Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation

The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.

Assisted Diagnosis of Cervical Intraepithelial Neoplasia (CIN)

This paper introduces an automated computer- assisted system for the diagnosis of cervical intraepithelial neoplasia (CIN) using ultra-large cervical histological digital slides. The system contains two parts: the segmentation of squamous epithelium and the diagnosis of CIN. For the segmentation, to reduce processing time, a multiresolution method is developed. The squamous epithelium layer is first segmented at a low (2X) resolution. The boundaries are further fine tuned at a higher (20X) resolution. The block-based segmentation method uses robust texture feature vectors in combination with support vector machines (SVMs) to perform classification. Medical rules are finally applied. In testing, segmentation using 31 digital slides achieves 94.25% accuracy. For the diagnosis of CIN, changes in nuclei structure and morphology along lines perpendicular to the main axis of the squamous epithelium are quantified and classified. Using multi-category SVM, perpendicular lines are classified into Normal, CIN I, CIN II, and CIN III. The robustness of the system in term of regional diagnosis is measured against pathologists' diagnoses and inter-observer variability between two pathologists is considered. Initial results suggest that the system has potential as a tool both to assist in pathologists' diagnoses, and in training.

A fast method for fuzzy neural modeling and refinement

In the identification of complex dynamic systems using fuzzy neural networks, one of the main issues is the curse of dimensionality, which makes it difficult to retain a large number of system inputs or to consider a large number of fuzzy sets. Moreover, due to the correlations, not all possible network inputs or regression vectors in the network are necessary and adding them simply increases the model complexity and deteriorates the network generalisation performance. In this paper, the problem is solved by first proposing a fast algorithm for selection of network terms, and then introducing a refinement procedure to tackle the correlation issue. Simulation results show the efficacy of the method.

Nitric oxide synthase gene therapy enhances the toxicity of cisplatin in cancer cells

BACKGROUND AIMS: Cell-based gene therapy is an alternative to viral and non-viral gene therapy. Emerging evidence suggests that mesenchymal stem cells (MSC) are able to migrate to sites of tissue injury and have immunosuppressive properties that may be useful in targeted gene therapy for sustained specific tissue engraftment. METHODS: In this study, we injected intravenously (i.v.) 1x10(6) MSC, isolated from green fluorescent protein (GFP) transgenic rats, into Rif-1 fibrosarcoma-bearing C3H/HeN mice. The MSC had been infected using a lentiviral vector to express stably the luciferase reporter gene (MSC-GFP-luci). An in vivo imaging system (IVIS 200) and Western blotting techniques were used to detect the distribution of MSC-GFP-luci in tumor-bearing animals. RESULTS: We observed that xenogenic MSC selectively migrated to the tumor site, proliferated and expressed the exogenous gene in subcutaneous fibrosarcoma transplants. No MSC distribution was detected in other organs, such as the liver, spleen, colon and kidney. We further showed that the FGF2/FGFR pathways may play a role in the directional movement of MSC to the Rif-1 fibrosarcoma. We performed in vitro co-culture and in vivo tumor growth analysis, showing that MSC did not affect the proliferation of Rif-1 cells and fibrosarcoma growth compared with an untreated control group. Finally, we demonstrated that the xenogenic MSC stably expressing inducible nitric oxide synthase (iNOS) protein transferred by a lentivirus-based system had a significant inhibitory effect on the growth of Rif-1 tumors compared with MSC alone and the non-treatment control group. CONCLUSIONS: iNOS delivered by genetically modified iNOS-MSC showed a significant anti-tumor effect both in vitro and in vivo. MSC may be used as a target gene delivery vehicle for the treatment of fibrosarcoma and other tumors

A hierarchical multiclass support vector machine incorporated with holistic triple learning units

This paper proposes a new hierarchical learning structure, namely the holistic triple learning (HTL), for extending the binary support vector machine (SVM) to multi-classification problems. For an N-class problem, a HTL constructs a decision tree up to a depth of A leaf node of the decision tree is allowed to be placed with a holistic triple learning unit whose generalisation abilities are assessed and approved. Meanwhile, the remaining nodes in the decision tree each accommodate a standard binary SVM classifier. The holistic triple classifier is a regression model trained on three classes, whose training algorithm is originated from a recently proposed implementation technique, namely the least-squares support vector machine (LS-SVM). A major novelty with the holistic triple classifier is the reduced number of support vectors in the solution. For the resultant HTL-SVM, an upper bound of the generalisation error can be obtained. The time complexity of training the HTL-SVM is analysed, and is shown to be comparable to that of training the one-versus-one (1-vs.-1) SVM, particularly on small-scale datasets. Empirical studies show that the proposed HTL-SVM achieves competitive classification accuracy with a reduced number of support vectors compared to the popular 1-vs-1 alternative.

