Molecular modelling assisted design and synthesis of cyclothialidine derivatives as DNA gyrase inhibitors

Since cyclothialidine was discovered as the most active DNA gyrase inhibitor in 1994, enormous efforts have been devoted to make it into a commercial medicine by a number of pharmaceutical companies and research groups worldwide. However, no serious breakthrough has been made up to now. An essential problem involved with cyclothialidine is that though it demonstrated the potent inhibition of DNA gyrase, it showed little activity against bacteria. This probably is attributable to its inability to penetrate bacterial cell walls and membranes. We applied the TSAR programme to generate a QSAR equation to the gram-negative organisms. In that equation, LogP is profoundly indicated as the key factor influencing the cyclothialidine activity against bacteria. However, the synthesized new analogues have failed to prove that. In the structure based drug design stage, we designed a group of open chain cyclothialidine derivatives by applying the SPROUT programme and completed the syntheses. Improved activity is found in a few analogues and a 3D pharmacophore of the DNA gyrase B is proposed to lead to synthesis of the new derivatives for development of potent antibiotics.

A corpus-assisted study of the discourse marker well as an indicator of judges' institutional roles in court cases with litigants in person

In this paper, I concentrate on court cases with litigants in person (lay people who act on their own behalf in legal proceedings without a counsel or solicitor) and discuss the challenges of building a corpus of courtroom discourse where it is crucial to distinguish between speakers due to their distinct institutional roles. The corpus incorporates seven sub-corpora of verbatim transcripts from different court cases with litigants in person and comprises over eleven-million tokens. The focus of this paper is on the interplay between the legal and lay discourse types and how judges project their institutional roles through well-initiated turns directed at litigants in person and counsels. As a versatile discourse marker, well provides a good opportunity to explore how judges have to adapt their roles to ensure lay litigants in person receive the necessary support and that their lack of competence does not impede on the fairness of the proceedings. Given the breadth and importance of the topic of litigation in person, I discuss how the tools and approaches of corpus linguistics can be helpful in this multi-disciplinary area where multiple functions and uses of individual linguistic features need to be explored in depth.

8-Hydroxydeoxyguanosine. A marker of oxidative DNA damage in systemic lupus erythematosus

8-Hydroxydeoxyguanosine (80HDG) is a specific marker of oxidative damage to DNA. We have observed that patients with SLE (systemic lupus erythematosus), have undetectable levels of urinary 80HDG by HPLC. Further analysis by GC-MS confirmed that levels of 80HDG in SLE urine were 10(3)-fold lower than in an age- and sex-matched control group. Experiments utilising cultures of SLE and normal lymphocytes exposed to H2O2 confirmed the impaired ability of SLE lymphocytes to repair 80HDG. We subsequently observed in SLE patients that 80HDG had accumulated in low molecular weight DNA associated with circulating immune complexes. We suggest that oxygen radicals may induce pathology in SLE by maintaining the presence of an antigenic form of DNA in the circulation.

Improving recombinant eukaryotic membrane protein yields in Pichia pastoris:the importance of codon optimization and clone selection

In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.

Isomer resolution by micelle-assisted diffusion-ordered spectroscopy

Diffusion-ordered NMR spectroscopy ("DOSY") is a useful tool for the identification of mixture components. In its basic form it relies on simple differences in hydrodynamic radius to distinguish between different species. This can be very effective where species have significantly different molecular sizes, but generally fails for isomeric species. The use of surfactant co-solutes can allow isomeric species to be distinguished by virtue of their different degrees of interaction with micelles or reversed micelles. The use of micelle-assisted DOSY to resolve the NMR spectra of isomers is illustrated for the case of the three dihydroxybenzenes (catechol, resorcinol, and hydroquinone) in aqueous solution containing sodium dodecyl sulfate micelles, and in chloroform solution containing AOT reversed micelles. © 2009 American Chemical Society.

