RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces

Background  

Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping.     

Experimental Study of Low Dose Ultrashortwave Promoting Nerve Regeneration after Acellular Nerve Allografts Repairing the Sciatic Nerve Gap of Rats

Objectives To observe the effect of ultrashortwave (USW) therapy on nerve regeneration after acellular nerve allografts(ANA) repairing the sciatic nerve gap of rats and discuss its acting mechanisms. Methods Sixteen Wistar rats weighing 180–220 g were randomly divided into four groups with four rats in each group: normal control group; acellular group (ANA, treated by hypotonic-chemical detergent, was applied for bridging a 10 mm-long sciatic nerve defect); USW group (After 24 h of ANA repairing the sciatic nerve gap, low dose USW was administrated for 7 min, once a day, 20 times a course of treatment, three courses of treatment in all); and autografts group. 12 weeks after operation, a series of examinations was performed, including electrophysiological methods, the restoring rate of tibialis anterior muscle wet weight, histopathological observation (myelinated nerve number, myelin sheath thickness, and axon diameter), vascular endothelial growth factor (VEGF) mRNA expression of spinal cord, and muscle at injury site, and analyzed statistically. Results Compared to acellular nerve allografts alone, USW therapy can increase nerve conductive velocity, the restoring rate of tibialis anterior muscle wet weight, myelinated nerve number, axon diameter, VEGF mRNA expression of spinal cord, and muscle at injury site, the difference is significant. There were no differences between USW group and autografts group except myelin sheath thickness. Conclusions USW therapy can promote nerve axon regeneration and Schwann cells proliferation after ANA repairing the sciatic nerve gap of rats, the upregulation of VEGF mRNA expression of spinal cord and muscle may play an important role.     

Amino acid variations resulting in functional and nonfunctional zebrafish P2X<Subscript>1</Subscript> and P2X<Subscript>5.1</Subscript> receptors

Several zebrafish P2X receptors (zP2X(1), zP2X(2), and zP2X(5.1)) have been reported to produce little or no current although their mammalian orthologs produce functional homomeric receptors. We isolated new cDNA clones for these P2X receptors that revealed sequence variations in each. The new variants of zP2X(1) and zP2X(5.1) produced substantial currents when expressed by Xenopus oocytes, however the new variant of zP2X(2) was still nonfunctional. zP2X(2) lacks two lysine residues essential for ATP responsiveness in other P2X receptors; however introduction of these two lysines was insufficient to allow this receptor to function as a homotrimer. We also tested whether P2X signaling is required for myogenesis or synaptic communication at the zebrafish neuromuscular junction. We found that embryonic skeletal muscle expressed only one P2X receptor, P2X(5.1). Antisense knockdown of P2X(5.1) eliminated skeletal muscle responsiveness to ATP but did not prevent myogenesis or behaviors that require functional transmission at the neuromuscular junction.     

Compositional reasoning of performance in component-based distributed systems

Rapid prototyping of distributed systems can be achieved by integrating commercial off-the-shelf (COTS) components. With components as the building blocks, it is important to predict the performance of the system based on the performance of individual components. In this paper, performance prediction of a system consisting of a small number of components is investigated under different inter-component communication patterns, and the number of threads provided by components. Based on the experimental results, it can be inferred that the proposed composition rules provide a reasonably accurate prediction of the performance of a system made out of these components.

Redox and temperature-sensitive changes in microbial communities and soil chemistry dictate greenhouse gas loss from thawed permafrost

Shelf seas and their associated benthic habitats represent key systems in the global carbon cycle. However, the quantification of the related stocks and flows of carbon are often poorly constrained. To address benthic carbon storage in the North–West European continental shelf, we have spatially predicted the mass of particulate organic carbon (POC) stored in the top 10 cm of shelf sediments in parts of the North Sea, English Channel and Celtic Sea using a Random Forest model, POC measurements on surface sediments from those seas and relevant predictor variables. The presented model explains 78% of the variance in the data and we estimate that approximately 250 Mt of POC are stored in surficial sediments of the study area (633,000 km²). Upscaling to the North–West European continental shelf area (1,111,812 km²) yielded a range of 230–882 Mt of POC with the most likely estimate being on the order of 476 Mt. We demonstrate that the largest POC stocks are associated with coarse-grained sediments due to their wide-spread occurrence and high dry bulk densities. Our results also highlight the importance of coastal sediments for carbon storage and sequestration. Important predictors for POC include mud content in surficial sediments, annual average bottom temperature and distance to shoreline, with the latter possibly a proxy for terrestrial inputs. Now that key variables in determining the spatial distribution of POC have been identified, it is possible to predict future changes to the POC stock, with the presented maps providing an accurate baseline against which to assess predicted changes.     

