In mammals, dosage compensation of X-linked genes is achieved by the transcriptional silencing of one X chromosome in the female (reviewed in ref. 1). This process, called X inactivation, is usually random in the embryo proper. In marsupials and the extra-embryonic region of the mouse, however, X inactivation is imprinted: the paternal X chromosome is preferentially inactivated whereas the maternal X is always active. Having more than one active X chromosome is deleterious to extra-embryonic development in the mouse. Here we show that the gene eed (embryonic ectoderm development), a member of the mouse Polycomb group (Pc-G) of genes, is required for primary and secondary trophoblast giant cell development in female embryos. Results from mice carrying a paternally inherited X-linked green fluorescent protein (GFP) transgene implicate eed in the stable maintenance of imprinted X inactivation in extra-embryonic tissues. Based on the recent finding that the Eed protein interacts with histone deacetylases, we suggest that this maintenance activity involves hypoacetylation of the inactivated paternal X chromosome in the extra-embryonic tissues.

Mammals co-exist with resident microbial ecosystem that is composed of an incredible number and diversity of bacteria, viruses and fungi. Owing to direct contact between resident microbes and mucosal surfaces, both parties are in continuous and complex interactions resulting in important functional consequences. These interactions govern immune homeostasis, host response to infection, vaccination and cancer, as well as predisposition to metabolic, inflammatory and neurological disorders. Here, we discuss recent studies on direct and indirect effects of resident microbiota on regulatory T cells (Tregs) and Th17 cells at the cellular and molecular level. We review mechanisms by which commensal microbes influence mucosa in the context of bioactive molecules derived from resident bacteria, immune senescence, chronic inflammation and cancer. Lastly, we discuss potential therapeutic applications of microbiota alterations and microbial derivatives, for improving resilience of mucosal immunity and combating immunopathology.

Many industrial processes used to produce chemicals and pharmaceuticals would benefit from enzymes that function under extreme conditions. Enzymes from extremophilic microorganisms have evolved to function in a variety of extreme environments, and bioprospecting for these microorganisms has led to the discovery of new enzymes with high tolerance to nonnatural conditions. However, bioprospecting is inherently limited by the diversity of enzymes evolved by nature. Protein engineering has also been successful in generating extremophilic enzymes by both rational mutagenesis and directed evolution, but screening for activity under extreme conditions can be difficult. This review examines the emerging synergy between bioprospecting and protein engineering in developing extremophilic enzymes. Specific topics include unnatural industrial conditions relevant to biocatalysis, biophysical properties of extremophilic enzymes, and industrially relevant extremophilic enzymes found either in nature or through protein engineering. 77 Annu. Rev. Chem. Biomol. Eng. 2012.3:77-102. Downloaded from www.annualreviews.org by University of Toronto on 11/15/12. For personal use only. Click here for quick links to Annual Reviews content online, including: • Other articles in this volume • Top cited articles • Top downloaded articles • Our comprehensive search Further ANNUAL REVIEWS T opt : optimum activity temperature 78 Liszka et al. Annu. Rev. Chem. Biomol. Eng. 2012.3:77-102. Downloaded from www.annualreviews.org by University of Toronto on 11/15/12. For personal use only. www.annualreviews.org • Enzymes Under Extreme Conditions 79 Annu. Rev. Chem. Biomol. Eng. 2012.3:77-102. Downloaded from www.annualreviews.org by University of Toronto on 11/15/12. For personal use only.greater potential for success. The answer to this question is not obvious and may depend strongly on the conditions required, the desired enzymatic reaction, and the type of enzyme used. EXTREME CONDITIONS RELEVANT TO INDUSTRIAL PROCESSES Conditions Found in NatureMicroorganisms exist in very different environments, and their enzymes and proteins have adapted to extreme temperatures, pressures, alkalinity/acidity, and/or osmolarity. Many of these extreme conditions mimic those found in industrial processes that currently employ enzymes or stand to benefit from them. Nature, therefore, is an abundant source of enzymes and proteins tolerant to extreme conditions. Extreme temperatures. Performing enzyme reactions at elevated temperatures has several potential advantages including higher substrate solubility, faster reaction rates, reduced risk of system contamination, lower solution viscosity, and increased solvent miscibility. However, there are many examples of enzymes used in processes that operate at lower temperatures as well, such as cold-active hydrolytic enzymes used in laundry detergents or for cleaning animal hides, proteases used for cleaning contact lenses, and pectinases for clarifying and extracting fruit juices (9). Mesophilic enzymes are less effective at...