Tissue-specific selection of reference genes is required for expression studies in the mouse model of maternal protein undernutrition

Suboptimal maternal nutrition during gestation results in the establishment of long-term phenotypic changes and an increased disease risk in the offspring. To elucidate how such environmental sensitivity results in physiological outcomes, the molecular characterisation of these offspring has become the focus of many studies. However, the likely modification of key cellular processes such as metabolism in response to maternal undernutrition raises the question of whether the genes typically used as reference constants in gene expression studies are suitable controls. Using a mouse model of maternal protein undernutrition, we have investigated the stability of seven commonly used reference genes (18s, Hprt1, Pgk1, Ppib, Sdha, Tbp and Tuba1) in a variety of offspring tissues including liver, kidney, heart, retro-peritoneal and inter-scapular fat, extra-embryonic placenta and yolk sac, as well as in the preimplantation blastocyst and blastocyst-derived embryonic stem cells. We find that although the selected reference genes are all highly stable within this system, they show tissue, treatment and sex-specific variation. Furthermore, software-based selection approaches rank reference genes differently and do not always identify genes which differ between conditions. Therefore, we recommend that reference gene selection for gene expression studies should be thoroughly validated for each tissue of interest. © 2011 Elsevier Inc.

EpiDOCK:a molecular docking-based tool for MHC class II binding prediction

Cellular peptide vaccines contain T-cell epitopes. The main prerequisite for a peptide to act as a T-cell epitope is that it binds to a major histocompatibility complex (MHC) protein. Peptide MHC binder identification is an extremely costly experimental challenge since human MHCs, named human leukocyte antigen, are highly polymorphic and polygenic. Here we present EpiDOCK, the first structure-based server for MHC class II binding prediction. EpiDOCK predicts binding to the 23 most frequent human, MHC class II proteins. It identifies 90% of true binders and 76% of true non-binders, with an overall accuracy of 83%. EpiDOCK is freely accessible at http://epidock.ddg-pharmfac. net. © The Author 2013. Published by Oxford University Press. All rights reserved.

Cellular and molecular consequences of S100A4-induced motility in rat breast tumour Rama 37 cells

Since the first discovery of S100 members in 1965, their expressions have been affiliated with numerous biological functions in all cells of the body. However, in the recent years, S100A4, a member of this superfamily has emerged as the central target in generating new avenue for cancer therapy as its overexpression has been correlated with cancer patients’ mortality as well as established roles as motility and metastasis promoter. As it has no catalytic activity, S100A4 has to interact with its target proteins to regulate such effects. Up to date, more than 10 S100A4 target proteins have been identified but the mechanical process regulated by S100A4 to induce motility remains vague. In this work, we demonstrated that S100A4 overexpression resulted in actin filaments disorganisation, reduction in focal adhesions, instability of filopodia as well as exhibiting polarised morphology. However, such effects were not observed in truncated versions of S100A4 possibly highlighting the importance of C terminus of S100A4 target recognition. In order to assess some of the intracellular mechanisms that may be involved in promoting migrations, different strategies were used, including active pharmaceutical agents, inhibitors and knockdown experiments. Treatment of S100A4 overexpressing cells with blebbistatin and Y-27632, non muscle myosin IIA (NMMIIA) inhibitors, as well as knockdown of NMMIIA, resulted in motility enhancement and focal adhesions reduction proposing that NMMIIA assisted S100A4 in regulating cell motility but its presence is not essential. Further work done using Cos 7 cell lines, naturally lacking NMMIIA, further demonstrated that S100A4 is capable of regulating cell motility independent of NMMIIA, possibly through poor maturation of focal adhesion. Given that all these experiments highlighted the independency of NMMIIA towards migration, a protein that has been put at the forefront of S100A4-induced motility, we aimed to gather further understanding regarding the other molecular mechanisms that may be at play for motility. Using high throughput imaging (HCI), 3 compounds were identified to be capable of inhibiting S100A4-mediated migration. Although we have yet to investigate the underlying mechanism for their effects, these compounds have been shown to target membrane proteins and the externalisation of S100 proteins, for at least one of the compounds, leading us to speculate that preventing externalisation of S100A4 could potentially regulate cell motility.