Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.

Atmospheric pressure plasmas : infection control and bacterial responses

Cold atmospheric pressure plasma (APP) is a recent, cutting-edge antimicrobial treatment. It has the potential to be used as an alternative to traditional treatments such as antibiotics and as a promoter of wound healing, making it a promising tool in a range of biomedical applications with particular importance for combating infections. A number of studies show very promising results for APP-mediated killing of bacteria, including removal of biofilms of pathogenic bacteria such as Pseudomonas aeruginosa. However, the mode of action of APP and the resulting bacterial response are not fully understood. Use of a variety of different plasma-generating devices, different types of plasma gases and different treatment modes makes it challenging to show reproducibility and transferability of results. This review considers some important studies in which APP was used as an antibacterial agent, and specifically those that elucidate its mode of action, with the aim of identifying common bacterial responses to APP exposure. The review has a particular emphasis on mechanisms of interactions of bacterial biofilms with APP.

Failure of amino acid homeostasis causes cell death following proteasome inhibition

The ubiquitin-proteasome system targets many cellular proteins for degradation and thereby controls most cellular processes. Although it is well established that proteasome inhibition is lethal, the underlying mechanism is unknown. Here, we show that proteasome inhibition results in a lethal amino acid shortage. In yeast, mammalian cells, and flies, the deleterious consequences of proteasome inhibition are rescued by amino acid supplementation. In all three systems, this rescuing effect occurs without noticeable changes in the levels of proteasome substrates. In mammalian cells, the amino acid scarcity resulting from proteasome inhibition is the signal that causes induction of both the integrated stress response and autophagy, in an unsuccessful attempt to replenish the pool of intracellular amino acids. These results reveal that cells can tolerate protein waste, but not the amino acid scarcity resulting from proteasome inhibition.

Mechanisms of mono- and poly-ubiquitination : ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/ E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.

Genome-wide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors

DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA-mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based "epigenetic unmasking" in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries.

F9 fimbriae of uropathogenic Escherichia coli are expressed at low temperature and recognise Galβ1-3GlcNAc-containing glycans

Uropathogenic Escherichia coli (UPEC) is the leading causative agent of urinary tract infections (UTI) in the developed world. Among the major virulence factors of UPEC, surface expressed adhesins mediate attachment and tissue tropism. UPEC strains typically possess a range of adhesins, with type 1 fimbriae and P fimbriae of the chaperone-usher class the best characterised. We previously identified and characterised F9 as a new chaperone-usher fimbrial type that mediates biofilm formation. However, the regulation and specific role of F9 fimbriae remained to be determined in the context of wild-type clinical UPEC strains. In this study we have assessed the distribution and genetic context of the f9 operon among diverse E. coli lineages and pathotypes and demonstrated that f9 genes are significantly more conserved in a UPEC strain collection in comparison to the well-defined E. coli reference (ECOR) collection. In the prototypic UPEC strain CFT073, the global regulator protein H-NS was identified as a transcriptional repressor of f9 gene expression at 37°C through its ability to bind directly to the f9 promoter region. F9 fimbriae expression was demonstrated at 20°C, representing the first evidence of functional F9 fimbriae expression by wild-type E. coli. Finally, glycan array analysis demonstrated that F9 fimbriae recognise and bind to terminal Galβ1-3GlcNAc structures.

