β‑Lactamases Evolve against Antibiotics by Acquiring Large Active-Site Electric Fields

A compound bound covalently to an enzyme active site can act either as a substrate if the covalent linkage is readily broken up by the enzyme or as an inhibitor if the bond dissociates slowly. We tracked the reactivity of such bonds associated with the rise of the resistance to penicillin G (PenG) in protein evolution from penicillin-binding proteins (PBPs) to TEM β-lactamases and with the development of avibactam (Avb) to overcome the resistance. We found that the ester linkage in PBP–PenG is resistant to hydrolysis mainly due to the small electric fields present in the protein active site. Conversely, the same linkage in the descendant TEM–PenG experiences large electric fields that stabilize the more charge-separated transition state and thus lower the free energy barrier to hydrolysis. Specifically, the electric fields were improved from −59 to −140 MV/cm in an ancient evolution dating back billions of years, contributing 5 orders of magnitude rate acceleration. This trend continues today in the nullification of newly developed antibiotic drugs. The fast linkage hydrolysis acquired from evolution is counteracted by the upgrade of PenG to Avb whose linkage escapes from the hydrolysis by returning to a low-field environment. Using the framework of electrostatic catalysis, the electric field, an observable from vibrational spectroscopy, provides a unifying physical metric to understand protein evolution and to guide the design of covalent drugs

Additional file 1: of Visualization of Miscanthus × giganteus cell wall deconstruction subjected to dilute acid pretreatment for enhanced enzymatic digestibility

Supplementary supporting data. Figure S1. Mass loss of dilute acid pretreated M. × giganteus under various conditions. Figure S2. TEM micrographs of M. × giganteus Sf after pretreatment with 1% H2SO4 at 170°C for 30 min. Figure S3. Explanation of void space calculation. Figure S4. Raman image of lignin distribution in treated M. × giganteus at 170°C, 1% H2SO4 for 30 min calculated by integrating over the spectral range from 1575 to 1620 cm−1

Copper-Catalyzed Cascade Radical Addition–Cyclization Halogen Atom Transfer between Alkynes and Unsaturated α‑Halogenocarbonyls

A Cu-catalyzed cascade radical addition/cyclization/halogen atom transfer between alkynes and α-halogeno-γ, δ-unsaturated carbonyl compounds for the synthesis of various substituted cyclopentenes is described. Since up to four Csp³–Csp² bonds, two Csp³–Br bonds, and two carbocycles can be established in a single reaction, this 100% atom-efficient reaction exhibits the advantages of wide substrate scope, high functional group tolerance, and step-economics, and it offers an entry of the atom transfer radical addition/cyclization (tandem ATRA-ATRC) process to the synthesis of substituted cyclopentenes

Isolation of a Heavier Cyclobutadiene Analogue: 2,4-Digerma-1,3-diphosphacyclobutadiene

The heavier cyclobutadiene analogue 2,4-digerma-1,3-diphosphacyclobutadiene ([L¹₂Ge₂P₂], <b>4</b>; L¹ = CH­{(CMe)­(2,6-^<i>i</i>Pr₂C₆H₃N)}₂), featuring a planar Ge₂P₂ four-membered ring, has been synthesized via the elimination of carbon monoxide from the corresponding phosphaketenyl germylene [L¹GePCO] (<b>2</b>) under UV irradiation

Isolation of a Heavier Cyclobutadiene Analogue: 2,4-Digerma-1,3-diphosphacyclobutadiene

The heavier cyclobutadiene analogue 2,4-digerma-1,3-diphosphacyclobutadiene ([L¹₂Ge₂P₂], <b>4</b>; L¹ = CH­{(CMe)­(2,6-^<i>i</i>Pr₂C₆H₃N)}₂), featuring a planar Ge₂P₂ four-membered ring, has been synthesized via the elimination of carbon monoxide from the corresponding phosphaketenyl germylene [L¹GePCO] (<b>2</b>) under UV irradiation

The τCstF-64 Polyadenylation Protein Controls Genome Expression in Testis

<div><p>The τCstF-64 polyadenylation protein (gene symbol <em>Cstf2t</em>) is a testis-expressed orthologue of CstF-64. Mice in which <em>Cstf2t</em> was knocked out had a phenotype that was only detected in meiotic and postmeiotic male germ cells, giving us the opportunity to examine CstF-64 function in an isolated developmental system. We performed massively parallel clonally amplified sequencing of cDNAs from testes of wild type and <em>Cstf2t^−/−</em> mice. These results revealed that loss of τCstF-64 resulted in large-scale changes in patterns of genome expression. We determined that there was a significant overrepresentation of RNAs from introns and intergenic regions in testes of <em>Cstf2t^−/−</em> mice, and a concomitant use of more distal polyadenylation sites. We observed this effect particularly in intronless small genes, many of which are expressed retroposons that likely co-evolved with τCstF-64. Finally, we observed overexpression of long interspersed nuclear element (LINE) sequences in <em>Cstf2t^−/−</em> testes. These results suggest that τCstF-64 plays a role in 3′ end determination and transcription termination for a large range of germ cell-expressed genes.</p> </div

ISGs are down-regulated and have increased read-through in <i>Cstf2t^−/−</i> mouse testes.

