A machine learning approach for prediction of DNA and peptide HPLC retention times

High performance liquid chromatography (HPLC) has become one of the most efficient methods for the separation of biomolecules. It is an important tool in DNA purification after synthesis as well as DNA quantification. In both cases the separability of different oligonucleotides is essential. The prediction of oligonucleotide retention times prior to the experiment may detect superimposed nucleotides and thereby help to avoid futile experiments. In 2002 Gilar et al. proposed a simple mathematical model for the prediction of DNA retention times, that reliably works at high temperatures only (at least 70°C). To cover a wider temperature rang we incorporated DNA secondary structure information in addition to base composition and length. We used support vector regression (SVR) for the model generation and retention time prediction. A similar problem arises in shotgun proteomics. Here HPLC coupled to a mass spectrometer (MS) is used to analyze complex peptide mixtures (thousands of peptides). Predicting peptide retention times can be used to validate tandem-MS peptide identifications made by search engines like SEQUEST. Recently several methods including multiple linear regression and artificial neural networks were proposed, but SVR has not been used so far

Repeatability of nerve fiber layer thickness measurements in patients with glaucoma and without glaucoma using spectral-domain and time-domain OCT

Background: The aim of this work is to assess the repeatability of spectral-domain-OCT (SD-OCT) retinal nerve fiber layer thickness (RNFL) thickness measurements in a non-glaucoma group and patients with glaucoma and to compare these results to conventional time-domain-OCT (TD-OCT). Methods: In a prospective, comparative, observational case-control study, 50 eyes of 25 non-glaucoma and 22 eyes of 11 patients with primary open angle glaucoma (POAG) were included. SD-OCT and TD-OCT circle scans were centered on the optic disc. In each eye, OCT scans were performed three times by two independent observers. RNFL thickness was measured in four quadrants around the optic disc. In addition, the overall mean RNFL thickness was assessed. Intraclass correlation coefficients (ICC) and coefficients of variation (COV) were calculated. Inter-observer and inter-OCT repeatability was visualized by using Bland-Altman analysis. Results: Intra-observer repeatability for TD- OCT was good with an ICCmean RNFL thickness of 0.939 in non-glaucomas and 0.980 in glaucomatous eyes. For SD-OCT, intra-observer repeatability was higher with an ICC of 0.989 for non-glaucomas and 0.997 for glaucomatous eyes. COVs for TD-OCT ranged from 2.9-7.7% in non-glaucomas and from 6.0-13.3% in glaucoma patients. COVs for SD-OCT ranged from 0.3-1% in non-glaucomas and from 0.9-2.3% in glaucomatous eyes. COVs were influenced by various factors. In the glaucoma group, COVs were significantly higher (p < 0.001) compared to the non-glaucoma group. COVs increased by a mean of 5.1% when TD-OCT was used instead of SD-OCT (p < 0.001). Conclusions: SD-OCT RNFL thickness measurements in healthy volunteers and glaucoma patients showed good intra- and inter-observer repeatability. Especially in glaucomatous eyes, repeatability of SD-OCT was superior to TD-OC

Combined influence of ectoine and salt: spectroscopic and numerical evidence for compensating effects on aqueous solutions

Ectoine is an important osmolyte, which allows microorganisms to survive in extreme environmental salinity. The hygroscopic effects of ectoine in pure water can be explained by a strong water binding behavior whereas a study on the effects of ectoine in salty solution is yet missing. We provide Raman spectroscopic evidence that the influence of ectoine and NaCl are opposing and completely independent of each other. The effect can be explained by the formation of strongly hydrogen-bonded water molecules around ectoine which compensate the influence of the salt on the water dynamics. The mechanism is corroborated by first principles calculations and broadens our understanding of zwitterionic osmolytes in aqueous solution. Our findings allow us to provide a possible explanation for the relatively high osmolyte concentrations in halotolerant bacteria

A general approach applicable to other radiation sources and biological targets

The determination of the microscopic dose-damage relationship for DNA in an aqueous environment is of a fundamental interest for dosimetry and applications in radiation therapy and protection. We combine geant4 particle- scattering simulations in water with calculations concerning the movement of biomolecules to obtain the energy deposit in the biologically relevant nanoscopic volume. We juxtaposition these results to the experimentally determined damage to obtain the dose-damage relationship at a molecular level. This approach is tested for an experimentally challenging system concerning the direct irradiation of plasmid DNA (pUC19) in water with electrons as primary particles. Here a microscopic target model for the plasmid DNA based on the relation of lineal energy and radiation quality is used to calculate the effective target volume. It was found that on average fewer than two ionizations within a 7.5-nm radius around the sugar-phosphate backbone are sufficient to cause a single strand break, with a corresponding median lethal energy deposit being E1/2=6±4 eV. The presented method is applicable for ionizing radiation (e.g., γ rays, x rays, and electrons) and a variety of targets, such as DNA, proteins, or cells

Polymers in disordered environments

Using a combination of analytical theory and newly developed numerical algorithms, we analyze the most pertinent conformational characteristics of three paradigmatic types of polymers in disordered environments: (i) flexible polymers in quenched, self-similar disorder as represented by a self-avoiding random walk on a critical percolation cluster, (ii) semiflexible polymers in quenched, steric disorder as represented by an equilibrium hard-disk fluid and (iii) semiflexible polymers subject to the random energy landscape that emerges from a surrounding network of similar semiflexible polymers

OpenMS - A Framework for Quantitative HPLC/MS-Based Proteomics

In the talk we describe the freely available software library OpenMS which is currently under development at the Freie Universität Berlin and the Eberhardt-Karls Universität Tübingen. We give an overview of the goals and problems in differential proteomics with HPLC and then describe in detail the implemented approaches for signal processing, peak detection and data reduction currently employed in OpenMS. After this we describe methods to identify the differential expression of peptides and propose strategies to avoid MS/MS identification of peptides of interest. We give an overview of the capabilities and design principles of OpenMS and demonstrate its ease of use. Finally we describe projects in which OpenMS will be or was already deployed and thereby demonstrate its versatility

Molecular Mechanisms

Ectoine, a compatible solute and osmolyte, is known to be an effective protectant of biomolecules and whole cells against heating, freezing and extreme salinity. Protection of cells (human keratinocytes) by ectoine against ultraviolet radiation has also been reported by various authors, although the underlying mechanism is not yet understood. We present the first electron irradiation of DNA in a fully aqueous environment in the presence of ectoine and at high salt concentrations. The results demonstrate effective protection of DNA by ectoine against the induction of single-strand breaks by ionizing radiation. The effect is explained by an increase in low-energy electron scattering at the enhanced free- vibrational density of states of water due to ectoine, as well as the use of ectoine as an hydroxyl-radical scavenger. This was demonstrated by Raman spectroscopy and electron paramagnetic resonance (EPR)

Direct electron irradiation of DNA in a fully aqueous environment. Damage determination in combination with Monte Carlo simulations

We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSBs) and double-strand breaks (DSBs), was determined by gel electrophoresis. The median lethal dose of D1/2 = 1.7 ± 0.3 Gy was found to be much smaller as compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of the DSBs to SSBs was found to be 1 : 12 as compared to 1 : 88 found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, co-solutes) for an electron energy range which is difficult to probe by standard methods

Implications for the Binding of the Protein G5P to DNA

Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-S-protein (G5P) to a single-stranded DNA (dT(25)). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration- dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonudeotide, which has important consequences for osmotic regulation mechanisms