92 research outputs found
Therapy: Metformin takes a new route to clinical efficacy.
International audienceMetformin is currently the first-line treatment option for patients with type 2 diabetes mellitus, yet its mechanism of action remains uncertain. A new study reveals the important role for the activation of a duodenal AMPK-dependent neuronal pathway in the acute antihyperglycaemic effect of metformin and the inhibition of hepatic glucose production
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Rational optimization of tolC as a powerful dual selectable marker for genome engineering
Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by âŒ40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is âŒ10-fold more efficient at achieving allelic diversity than previous best practices
Extracellular Ca2+ Is Required for Fertilization in the African Clawed Frog, Xenopus laevis
The necessity of extracellular Ca2+ for fertilization and early embryonic development in the African clawed frog, Xenopus laevis, is controversial. Ca2+ entry into X. laevis sperm is reportedly required for the acrosome reaction, yet fertilization and embryonic development have been documented to occur in high concentrations of the Ca2+ chelator BAPTA. Here we sought to resolve this controversy.Using the appearance of cleavage furrows as an indicator of embryonic development, we found that X. laevis eggs inseminated in a solution lacking added divalent cations developed normally. By contrast, eggs inseminated in millimolar concentrations of BAPTA or EGTA failed to develop. Transferring embryos to varying solutions after sperm addition, we found that extracellular Ca2+ is specifically required for events occurring within the first 30 minutes after sperm addition, but not after. We found that the fluorescently stained sperm were not able to penetrate the envelope of eggs inseminated in high BAPTA, whereas several had penetrated the vitelline envelope of eggs inseminated without a Ca2+ chelator, or with BAPTA and saturating CaCl2. Together these results indicate that fertilization does not occur in high concentrations of Ca2+ chelators. Finally, we found that the jelly coat includes >5 mM of readily diffusible Ca2+.Taken together, these data are consistent with requirement of extracellular Ca2+ for fertilization. Based on our findings, we hypothesize that the jelly coat surrounding the egg acts as a reserve of readily available Ca2+ ions to foster fertilization in changing extracellular milieu
Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli
The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as noncoding information for gene regulation and genome maintenance. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU codons, but the remaining 13 ârecalcitrantâ AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multidimensional âsafe replacement zoneâ (SRZ) within which alternative codons are more likely to be viable. To evaluate synonymous and nonsynonymous alternatives to essential AGRs further, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild-type allele, allowing us to evaluate exhaustively the fitness impact of all 64 codon alternatives. Using this method, we confirmed the relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes.United States. Department of Energy (DE-FG02-02ER63445
An inclusive search for free quarks at PEP
Abstract We report the results of a search for fractionally charged particles in e + e â reactions at a center of mass energy of 29 GeV. We find no evidence for such particles and present upper limitts on R q = Ï q q X/Ï ÎŒÎŒ for change 1 3 e and 2 3 e which range from 1 to 8 Ă 10 â2 for mass up to 12 GeV/ c 2
Direct Observation of Longitudinally Polarised W Bosons
The three different helicity states of W bosons, produced in the reaction
e+e- -> W+W- -> l nu q q~ are studied using leptonic and hadronic W decays at
sqrt{s}=183GeV and 189GeV. The W polarisation is also measured as a function of
the scattering angle between the W- and the direction of the e- beam. The
analysis demonstrates that W bosons are produced with all three helicities, the
longitudinal and the two transverse states. Combining the results from the two
center-of-mass energies and with leptonic and hadronic W decays, the fraction
of longitudinally polarised W bosons is measured to be 0.261 +/- 0.051(stat.)
+/- 0.016(syst.) in agreement with the expectation from the Standard Model
K0s K0s Final State in Two-Photon Collisions and Implications for Glueballs
The K0s K0s final state in two-photon collisions is studied with the L3
detector at LEP. The mass spectrum is dominated by the formation of the
f_2'(1525) tensor meson in the helicity-two state with a two-photon width times
the branching ratio into K Kbar of 76 +- 6 +- 11 eV. A clear signal for the
formation of the f_J(1710) is observed and it is found to be dominated by the
spin-two helicity-two state. No resonance is observed in the mass region around
2.2 GeV and an upper limit of 1.4 eV at 95% C.L. is derived for the two-photon
width times the branching ratio into K0s K0s for the glueball candidate
xi(2230)
Formation of the in Two-Photon Collisions at LEP
The two-photon width of the meson has been
measured with the L3 detector at LEP. The is studied in the decay
modes , KK, KK,
KK, , , and
using an integrated luminosity of 140 pb at GeV and
of 52 pb at GeV. The result is
(BR) keV. The dependence of the cross section is studied for
GeV. It is found to be better described by a Vector Meson
Dominance model form factor with a J-pole than with a -pole. In addition,
a signal of events is observed at the mass. Upper limits
for the two-photon widths of the , , and are also
given
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