Data Analysis and Processing in X-ray Diffraction Studies of Scattering From Myosin Heads in Muscle

The X-ray diffraction pattern of muscle tissue contains sharp reflections from ordered structures, and a region of diffuse scatter from disordered structures. The sharp reflections have been studied in detail over many years, but the diffuse scatter has received much less attention, apart from the pioneering work of J. Lowy and F. R. Poulsen, reviewed in the book 'Fibrous Protein Structure' edited by J. Squire and P. Vibert (Academic Press, 1987). In this thesis a set of FORTRAN programs were written to analyse diffraction data, with particular emphasis on the diffuse scatter. Film patterns from some synchrotron experiments and from some of J. Lowy and F. R. Poulsen’s archived material were analysed, to study the effects of varying osmolarity and/or sarcomere length on the sharp reflections and the diffuse scatter. Conclusions were drawn about the feasibility of using the diffuse scatter as an indication of changes in myosin subfragment 1 (the head of the myosin molecule), and of retrieving scattering data from the archived material which had been taken largely to observe sharp reflections. It was concluded that although the archived material could give interesting indications about physiological effects, new experiments were needed to confirm these indications, and these new experiments should be designed specifically towards the diffuse scattering data. In the course of this work, I have considered the possibility of using Guinier plots to derive information on head shapes from data on intact muscle, and the precision with which such information can be obtained. I conclude that the Guinier plots do contain information about the myosin heads in intact muscle, but that the accuracy of the information may not be much better than ±10%, and this may not be sufficient to confirm or deny possible conformation changes in the myosin head. However, I do not consider that the work done in this thesis reached the limit of accuracy, and further experiments may be worthwhile

Development of a novel cell-based assay system EPISSAY for screening epigenetic drugs and liposome formulated decitabine

Extent: 11 p.BACKGROUND: Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation. METHODS: A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. RESULTS: Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. CONCLUSIONS: The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.Sue Ping Lim, Raman Kumar, Yamini Akkamsetty, Wen Wang, Kristen Ho, Paul M. Neilsen, Diego J. Walther, Rachel J. Suetani, Clive Prestidge and David F. Calle

Liposome-encapsulated ISMN: a novel nitric oxide-based therapeutic agent against Staphylococcus aureus biofilms

Background: Staphylococcus aureus in its biofilm form has been associated with recalcitrant chronic rhinosinusitis with significant resistance to conventional therapies. This study aims to determine if liposomal-encapsulation of a precursor of the naturally occurring antimicrobial nitric oxide (NO) enhances its desired anti-biofilm effects against S. aureus, in the hope that improving its efficacy can provide an effective topical agent for future clinical use. Methodology: S. aureus ATCC 25923 biofilms were grown in-vitro using the Minimum Biofilm Eradication Concentration (MBEC) device and exposed to 3 and 60 mg/mL of the NO donor isosorbide mononitrate (ISMN) encapsulated into different anionic liposomal formulations based on particle size (unilamellar ULV, multilamellar MLV) and lipid content (5 and 25 mM) at 24 h and 5 min exposure times. Biofilms were viewed using Live-Dead Baclight stain and confocal scanning laser microscopy and quantified using the software COMSTAT2. Results: At 3 and 60 mg/mL, ISMN-ULV liposomes had comparable and significant anti-biofilm effects compared to untreated control at 24 h exposure (p = 0.012 and 0.02 respectively). ULV blanks also had significant anti-biofilm effects at both 24 h and 5 min exposure (p = 0.02 and 0.047 respectively). At 5 min exposure, 60 mg/mL ISMN-MLV liposomes appeared to have greater anti-biofilm effects compared to pure ISMN or ULV particles. Increasing liposomal lipid content improved the anti-biofilm efficacy of both MLV and ULVs at 5 min exposure. Conclusion: Liposome-encapsulated “nitric oxide” is highly effective in eradicating S. aureus biofilms in-vitro, giving great promise for use in the clinical setting to treat this burdensome infection. Further studies however are needed to assess its safety and efficacy in-vivo before clinical translation is attempted.Camille Jardeleza, Shasha Rao, Benjamin Thierry, Pratik Gajjar, Sarah Vreugde, Clive A. Prestidge, Peter-John Wormal

Distribution and Inhibition of liposomes on Staphylococcus aureus and Pseudomonas aeruginosa biofilm

