In vivo and in vitro characterization of Staphylococcus aureus and Bacillus subtilis polyglycerolphosphate lipoteichoic acid synthases

Staphylococcus aureus lipoteichoic acid (LTA) consists of a 1,3-linked polyglycerolphosphate chain retained in the bacterial membrane by a glycolipid anchor. The LTA backbone is produced by the lipoteichoic acid synthase LtaS, a membrane protein with five transmembrane helices and a large extracellular enzymatic domain (eLtaS). Proteomic studies revealed that LtaS is efficiently cleaved, and here it was demonstrated that the eLtaS domain is released into the culture supernatant as well as partially retained within the cell wall fraction. However, using an in vivo LtaS activity assay, it was shown that only the full-length LtaS enzyme is able to synthesize LTA. Neither expression of a secreted eLtaS variant, created by replacing the N-terminal membrane domain with a conventional signal sequence, nor expression of eLtaS fused to a single or multi-transmembrane domains of other staphylococcal proteins resulted in the production of LTA. These data indicate that the transmembrane domain of LtaS play an essential, yet unknown, role in LtaS enzyme function. In addition, the protease responsible for LtaS cleavage was identified. It was found that a S. aureus strain in which the gene encoding for the essential signal peptidase SpsB was cloned under inducible expression control showed an accumulation of the full-length LtaS enzyme in the absence of the inducer. These data suggest that SpsB is involved in LtaS cleavage. Four LtaS orthologues, YflE, YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay and purified protein, it was determined that all four B. subtilis proteins are Mn2+-dependent metal enzymes that use the lipid phosphatidylglycerol as substrate. It was shown that YflE, YfnI and YqgS are bonafide LTA synthases capable of producing polyglycerolphosphate chains, while YvgJ appears to function as an LTA primase, as indicated by the accumulation of a glycolipid with the expected chromatographic mobility of GroP-Glc2-DAG. Taken together, experimental evidence for the enzyme function of all four B. subtilis LtaStype proteins is provided in this work and it was shown that all four enzymes are involved in the LTA synthesis process

High-to-low CO2 acclimation reveals plasticity of the photorespiratory pathway and indicates regulatory links to cellular metabolism of Arabidopsis

Background: Photorespiratory carbon metabolism was long considered as an essentially closed and nonregulated pathway with little interaction to other metabolic routes except nitrogen metabolism and respiration. Most mutants of this pathway cannot survive in ambient air and require CO 2-enriched air for normal growth. Several studies indicate that this CO 2 requirement is very different for individual mutants, suggesting a higher plasticity and more interaction of photorespiratory metabolism as generally thought. To understand this better, we examined a variety of high- and low-level parameters at 1% CO 2 and their alteration during acclimation of wild-type plants and selected photorespiratory mutants to ambient air. Methodology and Principal Findings: The wild type and four photorespiratory mutants of Arabidopsis thaliana (Arabidopsis) were grown to a defined stadium at 1% CO 2 and then transferred to normal air (0.038% CO 2). All other conditions remained unchanged. This approach allowed unbiased side-by-side monitoring of acclimation processes on several levels. For all lines, diel (24 h) leaf growth, photosynthetic gas exchange, and PSII fluorescence were monitored. Metabolite profiling was performed for the wild type and two mutants. During acclimation, considerable variation between the individual genotypes was detected in many of the examined parameters, which correlated with the position of the impaired reaction in the photorespiratory pathway. Conclusions: Photorespiratory carbon metabolism does not operate as a fully closed pathway. Acclimation from high to low CO 2 was typically steady and consistent for a number of features over several days, but we also found unexpected short-term events, such as an intermittent very massive rise of glycine levels after transition of one particular mutant to ambient air. We conclude that photorespiration is possibly exposed to redox regulation beyond known substrate-level effects. Additionally, our data support the view that 2-phosphoglycolate could be a key regulator of photosynthetic-photorespiratory metabolism as a whole. © 2012 Timm et al

Aziridine-Metathesis based Approaches to Alkaloid Synthesis

The aim of the project is to synthesise (-)-morphine utilising aziridine and metathesischemistry. The thesis is divided into three chapters.Chapter 1 provides brief reviews on the subjects of total synthesis of morphine; ringrearrangementmetathesis (RRM) and regioselective ring-opening of aziridines.Chapter 2 focuses on the research findings in the past three years. Two routes, A and B,were investigated in attempts to synthesise morphine (Scheme 1). In route A, sulfonylcyclopentene II was prepared from ring-closing metathesis of a diene precursor, whichwas synthesised from lithiated cinnamylsulfone and butadiene monoxide. Subsequently,RRM reactions of several [alpha]-SO2Ph allyl derivatives of II were investigated and someinteresting results were obtained. The synthesis of 2,3-trans vinylaziridine III wasachieved in seven steps beginning with a Grignard reaction of (4-methoxyphenyl)magnesium bromide with butadiene monoxide. Subsequently, somehighly regioselective ring-opening reactions of III with sulfur-stabilised anionicnucleophiles were achieved. However, in an attempt to synthesise compound I from IIand III, no reaction was observed. This led to the investigation of route B, in which fivemethods for the synthesis of compound IV were investigated. The practical approachdeployed a novel Al-mediated substitution of the 4-tosyl group of the tosyltetrahydropyridine counterpart of IV, prepared from V and III, with a phenylthio group.Chapter 3 provides the experimental details and characterisation data.Imperial Users onl

