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Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6- d eoxy- D - a ltro- h eptose and GDP-6-O M e- L - g luco- h eptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes—C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC—are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad, and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134, and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization although DdahC preferred the pre-epimerized substrate and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesize novel sugars useful as glycobiology tools.

Background The purpose of this study was to compare short-term side effects of aspirin (ASA) 325 mg vs ASA 81 mg orally twice daily (PO BID) when used as thromboembolic prophylaxis following primary total joint arthroplasty. Methods A 1-year prospective cohort study was performed on 643 primary unilateral total joint arthroplasty patients operated on by a single surgeon. Two hundred eighty-two patients were administered ASA 325 mg PO BID and 361 patients were administered ASA 81 mg PO BID for 1 month. A questionnaire assessing the side effects of ASA intake was administered 1 month postoperatively. Results The overall rate of gastrointestinal side effects (GI upset and nausea) was 1.9%, but ASA 325 mg had a higher rate 9/282 (3.2%) than ASA 81 mg 3/361 (0.8%), P = .04. Overall GI bleeding was 0.9%, with 2/282 (0.7%) in the ASA 325 mg group, vs 4/361 (1.1%) in the ASA 81 mg group, P = .70. One patient in the ASA 81 mg group (0.3%) developed a deep vein thrombosis. No patient developed pulmonary embolism, periprosthetic joint infection, tinnitus, wheezing and/or shortness of breath, chest pain, or headaches. In the ASA 325 mg group, 9/282 (3.2%) discontinued ASA and in the ASA 81 mg group, 8/361 (2.2%) discontinued ASA, P = .47. Four patients in the ASA 325 group (1.4%) changed to ASA 81 mg. Conclusion ASA 81 mg is associated with significantly less GI distress and nausea compared with ASA 325 mg. GI bleeding was equally prevalent between the 2 dosing regimens, so patients need to be informed of this risk regardless of the ASA dose.

Background Sexual limitations in the setting of total knee arthroplasty (TKA) are poorly understood. Methods Surveys were designed to assess preoperative and postoperative sexual function, and limitations were retrospectively administered to 91 sexually active TKA patients at an average of 2.1 years (range, 0.5-4.0) after surgery. Preoperative and postoperative responses were compared using 1-tailed and 2-proportion z tests, with P Results Before TKA, sexual quality and/or frequency was limited in 45% of patients because of their knee. Patients experienced an average 17.1 months (range, 0-60) of sexual limitations before surgery, resulting largely from pain (87%) and diminished range of motion or flexibility (44%). Fifty-five percent of patients reported the need to change their sexual positions to accommodate their knee, with 97% of these patients indicating the need to avoid kneeling during sex. Postoperatively, fewer patients had to adjust their sexual positions to accommodate their knee (55% vs 28%, P = .0005), and avoid bearing weight on the afflicted knee during sex (97% vs 79%, P = .0213). Patients resumed sexual activity after an average of 2.4 months (range, 0-18). Despite these general improvements, 25% of individuals had less sex in the first year after surgery. After 1 year of recovery, however, 60% indicated that they more easily engaged in sexual activity than in the previous year, with 84% of these patients experiencing less pain, and 30% experiencing greater mobility or range of motion. Conclusion TKA does not eliminate sexual limitations, but it significantly decreases kneeling dysfunction and gives patients more liberty in selecting their sexual positions.

