Beamline characterization of a dielectric-filled reentrant cavity resonator as beam current monitor for a medical cyclotron facility

At PSI (Paul Scherrer Institute), Switzerland, a superconducting cyclotron called “COMET” delivers proton beam of 250 MeV pulsed at 72.85 MHz for proton radiation therapy. Measuring proton beam currents (0.1–10nA) is of crucial importance for the treatment safety and is usually performed with invasive monitors such as ionisation chambers (ICs) which degrade the beam quality. A new non-invasive beam current monitor working on the principle of electromagnetic resonance is built to replace ICs in order to preserve the beam quality delivered. The fundamental resonance frequency of the resonator is tuned to 145.7 MHz, which is the second harmonic of the pulse rate, so it provides signals proportional to beam current. The cavity resonator installed in the beamline of the COMET is designed to measure beam currents for the energy range 238–70 MeV. Good agreement is reached between expected and measured resonator response over the energy range of interest. The resonator can deliver beam current information down to 0.15 nA for a measurement integration time of 1 s. The cavity resonator might be applied serving as a safety monitor to trigger interlocks within the existing domain of proton radiation therapy. Low beam currents limit the abilities to detect sufficiently, however, with the potential implementation of FLASH proton therapy, the application of cavity resonator as an online beam-monitoring device is feasible.     

A temporal forecast of radiation environments for future space exploration missions

The understanding of future space radiation environments is an important goal for space mission operations, design, and risk assessment. We have developed a solar cycle statistical model in which sunspot number is coupled to space-related quantities, such as the galactic cosmic radiation (GCR) deceleration potential (φ) and the mean occurrence frequency of solar particle events (SPEs). Future GCR fluxes were derived from a predictive model, in which the temporal dependence represented by φ was derived from GCR flux and ground-based Climax neutron monitor rate measurements over the last four decades. These results showed that the point dose equivalent inside a typical spacecraft in interplanetary space was influenced by solar modulation by up to a factor of three. It also has been shown that a strong relationship exists between large SPE occurrences and φ. For future space exploration missions, cumulative probabilities of SPEs at various integral fluence levels during short-period missions were defined using a database of proton fluences of past SPEs. Analytic energy spectra of SPEs at different ranks of the integral fluences for energies greater than 30 MeV were constructed over broad energy ranges extending out to GeV for the analysis of representative exposure levels at those fluences. Results will guide the design of protection systems for astronauts during future space exploration missions.     

Interaction of phycobilisomes with photosystem II dimers and photosystem I monomers and trimers in the cyanobacterium Spirulina platensis.

Distribution of phycobilisomes between photosystem I (PSI) and photosystem II (PSII) complexes in the cyanobacterium Spirulina platensis has been studied by analysis of the action spectra of H2 and O2 photoevolution and by analysis of the 77 K fluorescence excitation and emission spectra of the photosystems. PSI monomers and trimers were spectrally discriminated in the cell by the unique 760 nm low-temperature fluorescence, emitted by the trimers under reductive conditions. The phycobilisome-specific 625 nm peak was observed in the action spectra of both PSI and PSII, as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 695 nm (PSII), 730 nm (PSI monomers), and 760 nm (PSI trimers). The contributions of phycobilisomes to the absorption, action, and excitation spectra were derived from the in vivo absorption coefficients of phycobiliproteins and of chlorophyll. Analyzing the sum of PSI and PSII action spectra against the absorption spectrum and estimating the P700:P680 reaction center ratio of 5.7 in Spirulina, we calculated that PSII contained only 5% of the total chlorophyll, while PSI carried the greatest part, about 95%. Quantitative analysis of the obtained data showed that about 20% of phycobilisomes in Spirulina cells are bound to PSII, while 60% of phycobilisomes transfer the energy to PSI trimers, and the remaining 20% are associated with PSI monomers. A relevant model of organization of phycobilisomes and chlorophyll pigment-protein complexes in Spirulina is proposed. It is suggested that phycobilisomes are connected with PSII dimers, PSI trimers, and coupled PSI monomers.     