BACKGROUND Staphylococcus aureus and Pseudomonas aeruginosa are major pathogens in chronic rhinosinusitis (CRS) and their biofilms have been associated with poorer postsurgical outcomes. This study investigated the distribution and anti-biofilm effect of cationic (+) and anionic (-) phospholipid liposomes with different sizes (unilamellar and multilamellar vesicle, ULV and MLV respectively) on S. aureus and P. aeruginosa biofilms. METHOD Specific biofilm models for S. aureus ATCC 25923 and P. aeruginosa ATCC 15692 were established. Liposomal distribution was determined by observing SYTO9 stained biofilm exposed to DiI labeled liposomes using confocal scanning laser microscopy, followed by quantitative image analysis. The anti-biofilm efficacy study was carried out by using the alamarBlue assay to test the relative viability of biofilm treated with various liposomes for 24 hours and five minutes. RESULTS The smaller ULVs penetrated better than larger MLVs in both S. aureus and P. aeruginosa biofilm. Except that +ULV and –ULV displayed similar distribution in S. aureus biofilm, the cationic liposomes adhered better than their anionic counterparts. Biofilm growth was inhibited at 24-hour and five-minute exposure time, although the decrease of viability for P. aeruginosa biofilm after liposomal treatment did not reach statistical significance. CONCLUSION The distribution and anti-biofilm effects of cationic and anionic liposomes of different sizes differed in S. aureus and P. aeruginosa biofilms. Reducing the liposome size and formulating liposomes as positively charged enhanced the penetration and inhibition of S. aureus and P. aeruginosa biofilms.Dong Dong, Nicky Thomas, Benjamin Thierry, Sarah Vreugde, Clive A. Prestidge, Peter-John Wormal

Casimir energy and variational methods in AdS spacetime

Following the subtraction procedure for manifolds with boundaries, we calculate by variational methods, the Schwarzschild-Anti-de Sitter and the Anti-de Sitter space energy difference. By computing the one loop approximation for TT tensors we discover the existence of an unstable mode at zero temperature, which can be stabilized by the boundary reduction method. Implications on a foam-like space are discussed.Comment: Submitted to Classical and Quantum Gravit

Classical and Thermodynamic Stability of Black Branes

It is argued that many non-extremal black branes exhibit a classical Gregory-Laflamme instability if, and only if, they are locally thermodynamically unstable. For some black branes, the Gregory-Laflamme instability must therefore disappear near extremality. For the black $p$-branes of the type II supergravity theories, the Gregory-Laflamme instability disappears near extremality for $p=1,2,4$ but persists all the way down to extremality for $p=5,6$ (the black D3-brane is not covered by the analysis of this paper). This implies that the instability also vanishes for the near-extremal black M2 and M5-brane solutions.Comment: 21 pages, LaTeX. v2: Various points clarified, typos corrected and reference adde

Ultraspinning instability: the missing link

We study linearized perturbations of Myers-Perry black holes in d=7, with two of the three angular momenta set to be equal, and show that instabilities always appear before extremality. Analogous results are expected for all higher odd d. We determine numerically the stationary perturbations that mark the onset of instability for the modes that preserve the isometries of the background. The onset is continuously connected between the previously studied sectors of solutions with a single angular momentum and solutions with all angular momenta equal. This shows that the near-extremality instabilities are of the same nature as the ultraspinning instability of d>5 singly-spinning solutions, for which the angular momentum is unbounded. Our results raise the question of whether there are any extremal Myers-Perry black holes which are stable in d>5.Comment: 19 pages. 1 figur

Casimir energy and black hole pair creation in Schwarzschild-de Sitter spacetime

Following the subtraction procedure for manifolds with boundaries, we calculate by variational methods, the Schwarzschild-de Sitter and the de Sitter space energy difference. By computing the one loop approximation for TT tensors we discover the existence of an unstable mode even for the non-degenerate case. This result seems to be in agreement with the sub-maximal black hole pair creation of Bousso-Hawking. The instability can be eliminated by the boundary reduction method. Implications on a foam-like space are discussed.Comment: 19 pages,RevTeX with package epsf and four eps figures. Added other references. Accepted for publication in Classical and Quantum Gravit

Some Aspects of Virtual Black Holes

In this paper we shall consistently third quantize modified gravity. Then we shall analyse certain aspects of virtual black holes in this third quantized modified gravity. We will see how a statistical mechanical origin for the Bekenstein-Hawking entropy naturally arises in this model. Furthermore, in this model the area and thus the entropy of a real macroscopic black hole is quantized. Virtual black holes cause loss of quantum coherence and this gives an intrinsic entropy to all physical systems which can be used to define a direction of time and hence provide a solution to the problem of time.Comment: 11 pages, 0 figures, accepted for publication in JET

Ultraspinning instability of anti-de Sitter black holes

Myers-Perry black holes with a single spin in d>5 have been shown to be unstable if rotating sufficiently rapidly. We extend the numerical analysis which allowed for that result to the asymptotically AdS case. We determine numerically the stationary perturbations that mark the onset of the instabilities for the modes that preserve the rotational symmetries of the background. The parameter space of solutions is thoroughly analysed, and the onset of the instabilities is obtained as a function of the cosmological constant. Each of these perturbations has been conjectured to represent a bifurcation point to a new phase of stationary AdS black holes, and this is consistent with our results.Comment: 22 pages, 7 figures. v2: Reference added. Matches published versio