Demonstration of regulatory cross-talk between P fimbriae and type 1 fimbriae in uropathogenic Escherichia coli

The majority of Escherichia coli strains isolated from urinary tract infections have the potential to express multiple fimbriae. Two of the most common fimbrial adhesins are type 1 fimbriae and pyelonephritis-associated pili (Pap). Previous research has shown that induced, plasmid-based expression of a Pap regulator, papB, and its close homologues can prevent inversion of the fim switch controlling the expression of type 1 fimbriae. The aim of the present study was to determine if this cross-regulation occurs when PapB is expressed from its native promoter in the chromosome of E. coli K-12 and clinical isolates. The regulation was examined in three ways: (1) mutated alleles of the pap regulatory region, including papB and papI, that maintain the pap promoter in either the off or the on phase were exchanged into the chromosome of both E. coli K-12 and the clinical isolate E. coli CFT073, and the effect on type 1 fimbrial expression was measured; (2) type 1 fimbrial expression was determined using a novel fimS : : gfp+ reporter system in mutants of the clinical isolate E. coli 536 in which combinations of complete fimbrial clusters had been deleted; (3) type 1 fimbrial expression was determined in a range of clinical isolates and compared with both the number of P clusters and their expression. All three approaches demonstrated that P expression represses type 1 fimbrial expression. Using a number of novel genetic approaches, this work extends the initial finding that PapB inhibits FimB recombination to the impact of this regulation in clinical isolates.

Whole-genome multiparametric screening to identify modulators of epithelial-to-mesenchymal transition

Metastasis accounts for the poor prognosis of the majority of solid tumors. The phenotypic transition of nonmotile epithelial tumor cells to migratory and invasive “mesenchymal” cells (epithelial-to-mesenchymal transition [EMT]) enables the transit of cancer cells from the primary tumor to distant sites. There is no single marker of EMT; rather, multiple measures are required to define cell state. Thus, the multiparametric capability of high-content screening is ideally suited for the comprehensive analysis of EMT regulators. The aim of this study was to generate a platform to systematically identify functional modulators of tumor cell plasticity using the bladder cancer cell line TSU-Pr1-B1 as a model system. A platform enabling the quantification of key EMT characteristics, cell morphology and mesenchymal intermediate filament vimentin, was developed using the fluorescent whole-cell-tracking reagent CMFDA and a fluorescent promoter reporter construct, respectively. The functional effect of genome-wide modulation of protein-coding genes and miRNAs coupled with those of a collection of small-molecule kinase inhibitors on EMT was assessed using the Target Activation Bioapplication integrated in the Cellomics ArrayScan platform. Data from each of the three screens were integrated to identify a cohort of targets that were subsequently examined in a validation assay using siRNA duplexes. Identification of established regulators of EMT supports the utility of this screening approach and indicated capacity to identify novel regulators of this plasticity program. Pathway analysis coupled with interrogation of cancer-related expression profile databases and other EMT-related screens provided key evidence to prioritize further experimental investigation into the molecular regulators of EMT in cancer cells.

Estrogen metabolizing enzymes in endometrium and endometriosis

BACKGROUND Estradiol (E-2) is an important promoter of the growth of both eutopic and ectopic endometrium. The findings with regard to the expression and activity of steroidogenic enzymes in endometrium of controls, in endometrium of endometriosis patients and in endometriotic lesions are not consistent. METHODS In this study, we have looked at the mRNA expression and protein levels of a range of steroidogenic enzymes [aromatase, 17 beta-hydroxysteroid dehydrogenases (17 beta-HSD) type 1, 2 and 4, estrogen sulfotransferase (EST) and steroid sulfatase (STS)l in eutopic and ectopic endometrium of patients (n = 14) with deep-infiltrative endometriosis as well as in disease-free endometrium (n = 48) using real-time PCR and immunocytochemistry. In addition, we evaluated their menstrual cycle-related expression patterns, and investigated their steroid responsiveness in explant cultures. RESULTS Aromatase and 17 beta-HSD type 1 mRNA levels were extremely low in normal human endometrium, while mRNAs for types 2 and 4 17 beta-HSD, EST and STS were readily detectable. Only 17 beta-HSD type 2 and EST genes showed sensitivity to progesterone in normal endometrium. Types 1 and 2 17 beta-HSD and STS protein was detected in normal endometrium using new polyclonal antibodies. CONCLUSIONS In endometriosis lesions, the balance is tilted in favor of enzymes producing E2. This is due to a suppression of types 2 and 4 17 beta-HSD, and an increased expression of aromatase and type 1 17 beta-HSD in ectopic endometrium.

