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Each of them is placed in a large spacecraft provisioned with a laboratory for human cloning, 6 months’ worth of basic food and water rations along with oxygen, and space to house 5000 people; the World Space Organization will send additional rations for the populations every 6 months, and once the colonies are at maximum capacity, the WSO will conduct scientific tests of a variety of parameters on the colony. After a lengthy journey, Bob and Flo arrive on their respective planets, set up their laboratories, and begin producing clones of themselves. Our initial testing of the inhabitants of Mars and Venus revealed stark differences between men and women for a variety of parameters. It is probably instantly obvious to most people that this experiment has a whole whack of problems that invalidate the scientists’ conclusions.
Turns out that Bob has a family history of genetic hypercholesterolemia and tall stature, and his allele for CYP2B6 is a variant that is very efficient in breaking down propofol. The genetic tendency to tallness of the Bob clones that was magnified by the lower gravity on Mars; although their bodies were larger, the lower gravity on Mars meant that their leg muscles did not have to be as well developed in order to support their (higher) body weight. Radiation exposure on the space journey to Venus caused a mutation in the CYP2B6 allele in the cell that Flo happened to use in her cloning; the mutation caused a 50% decrease in the activity of the enzyme, so propofol was metabolized more slowly in the clones than in Flo herself.
The findings on verbal reasoning fit with the researchers’ pre-existing perception that women are better communicators, so they didn’t consider whether there might be other factors that confounded their findings (ie.
The researchers are stretching their conclusions pretty far to argue that the difference seen in LDL cholesterol in a genetically homogeneous population under tightly controlled conditions explains a difference in the prevalence of a complex disease in a genetically heterogeneous population with little control over conditions.
You might be saying “well this is OBVIOUSLY a TOTALLY RIDICULOUS scenario and scientists would NEVER make these kinds of mistakes in interpretation.” Well, actually, what I’m suggesting is that people make these kinds of mistakes all the time when interpreting data from cell and animal studies (and also from human studies) that make crude comparisons of male vs. Most cells grown in vitro for laboratory use are ‘cell lines’: cells of the type we’re interested in that have a nearly limitless ability to proliferate. Most animals used in experiments are inbred, and have been adapted to laboratory conditions over many generations. The colonizers live in an atypical environment with the bare essentials they need to survive. Instead of living in a complex, dynamic human society made up of many different people, the colonizers live in a relatively static environment surrounded by others exactly like them. Instead of living in a complex, dynamic body made up of many kinds of cells, the cells live in a relatively static culture environment surrounded by other cells exactly like them. Instead of living in natural social groups and engaging in natural behaviour, animals are segregated into groups based on the convenience of the scientist, and live only with other animals of the same sex.
The living conditions of the residents of Mars are different from those of the residents of Venus. In mice (the most commonly used experimental mammal), males are usually housed at lower density than females because of the males’ tendency to aggression against one another. Differences observed between the inhabitants of Mars and Venus may be ascribable to a variety of factors, so making crude “male vs female” comparisons between the inhabitants of Mars and Venus is not very useful for understanding the influence of sex on the outcomes of interest.
Differences observed between male and female cell lines may be ascribable to a variety of factors, so making crude “male vs female” comparisons between male cell lines and female cell lines is not very useful for understanding the influence of sex on the outcomes of interest.
Differences observed between male and female animals may be ascribable to a variety of factors, so making crude “male vs female” comparisons between male cell lines and female cell lines is not very useful for understanding the influence of sex on the outcomes of interest. In the 14 May 2014 edition of Nature, the NIH announced that it intends to roll out policies beginning in October 2014 that will “require applicants to report their plans for the balance of male and female cells and animals in preclinical studies in all future applications, unless sex-specific inclusion is unwarranted, based on rigorously defined exceptions.” Although I applaud the motivation underlying these changes, I am far from convinced that simply requiring scientists to include male and female cells or animals in their work will be a significant advance in addressing sex and gender in medical research – in fact, I fear that a crude approach of this sort will not only fail to address concerns around equity, but it may in fact exacerbate them and serve to affirm our cultural bias that men are from Mars and women are from Venus.
Radiation-induced autophagy has been shown to play two different roles, in malignant glioma (MG) cells, cytocidal or cytoprotective.
We observed both temporal and dose-dependent response patterns of autophagy and apoptosis to radiation in MG cell lines. Autophagic activity measured by staining of acidic vesicle organelles and Western blotting of LC-3 protein increased in proportion to radiation dose from day 1 to 5 after irradiation. These findings suggest that autophagy appears earlier than apoptosis after irradiation and that a portion of the apoptotic population that appears later is autophagy-dependent. Radiation therapy, which is one of the primary treatment modalities for malignant glioma (MG), is considered standard therapy after cytoreductive surgery [1].
To define the cellular mechanism of radio-resistance, it is necessary to identify a responsible cell death pathway after irradiation. Autophagy is an augmented cellular protein recycling pathway in some cancer cells that was recently shown to lead to programmed cell death (PCD) type II in certain circumstances [6].
Herein, we investigated the temporal and dose-relationship of cell cycle change, autophagy and apoptosis in MG cells after irradiation with doses ranging from sublethal damage to lethal based on growth inhibition assay. U87, U373 MG, and LN229 human glioma cell lines were obtained from the American Type Culture Collection and maintained in Dulbeccoa€™s modified Eagle medium supplemented with 50 units of penicillin and streptomycin and 10% heat-inactivated fetal bovine serum at 37A°C under 5% CO2. To measure changes in the cell cycle, 1.5A—104 cells were plated on 60-mm plates and then subjected to radiation during the log growth phase. To detect and quantify autophagic activity, cells were plated and irradiated on 60 mm plates under the same conditions as for cell cycle measurement. U373 stably expressed GFP-LC3 cells were grown on 12-well glass-bottom dishes (Falcon, BD Biosciences) overnight (2A—104 cells per well). Quantitative data were expressed as the meanA±standard deviation or A±standard error of the mean, as indicated.
The radiation dose was as high as 20 Gy, which was equivalent to the dose prescribed for MG in fractionated conventional radiation therapy (60 Gy in 30 fractions) using the linear-quadratic formula [15].
To determine the radio-sensitivity of human MG cell lines by growth inhibition assay, radiation was applied to cells in 96-well plates at 20% to 30% confluency, which was the same confluency used in experiments measuring the cell cycle, autophagy and apoptosis. Based on these results, we set a target radiation dose of 10 Gy for a lethal condition to observe autophagy and apoptosis after irradiation. Both temporal and dose-dependent changes in the cell cycle were observed after irradiation upon flow cytometry analysis. We measured autophagy after irradiation by counting AVO containing cells in given time frames and dose ranges (Fig. We checked the expression of LC3 protein, which is known to be a standard indicator of autophagy. We also checked the amount of autophagy after irradiation using stable GFP-LC3 expression in U373 cells. Radiation-induced apoptosis based on the early apoptotic marker annexin-V was not observed 1 day after irradiation in any of the cell lines, which is in accordance with previous finding of no subG1 population upon cell cycle analysis (Fig. We next checked the expression of cleaved PARP, which is specific to late stage apoptosis, to determine if apoptosis measured by annexin-V staining continued to the late phase of apoptosis. It is well known that post-mitotic apoptosis after irradiation in radio-resistant solid cancer cell lines or sublethal dose in hematopoietic cancer cells is independent of caspase-inhibition [16]. The effects of autophagy-inhibition by knockdown of Atg5 on apoptosis were analyzed by flow cytometry with annexin-V staining (Fig.
These results indicated that at least some portion of radiation-induced apoptosis is dependent on the autophagic process in the tested cell lines, and that inhibition of autophagy could decrease cell death after irradiation.
To determine if cells bearing autophagosome-accumulation, an indicator of autophagy, undergo apoptosis after irradiation, we irradiated GFP-LC3 stable transfection U373 cell lines at a dose of 10 Gy. Cleaved-PARP, an indicator of late apoptosis, was typically observed in multi-nucleated cells following mitotic catastrophe (Fig.
