submucous fibrosis, a disease of collagen disorder, has been attributed to arecoline present in the saliva of betel quid chewers. However, the molecular basis of the action of arecoline in the pathogenesis of oral submucous fibrosis is poorly understood. The basic aim of our study was to elucidate the mechanism underlying the action of arecoline on the expression of genes in oral fibroblasts. Material and Methods: Human keratinocytes (HaCaT cells) and primary human gingival fibroblasts were treated with arecoline in combination with various pathway inhibitors, and the expression of transforming growth factor-beta isoform genes and of collagen isoforms was assessed using reverse transcription polymerase chain reaction analysis. Results: We observed the induction of transforming growth factor-beta2 by arecoline in HaCaT cells and this induction was found to be caused by activation of the M-3 muscarinic acid receptor via the induction of calcium and the protein kinase C pathway. Most importantly, we showed that transforming growth factor-beta2 was significantly overexpressed in oral submucous fibrosis tissues (p = 0.008), with a median of 2.13 (n = 21) compared with 0.75 (n = 18) in normal buccal mucosal tissues. Furthermore, arecoline down-regulated the expression of collagens 1A1 and 3A1 in human primary gingival fibroblasts; however these collagens were induced by arecoline in the presence of spent medium of cultured human keratinocytes. Treatment with a transforming growth factor-beta blocker, transforming growth factor-beta1 latency-associated peptide, reversed this up-regulation of collagen, suggesting a role for profibrotic cytokines, such as transforming growth factor-beta, in the induction of collagens. Conclusion: Taken together, our data highlight the importance of arecoline-induced epithelial changes in the pathogenesis of oral submucous fibrosis.