Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Beyond blue gene: Intellectual property and bioinformatics

This article considers the challenges posed to intellectual property law by the emerging field of bioinformatics. It examines the intellectual property strategies of established biotechnology companies, such as Celera Genomics, and information technology firms entering into the marketplace, such as IBM. First this paper argues that copyright law is not irrelevant to biotechnology, as some commentators would suggest. It claims that the use of copyright law and contract law is fundamental to the protection of biomedical and genomic databases. Second this article questions whether biotechnology companies are exclusively interested in patenting genes and genetics sequences. Recent evidence suggests that biotechnology companies and IT firms are patenting bioinformatics software and Internet business methods, as well as underlying instrumentation such as microarrays and genechips. Finally, this paper evaluates what impact the privatisation of bioinformatics will have on public research and scientific communication. It raises important questions about integration, interoperability, and the risks of monopoly. It finally considers whether open source software such as the Ensembl Project and peer to peer technology like DSAS will be able to counter this trend of privatisation.

Meeting the challenges of neuroimaging genetics

As research encompassing neuroimaging and genetics gains momentum, extraordinary information will be uncovered on the genetic architecture of the human brain. However, there are significant challenges to be addressed first. Not the least of these challenges is to accomplish the sample size necessary to detect subtle genetic influences on the morphometry and function of the healthy brain. Aside from sample size, image acquisition and analysis methods need to be refined in order to ensure optimum sensitivity to genetic and complementary environmental influences. Then there is the vexing issue of interpreting the resulting data. We describe how researchers from the east coast of Australia and the west coast of America have embarked upon a collaboration to meet these challenges using data currently being collected from a large-scale twin study, and offer some opinions about future directions in the field.

Circulating tumour cells in metastatic head and neck cancers

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer with 650,000 new cases p/a worldwide. HNSCC causes high morbidity with a 5-year survival rate of less than 60%, which has not improved due to the lack of early detection (Bozec et al. Eur Arch Otorhinolaryngol. 2013;270: 2745–9). Metastatic disease remains one of the leading causes of death in HNSCC patients. This review article provides a comprehensive overview of literature over the past 5 years on the detection of circulating tumour cells (CTCs) in HNSCC; CTC biology and future perspectives. CTCs are a hallmark of invasive cancer cells and key to metastasis. CTCs can be used as surrogate markers of overall survival and progression-free survival. CTCs are currently used as prognostic factors for breast, prostate and colorectal cancers using the CellSearch® system. CTCs have been detected in HNSCC, however, these numbers depend on the technique applied, time of blood collection and the clinical stage of the patient. The impact of CTCs in HNSCC is not well understood, and thus, not in routine clinical practice. Validated detection technologies that are able to capture CTCs undergoing epithelial–mesenchymal transition are needed. This will aid in the capture of heterogeneous CTCs, which can be compiled as new targets for the current food and drug administration-cleared CellSearch® system. Recent studies on CTCs in HNSCC with the CellSearch® have shown variable data. Therefore, there is an immediate need for large clinical trials encompassing a suite of biomarkers capturing CTCs in HNSCC, before CTCs can be used as prognostic markers in HNSCC patient management.