Olfactomedin-4 regulation by estrogen in the human endometrium requires epidermal growth factor signaling

Olfactomedin-4 (OLFM-4) is an extracellular matrix protein that is highly expressed in human endometrium. We have examined the regulation and function of OLFM-4 in normal endometrium and in cases of endometriosis and endometrial cancer. OLFM-4 expression levels are highest in proliferative-phase endometrium, and 17 beta-estradiol up-regulates OLFM-4 mRNA in endometrial explant cultures. Using the luciferase reporter under control of the OLFM-4 promoter, it was shown that both 17 beta-estradiol and OH-tamoxifen induce luciferase activity, and epidermal growth factor receptor-1 is required for this estrogenic response. In turn, EGF activates the OLFM-4 promoter, and estrogen receptor-alpha is needed for the complete EGF response. The cellular functions of OLFM-4 were examined by its expression in OLFM-4-negative HEK-293 cells, which resulted in decreased vimentin expression and cell adherence as well as increased apoptosis resistance. In cases of endometriosis and endometrial cancer, OLFM-4 expression correlated with the presence of epidermal growth factor receptor-1 and estrogen receptor-alpha (or estrogen signaling). An increase of OLFM-4 mRNA was observed in the endometrium of endometriosis patients. No change in OLFM-4 expression levels were observed in patients with endometrial cancer relative with controts. In conclusion, cross-talk between estrogen and EGF signaling regulates OLFM-4 expression. The role of OLFM-4 in endometrial tissue remodeling before the secretory phase and during the predisposition and early events in endometriosis can be postulated but requires additional investigation. (Am J Pathol 2010, 177:2495-2508: DOI: 10.2353/ajpath.2010.100026

Durability performance of carbon fibre-reinforced polymer strengthened circular hollow steel members under cold weather

The use of circular hollow steel members has attracted a great deal of attention during past few years because of having excellent structural properties, aesthetic appearance, corrosion and fire protection capability. However, no one can deny the structural deficiency of such structures due to reduction of strength when they are exposed to severe environmental conditions such as marine environment, cold and hot weather. Hence strengthening and retrofitting of structural steel members is now very imperative. This paper presents the findings of a research program that was conducted to study the bond durability of carbon fibre-reinforced polymer (CFRP) strengthened steel tubular members under cold weather and tested under four-point bending. Six number of CFRP-strengthened specimens and one unstrengthened specimen were considered in this program. The three specimens having sand blasted surface to be strengthened was pre-treated with MBrace primer and other three were remained untreated and then cured under ambient temperature at least four weeks and cold weather (3 C) for three and six months period of time. Quasi-static tests were then performed on beams to failure under four-point bending. The structural response of each specimen was predicted in terms of failure load, mid-span deflection, composite beam behaviour and failure mode. The research outcomes show that the cold weather immersion had an adverse effect on durability of CFRP-strengthened steel structures. Moreover, the epoxy based adhesion promoter was found to enhance the bond durability in plastic range. The analytical models presented in this study were found to be in good agreement in terms of predicting ultimate load and deflection. Finally, design factors are proposed to address the short-terms durability performance under cold weather.