Development and validation of an NMR-based identity assay for bacterial polysaccharides

A method utilizing NMR spectroscopy has been developed to confirm the identity of bacterial polysaccharides used to formulate a polyvalent pneumococcal polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units. The similarity of any two spectral profiles is evaluated using a correlation coefficient (rho), where rho >/= 0.95 between a sample and reference profile indicates a positive identification of the sample polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharide powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be readily adapted for use with other bacterial polysaccharide preparations as well.     

Single-cell confocal spectrometry of a filamentous cyanobacterium nostoc at room and cryogenic temperature. Diversity and differentiation of pigment systems in 311 cells

The fluorescence spectrum at 298 and 40 K and the absorption spectrum at 298 K of each cell of the filamentous cyanobacterium Nostoc sp. was measured by single-cell confocal laser spectroscopy to study the differentiation of cell pigments. The fluorescence spectra of vegetative (veg) and heterocyst (het) cells of Nostoc formed separate groups with low and high PSII to PSI ratios, respectively. The fluorescence spectra of het cells at 40 K still contained typical PSII bands. The PSII/PSI ratio estimated for the veg cells varied between 0.4 and 1.2, while that of het cells varied between 0 and 0.22 even in the same culture. The PSII/PSI ratios of veg cells resembled each other more closely in the same filament. 'pro-het' cells, which started to differentiate into het cells, were identified from the small but specific difference in the PSII/PSI ratio. The allophycocyanin (APC)/PSII ratio was almost constant in both veg and het cells, indicating their tight couplings. Phycocyanin (PC) showed higher fluorescence in most het cells, suggesting the uncoupling from PSII. Veg cells seem to vary their PSI contents to give different PSII/PSI ratios even in the same culture, and to suppress the synthesis of PSII, APC and PC to differentiate into het cells. APC and PC are gradually liberated from membranes in het cells with the uncoupling from PSII. Single-cell spectrometry will be useful to study the differentiation of intrinsic pigments of cells and chloroplasts, and to select microbes from natural environments.     

Recovery of bacillus spore contaminants from rough surfaces: a challenge to space mission cleanliness control

Microbial contaminants on spacecraft can threaten the scientific integrity of space missions due to probable interference with life detection experiments. Therefore, space agencies measure the cultivable spore load ("bioburden") of a spacecraft. A recent study has reported an insufficient recovery of Bacillus atrophaeus spores from Vectran fabric, a typical spacecraft airbag material (A. Probst, R. Facius, R. Wirth, and C. Moissl-Eichinger, Appl. Environ. Microbiol. 76:5148-5158, 2010). Here, 10 different sampling methods were compared for B. atrophaeus spore recovery from this rough textile, revealing significantly different efficiencies (0.5 to 15.4%). The most efficient method, based on the wipe-rinse technique (foam-spatula protocol; 13.2% efficiency), was then compared to the current European Space Agency (ESA) standard wipe assay in sampling four different kinds of spacecraft-related surfaces. Results indicate that the novel protocol out-performed the standard method with an average efficiency of 41.1% compared to 13.9% for the standard method. Additional experiments were performed by sampling Vectran fabric seeded with seven different spore concentrations and five different Bacillus species (B. atrophaeus, B. anthracis Sterne, B. megaterium, B. thuringiensis, and B. safensis). Among these, B. atrophaeus spores were recovered with the highest (13.2%) efficiency and B. anthracis Sterne spores were recovered with the lowest (0.3%) efficiency. Different inoculation methods of seeding spores on test surfaces (spotting and aerosolization) resulted in different spore recovery efficiencies. The results of this study provide a step forward in understanding the spore distribution on and recovery from rough surfaces. The results presented will contribute relevant knowledge to the fields of astrobiology and B. anthracis research.     

