Investigating the molecular basis of radiation resistance in proteins & Oxidative modifiactions of SARS-CoV-2 M^pro

The nature of biological radiation resistance is a question that has fascinated and inspired researchers since the first discovery of extremely radiation resistant organisms like Deinococcus radiodurans. To date many tightly coordinated mechanisms have been identified that lead to the emergence of the phenotype of extreme radiation resistance but only in the last decade was it found that proteome protection constitutes survival not DNA protection. This thesis aims to provide a molecular understanding of how protein sequence and structure evolved to mitigate the damaging effects of ionising radiation in the environment. To systematically study whether the amino acid composition (primary sequence) is the source of a protein radiation resistance free amino acids, specifically tryptophan, has been soaked into multiple protein crystals and the average response to radiation insult has been compared with their apo counterparts. By collecting dose series measurements and using online UV/Vis spectroscopy the effect on global and specific damage rates has been analysed, showing that for lysozyme, thaumatin, AcNiR and 4HB1 no significant radio protective effect could be achieved by supplementing a protein crystal with tryptophan. Interestingly, one 4HB1 crystal that had been soaked with 100 mM tryptophan survived a total absorbed dose of 182.3 MGy (half dose of 64.43 MGy). Although the exact conditions leading to this result were not reproducible, this result constitutes an unprecedented case of extreme radiation tolerance in an intense X-ray beam and was hence included in the analysis.A bioinformatics study has been performed, which analysed the amino acid composition radiation hard bacteria and compared them with 8000 bacteria proteomes. The amino acid distribution of radiation resistant organisms showed no common bias towards a particular amino acid or combination of amino acids. Clustering a subset of 200 proteomes from each domain showed that the phylogenetic filiation can be predicted from the amino acid composition, the phenomenon of extreme radiation resistance however can not be predicted. This result supports the conclusion that there is no single amino acid or combination thereof that are the source of a proteins radiation resistance.Oxidative modifications are not uncommon in cysteine proteases and have been shown affect and even inhibit enzymatic activity. Recent structures of the SARS-CoV-2 main protease (Mpro) show indications for similar modifications despite the fact that the enzyme is naturally expressed in the cytosol which is considered to be of reducing nature. To determine whether this modification is an artefact of the purification strategy and what impact such a modification on the enzyme activity as well as recent active-site drug screening efforts would have, Mpro was purified under aerobic conditions (as reported by most studies), aerobic conditions without the use of reducing agents and anaerobic conditions. X-ray diffraction data of Mpro from both aerobic purifications indicate oxidative modifications of the active site Cys145. Using mass spectrometry, we could show that in the presence of reducing agents Mpro is only oxidised when the effectiveness of reducing agents decays, e.g. during long crystallisation periods but not during the purification itself. Without reducing agents at latest after 12 days Mpro molecules can be expected to contain sulfenic acid (-SO) and sulfinic acid (-SO2) modifications at the active site Cys145. As a result the oxidised enzyme has a specificity constant approximately 50 % lower than unmodified Mpro for the substrate Ac-Abu-Tle-Leu-Gln-AMC. By purifying and crystallising Mpro under anaerobic conditions this study shows that the oxidation of the enzyme can be avoided and is therefore likely an artefact of the in vitro enzyme processing

Uncoupling of EGFR–RAS signaling and nuclear localization of YBX1 in colorectal cancer

The transcription factor YBX1 can act as a mediator of signals transmitted via the EGFR–RAS–MAPK axis. YBX1 expression has been associated with tumor progression and prognosis in multiple types of cancer. Immunohistochemical studies have revealed dependency between YBX1 expression and individual EGFR family members. We analyzed YBX1 and EGFR family proteins in a colorectal cancer (CRC) cohort and provide functional analyses of YBX1 in the context of EGFR–RAS–MAPK signaling. Immunohistochemistry for YBX1 and EGFR family receptors with two antibodies for YBX1 and EGFR were performed and related to clinicopathological data. We employed Caco2 cells expressing an inducible KRASV12 gene to determine effects on localization and levels of YBX1. Mouse xenografts of Caco2-KRASV12 cells were used to determine YBX1 dynamics in a tissue context. The two different antibodies against YBX1 showed discordant immunohistochemical stainings in cell culture and clinical specimens. Expression of YBX1 and EGFR family members were not correlated in CRC. Analysis of Caco2 xenografts displayed again heterogeneity of YBX1 staining with both antibodies. Our results suggest that YBX1 is controlled via complex regulatory mechanisms involving tumor stroma interaction and signal transduction processes. Our study highlights that YBX1 antibodies have different specificities, advocating their use in a combined manner

