First Report of Beet virus Q in Spain.

Beet virus Q (BVQ) is a member of the genus Pomovirus that is transmitted by Polymyxa betae Keskin. Initially described as the Wierthe serotype of Beet soilborne virus (BSBV), BVQ is now considered a distinct virus species based on its genomic properties (1). BVQ is commonly found in fields where BSBV and the causal agent of rhizomania disease, Beet necrotic yellow vein virus (BNYVV), are also present. Simultaneous infection of sugar beet plants with multiple virus species could affect disease symptom expression (4). For this reason, the pathogenicity of BVQ and its role in the epidemiology of rhizomania disease remain a subject of study. During 2004, six soil samples were collected from different sites in the Castilla-La Mancha Region in Spain (Albacete and Ciudad Real provinces) where rhizomania symptoms were observed in BNYVV-tolerant sugar beet cultivars. Soil from the Hainaut Region of Belgium, infected with BNYVV, BSBV, and BVQ and supplied by Prof. C. Bragard (Unité de Phytopathologie, Université Catholique de Louvain, Belgium) was used as a positive control. Sugar beet plants (cv. Asso) were grown in the soil samples for 45 days at 24°C and then root tissue was harvested. All samples were analyzed using enzyme-linked immunosorbent assay (ELISA) with commercial BNYVV antiserum (BIOREBA AG, Reinach, Switzerland) and BSBV/BVQ antisera (IC10 and 6G2) supplied by R. Koenig (Federal Biological Research Centre for Agriculture and Forestry, Braunschweig, Germany). Total RNA extracted from sugar beet roots as previously described (3) was tested using reverse transcription-polymerase chain reaction (RT-PCR). Primers BVQ3F (5'-GTT TTC AAA CTT GCC ATC CT-3') and BVQ3R2 (5'-CCA CAA TGG GCC AAT AGA-3'), which amplify a 690-bp fragment of the triple gene block region of BVQ RNA 3, were designed based on the published sequence (GenBank Accession No. AJ223598). The presence of BSBV and BNYVV was assayed using RT-PCR with previously described primers (2,3). BVQ was detected from plants grown in soil collected from La Roda (Albacete) in Spain and from Hainaut in Belgium. The fragments amplified from Spanish sample with BVQ3F and BVQ3R2 (GenBank Accession No. AY849375) showed 95.9% nucleotide sequence identity with the previously published sequence of BVQ (1). The La Roda BVQ isolate was mechanically transmitted to Chenopodium quinoa from infected sugar beet root tissue. BVQ was detected using RT-PCR in local lesions that appeared approximately 5 days after inoculation and subsequently spread along veins. To our knowledge, this is the first report of BVQ in soil from Spain, although it has been previously reported in Belgium, Bulgaria, France, Germany, Hungary, and the Netherlands (2). BSBV and BNYVV (type A) were detected in all six Spanish samples, as well as in the Belgian soil. References: (1) R. Koenig et al. J. Gen. Virol. 79:2027, 1998. (2) A. Meunier et al. Appl. Environ Microbiol. 69:2356, 2003. (3) C. Ratti et al. J. Virol. Methods 124:41, 2005. (4) C. Rush Annu. Rev Phytopathol 41:567, 2003.

Role of shielding in modulating the effects of solar particle events: Monte Carlo calculation of absorbed dose and DNA complex lesions in different organs.