EVA dosimetry in manned spacecraft

Extra Vehicular Activity (EVA) will become a large part of the astronaut's work on board the International Space Station (ISS). It is already well known that long duration space missions inside a spacecraft lead to radiation doses which are high enough to be a significant health risk to the crew. The doses received during EVA, however, have not been quantified to the same degree. This paper reviews the space radiation environment and the current dose limits to critical organs. Results of preliminary radiation dosimetry experiments on the external surface of the BION series of satellites indicate that EVA doses will vary considerably due to a number of factors such as EVA suit shielding, temporal fluctuations and spacecraft orbit and shielding. It is concluded that measurement of doses to crew members who engage in EVA should be done on board the spacecraft. An experiment is described which will lead the way to implementing this plan on the ISS. It is expected that results of this experiment will help future crew mitigate the risks of ionising radiation in space.     

Determining the limitations and regulation of photosynthetic energy transduction in leaves

The light-dependent production of ATP and reductants by the photosynthetic apparatus in vivo involves a series of electron and proton transfers. Consideration is given as to how electron fluxes through photosystem I (PSI), using absorption spectroscopy, and through photosystem II (PSII), using chlorophyll fluorescence analyses, can be estimated in vivo. Measurements of light-induced electrochromic shifts using absorption spectroscopy provide a means of analyzing the proton fluxes across the thylakoid membranes in vivo. Regulation of these electron and proton fluxes is required for the thylakoids to meet the fluctuating metabolic demands of the cell. Chloroplasts exhibit a wide and flexible range of mechanisms to regulate electron and proton fluxes that enable chloroplasts to match light use for ATP and reductant production with the prevailing metabolic requirements. Non-invasive probing of electron fluxes through PSI and PSII, and proton fluxes across the thylakoid membranes can provide insights into the operation of such regulatory processes in vivo.     

Resolution of the Photosystem I and Photosystem II contributions to chlorophyll fluorescence of intact leaves at room temperature

Green leaves illuminated with photosynthetically active light emit red fluorescence, whose time-dependent intensity variations reflect photosynthetic electron transport (the Kautsky effect). Usually, fluorescence variations are discussed by considering only the contribution of PSII-associated chlorophyll a, although it is known that the fluorescence of PSI-associated chlorophyll a also contributes to the total fluorescence [Aust. J. Plant Physiol. 22 (1995) 131]. Because the fluorescence emitted by each photosystem cannot be measured separately by selecting the emission wavelength in in vivo conditions, the contribution of PSI to total fluorescence at room temperature is still in ambiguity. By using a diode array detector, we measured fluorescence emission spectra corresponding to the minimal (F(O)) and maximal (F(M)) fluorescence states. We showed that the different shapes of these spectra were mainly due to a higher contribution of PSI chlorophylls in the F(O) spectrum. By exciting PSI preferentially, we recorded a reference PSI emission spectrum in the near far-red region. From the F(O) and F(M) spectra and from this PSI reference spectrum, we derived specific PSI and PSII emission spectra in both the F(O) and F(M) states. This enables to estimate true value of the relative variable fluorescence of PSII, which was underestimated in previous works. Accurate separation of PSI-PSII fluorescence emission spectra will also enable further investigations of the distribution of excitation energy between PSI and PSII under in vivo conditions.     

Is the risk for secondary cancers after proton therapy enhanced distal to the Planning Target Volume? A two-case report with possible explanations

