Development of an eight gene expression profile implicating human breast tumours of all grade

The goal of improving systemic treatment of breast cancers is to evolve from treating every patient with non-specific cytotoxic chemotherapy/hormonal therapy, to a more individually-tailored direct treatment. Although anatomic staging and histological grade are important prognostic factors, they often fail to predict the clinical course of this disease. This study aimed to develop a gene expression profile associated with breast cancers of differing grades. We extracted mRNA from FFPE archival breast IDC tissue samples (Grades I–III), including benign tumours. Affymetrix GeneChip� Human Genome U133 Plus 2.0 Arrays were used to determine gene expression profiles and validated by Q-PCR. IHC was used to detect the AXIN2 protein in all tissues. From the array data, an independent group t-test revealed that 178 genes were significantly (P B 0.01) differentially expressed between three grades of malignant breast tumours when compared to benign tissues. From these results, eight genes were significantly differentially expressed in more than one comparison group and are involved in processes implicated in breast cancer development and/or progression. The two most implicated candidates genes were CLD10 and ESPTI1 as their gene expression profile from the microarray analysis was replicated in Q-PCR analyses of the original tumour samples as well as in an extended population. The IHC revealed a significant association between AXIN2 protein expression and ER status. It is readily acknowledged and established that significant differences exist in gene expression between different cancer grades. Expansion of this approach may lead to an improved ability to discriminate between cancer grade and other pathological factors.

Modulation of in vitro platelet 5-HT release by species of Erythrina and Cymbopogon

Extracts of Australian plants were screened to detect constituents affecting adenosine di-phosphate (ADP) induced platelet aggregation and [14C]5-hydroxytryptamine (5-HT) release. Extracts of four tested plants including, Eremophila gilesii, Erythrina vespertilio, Cymbopogon ambiguus, and Santalum acuminatum, were found to cause significant inhibition of platelet 5-HT release. Inhibition levels ranged from 56-98%, and was not due to the non-specific effects of protein binding tannins. These extracts, and those we have previously identified as being active, were examined further to determine if they affect epinephrine (EPN), arachidonic acid (A.A) or collagen stimulated platelet aggregation and 5-HT release. Among those extracts investigated, we found that both the methanolic extract of E. vespertilio and the dichloromethane (DCM) extract of C. ambiguus were most potent and caused significant inhibition of platelet activation induced by EPN, A.A and to a lesser extent by collagen. Inhibition of ADP induced platelet 5-HT release by both of these extracts, was dose-dependent, with IC50 values for E. vespertilio and C. ambiguus estimated to be 20.4 microl (1.855 mg/ml) and 8.34 microl (0.758 mg/ml), respectively. Overall, C. ambiguus exhibited most activity and also caused dose-dependent inhibition of A.A induced platelet activation. These results indicate that inhibition may occur specifically at a site within the A.A pathway, and suggest the presence of a cyclo-oxygenase inhibitor. Both E. vespertilio and C. ambiguus are reported to be traditional headache treatments, with the present study providing evidence that they affect 5-HT release.

A Guide to Applying Road Safety within a Workplace : A Bilateral Approach to Organisational Road Safety in Australia and New Zealand

Corporate and organisational fleet and road safety is of strong interest to government and government agencies in Australia and New Zealand. It has been identified that there is great opportunity to engage and assist organisations and corporations in the delivery of road safety and road safety measures to achieve nationally significant road related trauma reductions. This guide has therefore been developed through public sector funding for use by any workplace within Australia and New Zealand. Significant road safety benefits can be achieved by road safety government agencies (Australia and New Zealand) that engage with private and public sectors at their workplaces to address work related road safety issues. It has also been noted that organisational road safety advancement creates effective and sustainable outcomes, safer places of employment, and safer communities. This can be achieved without totally relying upon traditional and often lengthy processes such as further public legislation and/ or community attitudinal and behavioural change programs. Currently, there is little in the way of robust guides or support for those organisations that are wishing to adopt road safety within their places of employment, supply chain and/ or community. Due to this identified gap in available resource and support, it has been recommended that a practical organisational road safety guide be produced; hence the development of this guide. A guide, or supporting documentation, that bridges the gap between government and road safety research knowledge, internationally endorsed road safety methodology, and assists industry as the end user. To achieve this, the guide is designed to be non-specific to any industry sector and usable for small or large organisations, public or private, and engaging for senior executives and the personnel on the ground responsible for its implementation. Therefore, this guide is based on methodology and principles so that it can be applicable in a scalable way to the greatest number of public and private organisations while providing enough detail and ‘how to’ advice to enable organisations to generate their own solutions to road safety issues.