To observe how radiation-induced autophagic cells change with time and determine if multi-nucleated cells have autophagic indicators in their initial stage, we observed GFPLC3 stable transfection U373 cells using a fluorescence live cell image analyzer from 3 to 5 days after irradiation at 10 Gy. Observation of changes in cell cycle after irradiation in MG cells are rare, but Yao et al. Our observation of aneuploidy cells after irradiation by FACS was confirmed with immunofluorescence staining. Autophagy and mitotic catastrophe are alternative PCD pathways to apoptosis, and these cell death pathways are independent of caspase, which is an essential executor of apoptosis. Caspase independent apoptosis can occur via one of the following routes: 1) late onset intracellular changes induces apoptosis via a detour in the caspase cascade, 2) early apoptotic marker of annexin-V is not only coupled to caspasedependent apoptosis, but also to PCD other than apoptosis, 3) time lapse between the duration of action of caspase inhibitor and initiation of caspase activation. Most studies of radiation-induced apoptosis mechanisms were performed before 2000, when autophagy was first considered, and most studies conducted in this period used cell of lymphoid lineages, in which apoptosis is the main pathway of cell death after irradiation. Therefore, we examined the relationship between autophagy and apoptosis in MG cells after irradiation by inhibition of autophagy using knockdown of autophagyrelated proteins.
In our experiment, inhibition of autophagy by knockdown of Atg5 reduced radiation-induced apoptosis, and in turn, cells were partially protected from the cell death after irradiation.
We demonstrated that autophagy appears earlier (1 day after irradiation) than apoptosis (3 or more days after irradiation) in MG cells subjected to radiation at clinically applicable doses. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology [2009-0089971; 2010-0016811]. Inhibition of autophagy significantly reduces apoptosis after irradiation and results in a cell survival benefit.
Live cell image analysis of autophagic cells bearing punctuated GFP-LC3 dots (arrows) after irradiation (10 Gy) (A— 200).
Cell survival after radiation measured by growth inhibition assay (A) and colony forming assay (B) after irradiation in malignant glioma cell lines. The aim of this work was to study the influence of biological and technical factors on the effect of flame weeding, as a basis for reducing the energy consumption and increasing the effective ground speed required for good weed control. Flame weeding belongs to a group of thermal weed control methods, which is a collective name for different physical methods using high or low temperatures or electrical fields.
There is currently a renewed interest in flame weeding as an alternative to chemical herbicides. Covered flamers were developed in the 1960s in the USA for non-selective weed control between the rows of cultivated crops (Parker et al.
American research reports on flaming from the 1960s and early 1970s were not particularly aimed at reducing the energy consumption for flaming, probably partly because fuel was cheaper then and partly because many researchers were sponsored by the petroleum industry. Interest in flame weeding increased during the 1950s but declined in most areas during the mid and late 1960s as a result of increasingly efficient chemical herbicides. Flame weeding is commonly believed to cause unwanted heating of the soil and thereby have a detrimental effect on beneficial soil borne organisms. The advantages of flaming are that the flame leaves no chemical residue in the crop, soil or ground water and there is no chemical carry-over effect on subsequent crops. The disadvantages of flaming include the short-term effect, the low selectivity and the need for repeated application. Although flame weeding has been used for a long time, the method poses problems such as high costs, low capacity and high energy consumption.
The general aim of this work was to study the influence of biological and technical factors on the effects of flame weeding in order to reduce the energy consumption and to increase the effective ground speed required to obtain good weed control. The first part of the work (Papers I and II) deals mainly with biological and biometrical issues, in order to develop and apply the dose-response concept, known from herbicide bioassay, to flame weeding. In the second part of the work (Papers III, IV and V), the general objective was to study the effect of technical factors on the efficacy of flame weeding. The work was limited to non-selective flaming of relatively small annual weeds and test plants, using propane as the fuel, and the results are therefore mainly relevant for such applications.
Naturally emerged weed mixtures were flamed in replicated experiments in the field (Papers II, III and V). Generally, the weeds and test plants in this study were flamed when the larger part of the population had emerged. Due to limited resources, only a few of the flamers could be purchased for this research project.
The propane consumption was determined in repeated tests by weighing the propane tanks before and after each test. In all dose-response experiments, the propane dose per unit area was varied with the ground speed. Dose regulation by adjusting the ground speed is considered a suitable method in flaming bioassay. Temperatures were measured above the ground in a rail track in the laboratory, with the same flamers and adjustments that were used in the field experiments, as described in Paper III and IV. Most of the field experiments were designed as dose-response experiments and were analysed with regression. The design of the dose-response experiments was inspired by the bioassay concept by Finney (1971, 1979).
From each replicate, one average temperature-time curve was calculated, which was analysed for the maximum temperature, the "exposure time" above a certain base temperature, and the temperature sum, based on the area between the time-temperature curve and a base temperature (Papers III, IV and V). The maximum temperature, the time above a certain temperature and the temperature sum correspond to those used by Harris et al. Non-linear regression analyses were also used to evaluate the correlation between the different thermal parameters measured in the laboratory and the weed reductions obtained in the field experiments (Papers IV and V). Several s-shaped models were tested, and three models were empirically found to reasonably well describe the relation between the different thermal parameters and the weed control. The effects of flaming are influenced by several factors including temperature, exposure time and energy input.
Several studies indicate that higher temperatures are more effective than lower for causing plant damage. Although the temperature measured in a stationary flame is essentially unaffected by fuel input (e.g. On the other hand, the heat losses may be greater at higher temperatures and energy inputs.
In flame weeding research, the flame treatments are usually compared at a few pre-set intensity levels, often quantified by e.g.
An example of the disadvantage of evaluating the effect at a single dose level can be seen in Paper III.
Another example of the problem associated with evaluating the effects at a single dose or ground speed is comparisons of flaming techniques. By using a series of doses and establishing dose-response curves, equipotent treatments can be compared by horizontal assessment, and effective doses and ground speeds can be estimated. This study shows that the logistic model used in herbicide bioassay (Streibig et al., 1993a) or modifications of this, can generally be used to describe plant responses to flaming at different doses (Papers I, II, IV, V). The re-expression to the right in model (2) makes it easier to see the similarities with other commonly used logistic models (Streibig, 1988, Ratkowsky, 1990) and to intuitively understand why the models are symmetric on log dose. If the dose-response curves are parallel, the horizontal distance between the curves is the same on a log dose scale, which means that there is a certain per cent difference in dose requirement regardless of response level.
Depending on the control situation, different response variables and control levels can be chosen. The same type of logistic model used for describing dose-responses, was also used for speed-responses. In herbicide bioassay, compounds with the same mode of action are assumed to have the same slope and thus have parallel dose-response curves (Streibig, 1988).
Generally, the slope of the dose-response curve supplies information about the variation in control effect within the plant stand. A large variation in control effect may be due to genetic differences in heat tolerance within and between species in the plant population. There is a principal difference between the dose-response curves for plant number and fresh weight, as Vester (1990) also pointed out.
A slight slope may also be attributed to variations in the control effect, due to uneven soil conditions, or irregular performance of the flame weeder. Flame weeding is effective mainly on small annual weed species, as a normal flame treatment only affects the plant parts above the soil.
Complete desiccation of plant parts above the ground is possible with most weed species as long as the flame dose is sufficiently high, although the necessary dose may vary considerably depending on weed species, developmental stage, flaming technique and environmental conditions. Weed species can be divided into four groups according to their tolerance to flaming (Paper II). The first group consists of sensitive species with unprotected growth points and thin leaves, such as Chenopodium album, Fumaria officinalis, Urtica urens and Stellaria media.
The second group contains moderately sensitive species that could also be completely killed with a single flame treatment, both at early and late developmental stages, but which required higher doses than the species in the first group.
The third group contains flame-tolerant species such as Capsella bursa- pastoris and Chamomilla suaveolens that could be killed completely by a single treatment only at early stages with less than four true leaves. The forth group contains very tolerant species with a creeping habit and protected growth points that could not be completely killed by one treatment, regardless of dose or developmental stage. At high flame doses, increased emergence and growth of Poa annua and Capsella bursa-pastoris was observed (Paper II). This result agrees with that of Parish (1990b), who found that a late flame treatment was more effective than an early one. The long-term effect of flaming depends largely on the extent of weed emergence after treatment (Parish, 1990b; Ascard, 1992b). However, split application or rather repeated applications of normal doses are necessary to starve larger plants and heat tolerant weeds such as grasses and perennial weeds, that will regrow after a single treatment. The tolerance of different plant parts of flaming depends on several biological factors such as protective layers of hair and wax, lignification, and conditions of water status. Young seedlings are often very sensitive to high temperatures; the region of the stem or hypocotyl close to the soil surface is often the most susceptible (Sutcliffe, 1977).