It is often assumed that radiation-induced secondary cancer after proton therapy forms preferentially close to the distal fall-off of the spread-out Bragg peak because of an increased relative biological effectiveness (RBE) with regard to cancer induction of low-energy protons. In this study we analyze to what extent dose gradients distal to the Planning Target Volume (PTV) may, independently from the RBE, contribute to enhanced radiation carcinogenesis. The study is based on two dogs which, out of 30 dogs treated with proton therapy at the Paul Scherrer Institute (PSI), developed a secondary cancer. Both dogs were originally diagnosed and treated for a fibrosarcoma and developed an osteosarcoma 48 and almost 60 months, respectively, after radiotherapy. From the dose distributions of the initial radiotherapy for both dogs three-dimensional maps of secondary cancer complication probability (SCCP) were computed. The SCCP maps were analyzed in the regions where the dogs developed a secondary cancer. The SCCP maps showed an enhanced risk in the regions of the femur where the secondary cancers were detected, as compared to the SCCP of the total femur. Excess risk of radiation-induced cancer at the distal part of proton radiation fields can thus be explained using SCCP calculations on the basis of the physical dose distributions. Therefore, the occurrence of secondary cancer close to the distal dose gradients of proton therapy is not necessarily due to an increased RBE of low-energy protons. More extensive studies based on more patients will be necessary to further elucidate the factors influencing the development of secondary tumors.     

A retrieval algorithm to evaluate the Photosystem I and Photosystem II spectral contributions to leaf chlorophyll fluorescence at physiological temperatures

A new computational procedure to resolve the contribution of Photosystem I (PSI) and Photosystem II (PSII) to the leaf chlorophyll fluorescence emission spectra at room temperature has been developed. It is based on the Principal Component Analysis (PCA) of the leaf fluorescence emission spectra measured during the OI photochemical phase of fluorescence induction kinetics. During this phase, we can assume that only two spectral components are present, one of which is constant (PSI) and the other variable in intensity (PSII). Application of the PCA method to the measured fluorescence emission spectra of Ficus benjamina L. evidences that the temporal variation in the spectra can be ascribed to a single spectral component (the first principal component extracted by PCA), which can be considered to be a good approximation of the PSII fluorescence emission spectrum. The PSI fluorescence emission spectrum was deduced by difference between measured spectra and the first principal component. A single-band spectrum for the PSI fluorescence emission, peaked at about 735?nm, and a 2-band spectrum with maxima at 685 and 740?nm for the PSII were obtained. A linear combination of only these two spectral shapes produced a good fit for any measured emission spectrum of the leaf under investigation and can be used to obtain the fluorescence emission contributions of photosystems under different conditions. With the use of our approach, the dynamics of energy distribution between the two photosystems, such as state transition, can be monitored in vivo, directly at physiological temperatures. Separation of the PSI and PSII emission components can improve the understanding of the fluorescence signal changes induced by environmental factors or stress conditions on plants.     

Effect of surfactants on apparent oxygen consumption of photosystem I isolated from Arthrospira platensis

Surfactants play a significant role in solubilization of photosystem I (PSI) in vitro. Triton X-100 (TX), n-Dodecyl-β-d-maltoside (DDM), and sodium dodecyl sulfate (SDS) were employed to solubilize PSI particles in MES buffer to compare the effect of surfactant and its dosage on the apparent oxygen consumption rate of PSI. Through a combined assessment of sucrose density gradient centrifugation, Native PAGE and 77 K fluorescence with the apparent oxygen consumption, the nature of the enhancement of the apparent oxygen consumption activity of PSI by surfactants has been analyzed. Aggregated PSI particles can be dispersed by surfactant molecules into micelles, and the apparent oxygen consumption rate is higher for surfactant-solubilized PSI than for integral PSI particles. For DDM, PSI particles are solubilized mostly as the integral trimeric form. For TX, PSI particles are solubilized as incomplete trimeric and some monomeric forms. For the much harsher surfactant, SDS, PSI particles are completely solubilized as monomeric and its subunit forms. The enhancement of the oxygen consumption rate cannot be explained only by the effects of surfactant on the equilibrium between monomeric and trimeric forms of solubililized PSI. Care must be taken when the electron transfer activity of PSI is evaluated by methods based on oxygen consumption because the apparent oxygen consumption rate is influenced by uncoupled chlorophyll (Chl) from PSI, i.e., the larger the amount of uncoupled Chl, the higher the rate of apparent oxygen consumption. 77 K fluorescence spectra can be used to ensure that there is no uncoupled Chl present in the system. In order to eliminate the effect of trace uncoupled Chl, an efficient physical quencher of ¹O₂, such as 1 mM NaN₃, may be added into the mixture.     

