A New TASK for Dipeptidyl Peptidase-like Protein 6

Dipeptidyl Peptidase-like Protein 6 (DPP6) is widely expressed in the brain where it co-assembles with Kv4 channels and KChIP auxiliary subunits to regulate the amplitude and functional properties of the somatodendritic A-current, I_SA. Here we show that in cerebellar granule (CG) cells DPP6 also regulates resting membrane potential and input resistance by increasing the amplitude of the I_K(SO) resting membrane current. Pharmacological analysis shows that DPP6 acts through the control of a channel with properties matching the K_2P channel TASK-3. Heterologous expression and co-immunoprecipitation shows that DPP6 co-expression with TASK-3 results in the formation of a protein complex that enhances resting membrane potassium conductance. The co-regulation of resting and voltage-gated channels by DPP6 produces coordinate shifts in resting membrane potential and A-current gating that optimize the sensitivity of I_SA inactivation gating to subthreshold fluctuations in resting membrane potential.     

Comparison and optimization of ancient DNA extraction

Ancient DNA analyses rely on the extraction of the tiny amounts of DNA remaining in samples that are hundreds to tens of thousands of years old. Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica. We have compared a number of these methods using quantitative PCR and then optimized each step of the most promising method. We found that most chemicals routinely added to ancient DNA extraction buffers do not increase, and sometimes even decrease, DNA yields. Consequently, our optimized method uses a buffer consisting solely of EDTA and proteinase K for bone digestion and binding DNA to silica via guanidinium thiocyanate for DNA purification. In a comparison with published methods, this minimalist approach, on average, outperforms all other methods in terms of DNA yields as measured using quantitative PCR. We also found that the addition of bovine serum albumin (BSA) to the PCR helps to overcome inhibitors in ancient DNA extracts. Finally, we observed a marked difference in the performance between different types of DNA polymerases, as measured by amplification success.     

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.     

The marine cyanobacterium Crocosphaera watsonii WH8501 synthesizes the compatible solute trehalose by a laterally acquired OtsAB fusion protein

Compatible solutes are small organic molecules that are involved in the acclimation to various stresses such as temperature and salinity. Marine or moderate halotolerant cyanobacteria accumulate glucosylglycerol, while cyanobacteria with low salt tolerance (freshwater strains) usually accumulate sucrose or trehalose as the main compatible solutes. The screening of the genome of the marine, unicellular N(2) -fixing cyanobacterium Crocosphaera watsonii WH8501 revealed that instead of genes for glucosylglycerol biosynthesis, a fusion protein for the synthesis of trehalose was found that displayed similarities to trehalose-phosphate-synthase and -phosphatase (OtsAB pathway) from enterobacteria. Accordingly, cells of Crocosphaera showed salt-stimulated expression of the otsAB gene as well as a salt-dependent trehalose accumulation. The biochemical characterization of recombinant full-length OtsAB and truncated OtsB versions revealed that the otsAB gene in Crocosphaera encodes for an active trehalose-phosphate-synthase/phosphatase fusion protein. Genes coding for such proteins were not found in the genomes of other cyanobacteria but were present in many other, non-related marine bacteria, suggesting that otsAB might have been acquired by lateral gene transfer into the Crocosphaera genome.     

Correlation between time reversal symmetry and chirality in certain molecular processes

If a molecule is identified not only by its static spatial constructions, but also by the motions at the sub-molecular level, application of time reversal symmetry operation to a certain molecule could lead to another distinguishable from the original in the sense of sub-molecular motions, a phenomenon now defined as time reversal isomerism. Assessment of the consideration of certain enantiomers as distinguishable time reversal isomers is suggested in order to evoke a comprehensive interpretation of a likely correlation between the two types of isomerisms. The conceptual basis of a connection between absolute asymmetric synthesis under the influence of external fields and the intrinsic time reversal symmetry violation at the molecular level is also established to encourage new experimental investigations on this theme. Chirality 10:289–293, 1998. © 1998 Wiley-Liss, Inc.     

Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants

To elucidate the history of living and extinct elephantids, we generated 39,763 bp of aligned nuclear DNA sequence across 375 loci for African savanna elephant, African forest elephant, Asian elephant, the extinct American mastodon, and the woolly mammoth. Our data establish that the Asian elephant is the closest living relative of the extinct mammoth in the nuclear genome, extending previous findings from mitochondrial DNA analyses. We also find that savanna and forest elephants, which some have argued are the same species, are as or more divergent in the nuclear genome as mammoths and Asian elephants, which are considered to be distinct genera, thus resolving a long-standing debate about the appropriate taxonomic classification of the African elephants. Finally, we document a much larger effective population size in forest elephants compared with the other elephantid taxa, likely reflecting species differences in ancient geographic structure and range and differences in life history traits such as variance in male reproductive success.     

Andromeda: a peptide search engine integrated into the MaxQuant environment

A key step in mass spectrometry (MS)-based proteomics is the identification of peptides in sequence databases by their fragmentation spectra. Here we describe Andromeda, a novel peptide search engine using a probabilistic scoring model. On proteome data, Andromeda performs as well as Mascot, a widely used commercial search engine, as judged by sensitivity and specificity analysis based on target decoy searches. Furthermore, it can handle data with arbitrarily high fragment mass accuracy, is able to assign and score complex patterns of post-translational modifications, such as highly phosphorylated peptides, and accommodates extremely large databases. The algorithms of Andromeda are provided. Andromeda can function independently or as an integrated search engine of the widely used MaxQuant computational proteomics platform and both are freely available at www.maxquant.org. The combination enables analysis of large data sets in a simple analysis workflow on a desktop computer. For searching individual spectra Andromeda is also accessible via a web server. We demonstrate the flexibility of the system by implementing the capability to identify cofragmented peptides, significantly improving the total number of identified peptides.