In general, the developmental stage has a major influence on the effective dose requirement (Papers I, II, IV and V).
The relationships between the lethal dose requirement and different variables describing the plant stands at treatment time were analysed with multiple regression (Paper I).
Plant density has only a minor influence on the dose required to achieve a certain percentage control (Paper I). Leaf surface moisture will prevent heat from a flame treatment penetrating different plant parts. Considerably higher doses were required in the dry year of 1992, than in the two previous years with moist weather (Paper I). The presence of inflammable dry materials that can be set alight during flaming is a factor indirectly related to the effect of flaming. Several heat transfer systems can be used for thermal weed control, as described in the introduction. Unfortunately, there is a great deal of confusion in the literature regarding the term "infrared" within thermal weed control, as the Dutch company that introduced the IR radiator for weed control in Europe is now marketing mainly thermal weeders with covered flame burners, but continues to market them as "infrared" weeders.
Some burners, with the air inlet close to the nozzles, designed to be operated as open burners, cannot easily be used on covered flamers. Commercials flame weeders usually have standard atmospheric burners without forced air assistance. There is a lack of consistent knowledge on how to adjust and improve the performance of a flame weeder. In the present study, the burners in the covered flamers were used at the fixed height determined by the manufacturer, which was between 5 and 15 cm for the different flamers (Paper V). In Paper III, the effects of burner angle were studied for an open burner similar to the Reinert burner. The use of flame weeding in Europe today, with mainly non-selective flaming of small weeds, is different from the selective flaming of larger weeds in the USA in the past. Burner power in this context means fuel input in terms of kw per metre working width, but it is here also expressed as the propane input in kg h-1 m-1. The burner power of a flamer can be increased by adding more burners to a given working width. The results in this study indicate that the effective ground speed generally increases with increasing burner power of the flamer. Generally, the results concerning the influence of burner power on the energy efficiency and effective ground speed are contradictory.
Information gathered from the literature indicates a relationship between burner power and effective ground speed. The increased effective ground speed with increased burner power can be explained by the fact that as the fuel pressure and number of burners increases, the gas velocity and energy density also increase and thereby more heat will be transferred from a dynamic flame through forced convection to the plants at any given time.
Greater changes in weed control effects are achieved by varying ground speeds than by varying rates of fuel pressures with the given burner. The results mentioned above indicate that the effective ground speed generally increases with increasing burner power of the flamer. By using multiple burner rows, the exposure time for the heat and thereby the weed control can be assumed to increase.
The results in Paper IV showed that the effective ground speed was not increased when tandem burners instead of a single burner row were used on a covered flamer.
Thermodynamic modelling by Bertram (1991, 1992) suggests that for a standard open flamer at a propane input of 50 kg ha-1, only about 15% of the combustion heat is actually transferred to the plants. Although covered flamers show several advantages, the use of covers may also involve problems such as oxygen deficiency in the propane combustion. Accurate measurement of leaf temperatures during flame treatment is very difficult as the temperature changes very rapidly. Many researchers, myself included, have avoided the problem of measuring the leaf temperatures during flame treatment and instead measured the temperatures in the vicinity of the plants or in an environment without plants.
Few basic results are available on the relation between increasing temperatures measured from dynamic flamers and weed control. In the present study, another approach was used; temperatures from dynamic flamers in the laboratory in weed-free conditions were recorded by thermocouples. This approach is related to the method used by Rahkonen & Vanhala (1993), who showed an s-shaped relationship between the maximum temperature measured in the vicinity of the weeds and the weight reduction of the weeds. When the effects of different burner angles were evaluated, there was a poor and non-significant correlation between the different temperature parameters obtained in the laboratory and the weed control in the field (Paper III). The weak correlation does not mean that temperature measurements are not useful in flame weeding research, but rather that the research methods have to be improved.
The correlation was generally high between the thermal parameters and the weed reduction, especially when maximum temperature and temperature sum were used (Papers IV and V).
No single thermal parameter showed the highest correlation with the weed control throughout this study. The sigmoidal relation between maximum temperature and weed control showed that an increasing maximum temperature corresponds to an almost linearly increasing weed control only up to a certain temperature or temperature sum, above which there is little increase in weed control.
Klooster (1983) also found generally increased weed control as the flame temperature and the exposure times increased, although the relative importance of these factors was not investigated.
The maximum temperature measured in the air is related to the convective heat transfer rate to the plants.
The temperature sum also takes into account the duration of the thermal treatment and is therefore probably a better measure of the amount of heat applied to the point of measurement and to the possible heat transfer to the plants, at least when the temperatures are measured by thin thermocouples.
The results were influenced by the height above the ground where the temperatures were measured. The sigmoidal relationship between the temperature and the weed control agrees with the results of Rahkonen & Vanhala (1993). The high correlation between the thermal parameters and the weed control is also partly a result of the wide dose range used in these experiments. Temperature measurements seem to be useful in flame weeding research, although improvements in the experimental methods are needed as is a better theoretical understanding of the relationship between temperatures and weed control. The main reason for using flaming is perhaps to avoid chemical herbicides, and thereby eliminate the risk of chemical residues in soil, water and in the crop.
A broadcast application of flaming, at a propane dose of 50 kg ha-1, corresponds to an energy content of 2300 MJ ha-1. Although flame weeding is an energy-requiring method, it should be viewed on the farm level. On the farm level, the total energy used to produce, for example, a maize crop is, according to Pimentel (1992), 47 900 MJ ha-1, and a major component of that support energy is nitrogen fertilizer (13 400 MJ ha-1). In the production of beet sugar, the total energy requirement for growing sugar beets is only about 22% of the total energy requirement for the production of sugar, i.e. After a worldwide search for the hardiest human beings, one man and one woman are selected to colonize other planets: Bob (a chemist from Finland) is sent to Mars, and Flo (a rice farmer from Thailand) is sent to Venus. They are very successful: by 2125 they are at maximum capacity, and the WSO sends scientists to study the populations. Men had significantly higher serum LDL cholesterol than women, which may explain the higher prevalence of cardiovascular disease in men. Given the way this was set up, it is OBVIOUSLY completely unreasonable to ascribe the differences observed in these tests to the sex of the populations. On the other hand, Flo comes from a somewhat short family with normal cholesterol, and her allele for CYP2B6 is a variant that has moderate efficiency in metabolizing propofol. Flo speaks fluent English and Thai, and so the clones on Venus learned both languages from her and are fluently bilingual.
Thus, gravity exerted less downward force on the bodies of the inhabitants of Mars, which would tend to allow them to grow taller than those living on Venus. In contrast, the genetic tendency to shortness of the Flo clones was magnified by the higher gravity; although their bodies were smaller, the higher gravity on Venus meant that their leg muscles had to be relatively more developed to support their (lower) body weight.
These are usually cancer cells, or cells that have been genetically modified to permit ongoing proliferation. However, neither the role of radiation-induced autophagy for cell death nor the existence of autophagy-induced apoptosis, a well-known cell-death pathway after irradiation, has been verified yet.
Additionally, we investigated the role of autophagy in apoptosis through knockdown of autophagy-related proteins.
Apoptosis measured by annexin-V staining and Western blotting of cleaved poly(ADP-ribose) polymerase demonstrated relatively late appearance 3 days after irradiation that increased for up to 7 days.
Although radiation has long been used, MGs easily acquire radioresistance, resulting in tumor recurrence, even in the radiation field [1,2]. Apoptosis is known to be a primary mechanism of cell death following radiation injury in cancer cells.
Through the specific knockdown of autophagy-related molecules (Vps34, autophagy-related gene 5 [Atg5], and Beclin1) with each shRNA-expressing lentivirus infection, we observed the effects of autophagy-inhibition on radiation-induced cell death or apoptosis.
All lentiviral vectors were transfected into 293T cells using Lipofectamine 2000 reagent according to the manufacturera€™s instructions.
