Wax boluses and accuracy of EBT and RTQA radiochromic film detectors in radiotherapy with the JINR Phasotron proton beam

Aim

To present the results obtained using radiochromic films EBT and RTQA 1010P for the reconstruction the dose distributions for targets irradiated by proton beam and modified by wax boluses.

Background

In Medico-Technical Complex at the Joint Institute for Nuclear Research in Dubna implemented technology of wax boluses.

Materials and methods

Wax boluses are easier to make and they give better dose distributions than boluses made from modeling clay previously used at our center. We irradiated two imaginary targets, one shaped as a cylinder and the other one as two cuboids. The evaluated calibration curve was used for calculation of the dose distributions measured by the EBT and RTQA radiochromic film. In both cases, the measured dose distributions were compared to the dose distributions calculated by the treatment planning system (TPS). We also compared dose distributions using three different conformity indices at a 95% isodose.

Results

Better target coverage and better compliance of measurements (semiconductor detectors and radiochromic films) with calculated doses was obtained for cylindrical target than for cuboidal target. The 95% isodose covered well the tumor for both target shapes, while for cuboidal target larger volume around the target received therapeutic dose, due to the complicated target shape. The use wax boluses provided to be effective tool in modifying proton beam to achieve appropriate shape of isodose distribution.

Conclusion

EBT film yielded the best visual matching. Both EBT and RTQA films confirmed good conformity between calculated and measured doses, thus confirming that wax boluses used to modify the proton beam resulted in good dose distributions.     

A library of recombinant Babesia microti cell surface and secreted proteins for diagnostics discovery and reverse vaccinology

Human babesiosis is an emerging tick-borne parasitic disease and blood transfusion-transmitted infection primarily caused by the apicomplexan parasite, Babesia microti. There is no licensed vaccine for B. microti and the development of a reliable serological screening test would contribute to ensuring the safety of the donated blood supply. The recent sequencing of the B. microti genome has revealed many novel genes encoding proteins that can now be tested for their suitability as subunit vaccine candidates and diagnostic serological markers. Extracellular proteins are considered excellent vaccine candidates and serological markers because they are directly exposed to the host humoral immune system, but can be challenging to express as soluble recombinant proteins. We have recently developed an approach based on a mammalian expression system that can produce large panels of functional recombinant cell surface and secreted parasite proteins. Here, we use the B. microti genome sequence to identify 54 genes that are predicted to encode surface-displayed and secreted proteins expressed during the blood stages, and show that 41 (76%) are expressed using our method at detectable levels. We demonstrate that the proteins contain conformational, heat-labile, epitopes and use them to serologically profile the kinetics of the humoral immune responses to two strains of B. microti in a murine infection model. Using sera from validated human infections, we show a concordance in the host antibody responses to B. microti infections in mouse and human hosts. Finally, we show that BmSA1 expressed in mammalian cells can elicit high antibody titres in vaccinated mice using a human-compatible adjuvant but these antibodies did not affect the pathology of infection in vivo. Our library of recombinant B. microti cell surface and secreted antigens constitutes a valuable resource that could contribute to the development of a serological diagnostic test, vaccines, and elucidate the molecular basis of host-parasite interactions.     

Redox control of proton transfers in membrane b-type cytochromes: an absorption and resonance Raman study on bis(imidazole) and bis(imidazolate) model complexes of iron-protoporphyrin

