Enriched sera protein profiling for detection of non-small cell lung cancer biomarkers

Background

Non Small Cell Lung Cancer (NSCLC) is the major cause of cancer related-death. Many patients receive diagnosis at advanced stage leading to a poor prognosis. At present, no satisfactory screening tests are available in clinical practice and the discovery and validation of new biomarkers is mandatory. Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-ToF-MS) is a recent high-throughput technique used to detect new tumour markers. In this study we performed SELDI-ToF-MS analysis on serum samples treated with the ProteoMiner? kit, a combinatorial library of hexapeptide ligands coupled to beads, to reduce the wide dynamic range of protein concentration in the sample. Serum from 44 NSCLC patients and 19 healthy controls were analyzed with IMAC30-Cu and H50 ProteinChip Arrays.

Results

Comparing SELDI-ToF-MS protein profiles of NSCLC patients and healthy controls, 28 protein peaks were found significantly different (p < 0.05), and were used as predictors to build decision classification trees. This statistical analysis selected 10 protein peaks in the low-mass range (2-24 kDa) and 6 in the high-mass range (40-80 kDa). The classification models for the low-mass range had a sensitivity and specificity of 70.45% (31/44) and 68.42% (13/19) for IMAC30-Cu, and 72.73% (32/44) and 73.68% (14/19) for H50 ProteinChip Arrays.

Conclusions

These preliminary results suggest that SELDI-ToF-MS protein profiling of serum samples pretreated with ProteoMiner? can improve the discovery of protein peaks differentially expressed between NSCLC patients and healthy subjects, useful to build classification algorithms with high sensitivity and specificity. However, identification of the significantly different protein peaks needs further study in order to provide a better understanding of the biological nature of these potential biomarkers and their role in the underlying disease process.     

Facilitating multimodal preclinical imaging studies in mice by using an immobilization bed

We have designed an immobilization bed that accommodates mice of all ages and sizes, to improve image registration for multimodal scans and for longitudinal preclinical imaging studies. Stationary pegs were placed such that they effectively immobilized mice and reduced set-up time. (22)Na fiducial markers were placed into the pegs at unique depths to provide 3D references to facilitate image registration. Multiple users registered positron emission tomographic (PET) and CT data obtained with and without the bed to examine the effect of the bed on registration accuracy and interuser variability. The image registrations performed by different users were evaluated for their similarity by using the Entropy Correlation Coefficient as a metric. The immobilization bed significantly reduced variations in body movement and interuser variability. Average differences in quantification of tumor PET signal among users when registering images without versus with the fiduciary-marker bed fell from 9.1% to 0.8% for maximal percentage injected dose per gram (%ID/g), from 15.6% to 2.3% for mean %ID/g, and from 9.4% to 0.7% for the 90th percentile of the maximum %ID/g. The bed improves animal immobilization, greatly reduces interuser variability, and supports registration of image data acquired from different imaging sessions.     

Hydrocarbon phytoremediation in the family Fabaceae--a review

Currently, studies often focus on the use of Poaceae species (grasses) for phytoremediation of hydrocarbon-contaminated soils. Research into the use of Fabaceae species (legumes) to remediate hydrocarbons in soils has been conducted, but these plants are commonly overlooked due to slower recorded rates of degradation compared with many grass species. Evidence in the literature suggests that in some cases Fabaceae species may increase total degradation of hydrocarbons and stimulate degradative capacity of the soil microbial community, particularly for contaminants which are normally more recalcitrant to degradation. As many recalcitrant hydrocarbons have negative impacts on human and ecosystem health, development of remediation options is crucial. Reconsideration of Fabaceae species for removal of such contaminants may lead to environmentally and economically sustainable technologies for remediation of contaminated sites.     

Viral CTL escape mutants are generated in lymph nodes and subsequently become fixed in plasma and rectal mucosa during acute SIV infection of macaques

SIV(mac239) infection of rhesus macaques (RMs) results in AIDS despite the generation of a strong antiviral cytotoxic T lymphocyte (CTL) response, possibly due to the emergence of viral escape mutants that prevent recognition of infected cells by CTLs. To determine the anatomic origin of these SIV mutants, we longitudinally assessed the presence of CTL escape variants in two MamuA*01-restricted immunodominant epitopes (Tat-SL8 and Gag-CM9) in the plasma, PBMCs, lymph nodes (LN), and rectal biopsies (RB) of fifteen SIV(mac239)-infected RMs. As expected, Gag-CM9 did not exhibit signs of escape before day 84 post infection. In contrast, Tat-SL8 escape mutants were apparent in all tissues by day 14 post infection. Interestingly LNs and plasma exhibited the highest level of escape at day 14 and day 28 post infection, respectively, with the rate of escape in the RB remaining lower throughout the acute infection. The possibility that CTL escape occurs in LNs before RBs is confirmed by the observation that the specific mutants found at high frequency in LNs at day 14 post infection became dominant at day 28 post infection in plasma, PBMC, and RB. Finally, the frequency of escape mutants in plasma at day 28 post infection correlated strongly with the level Tat-SL8-specific CD8 T cells in the LN and PBMC at day 14 post infection. These results indicate that LNs represent the primary source of CTL escape mutants during the acute phase of SIV(mac239) infection, suggesting that LNs are the main anatomic sites of virus replication and/or the tissues in which CTL pressure is most effective in selecting SIV escape variants.     