Strategies for the uses of lanthanide NMR shift probes in the determination of protein structure in solutio. Application to the EF calcium binding site of carp parvalbumin.

The homologous sequences observed for many calcium binding proteins such as parvalbumin, troponin C, the myosin light chains, and calmodulin has lead to the hypothesis that these proteins have homologous structures at the level of their calcium binding sites. This paper discusses the development of a nuclear magnetic resonance (NMR) technique which will enable us to test this structural hypothesis in solution. The technique involves the substitution of a paramagnetic lanthanide ion for the calcium ion which results in lanthanide induced shifts and broadening in the 1H NMR spectrum of the protein. These shifts are sensitive monitors of the precise geometrical orientation of each proton nucleus relative to the metal. The values of several parameters in the equation relating the NMR shifts to the structure are however known as priori. We have attempted to determine these parameters, the orientation and principal elements of the magnetic susceptibility tensor of the protein bound metal, by studying the lanthanide induced shifts for the protein parvalbumin whose structure has been determined by x-ray crystallographic techniques. The interaction of the lanthanide ytterbium with parvalbumin results in high resolution NMR spectra exhibiting a series of resonances with shifts spread over the range 32 to -19 ppm. The orientation and principal elements of the ytterbium magnetic susceptibility tensor have been determined using three assigned NMR resonances, the His-26 C2 and C4 protons and the amino terminal acetyl protons, and seven methyl groups; all with known geometry relative to the EF calcium binding site. The elucidation of these parameters has allowed us to compare the observed spectrum of the nuclei surrounding the EF calcium binding site of parvalbumin with that calculated from the x-ray structure. A significant number of the calculated shifts are larger than any of the observed shifts. We feel that a refinement of the x-ray based proton coordinates will be possible utilizing the geometric information contained in the lanthanide shifted NMR spectrum.     

Assessment of the orientation and conformation of pigments in protein binding sites from infrared difference spectra

Time resolved FTIR difference spectroscopy (DS) has been used to study photosystem I (PSI) with the disubstituted 1,4-naphthoquinones acequinocyl (AcQ) and lapachol (Lpc) incorporated into the A₁ binding site. AcQ is a 2-acetoxy-3-dodecyl-1,4-naphthoquinone, Lpc is a 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone. To assess whether the experimental spectra are specific to different orientations of the quinone and their substitutions ONIOM-type QM/MM vibrational frequency calculations were undertaken for various orientations of the pigments and side-chain conformations in the A₁ binding site.Comparison of calculated and experimental spectra for the reduced species (semiquinone anion) suggests that the orientation for the naphthoquinone ring in the binding site and specific side-chain conformations can be identified based on the spectra. In native PSI phylloquinone (PhQ) in the A₁ binding site binds with its phytyl chain ortho to the hydrogen bonded carbonyl group. This is not found to be the case for the hydrocarbon tail of AcQ, which is meta to the H-bonded carbonyl group. In contrast, Lpc in PSI binds with its hydrocarbon tail also ortho to the H-bonded carbonyl group. Furthermore, comparison of calculated and experimental spectra indicates which conformations the acetoxy group of AcQ and the hydroxy group of Lpc adopt in the A₁ binding site.     

Survival of Akinetes (Resting-State Cells of Cyanobacteria) in Low Earth Orbit and Simulated Extraterrestrial Conditions

