Measuring Dissociation Rate Constants of Protein Complexes through Subunit Exchange: Experimental Design and Theoretical Modeling

Protein complexes are dynamic macromolecules that constantly dissociate into, and simultaneously are assembled from, free subunits. Dissociation rate constants, koff, provide structural and functional information on protein complexes. However, because all existing methods for measuring koff require high-quality purification and specific modifications of protein complexes, dissociation kinetics has only been studied for a small set of model complexes. Here, we propose a new method, called Metabolically-labeled Affinity-tagged Subunit Exchange (MASE), to measure koff using metabolic stable isotope labeling, affinity purification and mass spectrometry. MASE is based on a subunit exchange process between an unlabeled affinity-tagged variant and a metabolically-labeled untagged variant of a complex. The subunit exchange process was modeled theoretically for a heterodimeric complex. The results showed that koff determines, and hence can be estimated from, the observed rate of subunit exchange. This study provided the theoretical foundation for future experiments that can validate and apply the MASE method

Evolution of Alternative Splicing Regulation: Changes in Predicted Exonic Splicing Regulators Are Not Associated with Changes in Alternative Splicing Levels in Primates

Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans) factors that bind to different sequence (cis) elements. cis-elements reside in both introns and exons and may either enhance or silence splicing. Differential combinations of cis-elements allows for a huge diversity of overall splicing signals, together comprising a complex ‘splicing code’. Many cis-elements have been identified, and their effects on exon inclusion levels demonstrated in reporter systems. However, the impact of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5′ splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little or no effect on splicing, and thus interspecific changes at short-time scales may primarily occur in these effectively neutral ESRs. These results underscore the difficulties of using current computational ESR prediction algorithms to identify truly functionally important motifs, and provide a cautionary tale for studies of the effect of SNPs on splicing in human disease

The use of cessation assistance among smokers from China: Findings from the ITC China Survey

<p>Abstract</p> <p>Background</p> <p>Stop smoking medications significantly increase the likelihood of smoking cessation. However, there are no population-based studies of stop-smoking medication use in China, the largest tobacco market in the world. This study examined stop-smoking medication use and its association with quitting behavior among a population-based sample of Chinese smokers.</p> <p>Methods</p> <p>Face-to-face interviews were conducted with 4,627 smokers from six cities in the ITC China cohort survey. Longitudinal analyses were conducted using Wave 1 (April to August, 2006) and Wave 2 (November 2007 to January 2008).</p> <p>Results</p> <p>Approximately 26% of smokers had attempted to quit between Waves 1 and 2, and 6% were abstinent at 18-month follow-up. Only 5.8% of those attempting to quit reported NRT use and NRT was associated with lower odds of abstinence at Wave 2 (OR = 0.11; 95%CI = 0.03-0.46). Visiting a doctor/health professional was associated with greater attempts to quit smoking (OR = 1.60 and 2.78; 95%CI = 1.22-2.10 and 2.21-3.49 respectively) and being abstinent (OR = 1.77 and 1.85; 95%CI = 1.18-2.66 and 1.13-3.04 respectively) at 18-month follow-up relative to the smokers who did not visit doctor/health professional.</p> <p>Conclusions</p> <p>The use of formal help for smoking cessation is low in China. There is an urgent need to explore the use and effectiveness of stop-smoking medications in China and in other non-Western markets.</p

Templating hydrogels

Templating processes for creating polymerized hydrogels are reviewed. The use of contact photonic crystals and of non-contact colloidal crystalline arrays as templates are described and applications to chemical sensing and device fabrication are illustrated. Emulsion templating is illustrated in the formation of microporous membranes, and templating on reverse emulsions and double emulsions is described. Templating in solutions of macromolecules and micelles is discussed and then various applications of hydrogel templating on surfactant liquid crystalline mesophases are illustrated, including a nanoscale analogue of colloidal crystalline array templating, except that the bead array in this case is a cubic array of nonionic micelles. The use of particles as templates in making core-shell and hollow microgel beads is described, as is the use of membrane pores as another illustration of confinement templating

A Global View of Cancer-Specific Transcript Variants by Subtractive Transcriptome-Wide Analysis

