Differential analysis of N-glycoproteome between hepatocellular carcinoma and normal human liver tissues by combination of multiple protease digestion and solid phase based labeling

Background

Dysregulation of glycoproteins is closely related with many diseases. Quantitative proteomics methods are powerful tools for the detection of glycoprotein alterations. However, in almost all quantitative glycoproteomics studies, trypsin is used as the only protease to digest proteins. This conventional method is unable to quantify N-glycosites in very short or long tryptic peptides and so comprehensive glycoproteomics analysis cannot be achieved.

Methods

In this study, a comprehensive analysis of the difference of N-glycoproteome between hepatocellular carcinoma (HCC) and normal human liver tissues was performed by an integrated workflow combining the multiple protease digestion and solid phase based labeling. The quantified N-glycoproteins were analyzed by GoMiner to obtain a comparative view of cellular component, biological process and molecular function.

Results/conclusions

An integrated workflow was developed which enabled the processes of glycoprotein coupling, protease digestion and stable isotope labeling to be performed in one reaction vessel. This workflow was firstly evaluated by analyzing two aliquots of the same protein extract from normal human liver tissue. It was demonstrated that the multiple protease digestion improved the glycoproteome coverage and the quantification accuracy. This workflow was further applied to the differential analysis of N-glycoproteome of normal human liver tissue and that with hepatocellular carcinoma. A total of 2,329 N-glycosites on 1,052 N-glycoproteins were quantified. Among them, 858 N-glycosites were quantified from more than one digestion strategy with over 99% confidence and 1,104 N-glycosites were quantified from only one digestion strategy with over 95% confidence. By comparing the GoMiner results of the N-glycoproteins with and without significant changes, the percentage of membrane and secreted proteins and their featured biological processes were found to be significant different revealing that protein glycosylation may play the vital role in the development of HCC.     

A systematic assessment of normalization approaches for the Infinium 450K methylation platform

The Illumina Infinium HumanMethylation450 BeadChip has emerged as one of the most popular platforms for genome wide profiling of DNA methylation. While the technology is wide-spread, systematic technical biases are believed to be present in the data. For example, this array incorporates two different chemical assays, i.e., Type I and Type II probes, which exhibit different technical characteristics and potentially complicate the computational and statistical analysis. Several normalization methods have been introduced recently to adjust for possible biases. However, there is considerable debate within the field on which normalization procedure should be used and indeed whether normalization is even necessary. Yet despite the importance of the question, there has been little comprehensive comparison of normalization methods. We sought to systematically compare several popular normalization approaches using the Norwegian Mother and Child Cohort Study (MoBa) methylation data set and the technical replicates analyzed with it as a case study. We assessed both the reproducibility between technical replicates following normalization and the effect of normalization on association analysis. Results indicate that the raw data are already highly reproducible, some normalization approaches can slightly improve reproducibility, but other normalization approaches may introduce more variability into the data. Results also suggest that differences in association analysis after applying different normalizations are not large when the signal is strong, but when the signal is more modest, different normalizations can yield very different numbers of findings that meet a weaker statistical significance threshold. Overall, our work provides useful, objective assessment of the effectiveness of key normalization methods.     

Predicting therapy response in live tumor cells isolated with the flexible micro spring array device

Cells disseminated from primary epithelial tumors into peripheral blood, called circulating tumor cells (CTCs), can be monitored to assess metastases and to provide a surrogate marker of treatment response. Here, we demonstrate how the flexible micro spring array (FMSA) device—a novel microfluidic device that enriches CTCs by two physical parameters: size and deformability—could be used in the rational development of treatment intervention and as a method to study the fundamental biology of CTCs. Cancer cells of different origins were spiked into healthy samples of donor blood to mimic blood samples of metastatic cancer patients. This spiked human blood was filtered using the FMSA device, and the recovered cells were successfully expanded in vitro and in a novel in vivo system. A series of experiments were performed to characterize these cells and to investigate the effect of chemotherapy on the resulting cultures. As few as 20 colon cancer cells in 7.5 mL blood could be isolated with the FMSA device, expanded both in vitro and in vivo and used at 25 cells per well to obtain significant and reliable chemosensitivity data. We also show that isolating a low number of viable patient CTCs and maintaining them in culture for a few weeks is possible. The isolation of viable cancer cells from human blood using the FMSA device provides a novel and realistic means for studying the biology of viable CTCs and for testing drug efficacy on these rare cells—a hypothesis that can be tested in future clinical trials.     

