Ocular blood flow analysis detects microvascular abnormalities in impaired glucose tolerance.

Objective: Waveform analysis has been used to assess vascular resistance and predict cardiovascular events. We aimed to identify microvascular abnormalities in patients with impaired glucose tolerance (IGT) using ocular waveform analysis. The effects of pioglitazone were also assessed. Methods: Forty patients with IGT and twenty-four controls were studied. Doppler velocity recordings were obtained from the central retinal, ophthalmic and common carotid arteries, and sampled at 200 Hz. A discrete wavelet-based analysis method was employed to quantify waveforms. The resistive index (RI),was also determined. Patients with IGT were randomised to pioglitazone or placebo and measurements repeated after 12 weeks treatment. Results: In the ocular waveforms, significant differences in power spectra were observed in frequency band four (corresponding to frequencies between 6.25 and 12.50 Hz) between groups (p

Networks for systems biology: conceptual connection of data and function

The purpose of this study is to survey the use of networks and network-based methods in systems biology. This study starts with an introduction to graph theory and basic measures allowing to quantify structural properties of networks. Then, the authors present important network classes and gene networks as well as methods for their analysis. In the last part of this study, the authors review approaches that aim at analysing the functional organisation of gene networks and the use of networks in medicine. In addition to this, the authors advocate networks as a systematic approach to general problems in systems biology, because networks are capable of assuming multiple roles that are very beneficial connecting experimental data with a functional interpretation in biological terms.

Network biology: a direct approach to study biological function

In this paper we discuss the dualism of gene networks and their role in systems biology. We argue that gene networks ( 1) can serve as a conceptual framework, forming a fundamental level of a phenomenological description, and ( 2) are a means to represent and analyze data. The latter point does not only allow a systems analysis but is even amenable for a direct approach to study biological function. Here we focus on the clarity of our main arguments and conceptual meaning of gene networks, rather than the causal inference of gene networks from data. (C) 2010 John Wiley & Sons, Inc. WIREs Syst Biol Med 2011 3 379-391 DOI: 10.1002/wsbm.134

Molecular subtyping of breast cancer: Opportunities for new therapeutic approaches

Evidence is accumulating that breast cancer is not one disease but many separate diseases. DNA microarray-based gene expression profiling has demonstrated subtypes with distinct phenotypic features and clinical responses. Prominent among the new subtypes is 'basal-like' breast cancer, one of the 'intrinsic' subtypes defined by negativity for the estrogen, progesterone, and HER2/neu receptors and positivity for cytokeratins-5/6. Focusing on basal-like breast cancer, we discuss how molecular technologies provide new chemotherapy targets, optimising treatment whilst sparing patients from un-necessary toxicity. Clinical trials are needed that incorporate long-term follow-up of patients with well-characterised tumour markers. Whilst the absence of an obvious dominant oncogene driving basallike breast cancer and the lack of specific therapeutic agents are serious stumbling blocks, this review will highlight several promising therapeutic candidates currently under evaluation. Thus, new molecular technologies should provide a fundamental foundation for better understanding breast and other cancers which may be exploited to save lives.

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly alpha-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

Molecular Dynamics of HIV1-Integrase in Complex with 93del - A Structural Perspective on the Mechanism of Inhibition

HIV1 integrase is an important target for the antiviral therapy. Guanine-rich quadruplex, such as 93del, have been shown to be potent inhibitors of this enzyme and thus representing a new class of antiviral agents. Although X-ray and NMR structures of HIV1 integrase and 93del have been reported, there is no structural information of the complex and the mechanism of inhibition still remains unexplored. A number of computational methods including automated protein-DNA docking and molecular dynamics simulation in explicit solvent were used to model the binding of 93del to HIV1 integrase. Analysis of the dynamic behaviour of the complex using principal components analysis and elastic network modelling techniques allow us to understand how the binding of 93del aptamer and its interactions with key residues affect the intrinsic motions of the catalytic loops by stabilising them in catalytically inactive conformations. Such insights into the structural mechanism of inhibition can aid in improving the design of anti-HIV aptamers.

