High-throughput profiling for discovery of non-coding RNA biomarkers of lung disease.

In respiratory medicine there is a need for clinical biomarkers for diagnosis, prognosis and assessment of response to therapy. Noncoding RNA (ncRNA) is expressed in all human cells; two major classes - long ncRNA and microRNA - are detectable extracellularly in the circulation and other biofluids. Altered ncRNA expression is associated with lung disease; collectively this indicates that ncRNA represents a potential biomarker class. This article presents and compares existing platforms for detection and quantification of ncRNA, specifically hybridization, qRT-PCR and RNA sequencing, and outlines methods for data interpretation and normalization. Each approach has merits and shortcomings, which can affect the choice of method when embarking on a biomarker study. Biomarker properties and pre-analytical considerations for ncRNA profiling are also presented. Since a variety of profiling approaches are available, careful study and experimental design are important. Finally, challenges and goals for reliable, standardized high-throughput ncRNA profiling in biofluids as lung disease biomarkers are reviewed

Non-coding RNA as lung disease biomarkers.

Biomarkers are quantifiable indicators of disease. These surrogates should be specific, sensitive, predictive, robust and easily accessible. A major class of RNA described as non-coding RNA fulfils many of these criteria, and recent studies have demonstrated that the two major subclasses of non-coding RNA, long non-coding RNA and, in particular, microRNA are promising potential biomarkers. The ability to detect non-coding RNAs in biofluids has highlighted their usefulness as non-invasive markers of lung disease. Because expression of specific non-coding RNAs is altered in many lung diseases and their levels in the circulation often reflect the changes in expression of their lung-specific counterparts, exploiting these biomolecules as diagnostic tools seems an obvious goal. New technology is driving developments in this area and there has been significant recent progress with respect to lung cancer diagnostics. The non-coding RNA biomarker field represents a clear example of modern-day bench-to-bedside research

Long noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis bronchial epithelium.

Long non-coding RNAs (lncRNAs) have emerged recently as key regulatory molecules with diverse roles at almost every level of the regulation of gene expression. The roles of these RNAs in the pathogenesis of cystic fibrosis (CF); a lethal multisystem, autosomal recessive disorder have yet to be explored. Our aim was to examine the expression profile of lncRNA, in the airway epithelium of people with CF. We examined the expression of 30,586 lncRNAs by microarray (Human LncRNA Array v3.0, Arraystar, Inc.), in vivo in bronchial cells isolated from endobronchial brushings obtained from CF and non-CF individuals. In total, we identified 1,063 lncRNAs with differential expression between CF and non-CF individuals (fold change ≥3, p≤0.001). The microarray also contained probes for ∼26,109 protein coding transcripts, of which 720 were differentially expressed between CF and non-CF brush samples (fold change ≥3, p≤0.001). Confirmation of a selection of differentially expressed coding mRNA and lncRNA transcripts such as XIST and TLR8 was achieved using qRT-PCR. Gene ontology bioinformatics analysis highlighted that many processes over-represented in the CF bronchial epithelium are related to inflammation. These data show a significantly altered lncRNA and mRNA expression profile in CF bronchial cells in vivo. Dysregulation of some of these lncRNAs may play important roles in the chronic infection and inflammation that exists in the lungs of people with CF

Crosslinking and Mechanical Properties Significantly Influence Cell Attachment, Proliferation, and Migration Within Collagen Glycosaminoglycan Scaffolds.

Crosslinking and the resultant changes in mechanical properties have been shown to influence cellular activity within collagen biomaterials. With this in mind, we sought to determine the effects of crosslinking on both the compressive modulus of collagen-glycosaminoglycan scaffolds and the activity of osteoblasts seeded within them. Dehydrothermal, 1-ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde crosslinking treatments were first investigated for their effect on the compressive modulus of the scaffolds. After this, the most promising treatments were used to study the effects of crosslinking on cellular attachment, proliferation, and infiltration. Our experiments have demonstrated that a wide range of scaffold compressive moduli can be attained by varying the parameters of the crosslinking treatments. 1-Ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde treatments produced the stiffest scaffolds (fourfold increase when compared to dehydrothermal crosslinking). When cells were seeded onto the scaffolds, the stiffest scaffolds also showed increased cell number and enhanced cellular distribution when compared to the other groups. Taken together, these results indicate that crosslinking can be used to produce collagen-glycosaminoglycan scaffolds with a range of compressive moduli, and that increased stiffness enhances cellular activity within the scaffolds