Optical absorption spectra and resonance Raman (RR) spectra, obtained with Soret excitation, are reported for bis(imidazole) and bis(imidazolate) complexes of iron(II)- and iron(III)-protoporphyrin IX, prepared in aqueous conditions. Perdeuteration experiments on the axial ligands permitted the assignment of the symmetric Fe-(ligand)₂ stretching mode of Fe[x]PP(L)₂ to RR bands at 203 (x = II; L = ImH), 212 (x = II; L = Im^–), 201 (x = III; L = ImH) and 226 cm^–1 (x = III; L = Im^–). These frequency differences indicate a strengthening of the axial bonds when the imidazole deprotonations occur. The larger difference observed for the ferric derivatives reflects the stronger -donor capability of the Im^– anion for iron(III) over iron(II). For the ferrous derivatives, the frequencies of several skeletal porphyrin modes (₄, ₁₀, ₁₁ and ₃₈) are downshifted by 2–10 cm^–1 upon deprotonation of the ligands. This effect corresponds to an increased back-bonding from the metal atom to the porphyrin ring when the axial ligand decreases its -acid strength. Bringing further support to this interpretation, an inverse linear relationship is established between the frequencies of (Fe(Il)-L₂) and ₁₁. This correlation is expected to monitor the overall H-bonding state of histidine ligands of reduced cytochromes b. On the other hand, absorption measurements have characterized large pK_a differences for the sequential imidazole ionizations of Fe[x]PP(ImH)₂ in aqueous cetyltrimethylammonium bromide (9.0 and 10.8 for x = 111; 13.0 and 14.1 for x = II). These titrations show that Fe(II)PP(Im^–)₂ and Fe(III)PP(ImH)₂ are good proton-acceptor and proton-donor, respectively, and suggest a model by which heme, located in a favorable environment inside a cytochrome, could couple a cycle of electron transfer with a proton transfer. Based on sequence data and structural models, it is further proposed that, in several membrane cytochromes b (b, b ₆, b ₅₅₉), a positively charged amino acid residue and an imidazolate ligand of the ferriheme could form an ion pair involved in a redox control of proton transfer.Abbreviations RR resonance Raman - EPR electron paramagnetic resonance - PP protoporphyrin IX - ImH imidazole - Im^– imidazolate - Im* imidazole or imidazolate - 1MeIm 1-methylimidazole - HisH histidine - His^– histidinate - CTABr cetyltrimethylammonium bromide - NaDS sodium dodecylsulphate - VLP very low potential - LP low potential - HP high potential     

Phenomenological and molecular models of biological proton transfers

In this paper, the main models describing the transfer of a proton in a molecular system are presented. Models valid when the intersite coupling is weak (non-adiabatic and electronically adiabatic regimes) and strong (adiabatic regime) are described. We distinguish molecular models in which the rate constant is obtained by considering explicitly various degrees of freedom of the system and simpler, phenomenological models built to account for the kinetic isotope effect. The relations between the various models are discussed. Their application to specific systems is illustrated by several studies reported in the literature, with a special emphasis on biological systems.     

Effect of brassinosteroids on growth and proton extrusion in the alga Chlorella vulgaris Beijerinck (Chlorophyceae)

Brassinolide, as a plant hormone, promotes growth of a number of plant species. Similar effects are induced by its epimer 24-epibrassinolide. In this paper we discuss the effects of brassinosteroids on the growth and proton extrusion in the green alga Chlorella vulgaris (Chlorophyceae). At concentrations between 10^–15 and 10^–8 m, brassinolide and 24-epibrassinolide induce a significant stimulation of growth and H⁺ extrusion. The growth was associated with an increase in the capability of algal cells to acidify the medium, where brassinolide is biologically more active than 24-epibrassinolide.Abbreviations BL brassinolide - BR(s) brassinosteroid(s) - epiBL 24-epibrassinolide - DW dry weight - IAA indole-3-acetic acid     

The effective molarity (EM) puzzle in proton transfer reactions

The DFT and HF calculation results for the proton transfer reactions of three different systems reveal that the reaction mechanism (transfer of a proton to a nucleophile) is largely determined by the distance between the two reactive centers (r).Systems with relatively large r values tend to abstract a proton from a molecule of water, whereas, these with a relatively small r values prefer to be engaged intramolecularly and their interaction with water is only via hydrogen bonding. Further, the results indicate that the effective molarity (log EM) for an intramolecular process is strongly correlated with the distance between the two reacting centers (r) in accordance with Menger’s “spatiotemporal hypothesis”.     

