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The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids.

Objective: To perform a meta-analysis of migraine biomarkers in cerebrospinal fluid (CSF) and of corresponding blood concentrations. Methods: We conducted a systematic search for studies that measured biochemical compounds in CSF of chronic or episodic migraineurs and non-headache controls. Subsequent searches retrieved studies with blood measurements of selected CSF biomarkers. If a compound was assessed in three or more studies, results were pooled in a meta-analysis with standardised mean differences (SMD) as effect measures. Results: Sixty-two compounds were measured in 40 CSF studies. Most important results include: increased glutamate (five studies, SMD 2.22, 95% CI: 1.30, 3.13), calcitonin gene-related peptide (CGRP) (three studies, SMD: 3.80, 95% CI: 3.19, 4.41) and nerve growth factor (NGF) (three studies, SMD: 6.47, 95% CI: 5.55, 7.39) in chronic migraine patients and decreased b-endorphin (b-EP) in both chronic (four studies, SMD: -1.37, 95% CI: -1.80, -0.94) and interictal episodic migraine patients (three studies, SMD: -1.12, 95% CI: -1.65, -0.58). In blood, glutamate (interictal) and CGRP (chronic, interictal and ictal) were increased and b-EP (chronic, interictal and ictal) was decreased. Conclusions: Glutamate, b-EP, CGRP and NGF concentrations are altered in CSF and, except for NGF, also in blood of migraineurs. Future research should focus on the pathophysiological roles of these compounds in migraine.

Experimental Autoimmune Encephalomyelitis (EAE) is the most commonly used animal model for Multiple Sclerosis (MScl). CSF metabolomics in an acute EAE rat model was investigated using targetted LC–MS and GC–MS. Acute EAE in Lewis rats was induced by co-injection of Myelin Basic Protein with Complete Freund’s Adjuvant. CSF samples were collected at two time points: 10 days after inoculation, which was during the onset of the disease, and 14 days after inoculation, which was during the peak of the disease. The obtained metabolite profiles from the two time points of EAE development show profound differences between onset and the peak of the disease, suggesting significant changes in CNS metabolism over the course of MBP-induced neuroinflammation. Around the onset of EAE the metabolome profile shows significant decreases in arginine, alanine and branched amino acid levels, relative to controls. At the peak of the disease, significant increases in concentrations of multiple metabolites are observed, including glutamine, O-phosphoethanolamine, branched-chain amino acids and putrescine. Observed changes in metabolite levels suggest profound changes in CNS metabolism over the course of EAE. Affected pathways include nitric oxide synthesis, altered energy metabolism, polyamine synthesis and levels of endogenous antioxidants.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-011-0306-3) contains supplementary material, which is available to authorized users.

Review A practical guide to nano-LC troubleshootingCapillary LC is one of the most powerful analytical tools available for separation scientists. Its unique analytical properties are associated with numerous technical issues that may cause operation of such systems to be somehow troublesome. Because of that, a good experience in capillary LC troubleshooting is required to keep the system in shape and, in effect, to obtain reliable results. In this paper, we summarize the most important issues of the capillary systems, including void and dead volumes, leakages, sample injection, and a multidimensional LC approach. The aim of this paper was to provide practical advise on system diagnosis, and to present solutions to problems discussed. Also, several exemplary nano-LC separations are included to demonstrate some typical problems encountered in our daily work.

The filamentous fungus Penicillium chrysogenum harbors an astonishing variety of nonribosomal peptide synthetase genes, which encode proteins known to produce complex bioactive metabolites from simple building blocks. Here we report a novel non-canonical tetra-modular nonribosomal peptide synthetase (NRPS) with microheterogenicity of all involved adenylation domains towards their respective substrates. By deleting the putative gene in combination with comparative metabolite profiling various unique cyclic and derived linear tetrapeptides were identified which were associated with this NRPS, including fungisporin. In combination with substrate predictions for each module, we propose a mechanism for a ‘trans-acting’ adenylation domain.

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A novel method for peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) is presented. A stable isotope label introduced in the peptide N-terminus by derivatization, using a 1:1 mixture of acetic anhydride and deuterated acetic anhydride, allows for easy and unambiguous identification of ions belonging either to the N- or the C-terminal ion series in the product ion spectrum, making sequence assignment significantly simplified. The good performance of this technique was shown by successful sequencing of the contents of several peptide maps. A similar approach was recently applied to nanoelectrospray ionization (nanoESI) and nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS). The MALDI-TOF/TOF technique allows for fast, direct sequencing of modified peptides in proteomics samples, and is complementary to the nanoESI and nanoLC/MS/MS approaches.