Next, 10 I?L of annexin-Va€“ FITC (BD Biosciences) and 5 I?L PI were added, and the cells incubated at room temperature for 15 minutes in the dark. Seiji Kondo [14] incorporated into the lentiviral vector using Lipofectamine 2000 reagent (Invitrogen Life Technologies). After incubation for 24 hours, U373 cells were irradiated with 10 Gy and then incubated for 5 days. Irradiation was conducted in a Gammacell chamber 1 day after plating as previously described.
The punctuated pattern of GFP-LC3 granules was identified as an indicator of autophagy after irradiation.
Cleaved PARP was not detected on day 1, but appeared at 3 days after irradiation at 10 Gy and increased for up to 7 days (Fig.
To evaluate whether observed apoptosis is caspase-dependent or not, we treated cells with 50 I?M of Z-VAD-FMK, a pan-caspase inhibitor, immediately after irradiation with 10 Gy. The percentage of AVO-positive cells after irradiation with 10 Gy was used to measure the degree of inhibition of autophagy at 3 days after irradiation.
The cells were fixed for immunofluorescence at 5 days after irradiation, when both autophagy and apoptosis were prominent in the previous experiment. Some autophagic cells showed condensation of their scattered GFP-LC3 granules and disruption of their cell membrane at the end of the experiment (Fig. Two different types of cell death according to radiation dose were evident with different possible cell death mechanisms.
Although we failed to co-localize cleaved PARP with GFPLC3 granules in these cells that failed to divide, we did observe a prominent GFP-LC3 punctuated pattern in the early phase of aneuploidy upon live cell image analysis, and most of these cells died later.
In the experiment conducted to investigate late apoptosis with non-small cell lung cancer cell lines, neither over-expression of apoptosis inhibitor (Bcl-2 or Bcl-xL) nor pan-caspase inhibitor (Z-VAD-FMK) showed any protective effect against cell death [19].
Thus, few studies investigating the relationship among late apoptosis, autophagy, and apoptosis have been conducted to date. Although autophagy after radiation is proportional to radiation dose, it is not clear that the degree of autophagy is related to radiosensitivity. Our experiment of MG cell lines with 20 Gy or less irradiation is similar to apoptosis-defective condition in terms of delayed appearance of annexin-V staining and lack of TUNEL staining. Inhibition of autophagy reduced apoptosis, which was independent of caspase activation, partially protected cells from radiation-induced cell death. Annexin-V and poly(ADPribose) polymerase (PARP) were used as markers of early and late apoptosis, respectively, at 5 days after irradiation with 10 Gy (A—400). Malignant glioma cells (U87, U373, and LN229) were plated at 96 wells at subconflucency or 60-mm plate at clonal density.
Whereas staurosporin (1 I?M), which is well-known apoptosis inducer reduces caspase-3 level dose and time dependently. By far the greatest amount of research and the most extensive application of flaming have been in selective flaming of small in-row weeds in cotton and other heat tolerant row crops. 1965), for pest insect control in alfalfa (Luttrell & Bennett, 1968) and for potato haulm desiccation (Hansen, 1969).

During the 1970s, flamers disappeared from the agricultural scene as the prices for petroleum rose (Kepner et al., 1978).
There are reports on the use of flaming for weed control in plant nurseries (Nyholm, 1950) and vineyards (Preuschen, 1968; Engel, 1969). However, soil is a very good insulator and can absorb a great deal of heat energy with little increase in temperature (Reeder, 1971).
The equipment and the labour costs for application are high compared with those for chemical application. The literature on flame weeding contains few basic studies on the energy required to kill weeds. Another aim was to measure temperatures from dynamic flamers in the laboratory and to correlate these with the weed control in the field. This concept can be used as a tool to evaluate the influence of different factors, in terms of effective propane dose required to achieve good control.
The weed flora was dominated by Capsella bursa- pastoris, Chamomilla suaveolens, Chenopodium album, Poa annua, Senecio vulgaris, Stellaria media and Urtica urens.
The weeds were intentionally allowed to emerge before treatment as the purpose was to study the effect on emerged weeds rather than the effect on weed emergence after treatment.
The variation of the fresh weights due to variations in the moisture content of the plants was kept to a minimum by recording the fresh weights within a few hours per replicate. The problem of ineffectual home-made flamers, obtained by covering burners designed to be used as open burners, or by uncovering burners designed to be used in covered flamers, was avoided by comparing different flaming systems designed for use either as open or covered flamers.
The propane consumption of all burners on a flamer unit, rather than single burners, was measured. The exceptions were the split application experiments (Paper II) and the field evaluation of burner angle (Paper III), that were designed as ordinary randomized block experiments. The appropriate range of ground speeds were based on preliminary experiments and previous experience. There is, however, no method for adjusting the propane dose, which does not also alter the overall performance of the flamer. In the laboratory, however, a series of ground speeds (0.5, 1, 2, 4, 8 and 12 km h-1) was used, similar for all flamer settings. The split application experiments (Paper II) and the field experiment on evaluation of burner angle (Paper III) were subject to standard analysis of variance. For each temperature recording obtained at a certain ground speed in the laboratory, the corresponding weed reduction was calculated according to the estimated speed- response model.
The same type of logistic model that was used for dose-response relationships was also used as a base model for describing the relation between the temperature sum and the weed control, as the temperature sum was often approximately linearly related to the dose.
Buttiglieri et al., 1967), in dynamic conditions, more heat will be transferred from the flame to the point of measurement at a given time as the energy input increases. Therefore, the energy efficiency may be both higher and lower for a flame weeder with a high energy input, depending on the design of the flamer. At that particular propane dose (49 kg ha-1), the reduction of Senecio vulgaris was not significant compared with the untreated control.
If the effect of two flamers with different burner power is evaluated at the same ground speed, an analysis of variance will probably show that the flamer with the higher burner power gives a better weed control than the flamer with a lower burner power.
Moreover, patterns in the mode of action, that will otherwise be difficult to reveal can be studied.
In general, the logistic models (1) and (2) gave good descriptions of the dose- response relationships.
The model has an upper limit of 100, a lower limit C, and is symmetric around its point of inflexion, a, on a log dose scale (Fig. The logistic dose-response models were modified to describe deviations from the symmetry (Paper I), and to describe increased emergence and growth at high flame doses (Paper II). However, as the dose-response curves in many cases were not parallel, the response curves were generally evaluated at a certain control level. In grain crops, 80% control effect on weed weight may be sufficient, but in most situations where flaming is used, for example, in flaming pre-emergence of the crop, it is desirable to kill as many weeds as possible, since surviving weeds need further control.
It may be tempting to apply the parallel-line assay to flame weeding, but there are several reasons for non-parallel curves, even though the "mode of action" may appear to be similar. Even a low, sublethal dose will result in a reduction in fresh weight, whereas a certain dose is needed to reach the sensitive parts and thereby kill a plant completely. An uneven application technique of flaming will probably influence the shape of the dose-response curves in the same manner as in herbicide application (Alness & Hagenwall, 1994).
Plant species with growing points below the soil surface will, therefore, survive and regrow after flaming.
The most important factor distinguishing sensitive and tolerant species is not the heat tolerance of the leaves, but rather the ability of plants to regrow after the flame treatment, which is decisive for obtaining a sufficient duration of effect (Paper II). Sensitive species have a taller stature with growth points located at the shoot apex and along the stem, where the flames can easily reach them. These species have a prostrate habit at early stages and, especially at later stages, protected growth points.
Chamomilla suaveolens was very tolerant and also showed a tendency towards increased growth at high doses in some experiments. Jensen, 1992), since they make it possible to target a larger proportion of weeds at the sensitive cotyledon stage than is targeted with a single full-dose application.
He obtained the highest weed reduction with a combination of early and late fall-dose treatments, but two half-dose treatments were not tried. Therefore, flaming before crop emergence is usually dose as late as possible in order to target as many weeds as possible, although not all will be at their most susceptible stage. When Sinapis alba plants at the six-leaf stage were treated, the fresh weight reduction was higher after two half-dose applications 3 or 13 days apart than after a single early full-dose treatment (Paper II). However, plant survival of high flame doses is largely dependent on the plant's ability to regrow after flaming (Vester, 1990, Paper II). For example, the thin and delicate hypocotyl of Chenopodium album and Sinapis alba is very heat sensitive at the cotyledon stage and once damaged, the plant cannot regrow (Vester, 1990, Papers I and II). However, Lalor & Buchele (1970) showed the opposite for soybeans at the cotyledon stage.
