A fast and simple, solvent-free matrix deposition protocol was developed for positive ionization mode phospholipid analysis in tissues. Finely ground 2,5-dihydroxybenzoic acid was deposited onto sagittal mouse brain sections using a dry-coating technique, in which solid matrix particles were filtered directly onto the tissue through a 20-m stainless steel sieve. Phospholipid signals were obtained directly off these sections, allowing acquisition of high-resolution MS images. These images were compared to those from serial sections that were spray-coated with a thin-layer chromatography (TLC) reagent sprayer. Signals obtained from the dry matrix deposition method were comparable to those from spray-coated sections, producing identical localization patterns with a simpler and faster sample preparation with virtually no analyte delocalization. This approach was found to yield highly reproducible results, eliminating much of the variance caused by operator differences, and making it an attractive alternative to the currently used matrix application methods. (J

Microbially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, longchain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli.Microbially derived free fatty acids (FFAs) are attractive intermediates for producing a wide range of high-energy-density biofuels from sustainable carbon sources, such as biomass (34). FFAs can be extracted from culture medium and catalytically converted to esters or alkanes (48, 55). Alternatively, enzymatic pathways exist for intracellular conversion to esters (42, 81), olefins (10, 59, 75), alkanes (78), or fatty aldehydes and fatty alcohols (22,81,82). These pathways can either be exploited in their native host or heterologously expressed in a genetically pliable microorganism (3). The physical and chemical properties of the resulting products are dependent on chain length and hydrophobicity; however, medium-chainlength (8-to 14-carbon) methyl esters, olefins, and alkanes exhibit many properties analogous to those of diesel and jet fuel and are therefore potential drop-in replacements (44,61).Several studies have demonstrated FFA overproduction in Escherichia coli (19,48,52,81,83). In each, the key strain modifications included overexpression of one or more cytosolic acyl-acyl carrier protein (ACP) thioesterases and deletion of fadD, or both fadD and fadE, which encode an acyl-coenzyme A (CoA) synthetase and acyl-CoA dehydrogenase, respectively. Overexpression of an acyl-ACP thioesterase depletes the level of acyl-ACP intermediates, which inhibit via feedback enzymes of fatty acid bi...

Early pregnancy failure often arises due to defects that occur before, during, or immediately after implantation. Even with in vitro fertilization and embryo-transfer techniques, implantation rates remain low in humans, most often as a result of embryos being transferred into nonreceptive uteri ( 1 ). Studying the molecular interactions that regulate implantation will provide a better understanding of these signaling pathways, eventually leading to new approaches to prevent implantation failure.There is evidence that phospholipid metabolism and signaling infl uences early pregnancy events ( 2 ). Phospholipids are vital structural and regulatory components of biological membranes and serve as precursors for many active biomolecules, such as eicosanoids and lysophospholipids ( 3, 4 ). Prostaglandins, one major group of eicosanoid lipid molecules, are produced from arachidonic acid (AA) that is released from membrane phospholipids by phospholipase A 2 . The released AA is acted upon by cyclooxygenases (COXs) to form prostaglandin H 2 , which is then converted to various prostaglandins by specifi c

Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both fieldcollected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus-bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.

Nanoparticle biological activity, biocompatibility and fate can be directly affected by layers of readily adsorbed host proteins in biofluids. Here, we report a study on the interactions between human blood plasma proteins and nanoparticles with a controlled systematic variation of properties using 18O-labeling and LC-MS-based quantitative proteomics. We developed a novel protocol to both simplify isolation of nanoparticle bound proteins and improve reproducibility. LC-MS analysis identified and quantified 88 human plasma proteins associated with polystyrene nanoparticles consisting of three different surface chemistries and two sizes, as well as, for four different exposure times (for a total of 24 different samples). Quantitative comparison of relative protein abundances was achieved by spiking an 18O-labeled “universal” reference into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantification across the entire sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive patterns that classified the nanoparticles based on their surface properties and size. In addition, temporal data indicated that the formation of the stable protein corona was at equilibrium within 5 min. The comprehensive quantitative proteomics results obtained in this study provide rich data for computational modeling and have potential implications towards predicting nanoparticle biocompatibility.