Experiences with a statistical quality control system (QCS) for coagulation diagnostics parameters

With the Quality-Control-Service (QCS) for blood coagulation a system for the statistical quality control of blood coagulation methods is presented. The system is based on the universal control plasma PreciClot which contains target values in the normal and abnormal range. As the control plasma is used daily from the participants for quality control exercises and datas are statistically analyzed each month this programme of quality assessment can be compared with a monthly ring trial. For the methods prothrombin time (PT/Quick), activated partial prothrombin time (APTT), fibrinogen assay (Fibrinogen) and thrombin time (Thrombin) datas of a survey period (January-December 1985) with 75 labs were evaluated. Calculated results for the methods are given and accuracy and precision of the methods are compared with the results of former ring trials. Based on the results the interlaboratory reliability of the methods is discussed and the advantages of QCS for blood Coagulation for a better information about quality of coagulation tests are presented.     

Mechanism for proton conduction of the M(2) ion channel of influenza A virus

The M(2) integral membrane protein of influenza A virus forms a proton-selective ion channel. We investigated the mechanism for proton transport of the M(2) protein in Xenopus oocytes using a two-electrode voltage clamp and in CV-1 cells using the whole cell patch clamp technique. Membrane currents were recorded while manipulating the external solution to alter either the total or free proton concentration or the solvent itself. Membrane conductance decreased by approximately 50% when D(2)O replaced H(2)O as the solvent. From this, we conclude that hydrogen ions do not pass through M(2) as hydronium ions, but instead must interact with titratable groups that line the pore of the channel. M(2) currents measured in solutions of low buffer concentration (<15 mM in oocytes and <0.15 mM in CV-1 cells) were smaller than those studied in solutions of high buffer concentration. Furthermore, the reversal voltage measured in low buffer was shifted to a more negative voltage than in high buffer. Also, at a given pH, M(2) current amplitude in 15 mM buffer decreased when pH-pK(a) was increased by changing the buffer pK(a). Collectively, these results demonstrate that M(2) currents can be limited by external buffer capacity. The data presented in this study were also used to estimate the maximum single channel current of the M(2) ion channel, which was calculated to be on the order of 1-10 fA.     

Influence of the physical states of membrane surface area and center area on lysosomal proton permeability

The physical state of the lysosomal membrane was modulated with the membrane fluidizers n-propanol and n-octanol and with the membrane rigidifiers cholesteryl hemisuccinate and cholesterol. Membrane fluidity was examined by the steady-state fluorescence anisotropy of 2-(9-anthroyloxy) palmitic acid and 16-(9-anthroyloxy) palmitic acid. Fluidizing the membranes at the surface and center areas increased the proton permeability coefficient by 92.8 and 18.0%, respectively. Rigidifying the membranes at the surface and center areas decreased the coefficient by 68.2 and 40.2%, respectively. Proton leakage of the lysosomes increased and decreased similar to the coefficient changes with the treatments. The results indicate that lysosomal proton permeability is affected by its membrane's physical state, and the physical state of the membrane surface area affects the proton permeability more markedly. The proton permeability coefficient of liposomes was similar to that of lysosomes, suggesting that efflux of lysosomal protons might occur through the lipid part of the bilayer but not transmembrane proteins.     

The determination of a dose deposited in reference medium due to (p,n) reaction occurring during proton therapy

AimThe aim of the investigation was to determine the undesirable dose coming from neutrons produced in reactions (p,n) in irradiated tissues represented by water.BackgroundProduction of neutrons in the system of beam collimators and in irradiated tissues is the undesirable phenomenon related to the application of protons in radiotherapy. It makes that proton beams are contaminated by neutrons and patients receive the undesirable neutron dose.Materials and methodsThe investigation was based on the Monte Carlo simulations (GEANT4 code). The calculations were performed for five energies of protons: 50 MeV, 55 MeV, 60 MeV, 65 MeV and 75 MeV. The neutron doses were calculated on the basis of the neutron fluence and neutron energy spectra derived from simulations and by means of the neutron fluence–dose conversion coefficients taken from the ICRP dosimetry protocol no. 74 for the antero-posterior irradiation geometry.ResultsThe obtained neutron doses are much less than the proton ones. They do not exceed 0.1%, 0.4%, 0.5%, 0.6% and 0.7% of the total dose at a given depth for the primary protons with energy of 50 MeV, 55 MeV, 60 MeV, 65 MeV and 70 MeV, respectively.ConclusionsThe neutron production takes place mainly along the central axis of the beam. The maximum neutron dose appears at about a half of the depth of the maximum proton dose (Bragg peak), i.e. in the volume of a healthy tissue. The doses of neutrons produced in the irradiated medium (water) are about two orders of magnitude less than the proton doses for the considered range of energy of protons.     

