Phragmites australis, Suaeda salsa and bare soil) in the Yellow River Estuary. During the growing season, there were significant linear relationships between soil respiration rates and shoot and root biomass, respectively. On the diurnal timescale, daytime soil respiration was more dependent on net photosynthesis. A positive correlation between soil respiration and net photosynthesis at the Phragmites australis site was found. There were exponential correlations between soil respiration and soil temperature, and the fitted Q10 values varied among different vegetation types . During the growing season, the mean soil respiration was consistently higher at the Phragmites australis site (1.11 µmol CO2 m−2 s−1), followed by the Suaeda salsa site (0.77 µmol CO2 m−2 s−1) and the bare soil site (0.41 µmol CO2 m−2 s−1). The mean monthly soil respiration was positively correlated with shoot and root biomass, total C, and total N among the three vegetation patches. Our results suggest that vegetation patchiness at a field scale might have a large impact on ecosystem-scale soil respiration. Therefore, it is necessary to consider the differences in vegetation types when using models to evaluate soil respiration in an estuary wetland.Vegetation type plays an important role in regulating the temporal and spatial variation of soil respiration. Therefore, vegetation patchiness may cause high uncertainties in the estimates of soil respiration for scaling field measurements to ecosystem level. Few studies provide insights regarding the influence of vegetation types on soil respiration and its temperature sensitivity in an estuary wetland. In order to enhance the understanding of this issue, we focused on the growing season and investigated how the soil respiration and its temperature sensitivity are affected by the different vegetation ( Vegetation type plays an important role in regulating the temporal and spatial variations of soil respiration through controlling a variety of environmental variables Q10) is also regulated by vegetation properties, including plant photosynthesis and productivity In ecosystems with steep physical/chemical gradients , vegetation distribution is markedly patchy and shows mosaic patterns The Yellow River Estuary is one of the most active regions of land-ocean interaction among the large river deltas in the world. Water and sand sedimentation from the Yellow River forms an important base for the extension and development of the Yellow River Delta Phragmites australis, Suaeda salsa and bare soil, in the Yellow River Estuary. Our objectives were to (1) examine seasonal patterns and spatial variation of soil respiration in the three adjacent vegetation types ; (2) evaluate the impact of vegetation types on the spatial variation of soil respiration at a field scale in the estuary ecosystem, and (3) quantify the effects of vegetation types on the temperature sensitivity of soil respiration during the growing season.To our knowledge, very few studies have been conducted on the influence of vegetation types on soil respiration and its temperature sensitivity in an estuary wetland. In this study, we sought to examine the relation between vegetation patch types and soil respiration in three adjacent vegetation types, We carried out the study at Yellow River Delta Ecological Research Station of Coastal Wetland, which belongs to the Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences. All necessary permits were obtained for the described field study. We confirm that our study had no harm to the environment and the field studies did not involve endangered or protected species.Phragmites australis, Suaeda salsa, Tamarix chinensis, and Imperata cylindrical var. major. In our research sites, the spatial distribution patterns of vegetation are mostly identified as patches of Phragmites australis, Suaeda salsa or bare soil on scales of meters to tens of meters because of the obvious gradient change of soil salt content. The main growing season of the estuary wetland ecosystem ranges from late April to late October.The study sites are located at Yellow River Delta Ecological Research Station of Coastal Wetland , Chinese Academy of Sciences, in Kenli County, Shandong Province, China. The climate is dominated by a warm-temperate and continental monsoon with distinctive seasons and a rainy summer. The annual mean air temperature and annual precipitation are 12.9°C and 560 mm, respectively. More than 50% of the annual precipitation occurs from July to September. The periodic surface ponding is often observed in the estuary wetlands, following heavy rainfall events. The soil type gradually varies from fluvo-aquic to saline soil. The soil texture is mainly identified as sandy clay loam. The most widespread vegetation types in the Yellow River Estuary are Phragmites australis, Suaeda salsa and bare soil) were established, each one was 15 m×15 m. The three sites were located no more than 30 m apart . The chamber was closed for 120 s and the linear increase of CO2 concentration in the chamber was used to estimate soil respiration every 15 days during the growing season. The measurements were taken every 2 h from 7∶00 to 19∶00  at clear days. For each site, seven measurements were performed each day.Three different vegetation sites , the rate of photosynthesis was measured at 2 h intervals from 7∶00 to 19∶00 on May 15, May 27, June 12 and July 9. All measurements were conducted in situ on the upper most fully expanded leaves of 5 individuals of Phragmites australis. The leaves of Suaeda salsa are line-shaped and fleshy, but there is no instrumentation for monitoring photosynthetic rates of fleshy leaves. Thus, the photosynthesis rates of Suaeda salsa were not measured in our present study.In order to evaluate the effect of photosynthesis on soil respiration, the daily course of photosynthesis was also measured simultaneously with soil respiration measurements at the During each soil respiration measurement, meteorological parameters were measured simultaneously with an array of sensors, including net radiation , air temperature , and precipitation . Soil temperature (every 10 cm from 0 to 50 cm depth) was measured with thermistors . Soil water content was measured by time domain reflectometry probes  at 10 cm, 20 cm and 40 cm depths below the surface. These data were logged every 30 min by a CR1000 datalogger .Phragmites australis and Suaeda salsa communities were measured by harvesting the vegetation approximately every 2 weeks. Five 0.5 m×0.5 m squares were randomly chosen in the Phragmites australis and Suaeda salsa communities, respectively. Live plants were clipped at 1 cm above the ground level, and root biomass was measured by taking five soil blocks (0.5 m wide×0.5 m long×0.3 m deep). Shoot and root biomass were oven dried at 80°C for 48 h and weighed.On each site of soil respiration measurement, every month we randomly selected six soil samples in each vegetation types to measure soil properties, including soil organic carbon (SOC), total carbon (C), and total nitrogen (N). SOC was determined using wet combustion, total soil C was analyzed using the potassium dichromate oxidation method, and total soil N was measured by the Kjeldahl method. In addition, the shoot and root biomass of Phragmites australis and Suaeda salsa sites. In the Phragmites australis community, correlation analysis was used to evaluate the effect of net photosynthesis rate on soil respiration during daytime on May 15, May 27, June 12 and July 9, respectively.Linear regression and correlation analysis were used to quantify the effect of shoot and root biomass on seasonal variations in soil respiration rate at SR is soil respiration, T is soil temperature, coefficient a is the intercept of soil respiration when temperature is zero, and coefficient b represents the temperature sensitivity of soil respiration.Exponential regression analysis between soil respiration and temperature used the data of soil respiration and soil temperature at 10 cm depth in the 3 different vegetation patches, respectively:Q10 can be estimated asPhragmites australis, Suaeda salsa and bare soil) with Tukey’s studentized range test using α = 0.05 for significance. In order to evaluate the impact of vegetation types on the spatial variation of soil respiration, the relationship between soil respiration and biotic factors  and soil chemical properties  were examined using correlations matrixes, and the Pearson’s coefficient was used to establish correlations between each pair of variables. All statistical analyses were performed using SPSS 11.5 .Data were first tested for normality and homogeneity of variance, then one-way analysis of variance (ANOVA) was used to test for significant differences of soil respiration, shoot biomass, root biomass, litter biomass, total C, total N and SOC at 0–20 cm depth among three vegetation patches (−2) in late June and a minimum (8.1 W m−2) in late October , soil temperature, soil water content (SWC), precipitation, and plant biomass are shown in  October . During  October . Precipi October . Seasona October .Phragmites australis and Suaeda salsa sites, the shoot biomass increased during the early growing season, but their seasonal variation patterns differed markedly between the two sites  and late October (363.8 g m−2), respectively  was significantly higher than that of the Suaeda salsa community (33.4 g m−2)  for soil respiration at the Phragmites australis, Suaeda salsa and bare soil sites were 77.2%, 