Computational epigenetic profiling of CpG islets in MTHFR

Computational epigenetics is a new area of research focused on exploring how DNA methylation patterns affect transcription factor binding that affect gene expression patterns. The aim of this study was to produce a new protocol for the detection of DNA methylation patterns using computational analysis which can be further confirmed by bisulfite PCR with serial pyrosequencing. The upstream regulatory element and pre-initiation complex relative to CpG islets within the methylenetetrahydrofolate reductase gene were determined via computational analysis and online databases. The 1,104 bp long CpG island located near to or at the alternative promoter site of methylenetetrahydrofolate reductase gene was identified. The CpG plot indicated that CpG islets A and B, within the island, contained 62 and 75 % GC content CpG ratios of 0.70 and 0.80–0.95, respectively. Further exploration of the CpG islets A and B indicates that the transcription start sites were GGC which were absent from the TATA boxes. In addition, although six PROSITE motifs were identified in CpG B, no motifs were detected in CpG A. A number of cis-regulatory elements were found in different regions within the CpGs A and B. Transcription factors were predicted to bind to CpGs A and B with varying affinities depending on the DNA methylation status. In addition, transcription factor binding may influence the expression patterns of the methylenetetrahydrofolate reductase gene by recruiting chromatin condensation inducing factors. These results have significant implications for the understanding of the architecture of transcription factor binding at CpG islets as well as DNA methylation patterns that affect chromatin structure.

Durability study of CFRP strengthened steel circular hollow section members under marine environment

Galvanic corrosion is a common phenomenon in Carbon Fibre Reinforced Polymer (CFRP) strengthened steel structures in wet environments and submerged conditions, which reduces durability by weakening the bond between the CFRP and steel substrate. CFRP materials have already been proven to have superior resistance to corrosion and chemical attacks but the adhesive and steel are generally affected by long-term exposure to moisture, especially in conjunction with salts resulting from deicing of ocean spray. This paper presents the results of a research program to improve the durability of CFRP strengthened steel circular hollow section (CHS) members by treating the steel surface with an epoxy based adhesion promoter and inserting Glass Fibre Reinforced Polymer (GFRP) as a galvanic corrosion barrier against simulated sea water. It also presents the effects of accelerated corrosion on the bond of CFRP strengthened hollow steel members. The program consisted of four CFRP strengthened steel beams and one unstrengthened steel beam. Two strengthened beams were used as control while the other two beams were exposed to a highly corrosive environment to induce accelerated corrosion. The corrosion rate was considered 10% which represents a moderate level of loss in the cross-sectional area of the steel tube throughout its intended service life. The beams were then loaded to failure under four-point bending. The research findings indicate that the accelerated corrosion adversely affected the ultimate strength of the conditioned beams and the embedded glass fibre enhanced the bond durability.

An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss

Background Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans. Results Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3′ untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass. Conclusions This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.

Inducible resistance to maize streak virus

Maize streak virus (MSV), which causes maize streak disease (MSD), is the major viral pathogenic constraint on maize production in Africa. Type member of the Mastrevirus genus in the family Geminiviridae, MSV has a 2.7 kb, single-stranded circular DNA genome encoding a coat protein, movement protein, and the two replication-associated proteins Rep and RepA. While we have previously developed MSV-resistant transgenic maize lines constitutively expressing ‘‘dominant negative mutant’’ versions of the MSV Rep, the only transgenes we could use were those that caused no developmental defects during the regeneration of plants in tissue culture. A better transgene expression system would be an inducible one, where resistance-conferring transgenes are expressed only in MSV-infected cells. However, most known inducible transgene expression systems are hampered by background or ‘‘leaky’’ expression in the absence of the inducer. Here we describe an adaptation of the recently developed INPACT system to express MSV-derived resistance genes in cell culture. Split gene cassette constructs (SGCs) were developed containing three different transgenes in combination with three different promoter sequences. In each SGC, the transgene was split such that it would be translatable only in the presence of an infecting MSV’s replication associated protein. We used a quantitative real-time PCR assay to show that one of these SGCs (pSPLITrepIII-Rb-Ubi) inducibly inhibits MSV replication as efficiently as does a constitutively expressed transgene that has previously proven effective in protecting transgenic maize from MSV. In addition, in our cell-culture based assay pSPLITrepIII-Rb-Ubi inhibited replication of diverse MSV strains, and even, albeit to a lesser extent, of a different mastrevirus species. The application of this new technology to MSV resistance in maize could allow a better, more acceptable product.