Distributions of absorbed dose and DNA clustered damage yields in various organs and tissues following the October 1989 solar particle event (SPE) were calculated by coupling the FLUKA Monte Carlo transport code with two anthropomorphic phantoms (a mathematical model and a voxel model), with the main aim of quantifying the role of the shielding features in modulating organ doses. The phantoms, which were assumed to be in deep space, were inserted into a shielding box of variable thickness and material and were irradiated with the proton spectra of the October 1989 event. Average numbers of DNA lesions per cell in different organs were calculated by adopting a technique already tested in previous works, consisting of integrating into "condensed-history" Monte Carlo transport codes--such as FLUKA--yields of radiobiological damage, either calculated with "event-by-event" track structure simulations, or taken from experimental works available in the literature. More specifically, the yields of "Complex Lesions" (or "CL", defined and calculated as a clustered DNA damage in a previous work) per unit dose and DNA mass (CL Gy-1 Da-1) due to the various beam components, including those derived from nuclear interactions with the shielding and the human body, were integrated in FLUKA. This provided spatial distributions of CL/cell yields in different organs, as well as distributions of absorbed doses. The contributions of primary protons and secondary hadrons were calculated separately, and the simulations were repeated for values of Al shielding thickness ranging between 1 and 20 g/cm2. Slight differences were found between the two phantom types. Skin and eye lenses were found to receive larger doses with respect to internal organs; however, shielding was more effective for skin and lenses. Secondary particles arising from nuclear interactions were found to have a minor role, although their relative contribution was found to be larger for the Complex Lesions than for the absorbed dose, due to their higher LET and thus higher biological effectiveness.

Estimating mixed field effects: an application supporting the lack of a non-linear component for chromosome aberration induction by neutrons.

The action of neutron fields on biological structures was investigated on the basis of chromosome aberration induction in human cells. Available experimental data on aberration induction by neutrons and their interaction products were reviewed. Present criteria adopted in neutron radiation protection were discussed. The linear coefficient alpha and the quadratic coefficient beta describing dose-response curves for dicentric chromosomes induced by neutrons of different energies were calculated via integration of experimental data on dicentric induction by photons and charged particles into the Monte Carlo transport code FLUKA. The predicted values of the linear coefficients for neutron beams of different energies showed good agreement with the corresponding experimental values, whereas the data themselves indicated that the neutron quadratic coefficient cannot be obtained by 'averaging' the beta values of recoil ions and other nuclear reaction products. This supports the hypothesis that neutron induced aberrations increase substantially linearly with dose, a question that has been object of debate for a long time and is still open.

Nuclear architecture and radiation induced chromosome aberrations: models and simulations.

Knowledge of radiation track structure and its interaction with biological targets is a fundamental starting point in understanding the mechanisms underlying the induction of biological damage. In this context Monte Carlo codes are a powerful tool of investigation, allowing one to simulate both track structure and the features of the target(s) of interest at different scales, from nanometres (linear dimensions of DNA) to micrometres (linear dimensions of human cell nuclei and interphase chromosome territories). In the light of recent experimental findings on nuclear architecture, different approaches in modelling chromosome structure and aberration induction are discussed. In particular, a model is presented in which chromosome territories were explicitly described as subnuclear regions and aberration induction was modelled by coupling the structure of the target with that of the radiation track. Comparisons between model predictions and experimental results from the literature are also reported.

Modelling chromosomal aberration induction by ionising radiation: the influence of interphase chromosome architecture.

Several advances have been achieved in the knowledge of nuclear architecture and functions during the last decade, thus allowing the identification of interphase chromosome territories and sub-chromosomal domains (e.g. arm and band domains). This is an important step in the study of radiation-induced chromosome aberrations; indeed, the coupling between track-structure simulations and reliable descriptions of the geometrical properties of the target is one of the main tasks in modelling aberration induction by radiation, since it allows one to clarify the role of the initial positioning of two DNA lesions in determining their interaction probability. In the present paper, the main recent findings on nuclear and chromosomal architecture are summarised. A few examples of models based on different descriptions of interphase chromosome organisation (random-walk models, domain models and static models) are presented, focussing on how the approach adopted in modelling the target nuclei and chromosomes can influence the simulation of chromosomal aberration yields. Each model is discussed by taking into account available experimental data on chromosome aberration induction and/or interphase chromatin organisation. Preliminary results from a mechanistic model based on a coupling between radiation track-structure features and explicitly-modelled, non-overlapping chromosome territories are presented.