Different in vitro activation methods for latent transforming growth factors (TGF)–β : considerable exogenous factors to promote higher mesenchymal-origin cell proliferation in a bioprocessing platform

Regenerative medicine includes two efficient techniques, namely tissue-engineering and cell-based therapy in order to repair tissue damage efficiently. Most importantly, huge numbers of autologous cells are required to deal these practices. Nevertheless, primary cells, from autologous tissue, grow very slowly while culturing in vitro; moreover, they lose their natural characteristics over prolonged culturing period. Transforming growth factors-beta (TGF-β) is a ubiquitous protein found biologically in its latent form, which prevents it from eliciting a response until conversion to its active form. In active form, TGF-β acts as a proliferative agent in many cell lines of mesenchymal origin in vitro. This article reviews on some of the important activation methods-physiochemical, enzyme-mediated, non-specific protein interaction mediated, and drug-induced- of TGF-β, which may be established as exogenous factors to be used in culturing medium to obtain extensive proliferation of primary cells.

The salt dependence of DNA recognition by NF-kappaB p50 : a detailed kinetic analysis of the effects on affinityand specificity

The binding kinetics of NF-kappaB p50 to the Ig-kappaB site and to a DNA duplex with no specific binding site were determined under varying conditions of potassium chloride concentration using a surface plasmonresonance biosensor. Association and dissociation rate constants were measured enabling calculation of the dissociation constants. Under previously established high affinity buffer conditions, the k a for both sequences was in the order of 10(7) M-1s-1whilst the k d values varied 600-fold in a sequence-dependent manner between 10(-1) and 10(-4 )s-1, suggesting that the selectivity of p50 for different sequences is mediated primarily through sequence-dependent dissociation rates. The calculated K D value for the Ig-kappaB sequence was 16 pM, whilst the K D for the non-specific sequence was 9.9 nM. As the ionic strength increased to levels which are closer to that of the cellular environment, the binding of p50 to the non-specific sequence was abolished whilst the specific affinity dropped to nanomolar levels. From these results, a mechanism is proposed in which p50 binds specific sequences with high affinity whilst binding non-specific sequences weakly enough to allow efficient searching of the DNA.

Optimization approaches for the design of additively manufactured scaffolds

Scaffolds play a pivotal role in tissue engineering, promoting the synthesis of neo extra-cellular matrix (ECM), and providing temporary mechanical support for the cells during tissue regeneration. Advances introduced by additive manufacturing techniques have significantly improved the ability to regulate scaffold architecture, enhancing the control over scaffold shape and porosity. Thus, considerable research efforts have been devoted to the fabrication of 3D porous scaffolds with optimized micro-architectural features. This chapter gives an overview of the methods for the design of additively manufactured scaffolds and their applicability in tissue engineering (TE). Along with a survey of the state of the art, the Authors will also present a recently developed method, called Load-Adaptive Scaffold Architecturing (LASA), which returns scaffold architectures optimized for given applied mechanical loads systems, once the specific stress distribution is evaluated through Finite Element Analysis (FEA).

