Discrimination of Nasopharyngeal Carcinoma from Noncancerous Ex Vivo Tissue Using Reflectance Spectroscopy

Reflectance spectroscopy is a low-cost, nondestructive, and noninvasive method for detection of neoplastic lesions of mucosal tissue. This study aims to evaluate the capability of reflectance spectroscopy system under white light (400-700 nm) with a multivariate statistical analysis for distinguishing nasopharyngeal carcinoma (NPC) from nasopharyngeal benign ex vivo tissues. High quality reflectance spectra were acquired from nasopharyngeal ex vivo tissues belonging to 18 noncancerous and 19 cancerous subjects, and the combination of principal component analysis-linear discriminant analysis (PCA-LDA) along with leave-onespectrum-out cross-validation (LOOCV) diagnostic algorithm was subsequently employed to classify different types of tissue group, achieving a diagnostic sensitivity of 73.7% and a specificity of 72.2%. Furthermore, in order to distinguish NPC from nasopharyngeal benign ex vivo tissues based on reflectance spectra simply, spectral intensity ratios of oxyhemoglobin ( 540/ 576) were used as an indicator of the carcinogenesis associated transformation in the hemoglobin oxygenation. This tentative work demonstrated the potential of reflectance spectroscopy for NPC detection using ex vivo tissue and has significant experimental and clinical value for further in vivo NPC detection in the future

GGQ methylation enhances both speed and accuracy of stop codon recognition by bacterial class-I release factors

Accurate translation termination in bacteria requires correct recognition of the stop codons by the class-I release factors (RFs) RF1 and RF2, which release the nascent peptide from the peptidyl tRNA after undergoing a "compact to open" conformational transition. These RFs possess a conserved Gly-Gly-Gln (GGQ) peptide release motif, of which the Q residue is posttranslationally methylated. GGQ-methylated RFs have been shown to be faster in peptide release than the unmethylated ones, but it was unknown whether this modification had additional roles. Using a fluorescence-based real-time in vitro translation termination assay in a stopped-flow instrument, we demonstrate that methylated RF1 and RF2 are two- to four-fold more accurate in the cognate stop codon recognition than their unmethylated variants. Using pH titration, we show that the lack of GGQ methylation facilitates the "compact to open" transition, which results in compromised accuracy of the unmethylated RFs. Furthermore, thermal melting studies using circular dichroism and SYPRO-orange fluorescence demonstrate that GGQ methylation increases overall stability of the RF proteins. This increased stability, we suspect, is the basis for the more controlled conformational change of the methylated RFs upon codon recognition, which enhances both their speed and accuracy. This GGQ methylation-based modulation of the accuracy of RFs can be a tool for regulating translational termination in vivo

Experimental Evolution of Escherichia coli Harboring an Ancient Translation Protein

The ability to design synthetic genes and engineer biological systems at the genome scale opens new means by which to characterize phenotypic states and the responses of biological systems to perturbations. One emerging method involves inserting artificial genes into bacterial genomes and examining how the genome and its new genes adapt to each other. Here we report the development and implementation of a modified approach to this method, in which phylogenetically inferred genes are inserted into a microbial genome, and laboratory evolution is then used to examine the adaptive potential of the resulting hybrid genome. Specifically, we engineered an approximately 700-million-year-old inferred ancestral variant of tufB, an essential gene encoding elongation factor Tu, and inserted it in a modern Escherichia coli genome in place of the native tufB gene. While the ancient homolog was not lethal to the cell, it did cause a twofold decrease in organismal fitness, mainly due to reduced protein dosage. We subsequently evolved replicate hybrid bacterial populations for 2000 generations in the laboratory and examined the adaptive response via fitness assays, whole genome sequencing, proteomics, and biochemical assays. Hybrid lineages exhibit a general adaptive strategy in which the fitness cost of the ancient gene was ameliorated in part by upregulation of protein production. Our results suggest that an ancient–modern recombinant method may pave the way for the synthesis of organisms that exhibit ancient phenotypes, and that laboratory evolution of these organisms may prove useful in elucidating insights into historical adaptive processes. Electronic supplementary material The online version of this article (doi:10.1007/s00239-017-9781-0) contains supplementary material, which is available to authorized users

Collateral toxicity limits the evolution of bacterial Release Factor 2 towards total omnipotence

When new genes evolve through modification of existing genes, there are often trade-offs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near cognate stop codons and highlight a functional trade-off that we term “collateral toxicity”. We suggest that this type of trade-off may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary trade-offs between “old” and “new” functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds not only to alterations in the demand for RF2 activity, but also for RF1 activity.The two first authors contributed equally to this work.</p

Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli

In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P-mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-P-K34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is tau(free) approximate to 16 ms. The typical residence time of an EF-P on the ribosome is very short, tau(bound) approximate to 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation. IMPORTANCE Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle

Effect of the Internal Humidity of Concrete on Frost Resistance and Air Void Structure under Different Low Temperature Conditions

From the perspective of combining macroscopic and microscopic properties, this paper simulates the freeze&ndash;thaw cycle process at different freezing low temperatures based on the climate simulation equipment and by setting the curing conditions with different temperatures and relative humidity to produce different moisture conditions in concrete. The frost resistance properties and microscopic air void performance of concrete with different internal water content under different freezing low temperatures in freeze&ndash;thaw cycles were systematically studied. The results show that the higher the internal water content of concrete, the more obvious the mass loss rate and dynamic elastic modulus loss of concrete in the freeze&ndash;thaw process, and the more serious the deterioration of the air void parameter performance of the air-entraining agent introduced into concrete, which is manifested as the average bubble diameter and bubble spacing factor become larger and the bubble specific surface area decreases. In addition, in the case of the same internal moisture content of concrete, the freezing temperature used in the freeze&ndash;thaw cycle also has an important impact on the frost resistance of concrete and air void parameters; the lower the freezing temperature used, the more significant the decline in the frost resistance of concrete, the more obvious the deterioration of air void parameters

An Integrated Indicator System and Evaluation Model for Regional Sustainable Development

Regional sustainable development has become a worldwide issue in recent years, but there is no single and universally agreed method of choosing indicators for sustainable development assessment. The subjective selection of indicators will affect the results of assessment. Each evaluation method has its own advantages and disadvantages, and the methods used to determine indicator weight also differ. Regional sustainable development is a complex system, which is difficult to evaluate objectively and scientifically using a single method. Therefore, a new integrated indicator system and evaluation model is constructed here to more accurately reflect regional sustainable development level. The indicator system and evaluation model were constructed using a case study of 17 cities in Shandong Province, China. The indicator system includes 4 subsystems, i.e., economy, society, resource, and environment. These indicators were selected through correlation analysis and discrimination analysis. A back propagation neural network was applied to evaluate the respective scores of the 4 subsystems. The comprehensive score for regional sustainable development was evaluated using the analytic hierarchy process with entropy correction. The results show that sustainable development levels in these 17 cities show a gradually decreasing trend from east to west and from coast to inland. Cities with an underdeveloped economy usually display poor levels of social development and serious environmental pollution. Through the improvement of indicator screening, evaluation model, and result correction, the error caused by a single evaluation method can be reduced significantly. This new methodology for indicator selection and comprehensive evaluation provides a new perspective for the assessment of regional sustainable development