Propane doses of 10-40 kg ha-1 (500- 1800 MJ ha-1) were required to achieve 95% control of plant number for sensitive weed species with 0-4 true leaves, whereas plants with 4-12 leaves required 40- 150 kg ha-1 (1800-6900 MJ ha-1), depending on species, developmental stage and treatment conditions (Paper II). An additional explanation for the higher dose requirement to kill larger plants is the greater leaf- and stem-surface and greater biomass to heat. There was a significant linear relation between fresh weight per plant and the effective dose for a plant number reduction of 95% (LD95) (Paper I).
Thus, as long as plant size remains the same and the growing points of the plants are not covered by neighbouring plants, the dose required to achieve a certain percentage control does not seem to be affected by varying plant densities.
Weeds can avoid the heat by being protected by water on the leaf surface or by being hidden by soil crumbs.
Thermodynamic modelling by Bertram (1991) showed an almost linear decrease in heat transfer to the plants with increasing leaf moisture in the range from 0 to 50 g m-2. When flaming is used in orchards and ornamental shrubberies, crop leaves arc easily damaged by the after-burning caused by fires starting in the dead weeds and rubbish (Ascard, 1988). The effect of this and other types of covered flamers is achieved both directly by the flames and to some extent indirectly by infrared radiation from the insulated cover, heated by the flames. Burners are commonly grouped according to the shape of the burner and the flame (flat or tubular) and according to whether they have a vapour chamber or not (liquid- or gas-phase burner). An inappropriate positioning of the burners and the cover can lead to oxygen deficiency and thus affect the combustion (Laguë et al, 1992).
These propane burners commonly produce a measurable flame temperature of 1200-1350°C (own measurements). The burner setting affects how the flame reaches the weeds and for how long a high temperature is maintained. If the burner is set too close to the ground, the flame will deflect upwards, especially if the burner angle is steep and the fuel pressure high. For the standard Reinert burner, an open flamer commonly used in some parts of Europe, Hoffmann (1989) recommends an angle of 45° at a burner height of 12 cm for flaming preemergence of the crop.
A burner height of 10 cm was chosen, similar for all burner angles, based on the recommendation by Hoffmann (1989) and on preliminary trials.
The fuel consumption of a burner can be increased by raising the fuel pressure and by using larger nozzles.
Thus, changes in the fuel pressure have relatively little effect on gas consumption and weed kill by flame, especially with some self-vaporizing burners (Matthews & Tupper, 1964). The effective ground speed depends on the treatment conditions (plant species, developmental stage, weather) and the flame weeder (burner power, burner type, cover design etc.).
This was most probably a result of an insufficient increase in the temperature and exposure time of the front burner row, due to a relatively low burner power of the first burner. For a standard covered flamer, however, the heat transferred to the plants is 30%, and with an optimal cover it can be increased to 60%, according to the model.
The open flamer required, on average, a dose 40% higher than that of the covered flamers to achieve good control, although there was a large variation from no difference up to the double-dose requirement. The temperature in a leaf will not exceed 100°C as long as moisture vaporises from the leaf surface.
Hansen et al., 1970), lower temperatures will be recorded in the centre than at the edges (Bertram, 1994), and such results may not be relevant for determining lethal temperatures in thin leaves. As there is no significant vaporization from a thermocouple, the temperatures in the thermocouple will rise considerably higher than 100°C. In a dynamic flame, however, lower temperatures will be recorded, due to the slowness of a thermocouple.
Some American researchers used thermal weeders constructed as hot air ovens, in which plants were exposed to a known air temperature for a specified length of time.
From the resulting temperature-time curves, a number of thermal parameters were calculated, i.e. The maximum temperature in particular was poorly correlated with the weed control as the burner angle of 45° directed forwards showed high maximum temperatures in the laboratory but relatively low weed control in the field. If the purpose is to evaluate the appropriate burner angle for field use, one probably either has to do the experiment in the field or create the field conditions in the laboratory. In several experiments, however, the maximum temperature generally showed the highest correlation with the fresh weight reduction in smaller plants, whereas the temperature sum generally showed higher correlation with the reduction in larger plants. The results of Klooster (1983) and Vriesema (1985) indicate that temperatures between 800 and 1000°C lasting for about one second were needed to obtain good weed control. More precisely, the convective heat transfer rate is linearly related to the temperature difference between the air and the plant. However, the maximum temperature often gave similar or higher correlation with the weed control than the temperature sum did, particularly when the temperatures were measured by the thicker 0.25 mm thermocouples (Papers IV and V).
For example, the correlation between the temperatures and the reduction in larger (taller) plants was significantly improved when the temperatures were measured at a height of 3.5 cm instead of 1 cm (Paper V). Therefore, the more robust 0.25 mm thermocouples seem to be more useful in flame weeding research owing to their greater durability.
A principal difference, however, is that they measured the temperatures in the vicinity of the weeds simultaneously with the flame treatment of the weeds, whereas in this study, the temperatures were measured in a weed-free environment.
Several smaller differences observed in the field, in particular not those between different types of flamers, could not be explained by the temperature measurements.
However, flaming is generally assumed to require too much energy to play a significant role or even be justifiable in future sustainable farming systems. If the energy for transport and processing of propane is added as well as diesel fuel for the application of flaming, the energy use will be about 2700 MJ ha-1, according to Jolliet (1993). Even in a crop with a lower nitrogen requirement such as carrots, a nitrogen fertilization of 100 kg ha-1 corresponds to about 6 000 MJ ha-1 (Loomis & Connors, 1992), and thereby far exceeds the energy use of a broadcast application of flaming. As expected, women had a much stronger aptitude for verbal reasoning than men, which is in line with the body of literature documenting that women have stronger communication skills than men. Bob speaks Finnish and broken English, and so the clones on Mars are not very proficient in English. Thus the net effect of these genetic and environmental influences resulted in similar lower body strength between the groups. Blocking of pan-caspase (Z-VAD-FMK) did not affect apoptosis after irradiation, but silencing of Atg5 effectively reduced radiation-induced autophagy, which decreased apoptosis significantly. However, radiation-induced apoptosis is delayed (within days) in solid tumors relative to the rapid response (within hours) that occurs in sensitive hematopoietic cell lines [3].
Specifically, autophagy helps cancer cells survive under nutrient-limiting conditions by recycling protein and protecting cancer cells from cellular damage caused by anti-cancer drugs or ionizing radiation, possibly by removing damaged macromolecules or organelles.
To evaluate if autophagy-bearing cells translate into apoptosis after irradiation, we stained green fluorescent protein (GFP)a€“light chain 3 (LC3) stably transfected cells with annexin-V, an early apoptosis marker, and then observed samples using immunofluorescence techniques. Virus particles were collected 2 days after the transfection of lentiviral plasmids and infected into U87, U373, and LN229 cells. Briefly, cells were plated at clonal density (1.0A—103 per plate) in a 60-mm dish and treated with radiation at a dose indicated in the results section the next day.
Cells (including non-adherent cells) were prepared at the indicated time after irradiation, fixed in 70% ethanol and kept overnight at 4A°C.
After trypsinization and washing, pelleted cells were suspended in PBS and subjected to fluorescence-activated cell sorter (FACS) analysis as previously described [14]. The stained cells were subsequently analyzed with a FACSCalibur flow cytometer and data were collected and analyzed using the CellQuest software. After selection with puromycin, expression of fluorescence was confirmed by microscopic evaluation before irradiation. Cells on the cover glass were washed with cold PBS and fixed with 4% paraformaldehyde for 10 minutes at 4A°C. Blots were washed and incubated with horseradish peroxidasea€“ conjugated secondary antibody. Increases in autophagy from 1 to 7 days after irradiation were demonstrated at 10 and 20 Gy and a dosedependent increase in autophagy after irradiation was confirmed at each designated time of measurement in all three cell lines.