73.5%, and 143.5%, respectively, over the whole measurement period.The seasonal variations of soil respiration at g period . From laPhragmites australis site, soil respiration rates showed no significant correlations with shoot and root biomass . However, during the early and peak growing season (from late April to late July), there was a good linear relationship between soil respiration and shoot biomass . Similarly, at the Suaeda salsa site, there was no significant relationship between soil respiration and shoot and root biomass during the entire growing season. However, during the initial growing season, there were significant linear relationships between soil respiration rates and shoot biomass  and root biomass , respectively.At the Phragmites australis community, the diurnal variation of net photosynthesis showed a bell shape with the peak almost exactly at noon on May 15, May 27, June 12 and July 9, respectively  and May 27 , respectively. In addition, the soil respiration rates showed no significant correlations with net photosynthesis rates on June 12  and July 9 .In the ectively . The diuPhragmites australis , Suaeda salsa  and bare soil site , respectively  of Eq. (1) for the Phragmites australis (0.47±0.15) was significantly greater (P<0.05) than those for the Suaeda salsa (0.22±0.01) and the bare soil (0.04±0.01). No significant difference in basal respiration between the Phragmites australis and bare soil sites was detected  at the bare site (0.12±0.01) was higher (P<0.05) than that at the Phragmites australis (0.06±0.01) and Suaeda salsa (0.08±0.01) sites (Q10 (P<0.05) than vegetated sites . However, there was no significant difference in the Q10 values between the Phragmites australis and Suaeda salsa sites  sites . Consequ P>0.05; .Phragmites australis, Suaeda salsa and bare soil sites was 1.59, 1.05 and 0.60 µmol CO2 m−2 s−1, respectively. The mean soil respiration of the Phragmites australis community was significantly higher than that at the bare soil site (P<0.05), whereas no significant differences in the soil respiration could be found neither between the Suaeda salsa community and the Phragmites australis community or between the Suaeda salsa community and the bare soil site  among the three vegetation patches . Plant growth itself is also under the influence of temporal changes in soil temperature and moisture. Consequently, temporal changes in soil temperature and moisture are likely to mask the effects of plant biomass and photosynthesis on soil respiration During the early and peak growing season of ectively . The res2 and carbonate dissolution chemistry. Ball et al. 2 fluxes. Increasing soil moisture allowed more CO2 to be dissolved in the soil solution, and when coupled with decreasing temperature, the largest negative CO2 flux rates were observed The relationships between soil respiration and soil temperature were best described by exponential equations at each site , which hQ10 of soil respiration was found to vary among different vegetation types in this study. The Q10 value of the bare soil (3.43) was significantly greater than that in two vegetated sites , which means that soil organic matter in bare soil is most sensitive to changes in soil temperature. Increasing evidence suggests that the apparent temperature response of soil respiration is closely related to vegetation type and activity. For example, Zheng et al. Q10 values of soil respiration in 49 ecosystems in China. By analyzing 384 field measurement data, Wang et al. Q10 of soil respiration.The Q10 at the bare soil site. Moreover, the temperature sensitivity of the organic matter should increase as the quality of the substrate decreases Q10 of soil respiration. Under optimum soil water conditions, Q10 decreased with increasing gross primary production (GPP), which could also be due to improved quality of substrate arising from increased supply of recent photosynthates Among the vegetation types observed in this study, the relative proportions of temperature-sensitive and -insensitive respiration fluxes among soil respiration might be different. Some studies have found that the mycorrhizal respiration, root respiration and the plant growth respiration are largely independent of temperature 2 efflux might be different, which may differentially regulates the temperature sensitivity of soil respiration. However, disagreement exists over the relative temperature sensitivities of microbial and root respiration, since the two components are difficult to partition in situ. Several contradictory results have suggested that the root respiration should be more sensitive to changes in temperature Q10 may cause a significant bias in the estimate of soil respiration Q10 of soil respiration at the regional scale studies.Meanwhile, among the different vegetation types, contribution of soil respiration components  to the overall soil COPhragmites australis site (1.59 µmol CO2 m−2 s−1), followed by the Suaeda salsa site (1.05 µmol CO2 m−2 s−1) and the bare soil site (0.60 µmol CO2 m−2 s−1). The differences in vegetation-related controls on soil respiration have been evaluated in different places for different ecosystems During the growing season, the mean soil respiration was consistently the highest at the The mean monthly rate of soil respiration was positively correlated with shoot and root biomass , which iPhragmites australis community likely possesses a higher organic C accumulation capability in Jiuduansha wetland Besides the influence of biotic factors, soil chemical properties in the present study showed large spatial variation among the three vegetation types . The meaAlthough SOC is also expected to be a major carbon supply to the soil microbial respiration, soil respiration was not correlated with SOC in this study. Previous researchers also found that soil respiration rates were not significantly correlated to SOC in other ecosystems Phragmites australis site. On the seasonal timescale, there were exponential correlations between soil respiration and soil temperature, and the fitted Q10 values varied among different vegetation types. Additional, there were significant linear relationships between soil respiration rates and shoot biomass and root biomass, respectively, during the early and peak growing season. At the field scale, the mean monthly soil respiration was positively correlated with shoot and root biomass, total C, and total N, while significant correlation was not found between soil respiration and litter biomass and SOC among the three vegetation patches. Our results suggest that vegetation patchiness at a field scale might have a large impact on ecosystem-scale soil respiration. Therefore, modeling soil respiration should not only take into account the simple soil temperature and moisture relationships, but also incorporate the patchy distribution of vegetation type as they affect the spatial and temporal variation of soil respiration.On the diurnal timescale, daytime soil respiration varied correspondingly with net photosynthesis, and there were positive correlations between soil respiration and net photosynthesis at the


The human oral microbiome is potentially related to diverse health conditions and high-throughput technology provides the possibility of surveying microbial community structure at high resolution. We compared two oral microbiome survey methods: broad-based microbiome identification by 16S rRNA gene sequencing and targeted characterization of microbes by custom DNA microarray.Oral wash samples were collected from 20 individuals at Memorial Sloan-Kettering Cancer Center. 16S rRNA gene survey was performed by 454 pyrosequencing of the V3–V5 region (450 bp). Targeted identification by DNA microarray was carried out with the Human Oral Microbe Identification Microarray (HOMIM). Correlations and relative abundance were compared at phylum and genus level, between 16S rRNA sequence read ratio and HOMIM hybridization intensity.Streptococcus, Veillonella, Leptotrichia, Prevotella, and Haemophilus; Correlation = 0.70–0.84).The major phyla, Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were identified with high correlation by the two methods (r = 0.70∼0.86). 