Vegetation patterns of floodplain meadows along the climatic gradient at the Middle Elbe River

Central European floodplain meadows are characterised by flooding mainly in winter/spring and dry conditions over the summer. They harbour many rare and endangered plant species. We studied the vegetation of floodplain meadows along the regional climatic gradient of the Middle Elbe River. This gradient exhibits rather continental conditions in the south-east and oceanic conditions in the north-west. We aimed at detecting the influence of climate on the vegetation of floodplain meadows along this gradient.     

Resolving complex mixtures: trilinear diffusion data

Complex mixtures are at the heart of biology, and biomacromolecules almost always exhibit their function in a mixture, e.g., the mode of action for a spider venom is typically dependent on a cocktail of compounds, not just the protein. Information about diseases is encoded in body fluids such as urine and plasma in the form of metabolite concentrations determined by the actions of enzymes. To understand better what is happening in real living systems we urgently need better methods to characterize such mixtures. In this paper we describe a potent way to disentangle the NMR spectra of mixture components, by exploiting data that vary independently in three or more dimensions, allowing the use of powerful algorithms to decompose the data to extract the information sought. The particular focus of this paper is on NMR diffusion data, which are typically bilinear but can be extended by a third dimension to give the desired data structure.     

A Unique C‐terminal Domain Allows Retention of Matrix Metalloproteinase‐27 in the Endoplasmic Reticulum

Matrix metalloproteinase‐27 (MMP‐27) is poorly characterized. Sequence comparison suggests that a C‐terminal extension (CTE) includes a potential transmembrane domain as in some membrane‐type (MT)‐MMPs. Having noticed that MMP‐27 was barely secreted, we investigated its subcellular localization and addressed CTE contribution for MMP‐27 retention. Intracellular MMP‐27 was sensitive to endoglycosidase H. Subcellular fractionation and confocal microscopy evidenced retention of endogenous MMP‐27 or recombinant rMMP‐27 in the endoplasmic reticulum (ER) with locked exit across the intermediate compartment (ERGIC). Conversely, truncated rMMP‐27 without CTE accessed downstream secretory compartments (ERGIC and Golgi) and was constitutively secreted. CTE addition to rMMP‐10 (a secreted MMP) caused ER retention and blocked secretion. Addition of a PKA target sequence to the cytosolic C‐terminus of transmembrane MT1‐MMP/MMP‐14 led to effective phosphorylation upon forskolin stimulation, but not for MMP‐27, excluding transmembrane anchorage. Moreover, MMP‐27 was protected from digestion by proteinase K. Finally, MT1‐MMP/MMP‐14 but neither endogenous nor recombinant MMP‐27 partitioned in the detergent phase after Triton X‐114 extraction, indicating that MMP‐27 is not an integral membrane protein. In conclusion, MMP‐27 is efficiently retained within the ER due to its unique CTE, which does not lead to stable membrane insertion. This could represent a novel ER retention system.      

ExpaRNA-P: simultaneous exact pattern matching and folding of RNAs

Background

Identifying sequence-structure motifs common to two RNAs can speed up the comparison of structural RNAs substantially. The core algorithm of the existent approach ExpaRNA solves this problem for a priori known input structures. However, such structures are rarely known; moreover, predicting them computationally is no rescue, since single sequence structure prediction is highly unreliable.

Results

The novel algorithm ExpaRNA-P computes exactly matching sequence-structure motifs in entire Boltzmann-distributed structure ensembles of two RNAs; thereby we match and fold RNAs simultaneously, analogous to the well-known “simultaneous alignment and folding” of RNAs. While this implies much higher flexibility compared to ExpaRNA, ExpaRNA-P has the same very low complexity (quadratic in time and space), which is enabled by its novel structure ensemble-based sparsification. Furthermore, we devise a generalized chaining algorithm to compute compatible subsets of ExpaRNA-P’s sequence-structure motifs. Resulting in the very fast RNA alignment approach ExpLoc-P, we utilize the best chain as anchor constraints for the sequence-structure alignment tool LocARNA. ExpLoc-P is benchmarked in several variants and versus state-of-the-art approaches. In particular, we formally introduce and evaluate strict and relaxed variants of the problem; the latter makes the approach sensitive to compensatory mutations. Across a benchmark set of typical non-coding RNAs, ExpLoc-P has similar accuracy to LocARNA but is four times faster (in both variants), while it achieves a speed-up over 30-fold for the longest benchmark sequences (≈400nt). Finally, different ExpLoc-P variants enable tailoring of the method to specific application scenarios. ExpaRNA-P and ExpLoc-P are distributed as part of the LocARNA package. The source code is freely available at http://www.bioinf.uni-freiburg.de/Software/ExpaRNA-P.