The number of autophagic cells, which was counted under a fluorescence microscope, increased from 3 to 5 days and showed a dosedependent increase at 3 days after irradiation from 5 to 10 Gy (Fig. At sublethal doses (2 Gy and 5 Gy), annexin-V was not stained until 7 days after irradiation in U87 and U373 cells, but the annexin-V staining population was increased to 18% in response to 5 Gy in LN229. Apoptosis was measured at 5 days after irradiation by calculating the percentage of annexin-V staining cells, and treatment with Z-VAD-FMK did not alter the fraction of apoptosis significantly (Fig. Among tested cell lines, U87 showed no significant inhibition of autophagy after silencing of autophagy-related proteins. In the U373 and LN229 cells, apoptosis after irradiation with 10 Gy decreased significantly to 65% and 35% of that in the parent cells, respectively, with the inhibition of autophagy occurring at 3 days and the inhibition of apoptosis being observed at 5 days. Cells with punctuated GFP-LC3 dots indicating autophagy were stained with annexin-V, representing early apoptosis (Fig.
These cells did not express a punctuated pattern of GFP-LC3 dots, but instead harbored condensed dots.
6A), while other GFP-LC3 granule bearing cells divided with time, after which the divided cells cleared up the LC3 dots (Fig. The former was called pre-mitotic apoptosis, while the latter was termed postmitotic apoptosis, which was not blocked by caspaseinhibitor.
However this transient G1 arrest, which had been evidence of pre-mitotic apoptosis in previous studies, was not converted to subG1 accumulation after one week, and they could find no evidence of apoptosis upon terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.
This observation of aneuploidy resulting in delayed apoptosis was also observed in relatively radio-resistant non-small cell lung cancer cell lines by Stuschke et al.
We observed apoptosis based on annexin-V staining after irradiation in MG cells and found that it was not influenced by caspase inhibitor.
However, in their experiments, the addition of caspase-inhibitor to the low-dose (5 Gy) post-mitotic apoptosis model could not reduce the PARP cleavage rate. Autophagy can be an indicator of accumulated intracellular stress as in starvation or exposure to noxious stimuli, including radiation.
Among the measurements of autophagy activity in this study, flow cytometry of AVO, the ratio of LC3 protein bands, and the observation of GFP-LC3 did not accurately reflect direct radiosensitivity since the degrees observed were relative and the basal levels differed between cell lines [22].
However, autophagy is a primary, dosedependent, cellular response to radiation of 20 Gy or less in MG cells. Immunofluorescence of GFP-LC3, an autophagy marker, co-localized with annexin-V, an early apoptotic indicator after irradiation at lethal doses (10 Gy). To measure acidic vesicular organelle (AVO), which is indicative of autophagy formation, cells were stained with acridine orange and analyzed quantitatively by flow cytometry.
Annexin-Va€“positive cells were not observed at 1 day after radiation, but appeared after 3 days and increased thereafter. New concepts in radiation-induced apoptosis: 'premitotic apoptosis' and 'postmitotic apoptosis'. Autophagy: a novel mechanism of synergistic cytotoxicity between doxorubicin and roscovitine in a sarcoma model. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Molecular response of human glioblastoma multiforme cells to ionizing radiation: cell cycle arrest, modulation of the expression of cyclin-dependent kinase inhibitors, and autophagy. Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells.
Different mechanisms between premitotic apoptosis and postmitotic apoptosis in X-irradiated U937 cells. Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint. Late activation of apoptotic pathways plays a negligible role in mediating the cytotoxic effects of discodermolide and epothilone B in non-small cell lung cancer cells.
Differential PARP cleavage: an indication of heterogeneous forms of cell death and involvement of multiple proteases in the infarct of focal cerebral ischemia in rat. Combined action of the dinuclear platinum compound BBR3610 with the PI3-K inhibitor PX-866 in glioblastoma.
Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes.
Autophagy for cancer therapy through inhibition of pro-apoptotic proteins and mammalian target of rapamycin signaling. Different dose of radiation as indicated on the abscissa was given to determine dose-response relationship and effective dose of killing 50% of cells (ED50). Cleaved caspase-3, a by-product of caspase-3 activation, is only observed in HT-29 colon cancer cell line 48 hours after exposure. In some heat-tolerant crops, such as onions and maize, selective post-emergence flaming is also used. The large-scale agricultural application of flaming began in the early 1940s for selective weed control in cotton fields in the USA. The method of using delayed sowing followed by non-selective flame weeding preemergence of the crop, was not common at this time. Research was conducted by the petroleum industry in various countries including the United Kingdom, Denmark, Belgium and Sweden, on flaming pre-emergence of the crop in sugar beets, and pre- and post-emergence as well as pre-harvest flaming in potatoes (Stewart, 1965).
Because the thermal treatment is brief, only the uppermost few millimetres of the soil are temporarily warmed up (Hoffmann, 1989, Balsari et al., 1994). Flaming controls a wide range of annual weed species, some of which are tolerant or resistant towards herbicides. Flaming usually has a low capacity due to narrow working widths and - for many flamers - the rather low effective ground speed.
In order to lower the energy requirement, more knowledge is needed on how to adjust the treatment intensity to the weed flora and to the stage of development. The main problem with the natural weed flora was the large spatial variation in weed distribution, which made any experimental work erroneous. In Paper II, the time span between tillage and early treatments was 19-27 days, which corresponds to the time from drilling to emergence of slow-emerging small-seeded crops such as carrots and onions in early spring in southern Sweden. The flamers used were chosen from commercial flamers and existing experimental flamers, to give a representative selection of different types of flamers with respect to burner type, cover, flame pattern and fuel input. To make the figures comparable, they were converted to propane consumption per hour and metre working width.
The aim was to obtain plant responses from 0 to 100%, evenly distributed on a log dose scale.
A flamer with a high burner power may have a sub-optimal combustion at low ground speeds (Paper IV and V). The flamers were carried by a tractor, equipped with an extractor fan for the diesel exhausts, guided along a 25 m long rail track (Fig. The response of the plants to varying ground speeds is a new concept, developed in this work (Papers IV and V). However, weed reductions were only calculated in the speed range corresponding to the ground speeds used in the field. The effect of flaming on plants depends both on a direct effect on the cell membranes and on an indirect effect during the subsequent desiccation of the tissue.
In fact, higher temperatures and weed control were obtained from flamers with high burner power than from those with lower fuel input at similar ground speeds (Papers IV and V).
When the effects of such treatments are compared by analysis of variance, a qualitative assessment is performed to see whether one treatment is significantly different from the other. Similar observations have been made by Swedish growers, who based on their observations have concluded that S. The use of dose-response curves has recently become quite popular in herbicide research, especially in Denmark (e.g. In this thesis, the doses needed for 95% control were estimated, in addition to those giving 50% control. Since the flame treatments in this study were often conducted in different plant stands and at different treatment times within the same experiment, differences in the prevailing treatment conditions caused different slopes of the response curves in many experiments (Papers I, II, IV and V). Therefore, the dose-response curves describing the effects on plant number arc displaced to the right.
However, a flat slope is not necessarily a sign of poor overall performance of the flame weeder.
Because of this superficial effect it is, however, possible to flame, for example, carrots just before crop emergence without damage to the crop. Tolerant species, on the other hand, have a prostrate growth habit with protected growth points often near the soil surface.
At early stages, when the plants had 0-4 true leaves, a 95% reduction of the plant number was achieved at 10-20 kg ha-1 and they were completely killed at doses of 20-50 kg ha-1, depending on species and treatment conditions. Even when the leaves were completely desiccated, regrowth took place from growth points at or below the soil surface. However, in the present study, the tolerant species can be divided into groups that can or cannot be killed by one flaming at early stages. However, split application with two half-dose treatments one week apart did not give a higher plant number reduction than a single late flame treatment at the same total dose, when naturally emerged weeds were flamed at early stages (Paper II). A split application cannot be generally recommended for flaming preemergence of the crop, but may be useful when the germination period is long, in order to target the weed seedlings of tolerant species before they grow out of the sensitive cotyledon stage. The higher thermal sensitivity of younger plants is generally caused by their thinner leaves and stems and less protected meristems than on older plants.
As the stem- and leaf-length increases, the heat transfer coefficient will decrease (Bertram, 1994). The fresh weight per unit area also showed a significant influence on the effective dose required for high fresh weight reduction. However, if the aim of the treatment is to get below a certain threshold in absolute numbers, the effective dose will of course be affected by the weed density.
Parish (1990a) found reduced effect of the flame treatment with increased level of water applied to the leaves in the range from 90 to 360 g m-2. The surface conditions are also important in non-selective flame weeding to avoid flame deflection upwards and to achieve an effective heat treatment close to the soil surface. Covered flamers can also be sensitive to wind, especially to wind penetrating under the flame cover from behind (Luttrell & Bennett, 1968).