16S rRNA gene pyrosequencing identified 77 genera and HOMIM identified 49, with 37 genera detected by both methods; more than 98% of classified bacteria were assigned in these 37 genera. Concordance by the two assays (presence/absence) and correlations were high for common genera (Microbiome community profiles assessed by 16S rRNA pyrosequencing and HOMIM were highly correlated at the phylum level and, when comparing the more commonly detected taxa, also at the genus level. Both methods are currently suitable for high-throughput epidemiologic investigations relating identified and more common oral microbial taxa to disease risk; yet, pyrosequencing may provide a broader spectrum of taxa identification, a distinct sequence-read record, and greater detection sensitivity. The NIH Human Microbiome Project, launched as part of the NIH Common Fund's Roadmap for Medical Research, pointed to the need to accelerate our understanding of how our bodies and microorganisms interact to influence health and disease With the characterization of microbial genetic profiles, molecular technologies can elucidate microbial community structure, including the identification and quantification of culturable and non-culturable organisms, at a much higher resolution than was previously possible with culture-based methods. Complete genetic sequencing of complex microbial ecosystems in humans have been accomplished 16S rRNA gene pyrosequencing has been applied in a wide range of human microbiome studies. Briefly, DNA primers to highly conserved regions in the 16SrRNA gene are designed for PCR amplification of DNA product, followed by DNA sequencing for characterization of microbial communities, including non-identifiable types, based on DNA sequence in the highly variable inter-primer regions. We (LY and ZP) have designed and validated a 16S rRNA pyrosequencing assay for the V3–V5 region of the gene and reported that 347F/803R is the most suitable pair of primers for classification of the foregut microbiome http://mim.forsyth.org/). The approach is based on 16S rRNA gene sequence hybridization and has been extensively validated The Human Oral Microbe Identification Microarray (HOMIM) is a custom array-based approach developed by one of us (BP) and others at the Forsyth Institute , using specially designed probes to detect ∼300 of the most prevalent oral bacterial species, initially identified from Sanger sequencing  as methods for microbiome characterization suitable for epidemiologic investigations.With 16S rRNA gene pyrosequencing, we recovered ∼79,000 sequences from the 20 oral wash samples , with 11HOMIM assay detected 7 phyla. As with pyrosequencing, Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were the major phyla identified by HOMIM, accounting for 96.5% of the distribution. No phyla were detected by HOMIM that were undetected in pyrosequencing, with the relative distribution being similar by the two methods . Correlations for abundance, comparing continuous level 16S rRNA sequence read ratio and HOMIM relative intensity, for the five major phyla ranged from r = 0.70 to r = 0.86. Relative abundance of phyla from pyrosequencing and HOMIM assay are shown for each individual in The 16S rRNA gene pyrosequencing detected 77 genera . In addiStreptococcus, Veillonella, and Leptotrichia, were positive by pyrosequencing and HOMIM in most study subjects  with 100% concordance . Moderate correlations in rarer taxa could also be due to the different quantitative estimation methods: HOMIM was based on discrete numbers in an intensity scale and 16S rRNA sequencing was based on sequence reads on a continuous scale. Furthermore, laboratory assay error . Five patients had oral cancer, 5 had premalignant oral lesions, and 10 were healthy controls. The study was approved by the institutional review boards at the Memorial Sloan-Kettering Cancer Center and NYU School of Medicine, and all participants provided informed written consent.20 subjects, ages 19–89 , were recruited at Oral wash saliva samples were obtained using saline from each subject, and immediately centrifuged to harvest cell pellets. DNA was extracted from the cell pellets using the QIAamp® DNA Mini Kit  according to the instructions of the manufacturer. The extracts were stored at −20°C until use.Bacterial 16S rRNA gene amplification, cloning, and sequencing of the polymerase chain reaction (PCR) products were performed as previously described 16S rRNA sequence data from pyrosequencing was downloaded, and multiplexed samples were deconvoluted computationally using customized Perl scripts, based on the presence of the unique barcodes assigned to each sample. Initial processing steps included trimming off the barcodes and primers, and removing sequences of low quality (<Q20). The sequence reads were binned to phyla and genera using the Classifier at RDP-II http://bioinformatics.forsyth.org/homim/) to determine the presence or absence of a particular microorganism, based on specific criteria set for that individual spot, thus generating microbial profile maps for each sample.HOMIM hybridization assay HOMIM output data were merged to the Human Oral Microbiome Database taxon table Ratios of total sample intensity, from HOMIM, were then compared with corresponding ratios of numbers of RDP-classified sequence reads, from pyrosequencing, for the same sample and taxa, making comparisons at the phylum and genera levels. The relative abundance of a specific taxonomic group was compared for the two assay methods by Chi-square test. Pearson coefficients were calculated as a measure of linear correlation between sequence and intensity ratios.Table S1Sequences recovered by 454 pyrosequencing for each subject samples.(DOC)Click here for additional data file.Table S2Genera detected by HOMIM and pyrosequencing.(DOC)Click here for additional data file.


Aponogeton madagascariensis) produces perforations in its leaves through PCD. The leaves of the plant consist of a latticework of longitudinal and transverse veins enclosing areoles. PCD occurs in the cells at the center of these areoles and progresses outwards, stopping approximately five cells from the vasculature. The role of mitochondria during PCD has been recognized in animals; however, it has been less studied during PCD in plants.Programmed cell death (PCD) is the regulated death of cells within an organism. The lace plant (in vivo in A. madagascariensis. A single areole within a window stage leaf (PCD is occurring) was divided into three areas based on the progression of PCD; cells that will not undergo PCD (NPCD), cells in early stages of PCD (EPCD), and cells in late stages of PCD (LPCD). Window stage leaves were stained with the mitochondrial dye MitoTracker Red CMXRos and examined. Mitochondrial dynamics were delineated into four categories (M1-M4) based on characteristics including distribution, motility, and membrane potential (ΔΨm). A TUNEL assay showed fragmented nDNA in a gradient over these mitochondrial stages. Chloroplasts and transvacuolar strands were also examined using live cell imaging. The possible importance of mitochondrial permeability transition pore (PTP) formation during PCD was indirectly examined via in vivo cyclosporine A (CsA) treatment. This treatment resulted in lace plant leaves with a significantly lower number of perforations compared to controls, and that displayed mitochondrial dynamics similar to that of non-PCD cells.The following paper elucidates the role of mitochondrial dynamics during developmentally regulated PCD in vivo as PCD progresses within the lace plant, and highlight the correlation of this organelle with other organelles during developmental PCD. To the best of our knowledge, this is the first report of mitochondria and chloroplasts moving on transvacuolar strands to form a ring structure surrounding the nucleus during developmental PCD. Also, for the first time, we have shown the feasibility for the use of CsA in a whole plant system. Overall, our findings implicate the mitochondria as playing a critical and early role in developmentally regulated PCD in the lace plant.Results depicted mitochondrial dynamics  A. madagascariensis) and Monstera obliqua. The mitochondrion is known to function in PCD in animal systems and the role of the organelle has been largely elucidated within this system; conversely, less is known regarding the mitochondria and PCD in plants [Programmed cell death (PCD) is the regulated death of a cell within an organism . In plann plants ,14.2+) in the cytosol or mitochondrial matrix [Within animal systems, mitochondria appear to undergo one of two physiological changes leading to the release of internal membrane space (IMS) proteins, allowing for the membrane permeability transition (MPT), inevitably aiding in PCD signaling. One hypothesized strategy involves the permeability transition pore (PTP), a multi-protein complex consisting of the voltage dependent ion channel (VDAC), the AdNT, and cyclophilin D (CyD) . The forl matrix ,19, incrl matrix ,21. In aThe second strategy is proposed to involve the Bcl-2 family of proteins and utilizes only the VDAC. The Bcl-2 family can be divided into two distinct groups based on functionality: the anti-apoptotic proteins including Bcl-2 and Bcl-xL, and the pro-apoptotic proteins including Bax, Bak, Bad and Bid ,22. If tA. madagascariensis) is an excellent model system for the study of developmental PCD in plants. It is one of forty species in the monogeneric family Aponogetonaceae, and is the only species in the family that forms perforations in its leaves via the PCD process [The aquatic freshwater lace plant  for 1 hour at room temperature in the dark. Following an intensive rinsing procedure, leaf pieces stained via this method displayed intense mitochondrial staining with little background staining, although it can be noted that a small amount of CMXRos dye is sequestered to the cell wall despite the presence or absence of mitochondria. This staining allowed the distribution of mitochondria to be easily identified within the cells, also permitting for the analysis of changes in mitochondria motility. Analysis of mitochondrial motility was completed by selecting still images from time-lapse videos of single epidermal cells at time 0 sec and 30 sec. Mitochondria at time 0 sec remain red, while mitochondria at time 30 sec were false colored green. These images were then overlaid to provide information on mitochondrial movement.Within a single areole of a stage 2 (window stage) leaf, mitochondrial dynamics were delineated into four categories M1-M4) based on the gradient of PCD. It is important to note that although these stages are seen simultaneously in a window stage leaf areole, if one was to examine a pre-perforation (stage 1) window, in which no cell death is yet visible, only stage M1 mitochondria would be