Seed germination may explain differences in invasiveness and prevalence : a case study using cat's claw creeper (Dolichandra unguis-cati)

High germination rates and rapid germination behavior in response to different environmental cues are traits that may be associated with invasiveness. Cat’s claw creeper (Dolichandra unguis-cati (L.) Lohmann (syn. Macfadyena unguis-cati (L.) Gentry), a Weed of National Significance has two forms, a long-pod (LP) form and a short-pod (SP) from. The LP form occurs in only a few localities in southeast Queensland while the SP form is widely distributed in Queensland and New South Wales. The aims of this investigation were: to evaluate whether there are significant differences in germination traits between the two forms of cat’s claw creeper; and if there are any significant differences, to find out whether the differences in germination can be related to prevalence and invasiveness levels for the two forms. Long pod and short pod seeds collected in 2009, 2010, 2011, 2012 and 2013 from various localities in Qld were germinated in growth chambers in early 2013. The growth chambers were set to 10/20 ºC, 15/25 ºC and 20/30 ºC temperature cycles. Seeds from 2009-2012 of either form did not germinate, while for the fresh seeds (2013), SP exhibited significantly higher total germination percentage and rates than LP. Assuming that the two forms were introduced in Australia at around the same period, these results could explain why SP is widely distributed (and therefore more invasive) in Qld and NSW while LP is only confined to a few localities in southeast Qld.

A puff of fluticasone for eosinophilic oesophagitis

Eosinophilic oesophagitis (EEl is most commonly observed in children and young males, and is estimated to affect one per 10,000 children, and lo/o of adults in Australia. EE causes inflammation of the oesophagus; and an endoscopy, and biopsy showing an increase in eosinophils through the oesophagus is used for diagnosis. People with EE often present with non-specific symptoms to varying degrees of severity that could also suggest swallowing difficulties, or acid reflux; the feeling of food sticking to the throat on the way down (sometimes completely), choking on food, regurgitation of food, and severe acid reflux that does not respond to stomach acid suppression treatment...

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.

The mechanical and inflammatory low back pain (MIL) index: development and validation

Abstract Background The purpose of this study was the development of a valid and reliable “Mechanical and Inflammatory Low Back Pain Index” (MIL) for assessment of non-specific low back pain (NSLBP). This 7-item tool assists practitioners in determining whether symptoms are predominantly mechanical or inflammatory. Methods Participants (n = 170, 96 females, age = 38 ± 14 years-old) with NSLP were referred to two Spanish physiotherapy clinics and completed the MIL and the following measures: the Roland Morris Questionnaire (RMQ), SF-12 and “Backache Index” (BAI) physical assessment test. For test-retest reliability, 37 consecutive patients were assessed at baseline and three days later during a non-treatment period. Face and content validity, practical characteristics, factor analysis, internal consistency, discriminant validity and convergent validity were assessed from the full sample. Results A total of 27 potential items that had been identified for inclusion were subsequently reduced to 11 by an expert panel. Four items were then removed due to cross-loading under confirmatory factor analysis where a two-factor model yielded a good fit to the data (χ2 = 14.80, df = 13, p = 0.37, CFI = 0.98, and RMSEA = 0.029). The internal consistency was moderate (α = 0.68 for MLBP; 0.72 for ILBP), test-retest reliability high (ICC = 0.91; 95%CI = 0.88-0.93) and discriminant validity good for either MLBP (AUC = 0.74) and ILBP (AUC = 0.92). Convergent validity was demonstrated through similar but weak correlations between the ILBP and both the RMQ and BAI (r = 0.34, p < 0.001) and the MLBP and BAI (r = 0.38, p < 0.001). Conclusions The MIL is a valid and reliable clinical tool for patients with NSLBP that discriminates between mechanical and inflammatory LBP. Keywords: Low back pain; Psychometrics properties; Pain measurement; Screening tool; Inflammatory; Mechanical

Functional and structural of the erector spinae muscle during isometric lumbar extension

Study Design Cross-sectional study. Objectives To compare erector spinae (ES) muscle fatigue between chronic non-specific lower back pain (CNLBP) sufferers and healthy subjects from a biomechanical perspective during fatiguing isometric lumbar extensions. Background Paraspinal muscle maximal contraction and fatigue are used as a functional predictor for disabilities. The simplest method to determine muscle fatigue is by evaluating the evolution during specific contractions, such as isometric contractions. There are no studies that evaluate the evolution of the ES muscle during fatiguing isometric lumbar extensions and analyse functional and architectural variables. Methods In a pre-calibrated system, participants performed a maximal isometric extension of the lumbar spine for 5 and 30 seconds. Functional variables (torque and muscle activation) and architecture (pennation angle and muscle thickness) were measured using a load cell, surface electromyography and ultrasound, respectively. The results were normalised and a reliability study of the ultrasound measurement was made. Results: The ultrasound measurements were highly reliable, with Cronbach’s alpha values ranging from 0.951 0.981. All measured variables shown significant differences before and after fatiguing isometric lumbar extension. Conclusion During a lumbar isometric extension test, architecture and functional variables of the ES muscle could be analised using ultrasound, surface EMG and load cell. In adition, during an endurance test, ES muscle suffers an acute effect on architectural and functional variables.