Conclusions

ExpaRNA-P’s novel ensemble-based sparsification reduces its complexity to quadratic time and space. Thereby, ExpaRNA-P significantly speeds up sequence-structure alignment while maintaining the alignment quality. Different ExpaRNA-P variants support a wide range of applications.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0404-0) contains supplementary material, which is available to authorized users.     

Homeostatic and injury‐induced microglia behavior in the aging brain

Microglia cells are essential for brain homeostasis and have essential roles in neurodegenerative diseases. Aging is the main risk factor for most neurodegenerative diseases, and age‐related changes in microglia may contribute to the susceptibility of the aging brain to dysfunction and neurodegeneration. We have analyzed morphology and dynamic behavior of neocortical microglia in their physiological environment in young adult (3‐month‐old), adult (11‐ to 12‐month‐old), and aged (26‐ to 27‐month‐old) C57BL/6J‐Iba1‐eGFP mice using in vivo 2‐photon microscopy. Results show that surveying microglial cells in the neocortex exhibit age‐related soma volume increase, shortening of processes, and loss of homogeneous tissue distribution. Furthermore, microglial process speed significantly decreased with age. While only a small population of microglia showed soma movement in adult mice, the microglia population with soma movement was increased in aged mice. However, in response to tissue injury, the dynamic microglial response was age‐dependently diminished. These results provide novel insights into microglial behavior and indicate that microglial dysfunction in the aging brain may contribute to age‐related cognitive decline and neurodegenerative diseases.     

The mzTab Data Exchange Format: Communicating Mass-spectrometry-based Proteomics and Metabolomics Experimental Results to a Wider Audience