present (data not shown). Stage M1 mitochondria were consistently found in healthy, NPCD cells  to aid in co-localization Figure . PreviouOur results indicate that mitochondria, as well as associated chloroplasts, appear to be moving on transvacuolar strands Figure , 7, 8, pin vivo perforation formation within the lace plant. Photographs of boxed plants and harvested leaves of control (just ethanol), and CsA (10 μM) treated plants clearly display a decrease in perforation formation  and 15 μM (P = 0.0007) compared to control plants  ; howeverArabidopsis protoplasts, and also by Lord and Gunawardena (2011) [Arabidopsis [The aggregation of mitochondria appears to be the first visible shift in mitochondrial dynamics during developmentally regulated PCD in the lace plant. This aggregation of mitochondria has also been demonstrated during induced cell death systems by Scott and Logan (2008) , Yao et a (2011)  in lace bidopsis , lace plbidopsis  and tobabidopsis  are locabidopsis  in develbidopsis ). HoweveArabidopsis protoplasts [Arabidopsis leaf discs [Following aggregation, mitochondria displayed a subsequent reduction in streaming. This cessation of streaming has also been demonstrated in mitochondria during several induced cell death examples in toplasts ,25, Arabaf discs , tobaccoaf discs ,30, and af discs . This imaf discs ,30,16.m appeared to occur between M3 and M4 mitochondria, possibly indicating that this is the first visible indication of membrane transition, and thus, possibly the first release of IMS proteins. The release of these IMS components at this time would correlate with the apparent degradation of the inner mitochondrial structure at this stage of PCD. This decrease in mitochondrial ΔΨm has been noted as a key characteristic of cell death in animal systems, and has also been demonstrated in a variety of other plant examples including induced cell death in Arabidopsis protoplasts [Following mitochondrial aggregation and cessation of streaming, they appear to undergo the MPT, characterized by a loss of CMXRos staining. The decrease in ΔΨtoplasts ,24,25, itoplasts , lace pltoplasts  and alsotoplasts .m are probably occurring simultaneously with the fragmentation of nuclear DNA, as noted by the presence of TUNEL-positive nuclei within these areas.A trend was noted within a single areole of a stage 2 (window stage) leaf; cells that contained TUNEL-positive nuclei were generally correlated with cells that contained M3 and M4 stage mitochondria. TUNEL-positive nuclei were not seen in NPCD cells that contained stage M1 mitochondria; this result was expected given that these cells are not pre-disposed to undergo cell death. TUNEL-positive nuclei were also absent in EPCD cells that contain stage M2 mitochondrial aggregates. This result clearly indicates that mitochondrial changes have begun prior to the fragmentation of nuclear DNA leading to TUNEL-positive nuclei. TUNEL-positive nuclei were consistently seen within LPCD cells that contained either M3 or M4 stage mitochondria. This trend also allows us to conclude that mitochondrial changes, particularly those seen in stage M3 and M4 stage mitochondria, including the cessation of mitochondrial movement and complete loss of ΔΨAn increased number of transvacuolar strands was noted in window stage leaf cells that were in the early stages of PCD (EPCD cells). This increased instance of transvacuolar strands is a common characteristic of PCD and has been noted previously during developmental cell death in the lace plant , in induin vivo. The inhibitor has been previously used during induced cell death examples on cell cultures [Zinnia, but this example is considered in vitro due to the cells being isolated from the plant prior to CsA treatment [The application of the PTP agonist CsA to the lace plant system marks the first time, to our knowledge, that this inhibitor has ever been applied cultures ,35, isolcultures ,17,24,26cultures ,36-39. Treatment .The application of CsA to lace plants in magenta boxes led to a reduction in perforation formation in leaves produced following the addition of the inhibitor. This significant decrease in perforation formation within the lace plant via CsA application indirectly indicates that the PTP pathway may play a role in cellular death within this system. Although the involvement of the PTP in animal PCD is well supported, it is controversial if a similar complex has a role in the release of IMS proteins in plant PCD, as shown by the following authors. Studies examining tobacco protoplasts , sycamorCsA concentrations in the lower range (2 μM and 4 μM) did not result in a significantly lower amount of perforations as compared to the controls (data not shown). This observation was expected, given that the inhibitor is dissolved in liquid and being applied to whole plants; therefore, higher concentrations may be required in order to affect the PTP. CsA at 10 μM significantly reduced the amount of perforations in the lace plant as compared to the control, but also maintained a healthy leaf appearance. The observations that no perforations formed at the 15 μM concentration, but did form in the controls, and that some transient leaf clearing occurred, indicates that this may be the lower limit of toxicity for CsA in the lace plant. The 20 μM CsA treatment resulted in brown and/or cleared leaves and therefore this concentration was considered very toxic and was not included in the subsequent statistical analysis. Overall, for further research it has been concluded that 10 μM CsA is the ideal concentration to inhibit the opening of the PTP during lace plant developmentally regulated PCD. This concentration has also been utilized as an optimal concentration in other plant examples, including sycamore cells .For this reason, 10 μM CsA treated leaves, four days following their emergence from the SAM, were chosen for examination; these leaves were therefore a similar developmental age as window stage leaves examined previously. CsA treated leaves depicted numerous, round mitochondria, which generally remained individual within the cytosol, and formed few aggregates in all cell types equal to NPCD-LPCD. These mitochondria also remained streaming within the entire areole and did not appear to undergo a membrane permeability transition causing loss of membrane potential and CMXRos staining. Given that treatment with CsA is hypothesized to block the release of IMS proteins from the mitochondria, we would anticipate variations in mitochondrial dynamics within this system. Intense mitochondrial fluorescence was anticipated, as this drug is hypothesized to inhibit the PTP and possibly the subsequent MPT. A round, and or swollen appearance of mitochondria following CsA treatment was also noted, although the reason behind this is unknown and needs to be further investigated. Overall CsA treated mitochondria display characteristics that most closely resemble M1 mitochondrial dynamics, where no PCD is occurring.A. madagascariensis. Developing leaves in which PCD was initiated (window stage) were stained with the mitochondrial membrane potential sensitive dye CMXRos and were examined via live cell imaging and confocal fluorescent microscopy. Observations of mitochondrial aggregation, motility and ΔΨm lead to the classification of mitochondria into one of four stages (M1-M4) based on their location in a window stage leaf areole. Our findings also indicate that within a single areole of a stage 2 (window stage) leaf a gradient of TUNEL-positive nuclei staining exists. TUNEL-positive nuclei were not seen in cells containing M1 and M2 stage mitochondria and were seen in cells with M3 and M4 stage mitochondria. These correlations suggest that the mitochondrial aggregation occurs prior to DNA fragmentation, whereas cessation of mitochondrial streaming and the membrane permeability transition resulting in complete loss of ΔΨm, based on CMXRos staining, probably occurs concurrently with the fragmentation of nuclear DNA. Mitochondria and chloroplasts were examined via live cell imaging, highlighting the role of transvacuolar strands in the movement of the organelles into a ring formation around the nucleus. The function of the mitochondrial PTP during PCD in developing lace plant leaves was also indirectly examined via CsA pre-treatment. Examination of CsA treated mitochondria revealed individual organelles, continued mitochondrial streaming and no loss in membrane potential over the same cellular areas (NPCD-LPCD) within one areole. Overall, results presented here detail organelle dynamics during developmentally regulated PCD in whole lace plant tissue and suggest that the mitochondria plays an important role in the early stages of PCD.The results presented here elucidate organelle dynamics, focused on mitochondria, during developmentally regulated PCD in the lace plant -2·s-1 at 23.5°C. All chemicals were purchased from Sigma , unless otherwise stated. All experiments were completed at least three times unless otherwise stated.Lace plants used for all experimental purposes were grown in sterile culture in magenta boxes and were maintained via subculture as described by Gunawardena et al.  optics and an eclipse 90i compound microscope  fitted with a digital camera (Nikon DXM 1200c) and using NIS Elements imaging and analysis software. This microscope was also used to acquire several of the additional file live cell imaging videos, all of which are in real time unless otherwise stated.i confocal microscope  fitted with a digital