This problem, encountered with flaming in several perennial crops, can be solved by mounting a sprayer to apply water immediately after flaming (Hansen, 1964).
There are no consistent results on which kind of burner is the most appropriate for weed control. Some covered flamers, therefore, use specially designed burners with the air inlet positioned at a distance from the nozzle and above the cover (Flamers No. Several technical factors influence the performance of the flame (Lewis & von Elbe, 1987).
The appropriate height and angle of the burner are influenced by many factors, including burner type, fuel pressure, flame length, surface conditions, weed height, ground speed and wind. A burner angle of 67° directed backwards showed the highest weed reduction, but there were no significant differences between the effects of the different burner angles. The fuel consumption increases with the square of the diameter of the nozzle and with the square root of the fuel pressure (Hoffmann, 1989). In dense weed areas, however, flame penetration is improved by higher pressures (Thomas, 1964).
Among the covered flamers with a propane input between 7 and 42 kg h-1 m-1 (90-540 kw m- 1), there was no clear indication of a generally poorer energy efficiency of the more powerful flamers, although the relative performance of the individual flamers was dependent on plant size (Paper V).
Thermodynamic modelling by Bertram (1991) showed an almost additive effect on the heat transfer rate of increased fuel input in the range of 50 to 100kg propane ha-1.
The advantage of using a covered flamer was small when sensitive weeds at early stages were flamed, and greater for tolerant species and larger plants.
Several other factors such as height, length and angle of the cover, also influence the performance of a covered flamer (Storeheier, 1991, 1994).
Several measuring errors may be involved as any sensor attached to or inserted in a thin leaf will itself act as a heat sink.
According to Thomas (1967), the moisture vaporization from the surface forms a very thin protective or cooling film which prevents a rise of temperature above about 93°C at the surface of a living succulent plant. Moreover, longer exposure times than, for example, the above-mentioned 0.13 s will be recorded. The focus on temperature patterns in the air, from 2.5 to 20 cm above the surface was relevant at the time when the main use was selective flaming of established weeds in row crops, where a heat deflection upwards was detrimental. However, in this particular study, one cannot expect a high correlation between temperature parameters and the weed control, as there were no significant differences between the effects of different burner angles on the weeds.
Temperature-time curve obtained from a dynamic open flamer at a ground speed of 3 km h-1 and at an angle of 45° aimed forwards. The generally high correlation between the thermal parameters and the weed control, agree with the results of Harris et al. However, these temperature levels differed depending not only on the species and developmental stage, but also on the thickness of the thermocouple.
Such indications of temperatures and exposure times are, as mentioned above, strongly dependent on the thickness of the thermocouple. In this study, however, it is of no importance to the correlation if the maximum temperature itself or the maximum temperature minus the temperature in the laboratory (20°C) is used. This may be explained by the way the temperature sum was calculated from the temperature-time curve. This is probably the reason why Rahkonen & Vanhala (1993) generally obtained lower temperatures.
It is true that flame weeding is energy-requiring in comparison with mechanical and chemical methods (e.g.
In practice, many growers use banded application of flaming and can thereby reduce the fuel consumption considerably. Women also required a 44% lower dose of propofol to achieve anaesthesia; anaesthesiologists should adjust their dosages of propofol accordingly for male and female patients. Inhibition of autophagy in Atg5 knockdown cells was shown to be beneficial for cell survival. Additionally, some anti-cancer therapy typically induces autophagy in proportion to noxious stimuli, and defective or excessive autophagy leads to autophagic cell death [7,8].
We also traced these GFP-LC3 bearing autophagic cells after irradiation using a live cell image analyzer to determine if these autophagic cells eventually die or overcome radiation-induced damage.
Louis, MO), while N-benzyloxycarbony-Val-Ala-Asp (O-methyl)a€“ fluoromethy-ketone (Z-VAD-FMK) was acquired from MBL International Corporation (Woburn, MA). Lentivirus-infected U87, U373, and LN229 cells were puromycin-selected and established stable cell lines were used experiments. Following irradiation at the indicated doses, cells were observed for the fluorescence of GFP-LC3 under a fluorescence microscope and LC3 punctate spots were counted.

For permeabilization and blocking, cells on the cover glass were incubated with 0.2% Triton X-100 on ice for 20 minutes, then with 1% bovine serum albumin for 1 hour at 37A°C. Antibody complexes were visualized using an enhanced chemo-luminescence Western blotting detection system (Pierce Biotechnology, Rockford, IL). At a dose range of 10 to 20 Gy (5 to 20 Gy in LN229), the subG1 population became apparent at 3 days after irradiation and it was proportional to the radiation dose.
At 2 Gy, significant increases in AVO containing cells were not observed any of the cell lines. At lethal doses (10 Gy and 20 Gy), this apoptotic marker became evident 3 days after irradiation and increased linearly for up to 7 days. In both U373 and LN229, knockdown of Atg5 resulted in an average of 46% and 60% of AVO-stained cells, respectively, relative to parent cell lines (Fig. To evaluate whether the observed decrease of apoptosis after autophagyinhibition is related to cell death, we conducted a cell survival assay of the Atg5 knockdown cell line. This finding suggests that few typical punctuated GFP-LC3 were observed in the late phase of apoptosis.
They suggested that a significant proportion of apoptosis induced by diverse toxicants is caspase-independent.
Thus, only chronological changes in a cell line or dose-dependency at a certain time point could be obtained. Thus, we could assume that autophagy in MG cell lines after irradiation is at least partially responsible for cell death, suggesting that it is important to sensitize MG cells to radiation to control autophagy after irradiation.
Additionally, tracking of GFP-LC3 bearing cells using a live cell image analyzer revealed that some autophagic cells clear-up autophagosomes after mitosis, while others failed to divide and condensed and died. Aneuploidy was observed at a position of 4n (circle) on 1 day after irradiation (upper, right panel).
ED50 is around 10 Gy in growth inhibition assay while it is less than 5 Gy in colony forming assay (Dotted lines are representing curve fit for sigmoid dose response model).
The sigmoidal dose-response and speed-response curves imply that propane dose and the ground speed can be adjusted to the required control effect, the weed flora and the developmental stage of the plants. In fact, this method was presented as an innovation in the late 1960s by Chappell & Ellwanger (1969). Flaming for potato haulm desiccation was investigated and used in practice in the Netherlands (Philipsen, 1970; de Leeuw, 1972). No significant damage to the microflora or microfauna in the soil can therefore be expected during normal flame treatment for weed control. In some cases, the effect of flaming is less subject to variation in weather conditions than that of herbicides, especially for pre-emergence herbicides applied to the soil.
Sown test plants of Brassica napus and Sinapis alba were therefore used in several field experiments to establish even plant stands.
However, especially in late spring and for faster emerging crops, flaming pre-emergence of the crop has to be done before most of the weeds have emerged, which will cause a lower long- term effect than that found in this study.
Another advantage of using available flamers is that the results can be related to other studies using similar equipment. In the early experiments, from 1989 to 1991, the range of ground speeds was determined individually for each experiment. Generally, direct heat injury involves denaturation and aggregation of membrane proteins causing an increase in cell permeability and death (Suteliffe, 1977; Levitt, 1980). In general, lethal temperature varies inversely with exposure time, and a negative exponential relationship between lethal temperature and lethal exposure time is reported (Sutcliffe, 1977; Levitt, 1980).
This is explained by the fact that as the fuel pressure or nozzle size of a burner is increased, the gas velocity and the energy density of the flame and thereby the forced convection to the plants also increase.
2 and 3), where the covered flamers with high burner power (440 and 540 kw m-1) generally showed either similar, lower or higher energy-efficiency than other covered flamers with lower burner power (90 to 150 kw m-1), depending on the treatment conditions. However, it is often more useful to do a quantitative assessment to determine the effective dose or ground speed required to obtain good control. Even higher control levels are interesting, but the calculations of LD99, which were estimated in a progress report (Ascard, 1992a), are more uncertain, especially if these estimates are beyond the observed dose range. When even plant stands of Sinapis alba were flame treated, the slopes of the curves describing the effect on plant number were steeper than those for fresh weight (Papers I, IV and V). The covered flamers gave flatter slopes of the dose-response curves than those for the open flamer (Paper V). The regrowth was rather quick in many tolerant species, even when the leaves were completely desiccated, although this regrowth required adequate soil moisture. At later stages (4-12 leaves), considerably higher rates (50-200 kg ha-1) were needed to kill the plants completely. One reason why split applications were not more effective than a single fall-dose application, is that the weed flora consisted predominantly of susceptible weed species, Chenopodium album and Fumaria officinalis, which could be easily controlled by a single later application.