A high-throughput screen for directed evolution of the natural product sulfotransferase LipB

In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia coli strain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natural desulfo-liposidomycin acceptor. The authors plan to use the screen to identify LipB variants with altered acceptor specificity and promiscuity for use in sulfation of natural products and other small-molecule therapeutics.     

Development and initial characterization of a HAPPY panel for mapping the X. tropicalis genome

HAPPY mapping was designed to pursue the analysis of approximately random HAPloid DNA breakage samples using the PolYmerase chain reaction for mapping genomes. In the present study, we improved the method and integrated two other molecular techniques into the process: whole genome amplification and the Sequenom SNP (single nucleotide polymorphism) genotyping assay in order to facilitate whole genome mapping of X. tropicalis. The former technique amplified enough DNA materials to genotype a large number of markers, while the latter allowed for relatively high throughput marker genotyping with multiplex assays on the HAPPY lines. A total of 58 X. tropicalis genes were genotyped on an initial panel of 383 HAPPY lines, which contributed to formation of a working panel of 146 lines. Further genotyping of 29 markers on the working panel led to construction of a HAPPY map for the X. tropicalis genome. We believe that our improved HAPPY method described in the present study has paved the way for the community to map different genomes with a simple, but powerful approach.     

Tactical release of a sexually-selected pheromone in a swordtail fish

Background

Chemical communication plays a critical role in sexual selection and speciation in fishes; however, it is generally assumed that most fish pheromones are passively released since most fishes lack specialized scent glands or scent-marking behavior. Swordtails (genus Xiphophorus) are widely used in studies of female mate choice, and female response to male chemical cues is important to sexual selection, reproductive isolation, and hybridization. However, it is unclear whether females are attending to passively produced cues, or to pheromones produced in the context of communication.

Methodology/Principal Findings

We used fluorescein dye injections to visualize pulsed urine release in male sheepshead swordtails, Xiphophorus birchmanni. Simultaneous-choice assays of mating preference showed that females attend to species- and sex-specific chemical cues emitted in male urine. Males urinated more frequently in the presence and proximity of an audience (conspecific females). In the wild, males preferentially courted upstream of females, facilitating transmission of pheromone cues.

Conclusions/Significance

Males in a teleost fish have evolved sophisticated temporal and spatial control of pheromone release, comparable to that found in terrestrial animals. Pheromones are released specifically in a communicative context, and the timing and positioning of release favors efficient signal transmission.     

Ancient antimicrobial peptides kill antibiotic-resistant pathogens: Australian mammals provide new options

Background

To overcome the increasing resistance of pathogens to existing antibiotics the 10×^''20 Initiative declared the urgent need for a global commitment to develop 10 new antimicrobial drugs by the year 2020. Naturally occurring animal antibiotics are an obvious place to start. The recently sequenced genomes of mammals that are divergent from human and mouse, including the tammar wallaby and the platypus, provide an opportunity to discover novel antimicrobials. Marsupials and monotremes are ideal potential sources of new antimicrobials because they give birth to underdeveloped immunologically naïve young that develop outside the sterile confines of a uterus in harsh pathogen-laden environments. While their adaptive immune system develops innate immune factors produced either by the mother or by the young must play a key role in protecting the immune-compromised young. In this study we focus on the cathelicidins, a key family of antimicrobial peptide genes.

Principal Finding

We identified 14 cathelicidin genes in the tammar wallaby genome and 8 in the platypus genome. The tammar genes were expressed in the mammary gland during early lactation before the adaptive immune system of the young develops, as well as in the skin of the pouch young. Both platypus and tammar peptides were effective in killing a broad range of bacterial pathogens. One potent peptide, expressed in the early stages of tammar lactation, effectively killed multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii.

Conclusions and Significance

Marsupial and monotreme young are protected by antimicrobial peptides that are potent, broad spectrum and salt resistant. The genomes of our distant relatives may hold the key for the development of novel drugs to combat multidrug-resistant pathogens.