camera (Nikon DS-Fi1) and using EZ-C1 3.80 imaging software and Ti-Control. Confocal microscope observations were performed using DIC optics with complimentary fluorescent images taken via a fluorescein isothiocyanate  or Tetramethyl Rhodamine Iso-Thiocyanate  laser. False color images were prepared by generating still images selected from time-lapse videos at time 0 and time 30 seconds following CMXRos staining. Mitochondria in time 30 sec images were then false colored green and overlaid onto time 0 images to demonstrate mitochondrial motility. This microscope was also used to acquire several of the additional file live cell imaging videos, all of which are in real time unless otherwise stated. All composite plates were assembled using Adobe Photoshop Elements version 6.0.Confocal observations were performed using a Nikon Eclipse T2 were excised from window stage leaves and fixed in 2% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 6.9, for 24 hours in a vacuum (20 psi). Following overnight incubation, samples were rinsed in buffer and post fixed in 2.5% aqueous osmium tetroxide for 4 h at room temperature. Tissues were then dehydrated in a graded ethanol series, and placed through ethanol:Spurr resin mixtures. Tissues were finally embedded in pure Spurr resin and polymerized at 70°C for 9 h. Gold sections were prepared on a Reichert-Jung ultra-microtome, collected onto formvar coated grids and stained with lead citrate and uranyl acetate. Observations were made using a Philips 201 transmission electron microscope  or a Philips Tecnai 12 transmission electron microscope  operated at 80 kV and fitted with a Kodak  Megaview II camera with software .Tissue pieces approximately 2 mm2 were excised from window stage leaves and fixed in FAA for 2 h, followed subsequently by 3 washes in phosphate buffered saline (PBS). The terminal deoxynu-cleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed according to the manufacturer's instructions . Nuclei were counterstained by incubation in 3% (w/v) propidium iodide for 2 min. Samples were observed via confocal microscopy. A negative control was performed without the terminal deoxynucleotidyl transferase enzyme, and a positive control was performed with DNase1.Tissue pieces approximately 5 mm2 were excised from window stage leaves and stained for one hour in 0.2 μM, 0.3 μM, 0.5 μM, 0.6 μM, 1 μM, and 2 μM CMXRos . Leaf sections were then rinsed with ddH20 eight times and shaken for 90 minutes in ddH20 at approximately 100 rpm. Leaf sections were then mounted on slides and excited with the TRITC cube (excitation 527-552 nm and emission 577-632 nm) to view mitochondrial fluorescence and the FITC cube (excitation 460-500 nm emission 510-560 nm) to view the corresponding chlorophyll autofluorescence using the confocal microscope.Tissue pieces approximately 5 mmThese four stages of mitochondrial dynamics (M1-M4) were consistent within a window stage leaf when viewing the surface of epidermal cells where mitochondria are pushed up against the plasma membrane. However, when looking deeper into an epidermal cell, where mitochondria are found within the thin ring of cytoplasm between the plasma membrane and tonoplast membrane, due to the differing focal plane and orientation, mitochondrial dynamics can vary in appearance. It was important therefore, that for quantitative measurements, that the videos and images were taken from the very top portion of epidermal cells. Mitochondrial staining was completed at least 15 times.Healthy plants between 3 to 4 weeks of age, containing at least 2 perforated leaves, were selected for use in CsA trials. Plants were divided at random into experimental or control groups and under sterile conditions, liquid medium was poured out of each magenta box and replaced with 200 mL of fresh liquid medium. For the treatment groups, CsA stock dissolved in 90% ethanol was added to the liquid medium to make a final concentration of 2 μM, 4 μM, 10 μM, 15 μM, or 20 μM CsA in the boxes. For control plants, an equivalent volume of ethanol was added to the liquid medium. The plants were then returned to the growth racks under normal light conditions until they were at the proper stage for harvesting. Digital photographs acquired with a Nikon Coolpix P5000 camera  were taken of each plant for each concentration at least twice a week in order to track growth of newly emerging leaves. For the examination of mitochondria in leaves that had been treated with CsA, CMXRos staining was carried out as described above.Plants were considered ready to harvest following approximately 3 to 4 weeks from the initiation of the inhibitor experiment. Using the successive images, which were taken each week, and labeled by means of Adobe Photoshop Elements 6, version 6.0, each leaf was identified and removed from the respective magenta box. The petiole of each leaf was cut to 1 cm in length and the number of perforations per leaf was counted as an indicator of PCD. Leaf length was also measured as an indicator of normal leaf development. Number of perforations in an individual leaf was then divided by individual leaf length to obtain the variable 'ratio of number of perforations per cm of leaf length'. This variable is a more inclusive measure of PCD than solely number of perforations due to it accounting for the assumption that number of perforations depends in part on leaf length. Each leaf was then individually blotted dry, flattened by hand, and aligned in chronological order of emergence for photography purposes.Data were assessed by a general linear model of variance (GLM ANOVA) and the means were compared by the Tukey test at 95% confidence intervals (P < 0.05). Statistical analyses were carried out using Minitab 15 Statistical Software English .CENL and JW carried out all experiments: pharmacological application and harvest, light, fluorescent, confocal and DIC microscopic observations and measurements, along with conceptual mitochondrial staging. CENL completed the statistical analysis, drafted and revised the final manuscript. JW also contributed to final manuscript and completed the revisions. SL completed transvacuolar strand observations using light microscopy, and live cell-imaging. AHLANG conceived the study, participated in its design and coordination, helped in drafting and revising the manuscript, and supervised all experimental work. All authors read and approved the final manuscript.Stage M1 mitochondrial dynamics. CMXRos stained NPCD cell, highlighting stage M1 mitochondrial dynamics. Note individual mitochondria actively streaming within the cytosol.Click here for fileStage M2 mitochondrial dynamics. CMXRos stained EPCD cell, highlighting stage M2 mitochondrial dynamics. Note the aggregation of mitochondria along with several individual mitochondria, all of which appear to be moving.Click here for fileStage M2-M3 mitochondrial transition. CMXRos stained EPCD cell and DIC overlay. Video highlights the transition from stage M2 to stage M3 mitochondria. Note mitochondrial aggregate moving towards the nucleus, followed by cessation of movement. Video is 15× normal speed.Click here for fileStage M3 mitochondrial dynamics. CMXRos stained LPCD cell, highlighting stage M3 mitochondrial dynamics. Note the absence of movement of the mitochondrial aggregate.Click here for fileStage M4 mitochondrial dynamics. CMXRos stained LPCD cell, aimed at highlighting stage M4 mitochondrial dynamics. Note the lack of mitochondrial staining by CMXRos possibly due to complete loss of ΔΨm.Click here for fileTransvacuolar strands in NPCD stage cells. NPCD stage cells showing several transvacuolar strands, and highlighting the close association and possible movement of mitochondria and chloroplasts along them. Video 20× normal speedClick here for fileTransvacuolar strands in EPCD stage cells. EPCD stage cells showing increased transvacuolar strands activity and highlighting the close association and possible movement of mitochondria and chloroplasts along them. Note the trajectory of most strands and organelles towards the nucleus. Video 20× normal speedClick here for fileTransvacuolar strands in LPCD stage cells. LPCD stage cells showing a decrease in the number of transvacuolar strands and absence of organelles. Note that although mitochondrial streaming in often ceased at this point, slight cytoplasmic streaming can be visualized in mesophyll cells below the point of focus. Video 20× normal speed.Click here for fileMitochondrial dynamics in CsA treated NPCD stage cells. CsA treated leaf subsequently stained with CMXROS, depicting a single cell that corresponds with an NPCD window stage cell. Note, individual mitochondria that are rapidly moving within the cytosol.Click here for fileMitochondrial dynamics in CsA treated EPCD stage cells. CsA treated leaf subsequently stained with CMXROS, depicting a single cell that corresponds with an EPCD window stage cell. Note, individual mitochondria that are rapidly moving in the cytosol.Click here for fileMitochondrial dynamics in CsA treated LPCD stage cells. CsA treated leaf subsequently stained with CMXROS, depicting a single cell that corresponds with an LPCD window stage cell. Note, many individual mitochondria, and several small aggregates that are rapidly moving.Click here for file