The essential requirement of an animal heme peroxidase protein during the wing maturation process in Drosophila

Background

Thus far, a handful of genes have been shown to be related to the wing maturation process in insects. A novel heme peroxidase enzyme known as curly suppressor (Cysu)(formerly CG5873), have been characterized in this report because it is involved in wing morphogenesis. Using bioinformatics tools we found that Cysu is remarkably conserved in the genus Drosophila (>95%) as well as in invertebrates (>70%), although its vertebrate orthologs show poor homology. Time-lapse imaging and histochemical analyses have confirmed that the defective wing phenotype of Cysu is not a result of any underlying cellular alterations; instead, its wings fail to expand in mature adults.

Results

The precise requirement of Cysu in wings was established by identifying a bona fide mutant of Cysu from the Bloomington Drosophila Stock Centre collection. Its requirement in the wing has also been shown by RNA knockdown of the gene. Subsequent transgenic rescue of the mutant wing phenotype with the wild-type gene confirmed the phenotype resulting from Cysu mutant. With appropriate GAL4 driver like engrailed-GAL4, the Cysu phenotype was compartmentalized, which raises a strong possibility that Cysu is not localized in the extracellular matrix (ECM); hence, Cysu is not engaged in bonding the dorsal and ventral cuticular layers. Finally, shortened lifespan of the Cysu mutant suggests it is functionally essential for other biological processes as well.

Conclusion

Cysu, a peroxinectin-like gene, is required during the wing maturation process in Drosophila because as a heme peroxidase, Cysu is capable of utilizing H₂O₂, which plays an essential role in post-eclosion wing morphogenesis.

     

Biotic and Abiotic Drivers of Peatland Growth and Microtopography: A Model Demonstration

Peatlands are important carbon reserves in terrestrial ecosystems. The microtopography of a peatland area has a strong influence on its carbon balance, determining carbon fluxes at a range of spatial scales. These patterned surfaces are very sensitive to changing climatic conditions. There are open research questions concerning the stability, behaviour and transformation of these microstructures, and the implications of these changes for the long-term accumulation of organic matter in peatlands. A simple two-dimensional peat microtopographical model was developed, which accounts for the effects of microtopographical variations and a dynamic water table on competitive interactions between peat-forming plants. In a case study of a subarctic mire in northern Sweden, we examined the consequences of such interactions on peat accumulation patterns and the transformation of microtopographical structure. The simulations demonstrate plausible interactions between peatland growth, water table position and microtopography, consistent with many observational studies, including an observed peat age profile from the study area. Our model also suggests that peatlands could exhibit alternative compositional and structural dynamics depending on the initial topographical and climatic conditions, and plant characteristics. Our model approach represents a step towards improved representation of peatland vegetation dynamics and net carbon balance in Earth system models, allowing their potentially important implications for regional and global carbon balances and biogeochemical and biophysical feedbacks to the atmosphere to be explored and quantified.     

Variability in the settlement of non-indigenous species in benthic communities from an oceanic island

The introduction of non-indigenous species (NIS) in new environments represents a major threat for coastal ecosystems. A good understanding of the mechanisms and magnitude of the impact of NIS colonisation on native ecosystems is becoming increasingly crucial to develop mitigation measures and prevent new invasions. In this present study, we asked if distinct coastal benthic communities from an oceanic island can have different vulnerability to NIS colonisation process. First, PVC settlement plates were deployed for 1 year on the rocky shore of two different locations of Madeira Island (North versus South coast). Then, we implemented a mesocosm experiment where recruited plate communities were maintained under different levels of NIS propagule pressure in order to assess their vulnerability to NIS colonisation process. Results showed that NIS colonisation success was not influenced by the level of propagule pressure, but however, final colonisation patterns varied depending on the origin of the communities. This variability can be attributed to major structural differences between the preponderant species of each community and therefore to the biotic substrate they offer to colonisers. This study highlights how biotic features can alter the NIS colonisation process and importantly, shows that in an urbanisation context, the nature of the resident communities facing invasions risks needs to be closely assessed.