A Monte Carlo code for a direct estimation of radiation risk.

An example of pragmatic approach for predicting mixed field effects is presented. The method was initially applied adopting the following, commonly used, assumptions: a) radiation risk (typically cancer) is correlated with chromosome aberration induction; b) radiation-induced chromosome-exchange yield can be well described by a linear-quadratic dependence on particle fluences (mostly linear with high-LET radiation), with parameters depending on particle types and energies. Information on monochromatic field radiobiological effects was integrated in a condensed-history Monte Carlo transport code (FLUKA), able to simulate nuclear interactions. The integrated code provides the chromosome aberration yield (and thus an estimation of radiation risk) in each voxel of any irradiated volume, given any external mixed-field irradiation; in the present work, the method was tested for neutron irradiation of a water phantom. FLUKA was then coupled with a geometrical human phantom provided with different radiation shielding, in order to apply this approach to estimate radiation risk in manned space missions.

Nuclear detecting systems at LNL and LNS: foreseen experiments to provide basic data for heavy-ion risk assessment.

The use of existing detecting systems developed for nuclear physics studies allows collecting data on particle and ion production cross-sections in reactions induced by Oxygen and Carbon beams, of interest for hadrontherapy and heavy-ion risk assessment. The MULTICS and GARFIELD apparatus, together with the foreseen experiments, are reviewed.

Mechanistic bases for modelling space radiation risk and planning radiation protection of astronauts.

The approaches generally adopted for planning radiation protection in ground-based facilities cannot be applied straightforward for astronaut protection in space. Indeed in such extreme conditions, modelling methods and shielding design must be based on a detailed mechanistic knowledge of the peculiar astronauts irradiation conditions. Great help can derive from mechanistic modelling, generally aimed to better understand the intermediate steps leading from the initial energy depositions to different biological endpoints, up to organ and organism level. In the present work, criteria will be illustrated for using mechanistic approaches in developing practical tools for astronauts radioprotection, once the external field and the interaction cross sections with the spacecraft walls are known; particular attention will be given to the treatment of mixed fields. Techniques for integrating into condensed-history codes stochastic information provided by event-by-event simulations will be presented.

Stochastic aspects and uncertainties in the prechemical and chemical stages of electron tracks in liquid water: a quantitative analysis based on Monte Carlo simulations.

A new physical module for the biophysical simulation code PARTRAC has recently been developed, based on newly derived electron inelastic-scattering cross-sections in liquid water. In the present work, two modules of PARTRAC describing the production, diffusion and interaction of chemical species were developed with the specific purpose of quantifying the role of the uncertainties in the parameters controlling the early stages of liquid water radiolysis. A set of values for such parameters was identified, and time-dependent yields and frequency distributions of chemical species produced by electrons of different energies were calculated. The calculated yields were in good agreement with available data and simulations, thus confirming the reliability of the code. As the primary-electron energy decreases down to 1 keV, the *OH decay kinetics were found to get faster, reflecting variations in the spatial distribution of the initial energy depositions. In agreement with analogous works, an opposite trend was found for energies of a few hundred eV, due to the very small number of species involved. The spreading effects shown at long times by *OH frequency distributions following 1 keV irradiation were found to be essentially due to stochastic aspects of the chemical stage, whereas for 1 MeV tracks the physical and pre-chemical stages also were found to play a significant role. Relevant differences in the calculated e(aq) -yields were found by coupling the physics of PARTRAC with descriptions of the pre-chemical and chemical stages adopted in different models. This indicates a strict interrelation of the various stages, and thus a strong dependence of the parameter values on the assumptions made for the preceding and subsequent stages of the process. Although equally acceptable results can be obtained starting from different assumptions, it is necessary to keep control of such uncertainties, since they can significantly influence the modeling of radical attack on DNA and, more generally, radiobiological damage estimation. This study confirms the need for new, independently derived data on specific steps of water radiolysis, to be included in comprehensive biophysical simulation codes.

