Directed evolution of transketolase, a carbon-carbon forming enzyme.

The enzyme transketolase (Enzyme Commission number: 2.2.1.1) has significant potential as a biocatalyst in the production of pharmaceuticals and fine chemicals. The enzyme catalyses the irreversible transfer of a C2 (1,2-dihydroxyethyl) moiety from p-hydroxypyruvate (P-HPA) to a wide range of acceptor substrates in a stereospecific reaction. However, commercial application of transketolase is currently restricted by the limited availability and expense of p-HPA. This project describes efforts to generate and identify variants of E. coli transketolase that are capable of accepting pyruvate: a related, but much cheaper compound. The variants were prepared by a novel directed evolution technique, "focused" error-prone PCR (fepPCR), and then screened for the desired activity: pyruvate (donor) and glycolaldehyde (acceptor) to (S)-3,4-dihydroxybutan-2-one (and carbon dioxide). The high-throughput screen consisted of the following steps: (1) transformation of a plasmid library into E. coli XLIO-Gold competent cells (2) culture of individual colonies in 384-well plates (3) lysis of the cultures (4) incubation of the lysates with cofactors and the target substrates and finally (5) high-throughput HPLC analysis measuring donor substrate (pyruvate) depletion. The first four steps were optimised to ensure the highest possible concentration of holotransketolase in each screening reaction. The HPLC method utilised a 50mm guard column as the separation matrix and was capable of processing one sample every 1.2 minutes. Several libraries of transketolase variants were generated using a novel mutagenesis technique: "focused" error-prone PCR (fepPCR). FepPCR uses knowledge of an enzyme as a map for targeting mutation to the most beneficial regions of the gene - in this case, three stretches of residues in the active site of E. coli transketolase (Ser24-His26, Gly99-Prol01, and Asp469- His473). Primers were designed to flank these small target sites (9-15bp) and they were PCR-amplified from the tkt gene under conditions that drastically lowered the fidelity of Taq DNA polymerase (0.14 misincorporations per nucleotide). The three mutated fepPCR products (50-54bp) were then cloned into the tkt vector pQR711 singly and in combination to create four distinct libraries. The PCR-based cloning techniques QuikChange Site-Directed Mutagenesis (Stratagene Ltd.) and QuikChange Multi Site-Directed Mutagenesis (Stratagene Ltd.) were both found to be effective for this step. The four libraries were screened for the target activity and the following levels of coverage were achieved: 100% of all possible single point mutations in the three targets sites and 22% of all possible combinations of double point mutations in the target sites. Careful analysis of the HPLC chromatograms failed to identify any variants with the desired activity. It is proposed that larger libraries may yield positive results

Intravenous versus inhalational maintenance of anaesthesia for postoperative cognitive outcomes in elderly people undergoing non-cardiac surgery