Synthesis of an analogue of the bisphosphonate drug Ibandronate for targeted drug-delivery therapeutic strategies

An analogue of the bisphosphonate drug Ibandronate was prepared and coupled via a cleavable ester function to a bromoacetyl linker with specific reactivity for thiol groups. This compound should find useful applications in therapeutic strategies aiming to deliver bisphosphonate drugs specifically to cancer cells making use of proteins as vectors. The specific delivery of bisphosphonates to cancer cells instead of bone, the usual site of accumulation of these cytotoxic drugs, could greatly widen their therapeutic applications.

Macroalgal introductions by hull fouling on recreational vessels: Seaweeds and sailors

Macroalgal invasions in coastal areas have been a growing concern during the past decade. The present study aimed to assess the role of hull fouling on recreational yachts as a vector for macroalgal introductions. Questionnaire and hull surveys were carried out in marinas in France and Spain. The questionnaires revealed that the majority of yacht owners are aware of seaweed introductions, usually undertake short range journeys, dry dock their boat at least once a year, and use antifouling paints. The hull survey showed that many in-service yachts were completely free of macroalgae. When present, fouling assemblages consisted mainly of one to two macroalgal species. The most commonly found species was the tolerant green seaweed Ulva flexuosa. Most of the other species found are also cosmopolitan and opportunistic. A few nonnative and potentially invasive Ceramiales (Rhodophyta) were found occasionally on in-service yachts. On the basis of the information gathered during interviews of yacht owners in the surveyed area, these occurrences are likely to be uncommon. However they can pose a significant risk of primary or secondary introductions of alien macroalgal species, especially in the light of the increase in yachting activities. With large numbers of recreational yachts and relatively rare occurrences of nonnative species on hulls, comprehensive screening programs do not seem justified or practical. The risks of transferring nonnative species may, however, be minimized by encouraging the behaviors that prevent fouling on hulls and by taking action against neglected boats before they can act as vectors.

C/EBP alpha Is Up-regulated in a Subset of Hepatocellular Carcinomas and Plays a Role in Cell Growth and Proliferation

BACKGROUND & AIMS: C/EBP alpha (cebpa) is a putative tumor suppressor. However, initial results indicated that cebpa was up-regulated in a subset of human hepatocellular carcinomas (HCCs). The regulation and function of C/EBP alpha was investigated in HCC cell lines to clarify its role in liver carcinogenesis. METHODS: The regulation of C/EBP alpha expression was studied by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemistry, methylation-specific PCR, and chromatin immunoprecipitation assays. C/EBP alpha expression was knocked-down by small interfering RNA or short hairpin RNA. Functional assays included colony formation, methylthiotetrazole, bromodeoxyuridine incorporation, and luciferase-reporter assays. RESULTS: Cebpa was up-regulated at least 2-fold in a subset (approximately 55%) of human HCCs compared with adjacent non tumor tissues. None of the up-regulated samples were positive for hepatitis C infection. The HCC cell lines Hep3B and Huh7 expressed high, PLC/PRF/5 intermediate, HepG2 and HCC-M low levels of C/EBP alpha, recapitulating the pattern of expression observed in HCCs. No mutations were detected in the CEBP alpha gene in HCCs and cell lines. C/EBP alpha was localized to the nucleus and functional in Hep3B and Huh7 cells; knocking-down its expression reduced target-gene expression, colony formation, and cell growth, associated with a decrease in cyclin A and CDK4 concentrations and E2F transcriptional activity. Epigenetic mechanisms including DNA methylation, and the binding of acetylated histone H3 to the CEBP alpha promoter-regulated cebpa expression in the HCC cells. CONCLUSIONS: C/EBP alpha is up-regulated in a subset of HCCs and has growth-promoting activities in HCC cells. Novel oncogenic mechanisms involving C/EBP alpha may be amenable to epigenetic regulation to improve treatment outcomes.

Family Histories and Rural Inheritance in Kenya

Inheritance systems and practices have a key role in people’s ability to exit poverty, or, conversely, plunging them further into it. As land is the major asset in low-income developing countries, how property is passed on and divided between future generations is a significant factor. This paper looks at inheritance through minimally-structured interviews with several generations of Kenyan families, seeking to explain that the how and why of poverty can be understood in the wider family context. It analyses their fortunes and misfortunes over a given time period in the context of property ownership rights. It also looks at the impact of education and the inheritance of cultural capital. When both fertility and survival are high, traditional patterns of land inheritance can lead to progression sub-division of land with long-term adverse implications for sustainability. While inheritance in Kenya is male dominated, the paper nonetheless examines the position of women in the chain as vectors of male property rights. The application of male-oriented customary law where inheritance is concerned, rather than the use of statutory legislation, was found to be the reality for the overwhelming majority of the participants in the study.

Applied aspects of Rhodococcus genetics

Eubacteria of the genus Rhodococcus are a diverse group of microorganisms commonly found in many environmental niches from soils to seawaters and as plant and animal pathogens. They exhibit a remarkable ability to degrade many organic compounds and their economic importance is becoming increasingly apparent. Although their genetic organisation is still far from understood, there have been many advances in recent years. Reviewed here is the current knowledge of rhodococci relating to gene transfer, recombination, plasmid replication and functions, cloning vectors and reporter genes, gene expression and its control, bacteriophages, insertion sequences and genomic rearrangements. Further fundamental studies of Rhodococcus genetics and the application of genetic techniques to the these bacteria will be needed for their continued biotechnological exploitation.

