We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.

We present here new evidence of cross-cultural agreement in the judgment of facial expression, Subjects in 10 cultures performed a more complex judgment task than has been used in previous cross-cultural studies. Instead of limiting the subjects to selecting only one emotion term for each expression, this task allowed them to indicate that multiple emotions were evident and the intensity of each emotion. Agreement was very high across cultures about which emotion was the most intense. The 10 cultures also agreed about the second most intense emotion signaled by an expression and about the relative intensity among expressions of the same emotion. However, cultural differences were found in judgments of the absolute level of emotional intensity.

The specification of SBML Level 1 is freely available from http://www.sbml.org/

The cell cycle-dependent nucleocytoplasmic transport of proteins is predominantly regulated by CDK kinase activities; however, it is currently difficult to predict the proteins thus regulated, largely because of the low prediction efficiency of the motifs involved. Here, we report the successful prediction of CDK1-regulated nucleocytoplasmic shuttling proteins using a prediction system for nuclear localization signals (NLSs). By systematic amino acid replacement analyses in budding yeast, we created activity-based profiles for different classes of importin-␣-dependent NLSs that represent the functional contributions of different amino acids at each position within an NLS class. We then developed a computer program for prediction of the classical importin-␣/␤ pathway-specific NLSs (cNLS Mapper, available at http//nls-mapper.iab.keio.ac.jp/) that calculates NLS activities by using these profiles and an additivity-based motif scoring algorithm. This calculation method achieved significantly higher prediction accuracy in terms of both sensitivity and specificity than did current methods. The search for NLSs that overlap the consensus CDK1 phosphorylation site by using cNLS Mapper identified all previously reported and 5 previously uncharacterized yeast proteins (Yen1, Psy4, Pds1, Msa1, and Dna2) displaying CDK1-and cell cycle-regulated nuclear transport. CDK1 activated or repressed their nuclear import activity, depending on the position of CDK1-phosphorylation sites within NLSs. The application of this strategy to other functional linear motifs should be useful in systematic studies of protein-protein networks.CDK ͉ computational method ͉ nuclear localization signal ͉ nuclear import ͉ phosphorylation R egulated nuclear import controls the nuclear function of many proteins that shuttle between the nucleus and cytoplasm or other cellular compartments in response to certain physiological conditions or extracellular stimuli (1). Nucleocytoplasmic shuttling comprises both nuclear import and nuclear export activities. Nuclear import is mediated by the interaction of nuclear localization signals (NLSs) with transport receptor importins, among which the importin-␣ family recognizes most major classes of NLSs with 1 or 2 basic stretches, called the classical NLSs (2-4). Six different classes of monopartite and bipartite NLSs bind to distinct binding grooves of importin-␣ (5-7). Nuclear export is mediated by nuclear export signals (NESs), which are recognized by Crm1/exportin or Msn5 in yeast (1, 8). The regulation of these 2 antagonistic activities directs a protein to either the nucleus or the cytoplasm. In general, these activities are regulated by the modification of the NLSs/NESs, by intermolecular or intramolecular interactions that lead to the structural masking of NLSs/NESs, or by the supply of an NLS/NES by another protein (9).Phosphorylation within or around an NLS is the usual strategy for the regulated nuclear transport of a protein (4, 10-13). It has been shown that acidic amino acids adjacent to the basic core ...