Cyanobacteria are photosynthetic organisms that have been considered for space applications, such as oxygen production in bioregenerative life support systems, and can be used as a model organism for understanding microbial survival in space. Akinetes are resting-state cells of cyanobacteria that are produced by certain genera of heterocystous cyanobacteria to survive extreme environmental conditions. Although they are similar in nature to endospores, there have been no investigations into the survival of akinetes in extraterrestrial environments. The aim of this work was to examine the survival of akinetes from Anabaena cylindrica in simulated extraterrestrial conditions and in Low Earth Orbit (LEO). Akinetes were dried onto limestone rocks and sent into LEO for 10 days on the ESA Biopan VI. In ground-based experiments, the rocks were exposed to periods of desiccation, vacuum (0.7 × 10⁻³ kPa), temperature extremes (−80 to 80°C), Mars conditions (−27°C, 0.8 kPa, CO₂) and UV radiation (325–400 nm). A proportion of the akinete population was able to survive a period of 10 days in LEO and 28 days in Mars simulated conditions, when the rocks were not subjected to UV radiation. Furthermore, the akinetes were able to survive 28 days of exposure to desiccation and low temperature with high viability remaining. Yet long periods of vacuum and high temperature were lethal to the akinetes. This work shows that akinetes are extreme-tolerating states of cyanobacteria that have a practical use in space applications and yield new insight into the survival of microbial resting-state cells in space conditions.     

A murine model for bone loss from therapeutic and space-relevant sources of radiation.

Cancer patients receiving radiation therapy are exposed to photon (gamma/X-ray), electron, and less commonly proton radiation. Similarly, astronauts on exploratory missions will be exposed to extended periods of lower-dose radiation from multiple sources and of multiple types, including heavy ions. Therapeutic doses of radiation have been shown to have deleterious consequences on bone health, occasionally causing osteoradionecrosis and spontaneous fractures. However, no animal model exists to study the cause of radiation-induced osteoporosis. Additionally, the effect of lower doses of ionizing radiation, including heavy ions, on general bone quality has not been investigated. This study presents data developing a murine model for radiation-induced bone loss. Female C57BL/6 mice were exposed to gamma, proton, carbon, or iron radiation at 2-Gray doses, representing both a clinical treatment fraction and spaceflight exposure for an exploratory mission. Mice were euthanized 110 days after irradiation. The proximal tibiae and femur diaphyses were analyzed using microcomputed tomography. Results demonstrate profound changes in trabecular architecture. Significant losses in trabecular bone volume fraction were observed for all radiation species: gamma, (-29%), proton (-35%), carbon (-39%), and iron (-34%). Trabecular connectivity density, thickness, spacing, and number were also affected. These data have clear implications for clinical radiotherapy in that bone loss in an animal model has been demonstrated at low doses. Additionally, these data suggest that space radiation has the potential to exacerbate the bone loss caused by microgravity, although lower doses and dose rates need to be studied.     

Photoaccumulation of the PsaB phyllosemiquinone in photosystem I of Chlamydomonas reinhardtii

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700(z.rad;+). Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700(z.rad;+), and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.     

Comparative risk assessment of secondary cancer incidence after treatment of Hodgkin's disease with photon and proton radiation

Probabilities for secondary cancer incidence have been estimated for a patient with Hodgkin's disease for whom treatment has been planned with different radiation modalities using photons and protons. The ICRP calculation scheme has been used to calculate cancer incidence from dose distributions. For this purpose, target volumes as well as critical structures have been outlined in the CT set of a patient with Hodgkin's disease. Dose distributions have been calculated using conventional as well as intensity-modulated treatment techniques using photon and proton radiation. The cancer incidence has been derived from the mean doses for each organ. The results of this work are: (a) Intensity-modulated treatment of Hodgkin's disease using nine photon fields (15 MV) results in nearly the same cancer incidence as treating with two opposed photon fields (6 MV). (b) Intensity-modulated treatment using nine proton fields (maximum energy 177.25 MeV) results in nearly the same cancer incidence as treating with one proton field (160 MeV). (c) Irradiation with protons using the spot scanning technique decreases the avoidable cancer incidence compared to photon treatment by a factor of about two. This result is independent of the number of beams used. Our work suggests that there are radiotherapy indications in which intensity-modulated treatments will result in little or no reduction of cancer incidence compared to conventional treatments. However, proton treatment can result in a lower cancer incidence than photon treatment.