Nudges and other moral technologies in the context of power: Assigning and accepting responsibility

Strawson argues that we should understand moral responsibility in terms of our practices of holding responsible and taking responsibility. The former covers what is commonly referred to as backward-looking responsibility , while the latter covers what is commonly referred to as forward-looking responsibility . We consider new technologies and interventions that facilitate assignment of responsibility. Assigning responsibility is best understood as the second- or third-personal analogue of taking responsibility. It establishes forward-looking responsibility. But unlike taking responsibility, it establishes forward-looking responsibility in someone else. When such assignments are accepted, they function in such a way that those to whom responsibility has been assigned face the same obligations and are susceptible to the same reactive attitudes as someone who takes responsibility. One family of interventions interests us in particular: nudges. We contend that many instances of nudging tacitly assign responsibility to nudgees for actions, values, and relationships that they might not otherwise have taken responsibility for. To the extent that nudgees tacitly accept such assignments, they become responsible for upholding norms that would otherwise have fallen under the purview of other actors. While this may be empowering in some cases, it can also function in such a way that it burdens people with more responsibility that they can (reasonably be expected to) manage

Aniline incorporated silica nanobubbles

We report the synthesis of stearate functionalized nanobubbles of SiO2 with a few aniline molecules inside, represented as C6H5NH2@SiO2@stearate, exhibiting fluorescence with red-shifted emission. Stearic acid functionalization allows the materials to be handled just as free molecules, for dissolution, precipitation, storage etc. The methodology adopted involves adsorption of aniline on the surface of gold nanoparticles with subsequent growth of a silica shell through monolayers, followed by the selective removal of the metal core either using sodium cyanide or by a new reaction involving halocarbons. The material is stable and can be stored for extended periods without loss of fluorescence. Spectroscopic and voltammetric properties of the system were studied in order to understand the interaction of aniline with the shell as well as the monolayer, whilst transmission electron microscopy has been used to study the silica shell

Characteristic promoter hypermethylation signatures in male germ cell tumors

BACKGROUND: Human male germ cell tumors (GCTs) arise from undifferentiated primordial germ cells (PGCs), a stage in which extensive methylation reprogramming occurs. GCTs exhibit pluripotentality and are highly sensitive to cisplatin therapy. The molecular basis of germ cell (GC) transformation, differentiation, and exquisite treatment response is poorly understood. RESULTS: To assess the role and mechanism of promoter hypermethylation, we analyzed CpG islands of 21 gene promoters by methylation-specific PCR in seminomatous (SGCT) and nonseminomatous (NSGCT) GCTs. We found 60% of the NSGCTs demonstrating methylation in one or more gene promoters whereas SGCTs showed a near-absence of methylation, therefore identifying distinct methylation patterns in the two major histologies of GCT. DNA repair genes MGMT, RASSF1A, and BRCA1, and a transcriptional repressor gene HIC1, were frequently methylated in the NSGCTs. The promoter hypermethylation was associated with gene silencing in most methylated genes, and reactivation of gene expression occured upon treatment with 5-Aza-2' deoxycytidine in GCT cell lines. CONCLUSIONS: Our results, therefore, suggest a potential role for epigenetic modification of critical tumor suppressor genes in pathways relevant to GC transformation, differentiation, and treatment response

Role of promoter hypermethylation in Cisplatin treatment response of male germ cell tumors

