Building a Better Fragment Library for De Novo Protein Structure Prediction

Fragment-based approaches are the current standard for de novo protein structure prediction. These approaches rely on accurate and reliable fragment libraries to generate good structural models. In this work, we describe a novel method for structure fragment library generation and its application in fragment-based de novo protein structure prediction. The importance of correct testing procedures in assessing the quality of fragment libraries is demonstrated. In particular, the exclusion of homologs to the target from the libraries to correctly simulate a de novo protein structure prediction scenario, something which surprisingly is not always done. We demonstrate that fragments presenting different predominant predicted secondary structures should be treated differently during the fragment library generation step and that exhaustive and random search strategies should both be used. This information was used to develop a novel method, Flib. On a validation set of 41 structurally diverse proteins, Flib libraries presents both a higher precision and coverage than two of the state-of-the-art methods, NNMake and HHFrag. Flib also achieves better precision and coverage on the set of 275 protein domains used in the two previous experiments of the the Critical Assessment of Structure Prediction (CASP9 and CASP10). We compared Flib libraries against NNMake libraries in a structure prediction context. Of the 13 cases in which a correct answer was generated, Flib models were more accurate than NNMake models for 10. “Flib is available for download at: http://www.stats.ox.ac.uk/research/proteins/resources”.     

Photoinactivation effects of hematoporphyrin monomethyl ether on Gram-positive and -negative bacteria detected by atomic force microscopy

The photodynamic antimicrobial chemotherapy as a promising approach for efficiently killing pathogenic microbes is attracting increasing interest. In this study, the cytotoxic and phototoxic effects of hematoporphyrin monomethyl ether (HMME) on the Gram-positive and Gram-negative bacteria were investigated. The cell viability was assessed by colony-forming unit method, and the results indicated that there was no significant cytotoxicity but high phototoxicity in the examined concentrations. Notably, the Gram-positive bacteria were more sensitive to HMME in phototoxicity. Simultaneously, an atomic force microscope (AFM) was used to detect the changes in morphological and nanomechanical properties of bacteria before and after HMME treatment. AFM images indicate that upon photoinactivation, the bacterial surface changed from a smooth, homogeneous architecture to a heterogenous, crackled morphology. The force spectroscopy measurements reveal that the cell wall became less rigid and the Young’s modulus decreased about 50%, whereas the tip-cell-surface adhesion forces increased significantly compared to those of native cells. It was speculated that the photodynamic effects of HMME induced the changes in the chemical composition of the outer membrane and exposure of some proteins inside the envelope. AFM can be utilized as a powerful and sensitive method for studying the interaction between bacteria and drugs.     

Differences in metabolite profile between blood plasma and serum

In metabolomic research, blood plasma and serum have been considered to possess similar compositions and properties. Their perceived equivalence has resulted in researchers choosing arbitrarily between serum and plasma for analysis. Here, routine serum and plasma were prepared and their low-molecular-weight compounds were determined using gas chromatography/time-of-flight mass spectrometry. Principal components analysis was applied to process the acquired data, and marked differences in metabolite profiles were observed between serum and plasma. Of the 72 identified compounds, 36 (50%) discriminate serum from plasma, with 29 and 7 metabolites showing a significantly higher abundance (t test, P < 0.05) in serum and plasma, respectively. Incubation of blood had distinct effects on the analyte peak areas, with the effects being more pronounced for plasma than for serum and more pronounced for a shorter incubation than for a longer incubation. These results highlight the importance in choosing serum or plasma as the analytical sample and in stipulating the incubation time. Because incubation affected the analyte peak areas less in serum than in plasma, we recommend serum as the sample of choice in metabolomic studies.     

Rapidly in situ-forming degradable hydrogels from dextran thiols through Michael addition

Thiol-functionalized dextrans (dex-SH) (M(n,dextran) = 14K or 31K) with degrees of substitution (DS) ranging from 12 to 25 were synthesized and investigated for in situ hydrogel formation via Michael type addition using poly(ethylene glycol) tetra-acrylate (PEG-4-Acr) or a dextran vinyl sulfone conjugate with DS 10 (dex-VS DS 10). Dex-SH was prepared by activation of the hydroxyl groups of dextran with 4-nitrophenyl chloroformate and subsequent reaction with cysteamine. Hydrogels were rapidly formed in situ under physiological conditions upon mixing aqueous solutions of dex-SH and either PEG-4-Acr or dex-VS DS 10 at polymer concentrations of 10 to 20 w/v%. Rheological studies showed that these hydrogels are highly elastic. By varying the DS, concentration, dextran molecular weight, and type of cross-linker, hydrogels with a broad range of storage moduli of 9 to 100 kPa could be obtained. Varying the ratio of thiol to vinyl sulfone groups from 0.9 to 1.1 did not alter the storage modulus of the hydrogels, whereas larger deviations from equimolarity (thiol to vinyl sulfone ratios of 0.75 and 1.5) considerably decreased the storage modulus. The plateau value of hydrogel storage modulus was reached much faster at pH 7.4 compared to pH 7, due to a higher concentration of the thiolate anion at higher pH. These hydrogels were degradable under physiological conditions. Degradation times were 3 to 7 weeks for dex-SH/dex-VS DS 10 hydrogels and 7 to over 21 weeks for dex-SH/PEG-4-Acr hydrogels, depending on the DS, concentration, and dextran molecular weight.     