The Arctic Summer Cloud Ocean Study (ASCOS): overview and experimental design

The climate in the Arctic is changing faster than anywhere else on earth. Poorly understood feedback processes relating to Arctic clouds and aerosol–cloud interactions contribute to a poor understanding of the present changes in the Arctic climate system, and also to a large spread in projections of future climate in the Arctic. The problem is exacerbated by the paucity of research-quality observations in the central Arctic. Improved formulations in climate models require such observations, which can only come from measurements in situ in this difficult-to-reach region with logistically demanding environmental conditions. The Arctic Summer Cloud Ocean Study (ASCOS) was the most extensive central Arctic Ocean expedition with an atmospheric focus during the International Polar Year (IPY) 2007–2008. ASCOS focused on the study of the formation and life cycle of low-level Arctic clouds. ASCOS departed from Longyearbyen on Svalbard on 2 August and returned on 9 September 2008. In transit into and out of the pack ice, four short research stations were undertaken in the Fram Strait: two in open water and two in the marginal ice zone. After traversing the pack ice northward, an ice camp was set up on 12 August at 87°21' N, 01°29' W and remained in operation through 1 September, drifting with the ice. During this time, extensive measurements were taken of atmospheric gas and particle chemistry and physics, mesoscale and boundary-layer meteorology, marine biology and chemistry, and upper ocean physics. ASCOS provides a unique interdisciplinary data set for development and testing of new hypotheses on cloud processes, their interactions with the sea ice and ocean and associated physical, chemical, and biological processes and interactions. For example, the first-ever quantitative observation of bubbles in Arctic leads, combined with the unique discovery of marine organic material, polymer gels with an origin in the ocean, inside cloud droplets suggests the possibility of primary marine organically derived cloud condensation nuclei in Arctic stratocumulus clouds. Direct observations of surface fluxes of aerosols could, however, not explain observed variability in aerosol concentrations, and the balance between local and remote aerosols sources remains open. Lack of cloud condensation nuclei (CCN) was at times a controlling factor in low-level cloud formation, and hence for the impact of clouds on the surface energy budget. ASCOS provided detailed measurements of the surface energy balance from late summer melt into the initial autumn freeze-up, and documented the effects of clouds and storms on the surface energy balance during this transition. In addition to such process-level studies, the unique, independent ASCOS data set can and is being used for validation of satellite retrievals, operational models, and reanalysis data sets.

Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion, −OCl) generated by the myeloperoxidase (MPO)–H2O2–Cl− system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC50 values of ~1 and ~6 µM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC50 values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed.

A model to explain specific cellular communications and cellular harmony: - a hypothesis of coupled cells and interactive coupling molecules

Background The various cell types and their relative numbers in multicellular organisms are controlled by growth factors and related extracellular molecules which affect genetic expression pathways. However, these substances may have both/either inhibitory and/or stimulatory effects on cell division and cell differentiation depending on the cellular environment. It is not known how cells respond to these substances in such an ambiguous way. Many cellular effects have been investigated and reported using cell culture from cancer cell lines in an effort to define normal cellular behaviour using these abnormal cells. A model is offered to explain the harmony of cellular life in multicellular organisms involving interacting extracellular substances. Methods A basic model was proposed based on asymmetric cell division and evidence to support the hypothetical model was accumulated from the literature. In particular, relevant evidence was selected for the Insulin-Like Growth Factor system from the published data, especially from certain cell lines, to support the model. The evidence has been selective in an attempt to provide a picture of normal cellular responses, derived from the cell lines. Results The formation of a pair of coupled cells by asymmetric cell division is an integral part of the model as is the interaction of couplet molecules derived from these cells. Each couplet cell will have a receptor to measure the amount of the couplet molecule produced by the other cell; each cell will be receptor-positive or receptor-negative for the respective receptors. The couplet molecules will form a binary complex whose level is also measured by the cell. The hypothesis is heavily supported by selective collection of circumstantial evidence and by some direct evidence. The basic model can be expanded to other cellular interactions. Conclusions These couplet cells and interacting couplet molecules can be viewed as a mechanism that provides a controlled and balanced division-of-labour between the two progeny cells, and, in turn, their progeny. The presence or absence of a particular receptor for a couplet molecule will define a cell type and the presence or absence of many such receptors will define the cell types of the progeny within cell lineages.