Bacteriophages as anti-infective agents: recent developments and regulatory challenges.

The biennial meeting on 'Exploiting Bacteriophages for Bioscience, Biotechnology and Medicine', held in London, UK, on 20 January 2012, and chaired by George Salmond (University of Cambridge, UK) hosted over 50 participants representing 13 countries. The highly multidisciplinary meeting covered a diverse range of topics, reflecting the current expansion of interest in this field, including the use of bacteriophages as the source of biochemical reagents for molecular biology, bacteriophages for the treatment of human and animal diseases, bacteriophage-based diagnostics and therapeutic delivery technologies and necessity for, and regulatory challenges associated with, robust clinical trials of phage-based therapeutics. This report focuses on a number of presentations from the meeting relating to cutting-edge research on bacteriophages as anti-infective agents.

Pharmacological profiling of store-operated Ca(2+) entry in retinal arteriolar smooth muscle

Objective: Pharmacological profiling of store-operated Ca(2+) entry (SOCE) and molecular profiling of ORAI and TRPC expression in arterioles. 
Methods: Fura-2 based microfluorimetry was used to assess CPA-induced SOCE in rat retinal arteriolar myocytes. Arteriolar ORAI and TRP transcript expression were screened using RT-PCR. 
Results: SKF96365 and LOE908 blocked SOCE (IC(50) s of 1.2µM and 1.4µM, respectively). Gd(3+) and La(3+) potently inhibited SOCE (IC(50) s of 21nM and 42nM, respectively), but Ni(2+) showed lower potency (IC(50) = 11.6µM). 2-aminoethyldiphenyl borate (2APB) inhibited SOCE (IC(50) = 3.7µM) but enhanced basal influx (>100µM). Verapamil and nifedipine had no effect at concentrations that inhibit L-type Ca(2+) channels, but diltiazem inhibited SOCE by approximately 40% (=0.1µM). RT-PCR demonstrated transcript expression for ORAI 1, 2 and 3, and TRPC1, 3, 4 and 7. Transcripts for TRPV1 and 2, which are activated by 2APB, were also expressed. 
Conclusion: The pharmacological profile of SOCE in retinal arteriolar smooth muscle appears unique when compared to other vascular tissues. This suggests that the molecular mechanisms underlying SOCE can differ, even in closely related tissues. Taken together, the pharmacological and molecular data are most consistent with involvement of TRPC1 in SOCE, although involvement of ORAI or other TRPC channels cannot be excluded. © 2012 John Wiley & Sons Ltd.

Eradication of Pseudomonas aeruginosa Biofilms by Atmospheric Pressure Non-Thermal Plasma

Bacteria exist, in most environments, as complex, organised communities of sessile cells embedded within a matrix of self-produced, hydrated extracellular polymeric substances known as biofilms. Bacterial biofilms represent a ubiquitous and predominant cause of both chronic infections and infections associated with the use of indwelling medical devices such as catheters and prostheses. Such infections typically exhibit significantly enhanced tolerance to antimicrobial, biocidal and immunological challenge. This renders them difficult, sometimes impossible, to treat using conventional chemotherapeutic agents. Effective alternative approaches for prevention and eradication of biofilm associated chronic and device-associated infections are therefore urgently required. Atmospheric pressure non-thermal plasmas are gaining increasing attention as a potential approach for the eradication and control of bacterial infection and contamination. To date, however, the majority of studies have been conducted with reference to planktonic bacteria and rather less attention has been directed towards bacteria in the biofilm mode of growth. In this study, the activity of a kilohertz-driven atmospheric pressure non-thermal plasma jet, operated in a helium oxygen mixture, against Pseudomonas aeruginosa in vitro biofilms was evaluated. Pseudomonas aeruginosa biofilms exhibit marked susceptibility to exposure of the plasma jet effluent, following even relatively short (~10's s) exposure times. Manipulation of plasma operating conditions, for example, plasma operating frequency, had a significant effect on the bacterial inactivation rate. Survival curves exhibit a rapid decline in the number of surviving cells in the first 60 seconds followed by slower rate of cell number reduction. Excellent anti-biofilm activity of the plasma jet was also demonstrated by both confocal scanning laser microscopy and metabolism of the tetrazolium salt, XTT, a measure of bactericidal activity.