<p>(<b>A</b>) Cumulative frequency of microarray log₂ mRNA expression changes of <i>Cstf2^−/−</i> (KO25) versus wild type (WT25) mouse testis at 25 dpp. Short genes were defined as the lowest 20% in length, with a cutoff of 6658 bp or shorter. Indicated are long multi-exon genes (11,451 genes, blue), short multi-exon genes (2,324 genes, green), and short single-exon genes (541 genes, red). There are 276 short single-exon genes in the region between -2 and 0 log₂ expression change. P values are 4.2×10⁻⁴ between short single-exon and short multi-exon genes and 1.0×10⁻¹⁵ between long multi-exon and short-multi-exon genes by a K-S test. (<b>B</b>) qRT-PCR was performed using primers specific for the indicated genes (see Table S1) normalized to <i>Rps16</i>. Each bar represents the amount (in percent) of the indicated mRNA in 25 dpp <i>Cstf2t^−/−</i> mouse testis RNA compared to wild type. The asterisks indicate values that are significantly different (P<0.001) from Rsp16 and Actb by ANOVA (Bonferroni multiple comparisons test). (<b>C</b>) Polyadenylation read-through assay. Random-primed cDNA is made from RNA from wild type or <i>Cstf2t^−/−</i> mouse testes. qRT-PCR is then performed using primer pairs within the body of the gene (“Upstream”) or downstream of the polyadenylation site (“Downstream”). An increase in read-through is measured as in increase in the downstream value compared to the upstream value in <i>Cstf2t^−/−</i> mice after normalization. (<b>D</b>) Read-through increases for ISGs in <i>Cstf2t^−/−</i> mouse testes. The polyadenylation read-through assay described in (C) was performed on the indicated genes and normalized to 1.0 in the wild type mice. The asterisk (P<0.05) and double asterisk (P<0.01) indicate values that differ significantly from the wild type by a Student's t-test.</p

High-throughput cDNA sequencing (RNA-seq) finds significant differences between wild type and <i>Cstf2t^−/−</i> mouse testis RNAs.

<p>(<b>A</b>) RNA was pooled from testes of five 25 dpp mice of either wild type or <i>Cstf2t^−/−</i> genotype, cDNA synthesized, and high-throughput sequencing performed (see Materials and Methods). (<b>B</b>) RNA-seq from wild type (∼55,000 reads) and <i>Cstf2t^−/−</i> (∼77,000 reads) mouse testis samples were not biased when mapped to the mouse genome. 454 sequencing reads were mapped to the mouse genome (Mouse Genome Assembly version mm8) using BLAT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048373#pone.0048373-Kent1" target="_blank">[24]</a>. Pie graphs show that similar proportions of reads mapped to either unique genomic regions (blue), multiple regions (non-unique, green), or could not be mapped to known regions (unmapped, tan) in samples from wild type or <i>Cstf2t^−/−</i> mouse testes. The proportion of uniquely mapped reads has no statistical difference between wild type and Cstf2t−/− mice (85.4% vs. 85.2%; P = 0.14, Fisher's exact test). (<b>C</b>) Introns and intergenic regions were more highly expressed in testes of <i>Cstf2t^−/−</i> mice, while exons were less expressed. Pie graphs show percentages of reads that were uniquely mapped to different regions of the genome for wild type and <i>Cstf2t^−/−</i> mice. Exon (blue), reads fully aligned to exons; exon & intron (green), reads aligned to both exonic and intronic regions; intron (tan), reads fully aligned to introns; 3′ UTR-ext (orange) and 5′ UTR-ext (purple), reads aligned to within 4 kb downstream of 3′ UTR or 1 kb upstream of the 5′ UTR, respectively; intergenic (grey), reads aligned to regions not within annotated genes or their extended regions. The difference of proportion of reads mapped to different genomic regions is significant: P <10–323 for both the intergenic region and intronic region (Fisher's exact test, exon region used as control).</p

Relative usage of distal poly(A) sites (RUD) decreases throughout testis development, but less so in <i>Cstf2t^−/−</i> mouse testes.

<p>The Y-axis is the mean RUD score which reflects relative usage of distal poly (A) sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048373#pone.0048373-Ji2" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048373#pone.0048373-Ji3" target="_blank">[27]</a>. RUD values were based on three replicates for the 17, 22 and 25 dpp time points and two replicates for the 85 dpp time point. Error bars indicate the standard deviation (P values for T-test are 0.69, 0.32, 0.006 and 0.15 for 17, 22, 25 and 85 dpp, respectively comparing KO and WT). A step-wise decrease can be seen, indicating progressive shortening of 3′ UTRs or more usage of proximal poly(A) sites from 17 to 85 days postpartum (dpp). However, <i>Cstf2t^−/−</i> differs from wild type starting at 25 dpp through 85 dpp.</p

Manganese-Doped Carbon Dots for Magnetic Resonance/Optical Dual-Modal Imaging of Tiny Brain Glioma

Brain gliomas are life-threatening diseases with low survival rates. Early detection and accurate intraoperative location of brain gliomas is vital to improving the prognosis. Herein, we synthesized manganese (Mn)-doped carbon dots (CDs) as magnetic resonance (MR)/optical dual-modal imaging nanoprobes by a one-pot green microwave-assisted route. These ultra-small-sized Mn-doped CDs possess distinct excitation-dependent photoluminescent emissions, high <i>r</i>₁ relaxivity, and low cytotoxicity. The in vivo MR imaging and ex vivo optical imaging of mouse brain with tiny glioma demonstrate that the Mn-doped CDs could lead to an enhanced MR <i>T</i>₁ contrast effect in the tiny brain glioma region, disclosing the great promise of these Mn-doped CDs as MR/optical dual-modal imaging nanoprobes for detection and intraoperative location of tiny brain gliomas