Synthesis, crystal structures and protease activity of amino acid Schiff base iron(III) complexes

Iron(III) complexes, (NHEt3)[Fe(III)(sal-met)(2)] and (NHEt3)[Fe(III)(sal-phe)(2)], of amino acid Schiffbase ligands, viz., N-salicylidene-L-methionine and N-salicylidene L-phenylalanine, have been prepared and their binding to bovine serum albumin (BSA) and photo-induced BSA cleavage activity have been investigated. The complexes are structurally characterized by single crystal X-ray crystallography. The crystal Structures of the discrete mononuclear rnonoanionic complexes show FeN2O4 octahedral coordination geometry in which the tridentate dianionic amino acid Schiff base ligand binds through phenolate and carboxylate oxygen and imine nitrogen atoms. The imine nitrogen atoms are trans to each other. The Fe-O and Fe-N bond distances range between 1.9 and 2.1 angstrom. The sal-met complex has two pendant thiomethyl groups. The high-spin iron(III) complexes (mu(eff) similar to 5.9 mu(B)) exhibit quasi-reversible Fe(III)/Fe(II) redox process near -0.6 V vs. SCE in water. These complexes display a visible electronic hand near 480 nm in tris-HCl buffer assignable to the phenolate-to-iron(III) charge transfer transition. The water soluble complexes bind to BSA giving binding constant values of similar to 10(5) M-1. The Complexes show non-specific oxidative cleavage of BSA protein on photo-irradiation with UV-A light of 365 nm.

Characterization of Ion-Exchange Fibers for Controlled Drug Delivery

Increasing attention has been focused on methods that deliver pharmacologically active compounds (e.g. drugs, peptides and proteins) in a controlled fashion, so that constant, sustained, site-specific or pulsatile action can be attained. Ion-exchange resins have been widely studied in medical and pharmaceutical applications, including controlled drug delivery, leading to commercialisation of some resin based formulations. Ion-exchangers provide an efficient means to adjust and control drug delivery, as the electrostatic interactions enable precise control of the ion-exchange process and, thus, a more uniform and accurate control of drug release compared to systems that are based only on physical interactions. Unlike the resins, only few studies have been reported on ion-exchange fibers in drug delivery. However, the ion-exchange fibers have many advantageous properties compared to the conventional ion-exchange resins, such as more efficient compound loading into and release from the ion-exchanger, easier incorporation of drug-sized compounds, enhanced control of the ion-exchange process, better mechanical, chemical and thermal stability, and good formulation properties, which make the fibers attractive materials for controlled drug delivery systems. In this study, the factors affecting the nature and strength of the binding/loading of drug-sized model compounds into the ion-exchange fibers was evaluated comprehensively and, moreover, the controllability of subsequent drug release/delivery from the fibers was assessed by modifying the conditions of external solutions. Also the feasibility of ion-exchange fibers for simultaneous delivery of two drugs in combination was studied by dual loading. Donnan theory and theoretical modelling were applied to gain mechanistic understanding on these factors. The experimental results imply that incorporation of model compounds into the ion-exchange fibers was attained mainly as a result of ionic bonding, with additional contribution of non-specific interactions. Increasing the ion-exchange capacity of the fiber or decreasing the valence of loaded compounds increased the molar loading, while more efficient release of the compounds was observed consistently at conditions where the valence or concentration of the extracting counter-ion was increased. Donnan theory was capable of fully interpreting the ion-exchange equilibria and the theoretical modelling supported precisely the experimental observations. The physico-chemical characteristics (lipophilicity, hydrogen bonding ability) of the model compounds and the framework of the fibrous ion-exchanger influenced the affinity of the drugs towards the fibers and may, thus, affect both drug loading and release. It was concluded that precisely controlled drug delivery may be tailored for each compound, in particularly, by choosing a suitable ion-exchange fiber and optimizing the delivery system to take into account the external conditions, also when delivering two drugs simultaneously.

Kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab) coat protein and its mutants with ssDNA

Gemini viral assembly and transport of viral DNA into nucleus for replication, ssentially involve DNA-coat protein interactions. The kinetics of interaction of Cotton LeafCtirl Kokhran Virus-Dabawali recombinant coat protein (rCP) with DNA was studied by electrophoretic mobility shift assay (EMSA) and Surface plasmon resonance (SPR). The rCP interacted with ssDNA with a K-A, of 2.6 +/- 0.29 x 10(8) M-1 in a sequence non-specific manner. The CP has a conserved C2H2 type zinc finger motif composed of residues C68, C72, H81 and H85. Mutation of these residues to alanine resulted in reduced binding to DNA probes. The H85A mutant rCP showed the least binding with approximately 756 fold loss in the association rate and a three order magnitude decrease in the binding affinity as compared to rCP. The CP-DNA interactions via the zinc finger motif could play a Crucial role ill Virus assembly and in nuclear transport. (C) 2009 Elsevier Inc.

Oral Immune Defense against Chronic Hyperplastic Candidosis

Candida yeast species are widespread opportunistic microbes, which are usually innocent opportunists unless the systemic or local defense system of the host becomes compromised. When they adhere on a fertile substrate such as moist and warm, protein-rich human mucosal membrane or biomaterial surface, they become activated and start to grow pseudo and real hyphae. Their growth is intricately guided by their ability to detect surface defects (providing secure hiding , thigmotropism) and nutrients (source of energy, chemotropism). The hypothesis of this work was that body mobilizes both non-specific and specific host defense against invading candidal cells and that these interactions involve resident epithelial cells, rapidly responding non-specific protector neutrophils and mast cells as well as the antigen presenting and responding den-dritic cell lymphocyte plasma cell system. It is supposed that Candida albicans, as a result of dar-winistic pressure, has developed or is utilizing strategies to evade these host defense reactions by e.g. adhering to biomaterial surfaces and biofilms. The aim of the study was to assess the host defense by taking such key molecules of the anti-candidal defense into focus, which are also more or less characteristic for the main cellular players in candida-host cell interactions. As a model for candidal-host interaction, sections of chronic hyperplastic candidosis were used and compared with sections of non-infected leukoplakia and healthy tissue. In this thesis work, neutrophil-derived anti-candidal α-defensin was found in the epithelium, not only diffusely all over in the epithelium, but as a strong α-defensin-rich superficial front probably able to slow down or prevent penetration of candida into the epithelium. Neutrophil represents the main host defence cell in the epithelium, to which it can rapidly transmigrate from the circulation and where it forms organized multicellular units known as microabscesses (study I). Neutrophil chemotactic inter-leukin-8 (IL-8) and its receptor (IL-8R) were studied and were surprisingly also found in the candidal cells, probably helping the candida to keep away from IL-8- and neutrophil-rich danger zones (study IV). Both leukocytes and resident epithelial cells contained TLR2, TLR4 and TLR6 receptors able to recognize candidal structures via utilization of receptors similar to the Toll of the banana fly. It seems that candida can avoid host defence via stimulation of the candida permissive TLR2 instead of the can-dida injurious TLR4 (study V). TLR also provides the danger signal to the immune system without which it will not be activated to specifically respond against candidal antigens. Indeed, diseased sites contained receptor activator of nuclear factor kappa B ligand (RANKL; II study), which is important for the antigen capturing, processing and presenting dendritic cells and for the T lymphocyte activation (study III). Chronic hyperplastic candidosis provides a disease model that is very useful to study local and sys-temic host factors, which under normal circumstances restrain C. albicans to a harmless commensal state, but failure of which in e.g. HIV infection, cancer and aging may lead to chronic infection.