Concurrent Whole-Genome Haplotyping and Copy-Number Profiling of Single Cells

Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cell’s alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones.     

Synthesis and structure-activity relationships of nitrobenzyl phosphoramide mustards as nitroreductase-activated prodrugs

A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.     

Tumor-Endothelial Cell Three-dimensional Spheroids: New Aspects to Enhance Radiation and Drug Therapeutics

Classic cancer research for several decades has focused on understanding the biology of tumor cells in vitro. However, extending these findings to in vivo settings has been impeded owing to limited insights on the impact of microenvironment on tumor cells. We hypothesized that tumor cell biology and treatment response would be more informative when done in the presence of stromal components, like endothelial cells, which exist in the tumor microenvironment. To that end, we have developed a system to grow three-dimensional cultures of GFP-4T1 mouse mammary tumor and 2H11 murine endothelial cells in hanging drops of medium in vitro. The presence of 2H11 endothelial cells in these three-dimensional cocultures was found to sensitize 4T1-GFP tumor cells to chemotherapy (Taxol) and, at the same time, protect cells from ionizing radiation. These spheroidal cultures can also be implanted into the dorsal skinfold window chamber of mice for fluorescence imaging of vascularization and disease progression/treatment response. We observed rapid neovascularization of the tumor-endothelial spheroids in comparison to tumor spheroids grown in nude mice. Molecular analysis revealed pronounced up-regulation of several proangiogenic factors in the tumor tissue derived from the tumor-endothelial spheroids compared with tumor-only spheroids. Furthermore, the rate of tumor growth from tumor-endothelial spheroids in mice was faster than the tumor cell-only spheroids, resulting in greater metastasis to the lung. This three-dimensional coculture model presents an improved way to investigate more pertinent aspects of the therapeutic potential for radiation and/or chemotherapy alone and in combination with antiangiogenic agents.     

Epidemiology, quality and reporting characteristics of systematic reviews of traditional Chinese medicine interventions published in Chinese journals

Background

Systematic reviews (SRs) of TCM have become increasingly popular in China and have been published in large numbers. This review provides the first examination of epidemiological characteristics of these SRs as well as compliance with the PRISMA and AMSTAR guidelines.

Objectives

To examine epidemiological and reporting characteristics as well as methodological quality of SRs of TCM published in Chinese journals.

Methods

Four Chinese databases were searched (CBM, CSJD, CJFD and Wanfang Database) for SRs of TCM, from inception through Dec 2009. Data were extracted into Excel spreadsheets. The PRISMA and AMSTAR checklists were used to assess reporting characteristics and methodological quality, respectively.

Results

A total of 369 SRs were identified, most (97.6%) of which used the terms systematic review or meta-analysis in the title. None of the reviews had been updated. Half (49.8%) were written by clinicians and nearly half (47.7%) were reported in specialty journals. The impact factors of 45.8% of the journals published in were zero. The most commonly treated conditions were diseases of the circulatory and digestive disease. Funding sources were not reported for any reviews. Most (68.8%) reported information about quality assessment, while less than half (43.6%) reported assessing for publication bias. Statistical mistakes appeared in one-third (29.3%) of reviews and most (91.9%) did not report on conflict of interest.

Conclusions

While many SRs of TCM interventions have been published in Chinese journals, the quality of these reviews is troubling. As a potential key source of information for clinicians and researchers, not only were many of these reviews incomplete, some contained mistakes or were misleading. Focusing on improving the quality of SRs of TCM, rather than continuing to publish them in great quantity, is urgently needed in order to increase the value of these studies.     

Metabonomic study of aristolochic acid-induced nephrotoxicity in rats

This paper describes a metabonomic study characterizing the nephrotoxicity induced by aristolochic acid (AA), a suspected kidney toxicant. For these studies, we examined the biochemical compositions of AA-treated rat urine using LC-MS and pattern recognition methods. The biochemical and histological patterns of rat groups treated with different AA sources showed distinct differences from those of the control group. Certain metabolic pathways, such as homocysteine formation and the folate cycle were significantly accelerated, while others, including arachidonic acid biosynthesis, were decreased. A subset-validation procedure using linear discriminant analysis (LDA) and selected predictive variables indicated that approximately 95% of the treated and nontreated rat urine samples were classified correctly into their respective treatment groups. The results suggested that this metabonomic approach is a promising methodology for the rapid in vivo screening of nephrotoxicity associated with ingesting multi-ingredient medicinal herb supplements, and provides a valid method for comprehending the chemical-induced perturbations in the metabolic network and the networked lesions.     

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.