Shock Acceleration of Cosmic Rays - a critical review

Motivated by recent unsuccessful efforts to detect the predicted flux of TeV gamma-rays from supernova remnants, we present a critical examination of the theory on which these predictions are based. Three crucial problems are identified: injection, maximum achievable particle energy and spectral index. In each case significant new advances in understanding have been achieved, which cast doubt on prevailing paradigms such as Bohm diffusion and single-fluid MHD. This indicates that more realistic analytical models, backed by more sophisticated numerical techniques should be employed to obtain reliable predictions. Preliminary work on incorporating the effects of anomalous transport suggest that the resulting spectrum should be significantly softer than that predicted by conventional theory.Comment: 8 pages, invited review presented at the 17th ECRS, Lodz, July 2000; to appear in Journal of Physics G: Nuclear and Particle Physic

Efficiency of Nonlinear Particle Acceleration at Cosmic Structure Shocks

We have calculated the evolution of cosmic ray (CR) modified astrophysical shocks for a wide range of shock Mach numbers and shock speeds through numerical simulations of diffusive shock acceleration (DSA) in 1D quasi- parallel plane shocks. The simulations include thermal leakage injection of seed CRs, as well as pre-existing, upstream CR populations. Bohm-like diffusion is assumed. We model shocks similar to those expected around cosmic structure pancakes as well as other accretion shocks driven by flows with upstream gas temperatures in the range $T_0=10^4-10^{7.6}$K and shock Mach numbers spanning $M_s=2.4-133$. We show that CR modified shocks evolve to time-asymptotic states by the time injected particles are accelerated to moderately relativistic energies (p/mc \gsim 1), and that two shocks with the same Mach number, but with different shock speeds, evolve qualitatively similarly when the results are presented in terms of a characteristic diffusion length and diffusion time. For these models the time asymptotic value for the CR acceleration efficiency is controlled mainly by shock Mach number. The modeled high Mach number shocks all evolve towards efficiencies $\sim 50$%, regardless of the upstream CR pressure. On the other hand, the upstream CR pressure increases the overall CR energy in moderate strength shocks ($M_s \sim {\rm a few}$). (abridged)Comment: 23 pages, 12 ps figures, accepted for Astrophysical Journal (Feb. 10, 2005

A Multi-Frequency Radio Study of Supernova Remnant G292.0+1.8 and its Pulsar Wind Nebula

(Abridged) We present a detailed radio study of the young supernova remnant (SNR) G292.0+1.8 and its associated pulsar PSR J1124-5916, using the Australia Telescope Compact Array at observing wavelengths of 20, 13 and 6 cm. We find that the radio morphology of the source consists of three main components: a polarized flat-spectrum central core coincident with the pulsar J1124-5916, a surrounding circular steep-spectrum plateau with sharp outer edges and, superimposed on the plateau, a series of radial filaments with spectra significantly flatter than their surroundings. HI absorption argues for a lower limit on the distance to the system of 6 kpc. The core clearly corresponds to radio emission from a pulsar wind nebula powered by PSR J1124-5916, while the plateau represents the surrounding SNR shell. The plateau's sharp outer rim delineates the SNR's forward shock, while the thickness of the plateau region demonstrates that the forward and reverse shocks are well-separated. Assuming a distance of 6 kpc and an age for the source of 2500 yr, we infer an expansion velocity for the SNR of ~1200 km/s and an ambient density ~0.9 cm^-3. We interpret the flat-spectrum radial filaments superimposed on the steeper-spectrum plateau as Rayleigh-Taylor unstable regions between the forward and reverse shocks of the SNR. The flat radio spectrum seen for these features results from efficient second-order Fermi acceleration in strongly amplified magnetic fields.Comment: 11 pages of text, plus 7 embedded EPS figures. Accepted to ApJ. Added missing units on x-axis of Fig