Architecture and functional groups of biofilms during composting with and without inoculation

Detailed knowledge of the architecture and molecular structure of biofilms during composting is important for understanding the underlying mechanisms of rapid composting with inoculation. In this study, multiple fluorescent labeling and two-dimensional Fourier transform infrared (FTIR) correlation spectroscopy were used to characterize biofilms during composting with and without inoculation with a cellulose-degrading strain, Aspergillus fumigatus F12. The results showed that inoculation with A. fumigatus F12 allowed the compost to rapidly reach the thermophilic phase. Further investigation demonstrated that the role of A. fumigatus F12 during composting was to destroy the network-like structure of cellulose and to increase the contact of other biopolymers with microorganisms, as observed in situ using multiple fluorescence labeling combined with confocal laser scanning microscopy (CLSM). Two-dimensional FTIR correlation spectroscopy supported the conclusion that the critical role of inoculation in composting was attributed to the degradation of cellulose (1420 cm^?1) prior to other biopolymers. In summary, multiple fluorescent labeling and two-dimensional FTIR correlation spectroscopy can be used as a novel tool for characterizing biofilms and the critical roles of microorganisms in composting.     

Parkinson's Disease: From Genetics to Clinical Practice

Breakthroughs in genetics over the last decade have radically advanced our understanding of the etiological basis of Parkinson''s disease (PD). Although much research remains to be done, the main genetic causes of this neurodegenerative disorder are now partially unraveled, allowing us to feel more confident that our knowledge about the genetic architecture of PD will continue to increase exponentially. How and when these discoveries will be introduced into general clinical practice, however, remains uncertain. In this review, we provide a general summary of the progress in the genetics of PD and discuss how this knowledge will contribute to the diagnosis and clinical management of patients with, or at risk of this disorder.     

Don’t Throw it Away!

This historical narrative tracks the evolution and devolution of visual arts education from Dewey's progressive era pedagogy and the theory of the arts as experience through the modern accountability movement. Archival material, state curricular documents, and conversations with policymakers show an increasing focus on core subject areas of reading, writing, and mathematics at the expense of arts education. Texas House Bill 3, the third generation of accountability legislation in the Lone Star State, provides a case study of the status of arts education after more than fifteen years of high-stakes testing and accountability. Policy considerations are offered for arts education and its future standing within the public educational curriculum.     

Comparison of computational modelling techniques for braided stent analysis

Abstract

Braided stents are associated with a number of complications in vivo. Accurate computational modelling of these devices is essential for the design and development of the next generation of these stents. In this study, two commonly utilised methods of computationally modelling filament interaction in braided stents are investigated: the join method and the weave method. Three different braided stent designs are experimentally tested and computationally modelled in both radial and v-block configurations. The results of the study indicate that while both methods are capable of capturing braided stent performance to some degree, the weave method is much more robust.     

Novel optical system for in vitro quantification of full surface strain fields in small arteries: II. Correction for refraction and illustrative results

In a companion paper, we described a theoretical foundation for and initial experimental implementation of a novel stereo-digital image correlation (DIC) method for quantifying the size, shape and deformation of small cylindrical specimens along their full length and around their entire circumference. In this paper, we further show that this panoramic-DIC method can be used to study mouse carotid arteries without affecting their native mechanical properties and show the advantage of the approach in studying more complex mouse aorta. In particular, we first resolve the ubiquitous issue of refraction in non-contacting optical measurements of strain while tissues are immersed in physiologic saline and we show that surface preparation for DIC does not affect the inferred mechanical properties either qualitatively or quantitatively, the latter via the use of a four-fibre family hyperelastic constitutive relation and associated estimation of material parameters using nonlinear regression. We thus submit that panoramic-DIC-based strain measurement has significant potential to increase our understanding of arterial mechanics in genetic models of arterial health and disease by allowing investigators to exploit advances in transgenic mice.     

Rate of progression of CT-quantified emphysema in male current and ex-smokers: a follow-up study

Background

Little is known about the factors associated with CT-quantified emphysema progression in heavy smokers. The objective of this study was to investigate the effect of length of smoking cessation and clinical / demographical factors on the rate of emphysema progression and FEV₁-decline in male heavy smokers.

Methods

3,670 male smokers with mean (SD) 40.8 (17.9) packyears underwent chest CT scans and pulmonary function tests at baseline and after 1 and 3 years follow-up. Smoking status (quitted ≥5, ≥1-<5, <1 years or current smoker) was noted. Rate of progression of emphysema and FEV₁-decline after follow-up were assessed by analysis of variance adjusting for age, height, baseline pulmonary function and emphysema severity, packyears, years in study and respiratory symptoms. The quitted ≥5 group was used as reference.

Results

Median (Q1-Q3) emphysema severity,<-950 HU, was 8.8 (5.1 – 14.1) and mean (SD) FEV₁ was 3.4 (0.73) L or 98.5 (18.5) % of predicted. The group quitted ‘>5 years’ showed significantly lower rates of progression of emphysema compared to current smokers, 1.07% and 1.12% per year, respectively (p<0.001). Current smokers had a yearly FEV₁-decline of 69 ml, while subjects quit smoking >5 years had a yearly decline of 57.5 ml (p<0.001).

Conclusion

Quit smoking >5 years significantly slows the rate of emphysema progression and lung function decline.

Trial registration

Registered at http://www.trialregister.nl with trial number ISRCTN63545820.