Speaker Recognition In Noisy Conditions With Limited Training Data

In this paper we present a novel method for performing speaker recognition with very limited training data and in the presence of background noise. Similarity-based speaker recognition is considered so that speaker models can be created with limited training speech data. The proposed similarity is a form of cosine similarity used as a distance measure between speech feature vectors. Each speech frame is modelled using subband features, and into this framework, multicondition training and optimal feature selection are introduced, making the system capable of performing speaker recognition in the presence of realistic, time-varying noise, which is unknown during training. Speaker identi?cation experiments were carried out using the SPIDRE database. The performance of the proposed new system for noise compensation is compared to that of an oracle model; the speaker identi?cation accuracy for clean speech by the new system trained with limited training data is compared to that of a GMM trained with several minutes of speech. Both comparisons have demonstrated the effectiveness of the new model. Finally, experiments were carried out to test the new model for speaker identi?cation given limited training data and with differing levels and types of realistic background noise. The results have demonstrated the robustness of the new system.

A novel dual-fluorescence strategy for functionally validating microRNA targets in 3-prime untranslated regions: regulation of the inward rectifier potassium channel Kir2.1 by miR-212.

Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 (KCNJ2) which is dysregulated in cardiac and vascular disorders. The 3'UTR was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK293 cells were co-transfected with the mCherry-3'UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3'UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known downregulator of Kir2.1 expression, and was used to investigate targeting of the Kir2.1 3'UTR by miR-212. Red/green ratio was lower in miR-212-expressing cells compared to non-targeting controls, an effect that was attenuated by mutating the predicted target site. MiR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.

Identification of essential operons with a rhamnose-inducible promoter in Burkholderia cenocepacia

Scanning of bacterial genomes to identify essential genes is of biological interest, for understanding the basic functions required for life, and of practical interest, for the identification of novel targets for new antimicrobial therapies. In particular, the lack of efficacious antimicrobial treatments for infections caused by the Burkholderia cepacia complex is causing high morbidity and mortality of cystic fibrosis patients and of patients with nosocomial infections. Here, we present a method based on delivery of the tightly regulated rhamnose-inducible promoter P(rhaB) for identifying essential genes and operons in Burkholderia cenocepacia. We demonstrate that different levels of gene expression can be achieved by using two vectors that deliver P(rhaB) at two different distances from the site of insertion. One of these vectors places P(rhaB) at the site of transposon insertion, while the other incorporates the enhanced green fluorescent protein gene (e-gfp) downstream from P(rhaB). This system allows us to identify essential genes and operons in B. cenocepacia and provides a new tool for systematically identifying and functionally characterizing essential genes at the genomic level.

Novel macrocyclic and acyclic cationic lipids for gene transfer: Synthesis and in vitro evaluation

The synthesis and in vitro evaluation of four cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic, and saturated or unsaturated hydrophobic regions, is described. The synthesis employed standard protocols, including ring-closing metathesis for macrocyclic lipid construction. All lipoplexes studied, formulated from plasmid DNA and a liposome composed of a synthesized lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol as co-lipid, exhibited plasmid DNA binding and protection from DNase I degradation, and concentration dependent cytotoxicity using Chinese hamster ovary-K1 cells. The transfection efficiency of formulations with cholesterol outperformed those with DOPE, and in many cases the EPC/cholesterol control, and formulations with a macrocyclic lipid (+/- 10:1) outperformed their acyclic counterparts (+/- 3:1).

Microneedle-mediated vaccine delivery:harnessing cutaneous immunobiology to improve efficacy

INTRODUCTION: Breaching the skin's stratum corneum barrier raises the possibility of the administration of vaccines, gene vectors, antibodies and even nanoparticles, all of which have at least their initial effect on populations of skin cells. AREAS COVERED: Intradermal vaccine delivery holds enormous potential for improved therapeutic outcomes for patients, particularly those in the developing world. Various vaccine-delivery strategies have been employed, which are discussed in this review. The importance of cutaneous immunobiology on the effect produced by microneedle-mediated intradermal vaccination is also discussed. EXPERT OPINION: Microneedle-mediated vaccines hold enormous potential for patient benefit. However, in order for microneedle vaccine strategies to fulfill their potential, the proportion of an immune response that is due to the local action of delivered vaccines on skin antigen-presenting cells, and what is due to a systemic effect from vaccines reaching the systemic circulation, must be determined. Moreover, industry will need to invest significantly in new equipment and instrumentation in order to mass-produce microneedle vaccines consistently. Finally, microneedles will need to demonstrate consistent dose delivery across patient groups and match this to reliable immune responses before they will replace tried-and-tested needle-and-syringe-based approaches.