Background: The use of anaesthetics in the elderly surgical population (more than 60 years of age) is increasing. Postoperative delirium, an acute condition characterized by reduced awareness of the environment and a disturbance in attention, typically occurs between 24 and 72 hours after surgery and can affect up to 60% of elderly surgical patients. Postoperative cognitive dysfunction (POCD) is a new-onset of cognitive impairment which may persist for weeks or months after surgery. Traditionally, surgical anaesthesia has been maintained with inhalational agents. End-tidal concentrations require adjustment to balance the risks of accidental awareness and excessive dosing in elderly people. As an alternative, propofol-based total intravenous anaesthesia (TIVA) offers a more rapid recovery and reduces postoperative nausea and vomiting. Using TIVA with a target controlled infusion (TCI) allows plasma and effect-site concentrations to be calculated using an algorithm based on age, gender, weight and height of the patient. TIVA is a viable alternative to inhalational maintenance agents for surgical anaesthesia in elderly people. However, in terms of postoperative cognitive outcomes, the optimal technique is unknown. Objectives: To compare maintenance of general anaesthesia for elderly people undergoing non-cardiac surgery using propofol-based TIVA or inhalational anaesthesia on postoperative cognitive function, mortality, risk of hypotension, length of stay in the postanaesthesia care unit (PACU), and hospital stay. Search methods: We searched the Cochrane Central Register of Controlled Trials (CENTRAL; 2017, Issue 11), MEDLINE (1946 to November 2017), Embase (1974 to November 2017), PsycINFO (1887 to November 2017). We searched clinical trials registers for ongoing studies, and conducted backward and forward citation searching of relevant articles. Selection criteria: We included randomized controlled trials (RCTs) with participants over 60 years of age scheduled for non-cardiac surgery under general anaesthesia. We planned to also include quasi-randomized trials. We compared maintenance of anaesthesia with propofol-based TIVA versus inhalational maintenance of anaesthesia. Data collection and analysis: Two review authors independently assessed studies for inclusion, extracted data, assessed risk of bias, and synthesized findings. Main results: We included 28 RCTs with 4507 randomized participants undergoing different types of surgery (predominantly cardiovascular, laparoscopic, abdominal, orthopaedic and ophthalmic procedures). We found no quasi-randomized trials. Four studies are awaiting classification because we had insufficient information to assess eligibility. All studies compared maintenance with propofol-based TIVA versus inhalational maintenance of anaesthesia. Six studies were multi-arm and included additional TIVA groups, additional inhalational maintenance or both. Inhalational maintenance agents included sevoflurane (19 studies), isoflurane (eight studies), and desflurane (three studies), and was not specified in one study (reported as an abstract). Some studies also reported use of epidural analgesia/anaesthesia, fentanyl and remifentanil. We found insufficient reporting of randomization methods in many studies and all studies were at high risk of performance bias because it was not feasible to blind anaesthetists to study groups. Thirteen studies described blinding of outcome assessors. Three studies had a high of risk of attrition bias, and we noted differences in the use of analgesics between groups in six studies, and differences in baseline characteristics in five studies. Few studies reported clinical trials registration, which prevented assessment of risk of selective reporting bias. We found no evidence of a difference in incidences of postoperative delirium according to type of anaesthetic maintenance agents (odds ratio (OR) 0.59, 95% confidence interval (CI) 0.15 to 2.26; 321 participants; five studies; very low-certainty evidence); we noted during sensitivity analysis that using different time points in one study may influence direction of this result. Thirteen studies (3215 participants) reported POCD, and of these, six studies reported data that could not be pooled; we noted no difference in scores of POCD in four of these and in one study, data were at a time point incomparable to other studies. We excluded one large study from meta-analysis because study investigators had used non-standard anaesthetic management and this study was not methodologically comparable to other studies. We combined data for seven studies and found low-certainty evidence that TIVA may reduce POCD (OR 0.52, 95% CI 0.31 to 0.87; 869 participants). We found no evidence of a difference in mortality at 30 days (OR 1.21, 95% CI 0.33 to 4.45; 271 participants; three studies; very low-certainty evidence). Twelve studies reported intraoperative hypotension. We did not perform meta-analysis for 11 studies for this outcome. We noted visual inconsistencies in these data, which may be explained by possible variation in clinical management and medication used to manage hypotension in each study (downgraded to low-certainty evidence); one study reported data in a format that could not be combined and we noted little or no difference between groups in intraoperative hypotension for this study. Eight studies reported length of stay in the PACU, and we did not perform meta-analysis for seven studies. We noted visual inconsistencies in these data, which may be explained by possible differences in definition of time points for this outcome (downgraded to very low-certainty evidence); data were unclearly reported in one study. We found no evidence of a difference in length of hospital stay according to type of anaesthetic maintenance agent (mean difference (MD) 0 days, 95% CI -1.32 to 1.32; 175 participants; four studies; very low-certainty evidence). We used the GRADE approach to downgrade the certainty of the evidence for each outcome. Reasons for downgrading included: study limitations, because some included studies insufficiently reported randomization methods, had high attrition bias, or high risk of selective reporting bias; imprecision, because we found few studies; inconsistency, because we noted heterogeneity across studies. Authors' conclusions: We are uncertain whether maintenance with propofol-based TIVA or with inhalational agents affect incidences of postoperative delirium, mortality, or length of hospital stay because certainty of the evidence was very low. We found low-certainty evidence that maintenance with propofol-based TIVA may reduce POCD. We were unable to perform meta-analysis for intraoperative hypotension or length of stay in the PACU because of heterogeneity between studies. We identified 11 ongoing studies from clinical trials register searches; inclusion of these studies in future review updates may provide more certainty for the review outcomes. © 2018 The Cochrane Collaboration