Selenocysteine (Sec) is the 21st genetically encoded amino acid in organisms across all domains of life. Although structurally similar to cysteine (Cys), the Sec selenol group has unique properties that are attractive for protein engineering and biotechnology applications. Production of designer proteins with Sec (selenoproteins) at desired positions is now possible with engineered translation systems in Escherichia coli. However, obtaining pure selenoproteins at high yields is limited by the accumulation of free Sec in cells, causing undesired incorporation of Sec at Cys codons due to the inability of cysteinyl-tRNA synthetase (CysRS) to discriminate against Sec. Sec misincorporation is toxic to cells and causes protein aggregation in yeast. To overcome this limitation, here we investigated a CysRS from the selenium accumulator plant Astragalus bisulcatus that is reported to reject Sec in vitro. Sequence analysis revealed a rare His → Asn variation adjacent to the CysRS catalytic pocket. Introducing this variation into E. coli and Saccharomyces cerevisiae CysRS increased resistance to the toxic effects of selenite and selenomethionine (SeMet), respectively. Although the CysRS variant could still use Sec as a substrate in vitro, we observed a reduction in the frequency of Sec misincorporation at Cys codons in vivo. We surmise that the His → Asn variation can be introduced into any CysRS to provide a fitness advantage for strains burdened by Sec misincorporation and selenium toxicity. Our results also support the notion that the CysRS variant provides higher specificity for Cys as a mechanism for plants to grow in selenium-rich soils.

Student attrition is a problem in universities. A systems perspective describing both departure and retention is helpful in explaining the circumstances in which student retention is increased, particularly for students traditionally excluded from university participation. Risk factors for departure include poor preparation for college and belonging to a demographic group traditionally excluded from academia. We sought to evaluate the impact of participation in a Learning Community on student retention at an urban public research university. We included all students who had been accepted into the College of Education at Level 1 for the fall 2007 semester. We found that involvement in a Learning Community, whether it is specifically formed to address and promote student success in a given area/major, or in a more general/university-wide Learning Community improves student retention.

The work is supported by the Chinese National Thousand Talents Program-, Wellcome Trust Senior Investigator Award (WT100209MA) and Royal Society Wolfson Merit Award. All domains of life make carbohydrate polymers and by anchoring them to lipid molecules they can decorate the outside of the cell. Polysaccharides are linked to proteins by glycosylation, a process found in both bacteria and in higher organisms. Bacteria do have other distinct uses for carbohydrate polymers; in gram-negative bacteria glycolipids form the outer leaflet of the outer membrane and in many pathogens (both gram-positive and gram-negative) sugar polymers are used to build a capsule or are secreted into the environment. There are parallels, but of course differences, in the biosynthesis of glycolipids between prokaryotes and eukaryotes, which occur at the membrane. The translocation of large sugar polymers across the outer membrane is unique to gram-negative bacteria. Recent progress in the molecular understanding of both the biosynthesis at the inner membrane and the translocation across the outer membrane are reviewed here. Publisher PDF

The production of hydrocarbons in nature has been documented for only a limited set of organisms, with many of the molecular components underpinning these processes only recently identified. There is an obvious scope for application of these catalysts and engineered variants thereof in the future production of biofuels. Here we present biochemical characterization and crystal structures of a cytochrome P450 fatty acid peroxygenase: the terminal alkene forming OleTJE (CYP152L1) from Jeotgalicoccus sp. 8456. OleTJE is stabilized at high ionic strength, but aggregation and precipitation of OleTJE in low salt buffer can be turned to advantage for purification, because resolubilized OleTJE is fully active and extensively dissociated from lipids. OleTJE binds avidly to a range of long chain fatty acids, and structures of both ligand-free and arachidic acid-bound OleTJE reveal that the P450 active site is preformed for fatty acid binding. OleTJE heme iron has an unusually positive redox potential (-103 mV versus normal hydrogen electrode), which is not significantly affected by substrate binding, despite extensive conversion of the heme iron to a high spin ferric state. Terminal alkenes are produced from a range of saturated fatty acids (C12-C20), and stopped-flow spectroscopy indicates a rapid reaction between peroxide and fatty acid-bound OleTJE (167 s(-1) at 200 μm H2O2). Surprisingly, the active site is highly similar in structure to the related P450BSβ, which catalyzes hydroxylation of fatty acids as opposed to decarboxylation. Our data provide new insights into structural and mechanistic properties of a robust P450 with potential industrial applications.