Incidence and mortality of female breast cancer in the Asia-Pacific region

Objective: To provide an overview of the incidence and mortality of female breast cancer for countries in the Asia-Pacific region. Methods: Statistical information about breast cancer was obtained from publicly available cancer registry and mortality databases (such as GLOBOCAN), and supplemented with data requested from individual cancer registries. Rates were directly age-standardised to the Segi World Standard population and trends were analysed using joinpoint models. Results: Breast cancer was the most common type of cancer among females in the region, accounting for 18% of all cases in 2012, and was the fourth most common cause of cancer-related deaths (9%). Although incidence rates remain much higher in New Zealand and Australia, rapid rises in recent years were observed in several Asian countries. Large increases in breast cancer mortality rates also occurred in many areas, particularly Malaysia and Thailand, in contrast to stabilising trends in Hong Kong and Singapore, while decreases have been recorded in Australia and New Zealand. Mortality trends tended to be more favourable for women aged under 50 compared to those who were 50 years or older. Conclusion: It is anticipated that incidence rates of breast cancer in developing countries throughout the Asia-Pacific region will continue to increase. Early detection and access to optimal treatment are the keys to reducing breast cancer-related mortality, but cultural and economic obstacles persist. Consequently, the challenge is to customise breast cancer control initiatives to the particular needs of each country to ensure the best possible outcomes.

Chlamydial infections of fish: Diverse pathogens and emerging causes of disease in aquaculture species

Chlamydial infections of fish are emerging as an important cause of disease in new and established aquaculture industries. To date, epitheliocystis, a skin and gill disease associated with infection by these obligate intracellular pathogens, has been described in over 90 fish species, including hosts from marine and fresh water environments. Aided by advances in molecular detection and typing, recent years have seen an explosion in the description of these epitheliocystis-related chlamydial pathogens of fish, significantly broadening our knowledge of the genetic diversity of the order Chlamydiales. Remarkably, in most cases, it seems that each new piscine host studied has revealed the presence of a phylogenetically unique and novel chlamydial pathogen, providing researchers with a fascinating opportunity to understand the origin, evolution and adaptation of their traditional terrestrial chlamydial relatives. Despite the advances in this area, much still needs to be learnt about the epidemiology of chlamydial infections in fish if these pathogens are to be controlled in farmed environments. The lack of in vitro methods for culturing of chlamydial pathogens of fish is a major hindrance to this field. This review provides an update on our current knowledge of the taxonomy and diversity of chlamydial pathogens of fish, discusses the impact of these infections on the health, and highlights further areas of research required to understand the biology and epidemiology of this important emerging group of fish pathogens of aquaculture species.

Behavior of simetryn and thiobencarb in rice paddy lysimeters and the effect of excess water storage depth in controlling herbicide run-off

Eight small-scale lysimeters with different excess water storage depths (EWSDs) were used to investigate the behavior of two herbicides, simetryn and thiobencarb, under paddy conditions. The concentration of simetryn dissipated similarly in all the lysimeters, while the thiobencarb concentration varied significantly because thiobencarb can adsorb onto the dissolved organic matter in a manure slurry, which was applied to six of the lysimeters. The herbicide losses (the percentage of the applied mass) from the lysimeters were reversely proportional with the EWSD. The correlation was stronger for simetryn than for thiobencarb. An appropriate EWSD is required to effectively prevent herbicide run-off from the paddy field, especially when a rainfall event occurs soon after herbicide application.

Impacts of tillage and application methods on atrazine and alachlor losses from upland fields: Research paper

The effects of tillage practises and the methods of chemical application on atrazine and alachlor losses through run-off were evaluated for five treatments: conservation (untilled) and surface (US), disk and surface, plow and surface, disk and preplant-incorporated, and plow and preplant-incorporated treatments. A rainfall simulator was used to create 63.5 mm h^-1 of rainfall for 60 min and 127 mm h^-1 for 15 min. Rainfall simulation occurred 24-36 h after chemical application. There was no significant difference in the run-off volume among the treatments but the untilled treatment significantly reduced erosion loss. The untilled treatments had the highest herbicide concentration and the disk treatments were higher than the plow treatments. The surface treatments showed a higher concentration than the incorporated treatments. The concentration of herbicides in the water decreased with time. Among the experimental sites, the one with sandy loam soil produced the greatest losses, both in terms of the run-off volume and herbicide loss. The US treatments had the highest loss and the herbicide incorporation treatments had smaller losses through run-off as the residue cover was effective in preventing herbicide losses. Incorporation might be a favorable method of herbicide application to reduce the herbicide losses by run-off.