A synthetic biology approach to probing nucleosome symmetry

The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell

Examining the Higgs boson potential at lepton and hadron colliders: a comparative analysis

We investigate inclusive Standard Model Higgs boson pair production at lepton and hadron colliders for Higgs boson masses in the range 120 GeV < m_H < 200 GeV. For m_H < 140 GeV we find that hadron colliders have a very limited capability to determine the Higgs boson self-coupling, \lambda, due to an overwhelming background. We also find that, in this mass range, supersymmetric Higgs boson pairs may be observable at the LHC, but a measurement of the self coupling will not be possible. For m_H > 140 GeV we examine ZHH and HH nu bar-nu production at a future e+e- linear collider with center of mass energy in the range of sqrt{s}=0.5 - 1 TeV, and find that this is likely to be equally difficult. Combining our results with those of previous literature, which has demonstrated the capability of hadron and lepton machines to determine \lambda in either the high or the low mass regions, we establish a very strong complementarity of these machines.Comment: Revtex, 25 pages, 2 tables, 10 figure

Determining the Higgs Boson Self Coupling at Hadron Colliders

Inclusive Standard Model Higgs boson pair production at hadron colliders has the capability to determine the Higgs boson self-coupling, lambda. We present a detailed analysis of the gg\to HH\to (W^+W^-)(W^+W^-)\to (jjl^\pm\nu)(jj{l'}^\pm\nu) and gg\to HH\to (W^+W^-)(W^+W^-)\to (jjl^\pm\nu)({l'}^\pm\nu {l''}^\mp\nu) (l, {l'}, {l''}=e, \mu) signal channels, and the relevant background processes, for the CERN Large Hadron Collider, and a future Very Large Hadron Collider operating at a center-of-mass energy of 200 TeV. We also derive quantitative sensitivity limits for lambda. We find that it should be possible at the LHC with design luminosity to establish that the Standard Model Higgs boson has a non-zero self-coupling and that lambda / lambda_{SM} can be restricted to a range of 0-3.8 at 95% confidence level (CL) if its mass is between 150 and 200 GeV. At a 200 TeV collider with an integrated luminosity of 300 fb^{-1}, lambda can be determined with an accuracy of 8 - 25% at 95% CL in the same mass range.Comment: 28 pages, Revtex3, 9 figures, 3 table

Current state of perinatal postmortem magnetic resonance imaging: European Society of Paediatric Radiology questionnaire-based survey and recommendations.

BACKGROUND: Postmortem magnetic resonance imaging (MRI) in perinatal and childhood deaths is increasingly used as a noninvasive adjunct or alternative to autopsy. Imaging protocols vary between centres and consensus guidelines do not exist. OBJECTIVE: Our aim was to develop practical, standardised recommendations for perinatal postmortem MRI. MATERIALS AND METHODS: Recommendations were based on the results of two surveys regarding local postmortem MRI practices sent electronically to all 14 members of the European Society of Paediatric Radiology (ESPR) Postmortem Imaging Task Force and 17 members of the International Society of Forensic Radiology and Imaging Task Force (25 different centres). RESULTS: Overall, 11/14 (78.6%) respondents from different institutions perform postmortem MRI. All of these centres perform postmortem MRI for perinatal and neonatal deaths, but only 6/11 (54.5%) perform imaging in older children. CONCLUSION: We propose a clinical standard for postmortem MRI sequences plus optional sequences for neuroimaging and cardiac anatomy depending on available scanning time and referral indications

Diverse Beliefs and Time Variability of Risk Premia

Why do risk premia vary over time? We examine this problem theoretically and empirically by studying the effect of market belief on risk premia. Individual belief is taken as a fundamental primitive state variable. Market belief is observable; it is central to the empirical evaluation and we show how to measure it. Our asset pricing model is familiar from the noisy REE literature but we adapt it to an economy with diverse beliefs. We derive equilibrium asset prices and implied risk premium. Our approach permits a closed form solution of prices; hence we trace the exact effect of market belief on the time variability of asset prices and risk premia. We test empirically the theoretical conclusions. Our main result is that, above the effect of business cycles on risk premia, fluctuations in market belief have significant independent effect on the time variability of risk premia. We study the premia on long positions in Federal Funds Futures, 3- and 6-month Treasury Bills (T-Bills). The annual mean risk premium on holding such assets for 1-12 months is about 40-60 basis points and we find that, on average, the component of market belief in the risk premium exceeds 50% of the mean. Since time variability of market belief is large, this component frequently exceeds 50% of the mean premium. This component is larger the shorter is the holding period of an asset and it dominates the premium for very short holding returns of less than 2 months. As to the structure of the premium we show that when the market holds abnormally favorable belief about the future payoff of an asset the market views the long position as less risky hence the risk premium on that asset declines. More generally, periods of market optimism (i.e. "bull" markets) are shown to be periods when the market risk premium is low while in periods of pessimism (i.e. "bear" markets) the market's risk premium is high. Fluctuations in risk premia are thus inversely related to the degree of market optimism about future prospects of asset payoffs. This effect is strong and economically very significant