Immunophilin FK506-binding protein 52 (FKBP52) is a cochaperone that binds to the progesterone receptor (PR) to optimize progesterone (P 4 )-PR signaling. We recently showed that Fkbp52-deficient (Fkbp52 −/− ) mice have reduced uterine PR responsiveness and implantation failure which is rescued by excess P 4 supplementation in a genetic background-dependent manner. This finding led us to hypothesize that FKBP52 has functions in addition to optimizing PR activity. Using proteomics analysis, we found that uterine levels of peroxiredoxin-6 (PRDX6), a unique antioxidant, are significantly lower in Fkbp52 −/− mice than in WT and PR-null (Pgr −/− ) mice. We also found that Fkbp52 −/− mice with reduced uterine PRDX6 levels are susceptible to paraquat-induced oxidative stress (OS), leading to implantation failure even with P 4 supplementation. The same dose of paraquat did not interfere with implantation in WT mice. Moreover, treatment with antioxidants α-tocopherol and N-acetylcysteine (NAC) attenuated paraquat-induced implantation failure in P 4 -treated Fkbp52 −/− mice. Functional analyses using mouse embryonic fibroblasts show that Fkbp52 deficiency associated with reduced PRDX6 levels promotes H 2 O 2 -induced cell death, which is reversed by the addition of NAC or by forced expression of PRDX6, suggesting that Fkbp52 deficiency diminishes the threshold against OS by reducing PRDX6 levels. These findings provide evidence that heightened uterine OS in Fkbp52 −/− females with reduced PRDX6 levels induces implantation failure even in the presence of excess P 4 . This study shows that FKBP52-PRDX6 signaling protects pregnancy from overt OS.embryo implantation | mouse | uterus

Mass spectrometry (MS) is an excellent technology for molecular imaging because of its high data dimensionality. MS can monitor thousands of individual molecular data channels measured as mass-to-charge (m/z). We describe the use of matrix-assisted laser desorption/ionization (MALDI) MS for the image analysis of proteins, peptides, lipids, drugs, and metabolites in tissues. We discuss the basic instrumentation and sample preparation methods needed to produce high-resolution images and high image reproducibility. Matrix-addition protocols are briefly discussed along with normal operating procedures, and selected biological and medical applications of MALDI imaging MS are described. We give examples of both two- and three-dimensional imaging, including normal mouse embryo implantation, sperm maturation in mouse epididymis, protein distributions in brain sections, protein alterations as a result of drug administration, and protein changes in brain due to neurodegeneration and tumor formation. Advantages of this technology and future challenges for its improvement are discussed.

A reciprocal interaction between the implantation-competent blastocyst and receptive uterus is an absolute requirement for implantation, a process crucial for pregnancy success. A comprehensive understanding of this interaction has yet to be realized. One major difficulty in clearly defining this discourse is the complexity of the implantation process involving heterogeneous cell types of both the uterus and blastocyst, each endowed with unique molecular signatures that show dynamic changes during the course of pregnancy. Whereas gene expression studies by in situ hybridization or immunohistochemistry have shown differential expression patterns of specific genes during implantation, there is no report how numerous signaling proteins are spatially displayed at specific times and stages of implantation in the context of blastocyst-uterine juxtaposition. Using in situ imaging (matrix assisted laser desorption/ionization) mass spectrometry directly on uterine sections, here we provide molecular composition, relative abundance, and spatial distribution of a large number of proteins during the periimplantation period. This approach has allowed us for the first time to generate in situ proteome profiles of implantation and interimplantation sites in mice in a region- and stage-specific manner with the progression of implantation. This application is reliable because patterns of expression of several proteins displayed by in situ imaging mass spectrometry correlate well with in situ hybridization results. More interestingly, the use of this approach has provided new insights regarding uterine biology of cytosolic phospholipase A(2alpha) null females that show implantation defects.