The HUPO Proteomics Standards Initiative has developed several standardized data formats to facilitate data sharing in mass spectrometry (MS)-based proteomics. These allow researchers to report their complete results in a unified way. However, at present, there is no format to describe the final qualitative and quantitative results for proteomics and metabolomics experiments in a simple tabular format. Many downstream analysis use cases are only concerned with the final results of an experiment and require an easily accessible format, compatible with tools such as Microsoft Excel or R.We developed the mzTab file format for MS-based proteomics and metabolomics results to meet this need. mzTab is intended as a lightweight supplement to the existing standard XML-based file formats (mzML, mzIdentML, mzQuantML), providing a comprehensive summary, similar in concept to the supplemental material of a scientific publication. mzTab files can contain protein, peptide, and small molecule identifications together with experimental metadata and basic quantitative information. The format is not intended to store the complete experimental evidence but provides mechanisms to report results at different levels of detail. These range from a simple summary of the final results to a representation of the results including the experimental design. This format is ideally suited to make MS-based proteomics and metabolomics results available to a wider biological community outside the field of MS. Several software tools for proteomics and metabolomics have already adapted the format as an output format. The comprehensive mzTab specification document and extensive additional documentation can be found online.Mass spectrometry (MS)¹ has become a major analysis tool in the life sciences (1). It is currently used in different modes for several “omics” approaches, proteomics and metabolomics being the most prominent. In both disciplines, one major burden in the exchange, communication, and large-scale (re-) analysis of MS-based data is the significant number of software pipelines and, consequently, heterogeneous file formats used to process, analyze, and store these experimental results, including both identification and quantification data. Publication guidelines from scientific journals and funding agencies'' requirements for public data availability have led to an increasing amount of MS-based proteomics and metabolomics data being submitted to public repositories, such as those of the ProteomeXchange consortium (2) or, in the case of metabolomics, the resources from the nascent COSMOS (Coordination of Standards in Metabolomics) initiative (3).In the past few years, the Human Proteome Organization Proteomics Standards Initiative (PSI) has developed several vendor-neutral standard data formats to overcome the representation heterogeneity. The Human Proteome Organization PSI promotes the usage of three XML file formats to fully report the data coming from MS-based proteomics experiments (including related metadata): mzML (4) to store the “primary” MS data (the spectra and chromatograms), mzIdentML (5) to report peptide identifications and inferred protein identifications, and mzQuantML (6) to store quantitative information associated with these results.Even though the existence of the PSI standard data formats represents a huge step forward, these formats cannot address all use cases related to proteomics and metabolomics data exchange and sharing equally well. During the development of mzML, mzIdentML, and mzQuantML, the main focus lay on providing an exact and comprehensive representation of the gathered results. All three formats can be used within analysis pipelines and as interchange formats between independent analysis tools. It is thus vital that these formats be capable of storing the full data and analysis that led to the results. Therefore, all three formats result in relatively complex schemas, a clear necessity for adequate representation of the complexity found in MS-based data.An inevitable drawback of this approach is that data consumers can find it difficult to quickly retrieve the required information. Several application programming interfaces (APIs) have been developed to simplify software development based on these formats (7–9), but profound proteomics and bioinformatics knowledge still is required in order to use them efficiently and take full advantage of the comprehensive information contained.The new file format presented here, mzTab, aims to describe the qualitative and quantitative results for MS-based proteomics and metabolomics experiments in a consistent, simpler tabular format, abstracting from the mass spectrometry details. The format contains identifications, basic quantitative information, and related metadata. With mzTab''s flexible design, it is possible to report results at different levels ranging from a simple summary or subset of the complete information (e.g. the final results) to fairly comprehensive representation of the results including the experimental design. Many downstream analysis use cases are only concerned with the final results of an experiment in an easily accessible format that is compatible with tools such as Microsoft Excel® or R (10) and can easily be adapted by existing bioinformatics tools. Therefore, mzTab is ideally suited to make MS proteomics and metabolomics results available to the wider biological community, beyond the field of MS.mzTab follows a similar philosophy as the other tab-delimited format recently developed by the PSI to represent molecular interaction data, MITAB (11). MITAB is a simpler tab-delimited format, whereas PSI-MI XML (12), the more detailed XML-based format, holds the complete evidence. The microarray community makes wide use of the format MAGE-TAB (13), another example of such a solution that can cover the main use cases and, for the sake of simplicity, is often preferred to the XML standard format MAGE-ML (14). Additionally, in MS-based proteomics, several software packages, such as Mascot (15), OMSSA (16), MaxQuant (17), OpenMS/TOPP (18, 19), and SpectraST (20), also support the export of their results in a tab-delimited format next to a more complete and complex default format. These simple formats do not contain the complete information but are nevertheless sufficient for the most frequent use cases.mzTab has been designed with the same purpose in mind. It can be used alone or in conjunction with mzML (or other related MS data formats such as mzXML (21) or text-based peak list formats such as MGF), mzIdentML, and/or mzQuantML. Several highly successful concepts taken from the development process of mzIdentML and mzQuantML were adapted to the text-based nature of mzTab.In addition, there is a trend to perform more integrated experimental workflows involving both proteomics and metabolomics data. Thus, we developed a standard format that can represent both types of information in a single file.     

Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays

Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody''s linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on- and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.Antibodies are used in proteomics both as imaging reagents for the analysis of tissue specificity (1) and subcellular localization (2) and as capturing agents for targeted proteomics (3), in particular for the enrichment of peptides for immunoaffinity methods such as Stable Isotope Standards and Capture by Anti-peptide Antibodies (4). In fact, the Human Proteome Project (5) has announced that one of the three pillars of the project will be antibody-based, with one of the aims being to generate antibodies to at least one representative protein from all protein-coding genes. Knowledge about the binding site (epitope) of an antibody toward a target protein is thus important for gaining basic insights into antibody specificity and sensitivity and facilitating the identification and design of antigens to be used for reagents in proteomics, as well as for the generation of therapeutic antibodies and vaccines (1, 6). With over 20 monoclonal-antibody-based drugs now on the market and over 100 in clinical trials, the field of antibody therapeutics has become a central component of the pharmaceutical industry (7). One of the key parameters for antibodies includes the nature of the binding recognition toward the target, involving either linear epitopes formed by consecutive amino acid residues or conformational epitopes consisting of amino acids brought together by the fold of the target protein (8).A large number of methods have therefore been developed to determine the epitopes of antibodies, including mass spectrometry (9), solid phase libraries (10, 11), and different display systems (12–14) such as bacterial display (15) and phage display (16). The most common method for epitope mapping involves the use of soluble and immobilized (tethered) peptide libraries, often in an array format, exemplified by the “Geysen Pepscan” method (11) in which overlapping “tiled” peptides are synthesized and used for binding analysis. The tiled peptide approach can also be combined with alanine scans (17) in which alanine substitutions are introduced into the synthetic peptides and the direct contribution of each amino acid can be investigated. Maier et al. (18) described a high-throughput epitope-mapping screen of a recombinant peptide library consisting of a total of 2304 overlapping peptides of the vitamin D receptor, and recently Buus et al. (19) used in situ synthesis on microarrays to design and generate 70,000 peptides for epitope mapping of antibodies using a range of peptides with sizes from 4-mer to 20-mer.So far it has not been possible to investigate on- and off-target binding in a proteome-wide manner, but the emergence of new methods for in situ synthesis of peptides on ultra-dense arrays has made this achievable. Here, we describe the design and use of peptide arrays generated with parallel in situ photolithic synthesis (20) of a total of 2.1 million overlapping peptides covering all human proteins with overlapping peptides. Miniaturization of the peptide arrays (21) has led to improved density of the synthesized peptides and consequently has improved the resolution and coverage of the epitope mapping. This has allowed us to study the specificity and cross-reactivity of both monoclonal and polyclonal antibodies across the whole “epitome” with the use of both proteome-wide arrays and focused-content peptide arrays covering selected antigen sequences to precisely map the contribution of each amino acid of the target protein for binding recognition of the corresponding antibodies. The results show the usefulness of proteome-wide epitope mapping, showing a path forward for high-throughput analysis of antibody interactions.     