Melanotropic peptides-lipid bilayer interaction. Comparison of the hormone alpha-MSH to a biologically more potent analog.

The interaction of the native peptide alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more active analog [Nle4, D-Phe7]-alpha-MSH(MSH-I) with lipid vesicles was studied by spin label electron spin resonance (ESR) spectroscopy and circular dichroism (CD). Using spin labels located at the membrane interface and at different depths along the acyl chain, it was shown that the binding of both peptides to the membrane induces tighter lipid packing at all the monitored positions. However, the effect of the analog on the spin label ESR parameters was much more evident, and suggested that it penetrates farthest into the lipid matrix than the native molecule. Lipid partition coefficients were calculated based on the effect the peptides cause on the ESR spectra of spin labels incorporated in the membrane. For the biologically more potent peptide, the partition coefficient was found to be about 4-times greater than that of the native hormone. For the same concentration of peptide bound to the membrane, MSH-I was found to cause a slightly greater effect on the membrane structure than alpha-MSH, in accord with its possible deeper penetration into the bilayer. CD spectra in aqueous solution and in the alpha-helix inducing solvent 2,2,2-trifluoroethanol showed that the two peptides have somewhat different structures in solution, though similar conformational changes occur in both peptides as a result of their interaction with negatively charged vesicles or micelles. The higher peptide-lipid association constant and the deeper penetration of the analog into lipid bilayers could be related to its greater activity and/or prolonged action.

How melatonin interacts with lipid bilayers: a study by fluorescence and ESR spectroscopies.

ESR spectra of spin labels placed at the membrane surface and at different depths of the bilayer core, and melatonin fluorescence in the presence of lipid vesicles, suggest an average shallow position for the hormone in the membrane. However, according to the melatonin ability to cross lipid bilayers, nitroxides placed deep in the bilayer were able to quench the melatonin fluorescence. Melatonin membrane partition coefficients were calculated for bilayers in different packing states, and similar and rather high values were found. The data presented here may be quite important to the understanding of melatonin physiological actions at the membrane level.

Spin label and 2H-NMR studies on the interaction of melanotropic peptides with lipid bilayers.

The interaction of the cationic tridecapeptide alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more active analog [Nle4, DPhe7]-alpha-MSH with lipid membranes was investigated by means of ESR of spin probes incorporated in the bilayer, and NMR of deuterated lipids. All spin labels used here, stearic acid and phospholipid derivatives labeled at the 5th and 12th position of the hydrocarbon chain, and the cholestane label, incorporated into anionic vesicles of DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) in the liquid-crystalline phase, indicated that both peptides decrease the motional freedom of the acyl chains. No peptide effect was detected with neutral lipid bilayers. Changes in the alpha-deuteron quadrupolar splittings and spin lattice relaxation time of DMPG deuterated at the glycerol headgroup paralleled the results obtained with ESR, showing that the peptides cause a better packing both at the headgroup and at the acyl chain bilayer regions. The stronger effect caused by the more potent analog in the membrane structure, when compared to the native hormone, is discussed in terms of its larger lipid association constant and/or its deeper penetration into the bilayer.

Analysis of urinary catecholamines by high-performance liquid chromatography in the presence of labetalol metabolites.

Common sample preparation methods for catecholamines lead to contamination with metabolites of labetalol, an anti-hypertensive drug. When the extracts are analyzed by cation-exchange high-performance liquid chromatography, these metabolites are separated from the catecholamines, but their strong retention lengthens the analysis time. A procedure has been developed for complete removal of these drug metabolites from acidified urine by the use of XAD-4 resin. Loss of catecholamines is monitored by an internal standard. This pretreatment can be combined with extraction by weak cation-exchange resin and borate elution to simplify catecholamine analysis for patients receiving labetalol.