In-vivo proton MR-spectroscopy of the human brain: assessment of N-acetylaspartate (NAA) reduction as a marker for neurodegeneration

Summary.  Proton magnetic resonance spectroscopy (¹H-MRS) is a non-invasive method to investigate changes in brain metabolite composition in different cerebral diseases. We performed proton spectroscopy in patients with dementia of the Alzheimer's type (AD) and in patients with motor neuron disease (MND) with the aim to detect the specific metabolic pattern for these neurodegenerative disorders. In the MND group we found a significant reduction of NAA/tCr metabolite ratios in the motor cortex, which correlates with the disease severity and the clinical lateralization of neurological symptoms and further decreases in the time course of the disease. In AD patients a reduction of NAA/tCr was observed in the medial temporal lobe. Since NAA is exclusively expressed in neurons as shown by immunohistochemical studies, reduced NAA levels suggest neuronal loss or dysfunction in the observed regions. The observed regional metabolic alterations reflect the neuronal basis of the characteristic neurological symptoms in AD (dementia) and MND (muscular palsy) and mirrors the disease progress over time. Received June 29, 2001 Accepted August 6, 2001 Published online August 9, 2002     

A model for the resistance of the proton channel formed by the proteolipid of ATPase

The conductance observed for single proton channels formed by proteolipids of ATPase (Schindler and Nelson 1982) is rationalized in terms of a hydrogen bonded network model. A simple algebraic expression for the resistance predicted for such a model is presented and the results are compared to the experimental observations. The comparision suggests that the conduction involves a series of bound water molecules and perhaps amino acid side groups.     

The proton ladder,a static mechanism for ion/proton coports and counterports

An ion/proton counterport is formed simply by locating a chain of ionizable residues connected by a proton conducting path near a passive ion pore which spans the membrane. The electric coupling between the ion in transit through the pore and the residues can ensure that for each ion passing through the pore in one direction a proton is driven along the chain of ionizable residues (the proton ladder) in the same or in the opposite direction. The mechanism is symmetrical in that a trans-membrane ion gradient may drive protons against their electrochemical potential gradient or a proton gradient may drive ions against theirs. The mechanism is applicable to cation or anion channels and to coports or counterports. No mechanical motion is required other than the motion of the ions and the protons. Monte Carlo computer simulations are performed on the model and its predicted properties are listed. The new type of counterport model is compared with currently used models. Offprint requests to: D. T Edmonds     

A hydroxide ion carrier in planar phospholipid bilayer membranes: (C6F5)2Hg (dipentafluorophenylmercury)

Although a number of molecules are known to function as current-carrying proton carriers across lipid bilayer membranes, no such hydroxide ion carriers have been found to date. We report that (C₆F₅)₂ Hg, which can function as a chloride ion carrier, can also carry a hydroxide ion. In 100 mM Na₂SO₄ solutions, membranes treated with (C₆F₅)₂Hg are almost ideally selective for H⁺/OH⁻ between pH 6.0 and 9.5. Membrane conductance varies linearly with [OH⁻] over this pH range and with the square of the (C₆F₅)₂Hg concentration. The presumed current-carrying species is the dimer [(C₆F₅)₂Hg]₂OH⁻, which, along with the neutral molecule (C₆F₅)₂Hg, shuttles back and forth within the bilayer. In 0.2 M NaCl at pH 9.5, the OH⁻ and Cl⁻ conductances are approximately equal. Thus, the carrier displays an approximately 10⁴-fold preference for OH⁻ over Cl⁻.     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a₃ as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK_a values during the pumping process are derived for comparison to experiments.     