The role of endothelin-1 in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare but debilitating disease, which if left untreated rapidly progresses to right ventricular failure and eventually death. In the quest to understand the pathogenesis of this disease differences in the profile, expression and action of vasoactive substances released by the endothelium have been identified in patients with PAH. Of these, endothelin-1 (ET-1) is of particular interest since it is known to be an extremely powerful vasoconstrictor and also involved in vascular remodelling. Identification of ET-1 as a target for pharmacological intervention has lead to the discovery of a number of compounds that can block the receptors via which ET-1 mediates its effects. This review sets out the evidence in support of a role for ET-1 in the onset and progression of the disease and reviews the data from the various clinical trials of ET-1 receptor antagonists for the treatment of PAH

Two-player preplay negotiation games with conditional offers

We consider an extension of strategic normal form games with a phase before the actual play of the game, where players can make binding offers for transfer of utilities to other players after the play of the game, contingent on the recipient playing the strategy indicated in the offer. Such offers transform the payoff matrix of the original game but preserve its non-cooperative nature. The type of offers we focus on here are conditional on a suggested 'matching offer' of the same kind made in return by the receiver. Players can exchange a series of such offers, thus engaging in a bargaining process before a strategic normal form game is played. In this paper we study and analyze solution concepts for two-player normal form games with such preplay negotiation phase, under several assumptions for the bargaining power of the players, such as the possibility of withdrawing previously made offers and opting out from the negotiation process, as well as the value of time for the players in such negotiations. We obtain results describing the possible solutions of such bargaining games and analyze the degrees of efficiency and fairness that can be achieved in such negotiation process

Identification of a serum biomarker panel for the differential diagnosis of cholangiocarcinoma and primary sclerosing cholagnitis

The non-invasive differentiation of malignant and benign biliary disease is a clinical challenge. Carbohydrate antigen 19-9 (CA19-9), leucine-rich α2-glycoprotein (LRG1), interleukin 6 (IL6), pyruvate kinase M2 (PKM2), cytokeratin 19 fragment (CYFRA21.1) and mucin 5AC (MUC5AC) have reported utility for differentiating cholangiocarcinoma (CCA) from benign biliary disease. Herein, serum levels of these markers were tested in 66 cases of CCA and 62 cases of primary sclerosing cholangitis (PSC) and compared with markers of liver function and inflammation. Markers panels were assessed for their ability to discriminate malignant and benign disease. Several of the markers were also assessed in pre-diagnosis biliary tract cancer (BTC) samples with performances evaluated at different times prior to diagnosis. We show that LRG1 and IL6 were unable to accurately distinguish CCA from PSC, whereas CA19-9, PKM2, CYFRA21.1 and MUC5AC were significantly elevated in malignancy. Area under the receiver operating characteristic curves for these individual markers ranged from 0.73–0.84, with the best single marker (PKM2) providing 61% sensitivity at 90% specificity. A panel combining PKM2, CYFRA21.1 and MUC5AC gave 76% sensitivity at 90% specificity, which increased to 82% sensitivity by adding gamma-glutamyltransferase (GGT). In the pre-diagnosis setting, LRG1, IL6 and PKM2 were poor predictors of BTC, whilst CA19-9 and C-reactive protein were elevated up to 2 years before diagnosis. In conclusion, LRG1, IL6 and PKM2 were not useful for early detection of BTC, whilst a model combining PKM2, CYFRA21.1, MUC5AC and GGT was beneficial in differentiating malignant from benign biliary disease, warranting validation in a prospective trial

Spectral functions of the uniform electron gas via coupled-cluster theory and comparison to GW and related approximations

We use, for the first time, ab initio coupled-cluster theory to compute the spectral function of the uniform electron gas at a Wigner-Seitz radius of rs = 4. The coupled-cluster approximations we employ go significantly beyond the diagrammatic content of state-of-the-art GW theory. We compare our calculations extensively to GW and GW-plus-cumulant theory, illustrating the strengths and weaknesses of these methods in capturing the quasiparticle and satellite features of the electron gas. Our accurate calculations further allow us to address the long-standing debate over the occupied bandwidth of metallic sodium. Our findings indicate that the future application of coupled-cluster theory to condensed phase material spectra is highly promising

Array signal processing robust to pointing errors

The objective of this thesis is to design computationally efficient DOA (direction-of- arrival) estimation algorithms and beamformers robust to pointing errors, by harnessing the antenna geometrical information and received signals. Initially, two fast root-MUSIC-type DOA estimation algorithms are developed, which can be applied in arbitrary arrays. Instead of computing all roots, the first proposed iterative algorithm calculates the wanted roots only. The second IDFT-based method obtains the DOAs by scanning a few circles in parallel and thus the rooting is avoided. Both proposed algorithms, with less computational burden, have the asymptotically similar performance to the extended root-MUSIC. The second main contribution in this thesis is concerned with the matched direction beamformer (MDB), without using the interference subspace. The manifold vector of the desired signal is modeled as a vector lying in a known linear subspace, but the associated linear combination vector is otherwise unknown due to pointing errors. This vector can be found by computing the principal eigen-vector of a certain rank-one matrix. Then a MDB is constructed which is robust to both pointing errors and overestimation of the signal subspace dimension. Finally, an interference cancellation beamformer robust to pointing errors is considered. By means of vector space projections, much of the pointing error can be eliminated. A one-step power estimation is derived by using the theory of covariance fitting. Then an estimate-and-subtract interference canceller beamformer is proposed, in which the power inversion problem is avoided and the interferences can be cancelled completely