BACKGROUND: Alternative pre-mRNA splicing (AS) plays a central role in generating complex proteomes and influences development and disease. However, the regulation and etiology of AS in human tumorigenesis is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: A Basic Local Alignment Search Tool database was constructed for the expressed sequence tags (ESTs) from all available databases of human cancer and normal tissues. An insertion or deletion in the alignment of EST/EST was used to identify alternatively spliced transcripts. Alignment of the ESTs with the genomic sequence was further used to confirm AS. Alternatively spliced transcripts in each tissue were then subtractively cross-screened to obtain tissue-specific variants. We systematically identified and characterized cancer/tissue-specific and alternatively spliced variants in the human genome based on a global view. We identified 15,093 cancer-specific variants of 9,989 genes from 27 types of human cancers and 14,376 normal tissue-specific variants of 7,240 genes from 35 normal tissues, which cover the main types of human tumors and normal tissues. Approximately 70% of these transcripts are novel. These data were integrated into a database HCSAS (http://202.114.72.39/database/human.html, pass:68756253). Moreover, we observed that the cancer-specific AS of both oncogenes and tumor suppressor genes are associated with specific cancer types. Cancer shows a preference in the selection of alternative splice-sites and utilization of alternative splicing types. CONCLUSIONS/SIGNIFICANCE: These features of human cancer, together with the discovery of huge numbers of novel splice forms for cancer-associated genes, suggest an important and global role of cancer-specific AS during human tumorigenesis. We advise the use of cancer-specific alternative splicing as a potential source of new diagnostic, prognostic, predictive, and therapeutic tools for human cancer. The global view of cancer-specific AS is not only useful for exploring the complexity of the cancer transcriptome but also widens the eyeshot of clinical research

Increased CD45RA+FoxP3low Regulatory T Cells with Impaired Suppressive Function in Patients with Systemic Lupus Erythematosus

BACKGROUND: The role of naturally occurring regulatory T cells (Treg) in the control of the development of systemic lupus erythematosus (SLE) has not been well defined. Therefore, we dissect the phenotypically heterogeneous CD4(+)FoxP3(+) T cells into subpopulations during the dynamic SLE development. METHODLOGY/PRINCIPAL FINDINGS: To evaluate the proliferative and suppressive capacities of different CD4(+) T cell subgroups between active SLE patients and healthy donors, we employed CD45RA and CD25 as surface markers and carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay. In addition, multiplex cytokines expression in active SLE patients was assessed using Luminex assay. Here, we showed a significant increase in the frequency of CD45RA(+)FoxP3(low) naive Treg cells (nTreg cells) and CD45RA(-)FoxP3(low) (non-Treg) cells in patients with active SLE. In active SLE patients, the increased proportions of CD45RA(+)FoxP3(low) nTreg cells were positively correlated with the disease based on SLE disease activity index (SLEDAI) and the status of serum anti-dsDNA antibodies. We found that the surface marker combination of CD25(+)CD45RA(+) can be used to defined CD45RA(+)FoxP3(low) nTreg cells for functional assays, wherein nTreg cells from active SLE patients demonstrated defective suppression function. A significant correlation was observed between inflammatory cytokines, such as IL-6, IL-12 and TNFα, and the frequency of nTreg cells. Furthermore, the CD45RA(+)FoxP3(low) nTreg cell subset increased when cultured with SLE serum compared to healthy donor serum, suggesting that the elevated inflammatory cytokines of SLE serum may promote nTreg cell proliferation/expansion. CONCLUSIONS/SIGNIFICANCE: Our results indicate that impaired numbers of functional CD45RA(+)FoxP3(low) naive Treg cell and CD45RA(-)FoxP3(low) non-suppressive T cell subsets in inflammatory conditions may contribute to SLE development. Therefore, analysis of subsets of FoxP3(+) T cells, using a combination of FoxP3, CD25 and CD45RA, rather than whole FoxP3(+) T cells, will help us to better understand the pathogenesis of SLE and may lead to the development of new therapeutic strategies

Surface Morphology Evolution Mechanisms of InGaN/GaN Multiple Quantum Wells with Mixture N2/H2-Grown GaN Barrier

This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Surface morphology evolution mechanisms of InGaN/GaN multiple quantum wells (MQWs) during GaN barrier growth with different hydrogen (H2) percentages have been systematically studied. Ga surface-diffusion rate, stress relaxation, and H2 etching effect are found to be the main affecting factors of the surface evolution. As the percentage of H2 increases from 0 to 6.25%, Ga surface-diffusion rate and the etch effect are gradually enhanced, which is beneficial to obtaining a smooth surface with low pits density. As the H2 proportion further increases, stress relaxation and H2 over- etching effect begin to be the dominant factors, which degrade surface quality. Furthermore, the effects of surface evolution on the interface and optical properties of InGaN/GaN MQWs are also profoundly discussed. The comprehensive study on the surface evolution mechanisms herein provides both technical and theoretical support for the fabrication of high-quality InGaN/GaN heterostructures.Peer reviewe