Genome-wide association studies

Genome-wide association studies (GWAS) are a powerful hypothesis-free tool for the dissection of susceptibility to common heritable human diseases, including osteoporosis. To date, more than 2000 loci for common human diseases have been identified by GWAS. Success using the GWAS model depends on genetic risk being determined by shared stretches of DNA carried with different frequencies in cases and controls, inherited from ancient ancestors, termed the “common disease–common variant” hypothesis. Not all disease risk is caused by common variants, however, and thus GWAS will not detect all variants involved. Successful GWAS performance requires careful quality control, especially as the effect sizes under study are modest, and there are multiple potential sources of error. Conservative interpretation, use of stringent significance thresholds, and replication in independent cohorts are required to ensure results are robust. Despite these challenging parameters, much has been learnt from GWAS and, as the approach matures and is modified to identify a wider range of variants, significantly more will be learnt about the etiopathogenesis of common diseases such as osteoporosis.

Developments in psoriasis and psoriatic arthritis

Psoriasis and psoriatic arthritis are common conditions for which treatment options have until recently been extremely limited. Recent advances in our understanding of the immunology and genetics underlying these conditions have been rapid, and have contributed to the development of new therapies for these diseases. This article discusses the current state of the art in our understanding of the aetiopathogenesis of psoriasis and psoriatic arthritis, and current therapies for the diseases.

Comparative leaf anatomy and micromorphology of the Chilean Myrtaceae: Taxonomic and ecological implications

The family Myrtaceae in Chile comprises 26 species in 10 genera. The species occur in a diverse rangeof environments including humid temperate forests, swamps, riparian habitats and coastal xeromorphicshrublands. Most of these species are either endemic to Chile or endemic to the humid temperate forestsof Chile and Argentina. Although many taxa have very restricted distributions and are of conservationconcern, little is known about their biology and vegetative anatomy. In this investigation, we describe andcompare the leaf anatomy and micromorphology of all Chilean Myrtaceae using standard protocols forlight and scanning electron microscopy. Leaf characters described here are related to epidermis, cuticle,papillae, stomata, hairs, mesophyll, crystals, secretory cavities and vascular system. Nearly all the specieshave a typical mesophytic leaf anatomy, but some species possess xerophytic characters such as doubleepidermis, hypodermis, pubescent leaves, thick adaxial epidermis and straight epidermal anticlinal walls,which correlate with the ecological distribution of the species. This is the first report on leaf anatomyand micromorphology in most of these species. We identified several leaf characters with potential tax-onomic and ecological significance. Some combinations of leaf characters can reliably delimitate genera,while others are unique to some species. An identification key using micromorphological and anatomicalcharacters is provided to distinguish genera and species.

Specific rolling circle amplification of low-copy human polyomaviruses BKV, HPyV6, HPyV7, TSPyV, and STLPyV

Eleven new human polyomaviruses have been recently discovered, yet for most of these viruses, little is known of their biology and clinical impact. Rolling circle amplification (RCA) is an ideal method for the amplification of the circular polyomavirus genome due to its high fidelity amplification of circular DNA. In this study, a modified RCA method was developed to selectively amplify a range of polyomavirus genomes. Initial evaluation showed a multiplexed temperature-graded reaction profile gave the best yield and sensitivity in amplifying BK polyomavirus in a background of human DNA, with up to 1 × 10(8)-fold increases in viral genomes from as little as 10 genome copies per reaction. Furthermore, the method proved to be more sensitive and provided a 200-fold greater yield than that of random hexamers based standard RCA. Application of the method to other novel human polyomaviruses showed successful amplification of TSPyV, HPyV6, HPyV7, and STLPyV from low-viral load positive clinical samples, with viral genome enrichment ranging from 1 × 10(8) up to 1 × 10(10). This directed RCA method can be applied to selectively amplify other low-copy polyomaviral genomes from a background of competing non-specific DNA, and is a useful tool in further research into the rapidly expanding Polyomaviridae family.