An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of ‘Pathogenic’, ‘Commensal’ and Antibiotic Resistant Strains

The Gram-positive bacterium Propionibacterium acnes is a member of the normal human skin microbiota and is associated with various infections and clinical conditions. There is tentative evidence to suggest that certain lineages may be associated with disease and others with health. We recently described a multilocus sequence typing scheme (MLST) for P. acnes based on seven housekeeping genes (http://pubmlst.org/pacnes). We now describe an expanded eight gene version based on six housekeeping genes and two ‘putative virulence’ genes (eMLST) that provides improved high resolution
typing (91eSTs from 285 isolates), and generates phylogenies congruent with those based on whole genome analysis. When compared with the nine gene MLST scheme developed at the University of Bath, UK, and utilised by researchers at Aarhus University, Denmark, the eMLST method offers greater resolution. Using the scheme, we examined 208 isolates from disparate clinical sources, and 77 isolates from healthy skin. Acne was predominately associated with type IA₁ clonal complexes CC1, CC3 and CC4; with eST1 and eST3 lineages being highly represented. In contrast, type IA₂ strains were recovered at a rate similar to type IB and II organisms. Ophthalmic infections were predominately associated with type IA₁ and IA₂ strains, while type IB and II were more frequently recovered from soft tissue and retrieved medical devices. Strains with rRNA mutations conferring resistance to antibiotics used in acne treatment were dominated by eST3, with some evidence for intercontinental spread. In contrast, despite its high association with acne, only a small number of resistant CC1 eSTs were identified. A number of eSTs were only recovered from healthy skin, particularly eSTs representing CC72 (type II) and CC77 (type III). Collectively our data lends support to the view that pathogenic versus truly commensal lineages of P. acnes may exist. This is likely to have important therapeutic and diagnostic implications.

Molecular basis of Yersinia enterocolitica temperature-dependent resistance to antimicrobial peptides

Antimicrobial peptides (APs) belong to the arsenal of weapons of the innate immune system against infections. In the case of gram-negative bacteria, APs interact with the anionic lipid A moiety of the lipopolysaccharide (LPS). In yersiniae most virulence factors are temperature regulated. Studies from our laboratory demonstrated that Yersinia enterocolitica is more susceptible to polymyxin B, a model AP, when grown at 37°C than at 22°C (J. A. Bengoechea, R. Díaz, and I. Moriyón, Infect. Immun. 64:4891-4899, 1996), and here we have extended this observation to other APs, not structurally related to polymyxin B. Mechanistically, we demonstrate that the lipid A modifications with aminoarabinose and palmitate are downregulated at 37°C and that they contribute to AP resistance together with the LPS O-polysaccharide. Bacterial loads of lipid A mutants in Peyer's patches, liver, and spleen of orogastrically infected mice were lower than those of the wild-type strain at 3 and 7 days postinfection. PhoPQ and PmrAB two-component systems govern the expression of the loci required to modify lipid A with aminoarabinose and palmitate, and their expressions are also temperature regulated. Our findings support the notion that the temperature-dependent regulation of loci controlling lipid A modifications could be explained by H-NS-dependent negative regulation alleviated by RovA. In turn, our data also demonstrate that PhoPQ and PmrAB regulate positively the expression of rovA, the effect of PhoPQ being more important. However, rovA expression reached wild-type levels in the phoPQ pmrAB mutant background, hence indicating the existence of an unknown regulatory network controlling rovA expression in this background.

Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids (SCFAs) and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from either poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective FFA2 agonists that interact with the orthosteric binding site. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 (ECL2) and the transmembrane domain (TM) regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in the regulation of lipolysis in murine 3T3-L1 adipocytes. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the orthosteric binding site of FFA2 that will be invaluable in future ligand development at this receptor.