Imaging in assessing hepatic and peritoneal metastases of gastric cancer: a systematic review

<p>Abstract</p> <p>Background</p> <p>Hepatic and peritoneal metastases of gastric cancer are operation contraindications. Systematic review to provide an overview of imaging in predicting the status of liver and peritoneum pre-therapeuticly is essential.</p> <p>Methods</p> <p>A systematic review of relevant literatures was performed in Pubmed/Medline, Embase, The Cochrane Library and the China Biological Medicine Databases. QUADAS was used for assessing the methodological quality of included studies and the bivariate model was used for this meta-analysis.</p> <p>Results</p> <p>Totally 33 studies were included (8 US studies, 5 EUS studies, 22 CT studies, 2 MRI studies and 5 18F-FDG PET studies) and the methodological quality of included studies was moderate. The result of meta-analysis showed that CT is the most sensitive imaging method [0.74 (95% CI: 0.59-0.85)] with a high rate of specificity [0.99 (95% CI: 0.97-1.00)] in detecting hepatic metastasis, and EUS is the most sensitive imaging modality [0.34 (95% CI: 0.10-0.69) ] with a specificity of 0.96 (95% CI: 0.87-0.99) in detecting peritoneal metastasis. Only two eligible MRI studies were identified and the data were not combined. The two studies found that MRI had both high sensitivity and specificity in detecting liver metastasis.</p> <p>Conclusion</p> <p>US, EUS, CT and ¹⁸F-FDG PET did not obtain consistently high sensitivity and specificity in assessing liver and peritoneal metastases of gastric cancer. The value of laparoscopy, PET/CT, DW-MRI, and new PET tracers such as ¹⁸F-FLT needs to be studied in future.</p

Minimum Free Energy Path of Ligand-Induced Transition in Adenylate Kinase

Large-scale conformational changes in proteins involve barrier-crossing transitions on the complex free energy surfaces of high-dimensional space. Such rare events cannot be efficiently captured by conventional molecular dynamics simulations. Here we show that, by combining the on-the-fly string method and the multi-state Bennett acceptance ratio (MBAR) method, the free energy profile of a conformational transition pathway in Escherichia coli adenylate kinase can be characterized in a high-dimensional space. The minimum free energy paths of the conformational transitions in adenylate kinase were explored by the on-the-fly string method in 20-dimensional space spanned by the 20 largest-amplitude principal modes, and the free energy and various kinds of average physical quantities along the pathways were successfully evaluated by the MBAR method. The influence of ligand binding on the pathways was characterized in terms of rigid-body motions of the lid-shaped ATP-binding domain (LID) and the AMP-binding (AMPbd) domains. It was found that the LID domain was able to partially close without the ligand, while the closure of the AMPbd domain required the ligand binding. The transition state ensemble of the ligand bound form was identified as those structures characterized by highly specific binding of the ligand to the AMPbd domain, and was validated by unrestrained MD simulations. It was also found that complete closure of the LID domain required the dehydration of solvents around the P-loop. These findings suggest that the interplay of the two different types of domain motion is an essential feature in the conformational transition of the enzyme

Clustering of classical swine fever virus isolates by codon pair bias

<p>Abstract</p> <p>Background</p> <p>The genetic code consists of non-random usage of synonymous codons for the same amino acids, termed codon bias or codon usage. Codon juxtaposition is also non-random, referred to as codon context bias or codon pair bias. The codon and codon pair bias vary among different organisms, as well as with viruses. Reasons for these differences are not completely understood. For classical swine fever virus (CSFV), it was suggested that the synonymous codon usage does not significantly influence virulence, but the relationship between variations in codon pair usage and CSFV virulence is unknown. Virulence can be related to the fitness of a virus: Differences in codon pair usage influence genome translation efficiency, which may in turn relate to the fitness of a virus. Accordingly, the potential of the codon pair bias for clustering CSFV isolates into classes of different virulence was investigated.</p> <p>Results</p> <p>The complete genomic sequences encoding the viral polyprotein of 52 different CSFV isolates were analyzed. This included 49 sequences from the GenBank database (NCBI) and three newly sequenced genomes. The codon usage did not differ among isolates of different virulence or genotype. In contrast, a clustering of isolates based on their codon pair bias was observed, clearly discriminating highly virulent isolates and vaccine strains on one side from moderately virulent strains on the other side. However, phylogenetic trees based on the codon pair bias and on the primary nucleotide sequence resulted in a very similar genotype distribution.</p> <p>Conclusion</p> <p>Clustering of CSFV genomes based on their codon pair bias correlate with the genotype rather than with the virulence of the isolates.</p