Sorting nexins 1 (Snx1) and 2 (Snx2) are homologues of the yeast gene VPS5 that is required for proper endosome-to-Golgi trafficking. The prevailing thought is that Vps5p is a component of a retrograde trafficking complex called the retromer. Genetic and biochemical evidence suggest mammals may have similar complexes, but their biological role is unknown. Furthermore, if SNX1 and SNX2 belong to such complexes, it is not known whether they act together or separately. Herein, we show that mice lacking SNX1 or SNX2 are viable and fertile, whereas embryos deficient in both proteins arrest at midgestation. These results demonstrate that SNX1 and SNX2 have a highly redundant and necessary function in the mouse. The phenotype of Snx1 Ϫ/Ϫ ;Snx2embryos is very similar to that of embryos lacking another retromer homologue, H␤58. This finding suggests that SNX1/SNX2 and H␤58 function in the same genetic pathway, providing additional evidence for the existence of mammalian complexes that are structurally similar to the yeast retromer. Furthermore, the viability of Snx1 Ϫ/Ϫ and Snx2 Ϫ/Ϫ mice demonstrates that it is not necessary for SNX1 and SNX2 to act together. Electron microscopy indicates morphological alterations of apical intracellular compartments in the Snx1 Ϫ/Ϫ ;Snx2 Ϫ/Ϫ yolk-sac visceral endoderm, suggesting SNX1 and SNX2 may be required for proper cellular trafficking. However, tetraploid aggregation experiments suggest that yolk sac defects cannot fully account for Snx1 Ϫ/Ϫ ; Snx2 Ϫ/Ϫ embryonic lethality. Furthermore, endocytosis of transferrin and low-density lipoprotein is unaffected in mutant primary embryonic fibroblasts, indicating that SNX1 and SNX2 are not essential for endocytosis in all cells. Although the two proteins demonstrate functional redundancy, Snx1 ϩ/Ϫ ;Snx2 Ϫ/Ϫ mice display abnormalities not observed in Snx1 Ϫ/Ϫ ;Snx2 ϩ/Ϫ mice, revealing that SNX1 and SNX2, or their genetic regulation, are not equivalent. Significantly, these studies represent the first mutations in the mammalian sorting nexin gene family and indicate that sorting nexins perform essential functions in mammals.

A highly consistent trait of tumor stromal fibroblasts is the induction of the membrane-bound serine protease fibroblast activation protein-α (FAP), which is overexpressed on the surface of reactive stromal fibroblasts present within the stroma of the majority of human epithelial tumors. In contrast, FAP is not expressed by tumor epithelial cells or by fibroblasts or other cell types in normal tissues. The proteolytic activity of FAP, therefore, represents a potential pan-tumor target that can be exploited for the release of potent cytotoxins from inactive prodrugs consisting of an FAP peptide substrate coupled to a cytotoxin. To identify FAP peptide substrates, we used liquid chromatography tandem mass spectroscopy based sequencing to generate a complete map of the FAP cleavage sites within human collagen I derived gelatin. Positional analysis of the frequency of each amino acid at each position within the cleavage sites revealed FAP consensus sequences PPGP and (D/E)-(R/K)-G-(E/D)-(T/S)-G-P. These studies further demonstrated that ranking cleavage sites based on the magnitude of the LC/MS/MS extracted ion current predicted FAP substrates that were cleaved with highest efficiency. Fluorescence-quenched peptides were synthesized on the basis of the cleavage sites with the highest ion current rankings, and kinetic parameters for FAP hydrolysis were determined. The substrate DRGETGP, which corresponded to the consensus sequence, had the lowest K m of 21 μM. Overall the K m values were relatively similar for both high and low ranked substrates, whereas the k cat values differed by up to 100-fold. On the basis of these results, the FAP consensus sequences are currently being evaluated as FAPselective peptide carriers for incorporation into FAP-activated prodrugs.The growth of epithelial neoplasms requires the formation of a supporting tumor stroma to supply nutrients and growth factors for tumor cell survival and continued growth. This invasive growth is associated with characteristic changes in the supporting stroma that include induction of tumor blood vessel formation, the recruitment of reactive stromal † This work supported by funding from the Susan G. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript myofibroblasts, lymphocytes, and macrophages, the release of peptide signaling molecules and proteases, and the production of an altered extracellular matrix (1-5). The tumor stroma compartment represents a major component of the mass of most carcinomas, with 20-50% commonly seen in breast, lung, and colorectal cancers and reaching >90% in carcinomas that have desmoplastic reactions such as breast and pancreatic cancers (5, 6).Unlike malignant epithelial cells, activated tumor stromal fibroblasts are not transformed genetically and do not demonstrate the genetic and phenotypic heterogeneity seen in malignant cells. Reactive tumor stromal fibroblasts differ from fibroblasts of normal adult tissues in regard to morphology, gene expression profiles, and production of important biological ...