Furthermore, separate boost plans  were created to boost PTVboost up to 70 Gy (IsoE) and 78 Gy (IsoE) for H&N and PC cases, respectively. Doses to brainstem, myelon, larynx and parotid glands were assessed for H&N cases. Additionally, various OARs  were evaluated that are currently discussed with respect to quality of life after treatment. For PC cases, bladder, rectum and femoral heads were considered as OARs. For both tumour sites target goals were easily met. Looking at OAR sparing, generally VMAT + VMAT was worst. VMAT + IMIT had the potential to spare some structures in very close target vicinity  significantly better than VMAT + IMPT. Mean doses for rectal and bladder wall were on average 4 Gy (IsoE) and 1.5 Gy (IsoE) higher, respectively, compared to photons plus particles scenarios. Similar results were found for parotid glands and larynx. Concerning target coverage, no significant differences were observed between the three treatment concepts. Clear dosimetric benefits were observed for particle beam therapy as boost modality. However, the clinical benefit of combined modality treatments remains to be demonstrated.The purpose of the study was to determine the dosimetric difference between three emerging treatment modalities—volumetric-modulated arc therapy (VMAT), intensity-modulated proton beam therapy (IMPT) and intensity-modulated carbon ion beam therapy (IMIT)—for two tumour sites where selective boosting of the tumour is applied. For 10 patients with locally advanced head and neck (H&N) cancer and 10 with high-risk prostate cancer (PC) a VMAT plan was generated for PTV It has been shown that dose escalation in the tumour volume can significantly increase local control , 2. For et al. compared VMAT with IMRT for 10 prostate cancer (PC) patients and the VMAT technique resulted in more favourable dose distributions and reduced the monitor units required compared with IMRT [et al. for patients with head and neck (H&N) tumours [The rapidly evolving field of particle therapy (protons as well as carbon ions), and the increasing number of facilities worldwide make this modality more commonly available and hence easier to incorporate into a boost treatment regimen. Nevertheless, photon beam therapy does not stand still and the recent development of rotational intensity-modulated radiation therapy (IMRT) like volumetric-modulated arc therapy (VMAT) is very promising, both with respect to treatment plan quality and treatment efficiency, i.e. highly conformal dose distributions can be achieved within a few minutes , 8. Palmith IMRT . SimilarThe scope of this study was to determine and assess the dosimetric differences between three emerging treatment modalities for two treatment sites where selective boosting of the tumour is applied, i.e. advanced H&N and prostate cancer (PC). More specifically, VMAT, intensity-modulated proton beam therapy (IMPT) and intensity-modulated carbon ion beam therapy (IMIT) were explored for these scenarios. Furthermore, their combinations were evaluated to answer the question if there is room for combined modality treatment.This retrospective study was performed for 10 patients with locally advanced H&N cancer and 10 patients with high-risk PC. All of them underwent IMRT treatment at the Medical University of Vienna/AKH Vienna.™ mask combined with the BiteSTEP™ system . For prostate cases the ProSTEP™ system  was utilized for external immobilization of the legs, while simultaneously an endorectal balloon (filled with 40 cm3 air) was used for pushing the posterior part of the rectal wall out of the high dose region. For treatment planning, images were acquired with a multi-slice CT scanner . In general, 4-mm slice thickness was used but treatment site-specific scanning protocols were applied.Patients with H&N tumours were immobilized with the double-layered HeadSTEPinitial consisted of the visible tumour itself, affected lymph nodes and additional areas of potential risk while CTVboost comprised only the tumour. Following our clinical practice both CTVs were expanded by a uniform 5-mm margin in order to create the planning target volumes . A dose of 50 Gy (IsoE) was prescribed to the PTVinitial and an additional 20 Gy (IsoE) was prescribed to the PTVboost. It should be emphasized that all doses in this manuscript are expressed as isoeffective doses, which are the absorbed doses that, if delivered under the reference treatment conditions, would produce the same effects on a given biological system as the dose delivered under the actual treatment conditions, with all other treatment conditions being identical. This is in accordance with the most recent recommendation of the ICRU and the IAEA in order to make doses delivered by photon as well as particle modalities comparable [On individual planning CTs, two clinical target volumes (CTVs) were delineated. The CTVmparable .Furthermore, the following organs at risk (OARs) were contoured, for which our in-house-developed constraints were applied to drive treatment plan optimization  was created of the CTVboost plus an additional safety margin of 10 mm in all directions. Finally, the PTVboost was defined as a uniform extension of the CTVboost by 5 mm. The intended dose to the PTVinitial was 50.4 Gy (IsoE), with a simultaneously integrated dose boost of 56 Gy (IsoE) administered to the PTV10mm. An additional dose of 22 Gy (IsoE) was prescribed to the PTVboost with a sequential boost.Similar to the H&N group, two CTVs were defined. The CTVRegarding OARs, bladder, rectum and femoral heads were considered. The rectal wall was derived from the rectum structure by extracting 3 mm from the external wall of the rectum. In a similar manner the bladder wall was defined. Furthermore, to simulate the bladder without contrast agent, the electron density of the opacified bladder was virtually changed to 1 (relative to water). The rectal balloon  was modified to be water equivalent, in order to avoid range uncertainties related to heterogeneity changes in the path of the particles and to mimic clinical particle beam therapy scenarios. OAR constraints for plan optimization are given in Table initial that included lymph node regions, i.e. of 50 Gy (IsoE) for H&N cases and 50.4/56 Gy (IsoE) to the PTVinitial/PTV10mm for PC cases. Thereafter, three separate boost plans were created  to deliver the additional dose to the PTVboost. Treatment plans were considered clinically acceptable if 95% of the respective target volume was covered with at least 95% of the prescribed dose and simultaneously all OAR constraints were fulfilled [For each patient an initial VMAT plan was generated to deliver the prescribed dose to the large PTVulfilled .initial was delivered within two arcs (2 × 360°). Due to the much simpler geometry of the PTVboost, a single arc (360°) was found to be sufficient for the boost plans. For all VMAT plans 10-MV photon beams were used. The maximum number of control points was 150 while the dose rate was defined as 150 MU/min with a minimum segment width of 0.5 cm. The dose was calculated using a Monte Carlo (MC) algorithm with a standard deviation of 3% on a grid size of 3 × 3 × 3 mm3.For the VMAT technique, the treatment planning system (TPS) Monaco  was used. The first dose level for the large PTV3. The energy-dependent spot sigma was calculated internally by the planning software based on an initial spot sigma of 3 mm in air. The lateral spot spacing of the pencil beams was 5 mm. The energy-dependent distance between two spot layers was designed to be in the order of a single Bragg peak (width at 80% of the peak maximum) at the respective depth. A global RBE of 1.1 (relative high energy photons) was assumed.Proton boost plans were generated with the TPS XiO . In general two beam ports were used. While for H&N cases the incident beam angles were 45°/90° or 315°/270°, depending on the respective tumour location, in the prostate group two lateral opposed beams (90° and 270°) were applied. Dose calculation was performed with a pencil-beam algorithm on the grid size 3 × 3 × 3 mmFor carbon ion boost plans the TPS TRiP98  was used. This software was especially developed for optimization and calculation of carbon ion dose distributions, both physical and biological ones. The software was designed to be compatible with 3D active dose-shaping devices like a raster scan system . The phy3. For evaluation purposes it was afterwards recalculated to a 3 × 3 × 3 mm3 grid.Beam arrangements were the same as for IMPT. In order to ensure comparable proton and carbon ion delivery scenarios, similar beam spot-defining parameters were chosen. The sigma value for the beam spots was 3.4 mm, while the distance between two successive layers was set to 3 mm. The lateral spot spacing of the pencil beams was 5 mm. Dose computation was carried out on the CT grid of 1 × 1 × 4 mmWith an in-house adapted version of the software platform CERR (V4.1) dose matrices of the initial VMAT plans and the boost plans were mapped together in order to sum up the doses on a voxel-by-voxel basis. In other words, evaluation of the final results was performed by creating dose-volume histograms (DVHs) for the whole treatment, independently from the TPS. Hence, as an outcome for each patient, three DVHs were generated consisting of summed doses from VMAT plus the VMAT boost plan (VMAT + VMAT), the VMAT plus the IMPT boost plan (VMAT + IMPT) and the VMAT plus the IMIT boost plan (VMAT + IMIT). The dosimetric differences between the three different treatment approaches were evaluated. Average DVHs were created over the entire patient populations .median), dose to 95% of the volume (D95%), volume covered by 95% of the prescribed dose (V95%), homogeneity index (HI) as well as conformity number (CN). HI was defined as the ratio of the difference between the D2% and D98% and the median dose. CN was calculated as a ratio of the squared target volume covered by 95% isodose and the total target volume multiplied by the volume covered by the 95% isodose. More detailed information regarding these indices can be found in the literature [mean) as well as near maximum doses (D2%) were investigated. For parallel OARs  other DVH parameters (e.g. V30 Gy (IsoE), V60 Gy (IsoE), D30%, D90%) were derived additionally.For each of the composite treatment plans the following dose indices regarding target volumes were reported: median dose . The software SPSS Statistics  was utilized for this purpose [In total 60 plans were analysed. The data were assumed to follow a normal distribution and a paired  purpose , 25.n = 3) where averaged doses exceeded the constraints.Each of the created plans fulfilled the given target constraints. median was on average up to 10% higher for the VMAT + VMAT approach, compared to the others. Therefore, systematically lower values of HI were observed for this treatment option. On the other hand, DVHs of the respective initial PTVs and CTVs revealed that differences between the particle modalities were marginal.95% and V95% for the PTVinitial and the CTVinitial were found to be significantly different in favour of sole photon treatments compared to combined photon and particle therapy modalities, although being only higher by up to 2.5%. For the PTVboost and CTVboost no significant difference could be obtained for those indices. Near minimum as well as maximum doses were very similar with deviations of less than 1% for all modalities. Due to the specific physical characteristics of particles, combined modality treatments  resulted in significantly higher CN than VMAT + VMAT.Dmean and D2% for the VMAT + VMAT treatment were found to be significantly worse compared with the other methods. Moreover, the VMAT + IMIT modality spared the myelon, brainstem and the larynx the most. By adopting particle beams for the boost, maximum doses to the myelon as well as to the brainstem could be constrained on average below 37 Gy (IsoE), compared to up to 40 Gy (IsoE) for sole photon treatment.mean, D2%, V5 Gy (IsoE) and V35 Gy (IsoE) of the body structure, a significant increase in the dose was observed for the VMAT + VMAT method compared to VMAT + IMPT and VMAT + IMIT (on average 1 Gy (IsoE) for all the indices, but for D2% it was 6 Gy (IsoE) higher). Furthermore, concerning Dmean and D2% VMAT + IMIT was superior over VMAT + IMPT.Looking at Dmean and D2% were significantly higher, with mean dose differences mostly around 3 Gy (IsoE). Additionally, for some of the structures like ipsilateral cochlea, ipsilateral middle ear, base of tongue, upper lip etc., VMAT + IMIT showed significant benefits even over VMAT + IMPT. The detailed results are summarized in Concerning the sparing of additional OARs, which might have an impact on the quality of life after treatment, the VMAT + VMAT method appeared to be the worst in most of the cases. For almost all OARs considered, Dmedian. The respective dosimetric difference was not that pronounced (within 2%) and hence considered to be not clinically relevant. This was caused by the much larger volumetric difference between the PTVinitial and the PTVboost between H&N and PC cases. Statistically significant differences between sole photon treatments and combined photon and particle beam treatments were observed for the PTVinitial, where for D95% differences were up to 0.8 Gy (IsoE) and for V95% up to 1.5%. Again, for the PTVboost, the CTVboost and the PTV10 mm no significant differences were observed for those indices.In initial), except for the PTVboost and the CTVboost. Moreover, combined treatments  resulted in significantly higher CN.Variations of minimum as well as maximum doses to target volumes were found to be within 1% for all scenarios. On the other hand, on average systematically lower HI values were obtained for sole photon treatments , V60 Gy (IsoE), D30%, D90%) VMAT + VMAT plans were significantly worse compared to photons combined with particles, except for those for the bladder wall (V60 Gy (IsoE), D30%, D90%). Moreover, for some indices of the rectal wall (V30 Gy (IsoE), D30%, D90%) and for V30 Gy (IsoE) of the bladder wall, combining photons with carbon ions spared the organs significantly better than VMAT + IMPT.No significant differences were observed for Dmean, D2%, V5 Gy (IsoE) and V39 Gy (IsoE) of the body structure, a significant increase in the dose was observed for the VMAT + VMAT method compared with the other techniques, for example for Dmean the difference was almost 7% and for D2% over 4%.Regarding Dmean, D2%, and V35 Gy (IsoE) was found to be the worst for VMAT + IMIT, while the values of V40 Gy (IsoE), V50 Gy (IsoE) and V60 Gy (IsoE) were highest for combined VMAT + VMAT plans. However, almost none of those differences reached statistical significance.The femoral heads were spared best with VMAT + IMPT (results not shown). Dinitial in In the case of dose-escalated treatments in the H&N region clear differences between sole photon treatment and treatments combining photons and particle beam therapy were observed. First, the dosimetric characteristics of particle beam therapy resulted in improved dose conformity. While for the boost target no dosimetric differences were observed, reduced doses outside the boost target could be achieved. That feature of combined treatment techniques can be seen in et al. [a priori included in the optimization procedure but analyzed in the following treatment plan evaluation. For structures which were situated very closely around the targets , carbon ions showed advantages over protons due to their much smaller lateral penumbra. In general, there is not much dosimetric data available in recent literature regarding this issue. However, Schwartz et al. [Furthermore, for OARs VMAT + IMPT as well as VMAT + IMIT showed systematic benefits over VMAT + VMAT. For H&N cases, on average a 10% reduction of the mean dose to the contralateral parotid gland was obtained for these combined modality treatments. Such dose limitation can cause clinically relevant reduction of xerostomia, which often burdens the patients' quality of life after radiation treatment . Moreoveet al.  associatz et al.  focused For prostate cases similar characteristics concerning the overall dose distributions were found as for H&N cases. Target volume parameters showed better results for combined modality treatments employing particles, although target median dose differences between the various treatment concepts were not that pronounced. This can be explained by the larger difference of the volumes of the initial and the boost target. Consequently, DVH curves varied more strongly for smaller volumes than for larger volumes, and the overall DVH was dictated by that of the large PTV. Dosimetric differences regarding the rectal as well as the bladder wall were predominantly obtained in the low- and medium dose area, while in the hight dose range no beneficial effect of one or the other boost modality could be observed. Highest dose values to the femoral head were obtained for VMAT + IMIT, which was associated with an overestimated RBE in the entrance channel of the beams by the LEM1 model, as well as the optimization process of the TRiP98 version used. These limitations were counteracted in newer versions of the planning system incorporating the new LEM4 model for biological optimization , 28.et al. [et al. [A uniform dose-escalation approach to the entire boost volumes was pursued in this study, which represents the current standard. This concept of delivering a homogeneous dose to all areas of proven or potential risk is currently under discussion . Selectiet al.  and Lühr [et al.  proposed [et al. ).In this study standard fractionation schedules, i.e. 2 Gy (IsoE) fractions, were assumed in order to allow a direct comparison of resulting dose distributions. Dose effects, originating from different fractionation schemes for tumour and OAR volumes are challenging. The investigation of altered fractionation for the boost volume was beyond the scope of our investigation. However, the potential of hypo-fractionated treatment regimens was promoted especially for PC , 36 and Another slight limitation of the current study is that not all potentially available boost techniques were considered, since brachytherapy (BT) was not included. The combination of external beam radiotherapy (EBRT) and BT has become a common treatment approach, especially for prostate and gynaecological cancer , 41. TheIn summary, dosimetric differences between multi-modal boosting strategies and their potential impact on the patients' quality of life after treatment were investigated. Sole photon treatments based on rotational IMRT (VMAT + VMAT) performed worse compared to treatment regimens employing a combination of photon and charged particle beam therapy . VMAT + IMIT had the potential to spare some structures in very close target vicinity significantly better than VMAT + IMPT. However, the clinical benefit of combined modality treatments remains to be demonstrated.Financial support by The European Community's Seventh Framework Programme (FP7 2007–2013) under Grant Agreement No. 215849-2 (Project PARTNER), and the Federal Ministry of Economy, Family and Youth, and the National Foundation for Research, Technology and Development is gratefully acknowledged.