Observation of inverse Compton emission from a long γ-ray burst.

Long-duration γ-ray bursts (GRBs) originate from ultra-relativistic jets launched from the collapsing cores of dying massive stars. They are characterized by an initial phase of bright and highly variable radiation in the kiloelectronvolt-to-megaelectronvolt band, which is probably produced within the jet and lasts from milliseconds to minutes, known as the prompt emission1,2. Subsequently, the interaction of the jet with the surrounding medium generates shock waves that are responsible for the afterglow emission, which lasts from days to months and occurs over a broad energy range from the radio to the gigaelectronvolt bands1-6. The afterglow emission is generally well explained as synchrotron radiation emitted by electrons accelerated by the external shock7-9. Recently, intense long-lasting emission between 0.2 and 1 teraelectronvolts was observed from GRB 190114C10,11. Here we report multi-frequency observations of GRB 190114C, and study the evolution in time of the GRB emission across 17 orders of magnitude in energy, from 5 × 10-6 to 1012 electronvolts. We find that the broadband spectral energy distribution is double-peaked, with the teraelectronvolt emission constituting a distinct spectral component with power comparable to the synchrotron component. This component is associated with the afterglow and is satisfactorily explained by inverse Compton up-scattering of synchrotron photons by high-energy electrons. We find that the conditions required to account for the observed teraelectronvolt component are typical for GRBs, supporting the possibility that inverse Compton emission is commonly produced in GRBs

Mapping geographical inequalities in childhood diarrhoeal morbidity and mortality in low-income and middle-income countries, 2000–17 : analysis for the Global Burden of Disease Study 2017

Background Across low-income and middle-income countries (LMICs), one in ten deaths in children younger than 5 years is attributable to diarrhoea. The substantial between-country variation in both diarrhoea incidence and mortality is attributable to interventions that protect children, prevent infection, and treat disease. Identifying subnational regions with the highest burden and mapping associated risk factors can aid in reducing preventable childhood diarrhoea. Methods We used Bayesian model-based geostatistics and a geolocated dataset comprising 15 072 746 children younger than 5 years from 466 surveys in 94 LMICs, in combination with findings of the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017, to estimate posterior distributions of diarrhoea prevalence, incidence, and mortality from 2000 to 2017. From these data, we estimated the burden of diarrhoea at varying subnational levels (termed units) by spatially aggregating draws, and we investigated the drivers of subnational patterns by creating aggregated risk factor estimates. Findings The greatest declines in diarrhoeal mortality were seen in south and southeast Asia and South America, where 54·0% (95% uncertainty interval [UI] 38·1–65·8), 17·4% (7·7–28·4), and 59·5% (34·2–86·9) of units, respectively, recorded decreases in deaths from diarrhoea greater than 10%. Although children in much of Africa remain at high risk of death due to diarrhoea, regions with the most deaths were outside Africa, with the highest mortality units located in Pakistan. Indonesia showed the greatest within-country geographical inequality; some regions had mortality rates nearly four times the average country rate. Reductions in mortality were correlated to improvements in water, sanitation, and hygiene (WASH) or reductions in child growth failure (CGF). Similarly, most high-risk areas had poor WASH, high CGF, or low oral rehydration therapy coverage. Interpretation By co-analysing geospatial trends in diarrhoeal burden and its key risk factors, we could assess candidate drivers of subnational death reduction. Further, by doing a counterfactual analysis of the remaining disease burden using key risk factors, we identified potential intervention strategies for vulnerable populations. In view of the demands for limited resources in LMICs, accurately quantifying the burden of diarrhoea and its drivers is important for precision public health