BACKGROUND: Male germ cell tumor (GCT) is a highly curable malignancy, which exhibits exquisite sensitivity to cisplatin treatment. The genetic pathway(s) that determine the chemotherapy sensitivity in GCT remain largely unknown. RESULTS: We studied epigenetic changes in relation to cisplatin response by examining promoter hypermethylation in a cohort of resistant and sensitive GCTs. Here, we show that promoter hypermethylation of RASSF1A and HIC1 genes is associated with resistance. The promoter hypermethylation and/or the down-regulated expression of MGMT is seen in the majority of tumors. We hypothesize that these epigenetic alterations affecting MGMT play a major role in the exquisite sensitivity to cisplatin, characteristic of GCTs. We also demonstrate that cisplatin treatment induce de novo promoter hypermethylation in vivo. In addition, we show that the acquired cisplatin resistance in vitro alters the expression of specific genes and the highly resistant cells fail to reactivate gene expression after treatment to demethylating and histone deacetylase inhibiting agents. CONCLUSIONS: Our findings suggest that promoter hypermethylation of RASSF1A and HIC1 genes play a role in resistance of GCT, while the transcriptional inactivation of MGMT by epigenetic alterations confer exquisite sensitivity to cisplatin. These results also implicate defects in epigenetic pathways that regulate gene transcription in cisplatin resistant GCT

Sheep Updates 2007 - part 5

This session covers six papers from different authors: GENETIC IMPROVEMENT 1. Breech Strike Resistance: Selecting for resistance traits reduces breech strike, Bindi Murray, John Karlsson, Johan Greeff, Department of Agriculture and Food, Western Australia 2. Breeding Merino Sheep for Worm Resistance increases profit in a Mediterranean Environment, John Karlsson and Johan Greeff Department of Agriculture and Food, Western Australia FEEDING 3. Embryo lasses were not increased when Merino ewes that had lost weight were supplemented with lupins, C. Viñoles Gil, B.L. Paganoni, K.M.M. Glover, J.T.B. Milton & G.B. Martin, School of Animal Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia, Crawley, WA 4. Mineral nutrition of sheep grazing dual-purpose wheats, Hugh Dove, CSIRO Plant Industry, Canberra, ACT BEEF PRODUCTIVITY 5. The effect of genetic potential and pre feedlot growth path on beef eating quality, Bill McKiernan and John WilkinsNSW Department of Primary Industries 6. Long-term consequences of growth and nutrition of cattle early in life for beef production, Paul Greenwood and Linda Cafe, Cooperative Research Centre for Beef Genetic Technologies, and NSW Department of Primary Industries Beef Industry Centre of Excellence, University of New England, Armidale NS

Bmf upregulation through the AMP-activated protein kinase pathway may protect the brain from seizure-induced cell death.

Prolonged seizures (status epilepticus, SE) can cause neuronal death within brain regions such as the hippocampus. This may contribute to impairments in cognitive functioning and trigger or exacerbate epilepsy. Seizure-induced neuronal death is mediated, at least in part, by apoptosis-associated signaling pathways. Indeed, mice lacking certain members of the potently proapoptotic BH3-only subfamily of Bcl-2 proteins are protected against hippocampal damage caused by status epilepticus. The recently identified BH3-only protein Bcl-2-modifying factor (Bmf) normally interacts with the cytoskeleton, but upon certain cellular stresses, such as loss of extracellular matrix adhesion or energy crisis, Bmf relocalizes to mitochondria, where it can promote Bax activation and mitochondrial dysfunction. Although Bmf has been widely reported in the hematopoietic system to exert a proapoptotic effect, no studies have been undertaken in models of neurological disorders. To examine whether Bmf is important for seizure-induced neuronal death, we studied Bmf induction after prolonged seizures induced by intra-amygdala kainic acid (KA) in mice, and examined the effect of Bmf-deficiency on seizures and damage caused by SE. Seizures triggered an early (1-8 h) transcriptional activation and accumulation of Bax in the cell death-susceptible hippocampal CA3 subfield. Bmf mRNA was biphasically upregulated beginning at 1 h after SE and returning to normal by 8 h, while again being found elevated in the hippocampus of epileptic mice. Bmf upregulation was prevented by Compound C, an inhibitor of adenosine monophosphate-activated protein kinase, indicating Bmf expression may be induced in response to bioenergetic stress. Bmf-deficient mice showed normal sensitivity to the convulsant effects of KA, but, surprisingly, displayed significantly more neuronal death in the hippocampal CA1 and CA3 subfields after SE. These are the first studies investigating Bmf in a model of neurologic injury, and suggest that Bmf may protect neurons against seizure-induced neuronal death in vivo