Instrumentation methodology for recording and feeding-back surface electromyographic (SEMG) signals

The use of biofeedback devices has been plagued by the garbage in—garbage out problem since its inception. This paper reviews the basics of how to use biofeedback devices for surface electromyographic applications at each stage in the process from sensors to displays so meaningful information can be gathered and utilized.     

Heavy particle beams (proton) in oncology (35-year practice of N.N. Blokhin Cancer Research Centre)

Clinically proved "alternative breast-preserving method" applicable for patients with locally advanced nodal breast cancer. These patients refuse to go in to surgery or have indications for surgery (183 patients--T(1-4)N(0-3)M(0-1)). This method consists of the combination of the traditional method of the whole breast and/or nodal photon irradiation with local highly concentrated proton irradiation in the dosage iso-equivalent to tumor (proton with energy from 130 to 180 MeV, with stop in the target in notice depth). We used the irradiation of the adenohypophysis with narrow proton beams energy 200 MeV. The aim of the first combination of the components is the selectively influence on the target and the channeling to the tumor and its subclinical substances the dosages sufficient for the total irradiation of tumor with sparing surrounding tissues and parts of body. The goal of irradiation of adenohypophysis with protons is the normalization of its hormonal activity and elimination of factors stimulating growth of tumor cells in case of dishormonal cancer. The suggested method didu't only improve the results of the treatment of locally advanced breast cancer, but also contributed to the reducing of the emotional stress. We received high results of the patients with locally advanced nodal breast cancer of criteria: local control rate (96%), long remission (more then 40%) and 5-year actuarial survival rate (83%). The patients examined during 5 and 18 years. It made a good cosmetic effect and high quality life of the patients with breast cancer.     

Cytochrome oxidase: pathways for electron tunneling and proton transfer

 Electrons from cytochrome c, the substrate of cytochrome oxidase, a redox-linked proton pump, are accepted by Cu_A in subunit II. From there they are transferred to the proton pumping machinery in subunit I, cytochrome a and cytochrome a ₃–Cu_B. The reduction of the latter site, which is the dioxygen reducing unit, is coupled to proton uptake. Dioxygen reduction involves a peroxide and a ferryl ion intermediate, and it is the transition between these and back to the resting oxidized enzyme that are coupled to proton pumping. The X-ray structures suggest electron–transfer pathways that can account for the observed rates provided that the reorganization energies are small. They also reveal two proton-transfer pathways, and mutagenesis experiments have shown that one is used for proton uptake during the initial reduction of cytochrome a ₃–Cu_B, whereas the other mediates transfer of the pumped protons. Received: 23 March 1998 / Accepted: 11 May 1998     

Reduction of intrinsic kinetic and thermodynamic barriers for enzyme-catalysed proton transfers from carbon acid substrates

Many enzymes catalyse the heterolytic abstraction of the alpha-proton from a carbon acid substrate. Gerlt and Gassman have applied Marcus formalism to such proton transfer reactions to argue that transition states for concerted general acid-general base catalysed enolization at enzyme active sites occur late on the reaction coordinate (J. Am. Chem. Soc. 115 (1993) 11552). We postulate that as an enzyme evolves, it may decrease deltaG++ for a proton transfer step associated with substrate enolization by following the path of steepest descent on the two-dimensional surface corresponding to deltaG++, as defined by Marcus formalism. We show that for an enzyme that has decreased deltaG++ following the path of steepest descent, the values of the intrinsic kinetic (deltaG++(int,E)) and thermodynamic (deltaG(E)0) barriers for proton transfer reactions on the enzyme may be predicted from the known values of deltaG++(int,N) and deltaG(N)0 for the corresponding non-enzymic reaction and the free energy of activation on the enzyme (deltaG++(E)). In addition, the enzymic transition state will occur later on the reaction coordinate than the corresponding non-enzymic transition state (i.e. x++(E)>x++(N)) if the condition (6 - square root 2)/82deltaG++(int,N).