Resting Heart Rate Variability Predicts Safety Learning and Fear Extinction in an Interoceptive Fear Conditioning Paradigm

This study aimed to investigate whether interindividual differences in autonomic inhibitory control predict safety learning and fear extinction in an interoceptive fear conditioning paradigm. Data from a previously reported study (N = 40) were extended (N = 17) and re-analyzed to test whether healthy participants'' resting heart rate variability (HRV) - a proxy of cardiac vagal tone - predicts learning performance. The conditioned stimulus (CS) was a slight sensation of breathlessness induced by a flow resistor, the unconditioned stimulus (US) was an aversive short-lasting suffocation experience induced by a complete occlusion of the breathing circuitry. During acquisition, the paired group received 6 paired CS-US presentations; the control group received 6 explicitly unpaired CS-US presentations. In the extinction phase, both groups were exposed to 6 CS-only presentations. Measures included startle blink EMG, skin conductance responses (SCR) and US-expectancy ratings. Resting HRV significantly predicted the startle blink EMG learning curves both during acquisition and extinction. In the unpaired group, higher levels of HRV at rest predicted safety learning to the CS during acquisition. In the paired group, higher levels of HRV were associated with better extinction. Our findings suggest that the strength or integrity of prefrontal inhibitory mechanisms involved in safety- and extinction learning can be indexed by HRV at rest.     

Visualizing Typical Features of Breast Fibroadenomas Using Phase-Contrast CT: An Ex-Vivo Study

Background

Fibroadenoma is the most common benign solid breast lesion type and a very common cause for histologic assessment. To justify a conservative therapy, a highly specific discrimination between fibroadenomas and other breast lesions is crucial. Phase-contrast imaging offers improved soft-tissue contrast and differentiability of fine structures combined with the potential of 3-dimensional imaging. In this study we assessed the potential of grating-based phase-contrast CT imaging for visualizing diagnostically relevant features of fibroadenomas.

Materials and Methods

Grating-based phase-contrast CT was performed on six ex-vivo formalin-fixed breast specimens containing a fibroadenoma and three samples containing benign changes that resemble fibroadenomas using Talbot Lau interferometry and a polychromatic X-ray source. Phase-contrast and simultaneously acquired absorption-based 3D-datasets were manually matched with corresponding histological slices. The visibility of diagnostically valuable features was assessed in comparison with histology as the gold-standard.

Results

In all cases, matching of grating-based phase-contrast CT images and histology was successfully completed. Grating-based phase-contrast CT showed greatly improved differentiation of fine structures and provided accurate depiction of strands of fibrous tissue within the fibroadenomas as well as of the diagnostically valuable dilated, branched ductuli of the fibroadenomas. A clear demarcation of tumor boundaries in all cases was provided by phase- but not absorption-contrast CT.

Conclusions

Pending successful translation of the technology to a clinical setting and considerable reduction of the required dose, the data presented here suggest that grating-based phase-contrast CT may be used as a supplementary non-invasive diagnostic tool in breast diagnostics. Phase-contrast CT may thus contribute to the reduction of false positive findings and reduce the recall and core biopsy rate in population-based screening. Phase-contrast CT may further be used to assist during histopathological workup, offering a 3D view of the tumor and helping to identify diagnostically valuable tissue sections within large tumors.