In this paper we discuss the use of Cupron copper oxide impregnated non-porous solid surface in the hospital setting and present The two countertops tested passed all the acceptance criteria by the EPA (>99.9% kill within 2 hours of exposure) killing a range of bacterial pathogens on the surface of the countertops even after repeated exposure of the countertops to the pathogen, and multiple wet and dry abrasion cycles.Cupron enhanced EOS countertops thus may be an important adjunct to be used in hospital settings to reduce environmental bioburden and potentially nosocomial infections. Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter species [S. aureus accounts for 50% of HAI associated with multidrug resistant pathogens [P. aeruginosa[Hospital Acquired Infections (HAI) have exacted a heavy toll worldwide with over 2 million patients annually contracting an infection in the US , being o species . Methiciathogens . The Exteruginosa. HAI areeruginosa. Recent eruginosa-17, haveeruginosa-28 and aThus, our objective was to demonstrate the efficacy of two solid surface countertops incorporating copper (I) oxide in killing a range of pathogens according to protocols that were approved by the USA Environmental Protection Agency (EPA).Staphylococcus aureus (ATCC 6538); Enterobacter aerogenes (ATCC 13048); Pseudomonas aeruginosa (ATCC 15442); Methicillin resistant Staphylococcus aureus (MRSA)(ATCC 33592); and Escherichia coli 0157:H7 (ATCC 35150).The following bacterial strains were tested: The studied countertops were composed of homogenous blends of polyester, acrylic alloys and fillers, inert pigment and dyes, with (test samples) or without (control samples) Cupron’s 16% copper (I) oxide weight/weight. Three and two separate manufacturing lots of the test and control countertop samples were tested, respectively. A total of 1500 pieces, cut into one inch by one inch squares  according to protocols pre-approved by the USA EPA.−1 to 10−4) were made in PBS and plated using TSA and incubated for 48 hours at 35-37°C for colony observation and enumeration, taking into account also the 20 fold dilution used to retrieve the bacteria from the carriers. The following controls were performed: culture purity control - each prepared culture was streaked using TSA for purity control; organic soil purity control - duplicate 1 ml aliquots of organic soil were plated in TSA pour plates for sterility control; neutralizer sterility control – a jar containing the neutralizer was incubated with the test plates and observed for growth or no growth; carrier sterility control – an uninoculated test (per lot) and control carrier was put in independent jars containing the neutralizer, incubated and observed for growth or no growth; carrier viability control – for each challenge microorganism, a single inoculated control carrier was subcultured in a jar containing the neutralizer, incubated and the neutralizer observed for growth or no growth; and neutralization confirmation control – for each challenge microorganisms, per lot of the test article, a single sterile test carrier was put in individuals jars containing 20 ml of the neutralizer. To each jar a 1 ml aliquot of the diluted inoculum was added to reach ~100 colony forming units (CFU)/ml in the neutralizer. The jar was mixed and the 1 ml inoculum was removed and plated in duplicate.The carriers were cleaned with 70% isopropyl alcohol, rinsed with deionized water, and allowed to air dry. After steam sterilization for 15 minutes at 121°C, each carrier was placed into a plastic Petri dish matted with two pieces of filter paper using sterile forceps. Two or three lots of manufacturing of each test material were tested per microorganism. Five replicates of each material were used in each test. Organisms from stock cultures were transferred to Tryptic Soy Broth and incubated for 24 hours at 35-37°C (25-30°C for ATCC 13048). Two loopfuls of culture were transferred consecutively daily for three days for the inoculation stocks and the pellicle of bacteria were aspirated. Daily transfers were done for at least 3 consecutive days but for no more than 10 days. To this culture 0.25 ml of heat-inactivated fetal bovine serum (FBS) and 0.05 ml of Triton X-100 were added to 4.7 ml bacteria suspension to yield 5% FBS and 0.01% Triton X-100 organic soil load. The challenge microorganism titer was determined by serially diluting a final 48 hour culture using phosphate buffered solution (PBS) and selected dilutions were plated in duplicate using Tryptic Soy Agar (TSA) pour plates. Carriers were inoculated with 0.02 ml of the 48 hour culture. The bacterial inoculum per experiment is detailed in Table A sanitization test was followed as described above (Protocol 1) using 4 replicates per material. Post this initial test a Gardner apparatus was used to simulate surface wear of the test and control samples. The abrasion tester was used at a speed of 2.25 to 2.5 for a total contact time of 4–5 seconds for one complete cycle. A wear cycle equals one pass to the left and a return pass to the right. After a minimum of 15 minutes after the wear cycle each carrier was reinoculated as described above and dried for a minimum of 30 minutes. After each set of surface wear, absolute ethanol was used to sterilize the apparatus and the foam liner and cotton cloth were changed after each wear test. Wet cycles and dry cycles were alternated and for wet wear cycles the boat assembly included a new foam liner and dry cotton cloth sprayed with sterile deionized water using a preval sprayer from a distance of 75±1 cm for not more than one second. At least 24 hours passed between the initial inoculation and final sanitizer. Overall 12 wear cycles were completed before sanitizer activity was assessed using the method outlined above. All the controls as outlined for Protocol 1 were performed.−1 to 10−4) were spread on plates using appropriate media and incubated for 48 hours for colony observation and enumeration. All the controls as outlined for Protocol 1 were performed.A sanitization test was followed as described above (Protocol 1) using 5 replicates per each material tested. The carriers were consecutively inoculated for 8 times by adding the challenge microorganism at 0, 3, 6, 9, 12, 15, 18 and 21 hours. Efficacy was assessed at 2, 6, 12, 18 and 24 hours, which corresponds to 1, 2, 4, 6, and 8 inoculations. After exposure the carriers were transferred to a neutralizer solution and sonicated and rotated to mix. Within one hour, serial dilutions  recovered from the control samples following the different protocols, which included air drying, sonication, and recovering the bacteria from the exposed carrier, are summarized in Table sa Table , which en Figure D and E an Figure B and C oBacteria can persist on inanimate surfaces for months  and can Copper has a long history as an antimicrobial and preventative measure and metallic copper countertops have previously been approved for EPA public health claims . Field tStaphylococcus aureus (ATCC 6538), Enterobacter aerogenes (ATCC 13048), MRSA (ATCC 33592), Escherichia coli O157:H7 (ATCC 35150) and Pseudomonas aeruginosa (ATCC 15442).Based on the data presented in this publication, Cupron Enhanced EOS Surfaces infused with copper have been approved for public health claims relating to their anti bacterial efficacy. Some of the approved health claims are a) “This surface continuously reduces bacterial* contamination achieving a 99.9% reduction within two hours of exposure.”; b) “This surface kills greater than 99.9% of Gram negative and Gram positive bacteria* within two hours of exposure.”; c) “This surface kills greater than 99.9% of bacteria* within two hours and continues to kill 99% of bacteria* even after repeated contamination.”; and d) “This surface helps inhibit the buildup and growth of bacteria* within two hours of exposure between routine cleaning and sanitizing steps”. *Testing demonstrates effective antibacterial activity against These non-porous Cupron copper oxide containing solid surfaces possess comparable efficacy to the metallic hard surfaces, however represent a much more feasible alternative to the metallic surfaces as they are expected to be significantly more affordable and aesthetically more pleasing. Field trials are ongoing at the time of publication to demonstrate the efficacy of these countertops in a “real world” setting . The biocidal properties of copper oxide against a range of organisms have also been previously demonstrated -43.Limitations of this study include that the data is based upon ATCC laboratory strains and conducted in a controlled setting such as a laboratory, however further work is ongoing utilizing field trials of the surface to demonstrate the efficacy in real world applications.in vitro when repeatedly and consecutively exposed to fabrics containing copper oxide particles [2+ and Cu1+, which can catalyze the production of highly reactive hydroxyl radicals, can subsequently damage lipids, proteins, DNA and other biomolecules [Bacterial resistance to biocidal control agents is of concern in infection prevention and can be exemplified by highly antibiotic resistant bacteria (with up to 2200-fold decreased sensitivity to the antibiotic (e.g. ) that haarticles . The reaarticles ,49. Botharticles ,49. It iarticles , alterinarticles ,52. Copparticles ,54). Furolecules ,55. In aolecules -58. AppaMicroorganisms cannot cope when exposed to high concentrations of copper and are irreversibly damaged, as demonstrated also in this study. Thus, the development of resistant bacteria to copper due to the introduction of the copper containing countertops to the hospital environment is not a concern.With the ongoing HAI problem and the role of fomites and the environment being more clearly defined, the role of antimicrobial products with EPA approved public health claims, above and beyond the treated article claims and with clinical data supporting their role in HAI prevention, will become more important.The tested Cupron Enhanced EOS Surfaces containing copper oxide kill above 99.9% of a wide range of bacteria within two hours of exposure and continue to do so even after repeated contamination and multiple wet and dry abrasion cycles, passing all the acceptance criteria required by the EPA. These biocidal surfaces thus may be an important adjunct to be used in hospital settings to reduce environmental bioburden and potentially nosocomial infections.CFU: Colony forming units; EDS: Energy-dispersive X-ray spectroscopy; EPA: Environmental Protection Agency; FBS: Fetal bovine serum; GLP: Good Laboratory Practice; HAI: Hospital Acquired Infections; MRSA: Methicillin resistant Staphylococcus aureus; PBS: Phosphate buffered solution; SEM: Scanning Electron Microscopy; TSA: Tryptic Soy Agar.KT is an employee of EOS Surfaces. ABM, VK and GB are employees of Cupron Inc. This study was funded by Cupron Inc. and EOS Surfaces that developed the antimicrobial surfaces.ABM and GB made substantial contributions to conception, design, analysis and interpretation of data of the study, and writing the manuscript; VK and KT were key in designing and developing the test materials studied, and revising the manuscript critically for important intellectual content. All authors read and approved the final manuscript.