Brownian Carnot engine

The Carnot cycle imposes a fundamental upper limit to the efficiency of a macroscopic motor operating between two thermal baths. However, this bound needs to be reinterpreted at microscopic scales, where molecular bio-motors and some artificial micro-engines operate. As described by stochastic thermodynamics, energy transfers in microscopic systems are random and thermal fluctuations induce transient decreases of entropy, allowing for possible violations of the Carnot limit. Despite its potential relevance for the development of a thermodynamics of small systems, an experimental study of microscopic Carnot engines is still lacking. Here we report on an experimental realization of a Carnot engine with a single optically trapped Brownian particle as working substance. We present an exhaustive study of the energetics of the engine and analyze the fluctuations of the finite-time efficiency, showing that the Carnot bound can be surpassed for a small number of non-equilibrium cycles. As its macroscopic counterpart, the energetics of our Carnot device exhibits basic properties that one would expect to observe in any microscopic energy transducer operating with baths at different temperatures. Our results characterize the sources of irreversibility in the engine and the statistical properties of the efficiency -an insight that could inspire novel strategies in the design of efficient nano-motors.Comment: 7 pages, 7 figure

Canonical Particle Acceleration in FRI Radio Galaxies

Matched resolution multi-frequency VLA observations of four radio galaxies are used to derive the asymptotic low energy slope of the relativistic electron distribution. Where available, low energy slopes are also determined for other sources in the literature. They provide information on the acceleration physics independent of radiative and other losses, which confuse measurements of the synchrotron spectra in most radio, optical and X-ray studies. We find a narrow range of inferred low energy electron energy slopes, n(E)=const*E^-2.1 for the currently small sample of lower luminosity sources classified as FRI (not classical doubles). This distribution is close to, but apparently inconsistent with, the test particle limit of n(E)=const*E^-2.0 expected from strong diffusive shock acceleration in the non-relativistic limit. Relativistic shocks or those modified by the back-pressure of efficiently accelerated cosmic rays are two alternatives to produce somewhat steeper spectra. We note for further study the possiblity of acceleration through shocks, turbulence or shear in the flaring/brightening regions in FRI jets as they move away from the nucleus. Jets on pc scales and the collimated jets and hot spots of FRII (classical double) sources would be governed by different acceleration sites and mechanisms; they appear to show a much wider range of spectra than for FRI sources.Comment: 16 figures, including 5 color. Accepted to Astrophysical Journa

Tissue invasion and metastasis: molecular, biological and clinical perspectives

Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. While advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarizes recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. This review lists the disruption of E-cadherin and tight junctions, key signaling pathways, including urokinase type plasminogen activator (uPA), phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT), focal adhesion kinase (FAK), β-catenin/zinc finger E-box binding homeobox 1 (ZEB-1) and transforming growth factor beta (TGF-β), together with inactivation of activator protein-1 (AP-1) and suppression of matrix metalloproteinase-9 (MMP-9) activity as key targets and the use of phytochemicals, or natural products, such as those from Agaricus blazei, Albatrellus confluens, Cordyceps militaris, Ganoderma lucidum, Poria cocos and Silybum marianum, together with diet derived fatty acids gamma linolenic acid (GLA) and eicosapentanoic acid (EPA) and inhibitory compounds as useful approaches to target tissue invasion and metastasis as well as other hallmark areas of cancer. Together, these strategies could represent new, inexpensive, low toxicity strategies to aid in the management of cancer metastasis as well as having holistic effects against other cancer hallmarks.W.G. Jiang ... S.K. Thompson ... et al

Numerical Studies of Cosmic Ray Injection and Acceleration

A numerical scheme that incorporates a thermal leakage injection model into a combined gas dynamics and cosmic ray (CR, hereafter) diffusion-convection code has been developed. The particle injection is followed numerically by filtering the diffusive flux of suprathermal particles across the shock to the upstream region according to a velocity-dependent transparency function that controls the fraction of leaking particles. We have studied CR injection and acceleration efficiencies during the evolution of CR modified planar shocks for a wide range of initial shock Mach numbers, $M_0$, assuming a Bohm-like diffusion coefficient. The injection process is very efficient when the subshock is strong, leading to fast and significant modification of the shock structure. As the CR pressure increases, the subshock weakens and the injection rate decreases accordingly, so that the subshock does not disappear. Although some fraction of the particles injected early in the evolution continue to be accelerated to ever higher energies, the postshock CR pressure reaches an approximate time-asymptotic value due to a balance between fresh injection/acceleration and advection/diffusion of the CR particles away from the shock. We conclude that the injection rates in strong parallel shocks are sufficient to lead to rapid nonlinear modifications to the shock structures and that self-consistent injection and time-dependent simulations are crucial to understanding the non-linear evolution of CR modified shocks.Comment: 28 pages, To appear in ApJ November 1, 2002 issu