However, in some species, the location of these sensitive plant parts is different during different developmental stages. Although this kind of comparison has to be done with great caution, it is a possible method to compare lethal doses between assays in future studies.
For example, to reach a control level of 2 surviving plants per m2, the effective propane dose was 60 kg ha-1 at a plant density of 195 plants m-2 and had to be raised by 17% when the plant density was twice as high (Paper I). The reason for the high dose requirement in 1992 may, therefore, be some physiological difference, such as a more dense covering of hair and wax on the plant surface and a lower moisture content in the plants due to the dry weather. The risk of fire will also be eliminated if hot water is used as a heat carrier (Berling, 1993).
In interaction between burner height and burner angle was found by Storeheier (1991, 1994). The higher weed reduction at an angle of 67° backwards supports the visual field observations during flaming, which showed that the flame in operation hit the ground and then flattened out with little deflection upwards. However, the American recommendations on flame weeding from the 1950s and 1960s, are not necessarily relevant today. Higher fuel pressures or nozzles with larger diameters produce more heat farther away from the nozzle tips.
In selective flaming, higher fuel pressures may result in less efficient weed control because the increased flame velocity pushes the heat across the row to the next middle (Thomas, 1964). The lower energy-efficiency at the highest fuel pressure was probably caused by exceeding the optimal fuel pressure. However, it seems that there are no benefits achieved in increasing the burner power above a certain level, if the optimum fuel input of the burner is exceeded. Klooster (1983) and Vester (1987b, 1988, 1990) showed that flamers with high burner power gave similar or better weed control per unit propane than burners with lower power. When liquid propane spray was used, effective ground speeds up to 24 km h-1 were reported (Pivonka, 1968). This conclusion was based on experiments with tall cotton plants with corky layers on the stem, treated with open flame burners perpendicular to the row.
Moreover, the temperature recorded will be affected by where, in the living plant, the sensor is placed.
As a normal flame treatment does not burn, but rather blanches the plant tissue, leaf temperatures above 100°C such as those reported by Albrecht (1985), Hege (1989) and Hoffmann (1989) are probably a result of measuring errors. The deviation from the true temperature is dependent on the time constant of the thermocouple. The temperature pattern at the surface level, where cotyledon weeds arc found, and the effect of different burner angles on weeds were, however, not studied. However, in these experiments, the temperatures were considerably lower and the exposure time longer than in normal flame treatments. The maximum temperature at 0.5 s was recorded when the rear part of the flame passed the thermocouple. For example, when temperatures were measured from a flamer at a ground speed of 2 km h-1, giving 95% weed control (Fig.
Moreover, the experimental error in these measurements was such that the temperature difference between two replicates was often in the range from 20 to 100°C. A large part of the temperature sum corresponds to temperatures that are considerably lower than the maximum temperature and that were recorded after the actual thermal treatment, when the temperature in the thermocouple dropped to the air temperature in the laboratory. The higher correlation in this study is partly because different models were used for curve fitting and partly because the correlation was done on the average temperatures of two replicates versus the estimated response according to the speed-response curve. Although this experimental method is quite common in flame weeding research, the use of naked thermocouples in the laboratory is probably not the best method to model the response of a plant to a flame treatment in the field. In row crops, flaming can be restricted to a narrow strip over the row, since weeds between the rows can be mechanically tilled very close to the row (Ascard & Mattsson, 1993). Flaming is then used when mechanical methods are less effective and chemical herbicides not available due to legislation or consumer demand.
Just like on Earth, men were taller than women, but surprisingly there were no significant differences in lower body strength. This post-mitotic cell death after irradiation has been known to reflect inadequate DNA damage, which could not provoke immediate apoptosis, in cells with defective apoptotic machinery or following exposure to sublethal doses of radiation [4,5]. Although its role in radiation response remains obscure, autophagy can be observed in cancer cells, including MG cells, after irradiation [9,10]. Annexin-Va€“fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining solution were obtained from BD Biosciences (San Diego, CA), while Lipofectamine 2000 was from Invitrogen Life Technologies (Grand Island, NY).
Colonies were fixed with ice-cold methanol for 15 minutes, then stained with 0.4% crystal violet after washing with distilled water.
DNA content was analyzed using a fluorescence-activated cell sorter (FACSCalibur, BD Biosciences) and the CellQuest software (BD Biosciences). Rabbit polyclonal antia€“annexin-V or antia€“cleaved poly(ADP-ribose) polymerase (PARP) antibody was used as the primary antibody, and Alexa Flour594a€“conjugated anti-rabbit antibody was used as the secondary ant body. The microscope was housed in a custom-designed 37A°C chamber with a secondary internal chamber that delivered humidified 5% CO2. Statistical significance of differences was assessed using a two-tailed unpaired Studenta€™s t test and one-way ANOVA (ver.
At 5 Gy, a discernible increase in AVO containing cells in proportion to days after irradiation was only observed in LN229 cells. Western blotting of caspase-3, a common final executor of prototype apoptosis, was conducted to evaluate any change after radiation. The Atg5 knockdown cells showed significantly increased cell survival relative to the control group after irradiation (Fig. These results suggest that cells bearing autophagic indicators underwent apoptosis and that autophagy could be an interim state leading to apoptosis after irradiation. Although this observation was not a quantitative measurement, there was a tendency for cells bearing more dense GFP-LC3 dots to have a higher probability of undergoing cell death. To investigate this, we treated cells with caspase inhibitor 2 days after irradiation; however, we still saw no change in apoptosis rate (data not shown). Nevertheless, our study demonstrated knockdown of Atg5 decreased autophagy and reduced autophagy measured by AVO formation, resulting in decreased apoptosis.
These findings suggest that autophagy is a cell death pathway after irradiation in MG cells. The error bars indicate A±standard error of mean.(B) Cleaved poly(ADP-ribose) polymerase (PARP), a marker of late apoptosis, was not detected at 1 day after irradiation with 10 Gy.
Data from three independent experiments were combined and error bars indicate A±standard error of means. A 95% reduction in susceptible annual weed species, with 0-4 true leaves, was achieved at propane doses of 10-20 kg ha- 1, and the weeds were completely killed at 20-50 kg ha-1 (900-2300 MJ ha-1).
In fact, Dierauer & Pfiffner (1993) found no species-specific effect of flame weeding on carabide beetles, in early flaming before crop emergence in vegetables. For weed control in vegetables, labour costs for supplementary manual weed control make up a large part of the total cost of using flame weeding as opposed to herbicides (Ascard, 1988). From a resource and environmental point of view, the high energy requirement and the release of carbon dioxide can be seen as a disadvantage, although propane combustion is relatively clean compared with other fossil fuels. The disadvantage of evaluating different flaming systems, as in Paper V, is that any differences between flamers cannot be attributed to cover, to burner power or to burner type only, but rather to the whole flaming system.
In later experiments, in 1992 and 1993, the ground speeds were chosen from a fixed number of ground speeds (0.5, 1, 2, 4, 6, 8 and 12 km h-1) in order to facilitate the experimental procedures. Vester (1990) and Rahkonen & Vanhala (1993) used a combination of fuel pressure and ground speed to obtain a wide dose range.
The temperatures were measured 1 cm above ground in replicated experiments, by thermocouples of type K (Chromel-Alumel).
The methods and models are further described in the section "Quantitative assessment" in this thesis, and in Papers I, II and IV. The lethal exposure time was also reduced to a certain extent, measured as an increased effective ground speed, by using the more powerful flamers. However, when a series of doses was applied and a dose-response curve was established, it was clearly shown that S.
If instead, the same flamers are evaluated at the same dose level, a relevant comparison of the weed control effect can be made, but this comparison will not be useful for determining the effective dose either.
Parameter a is the effective dose that gives a response half-way between the upper and lower limit.
However, the slopes of the dose-response curves for natural weed mixtures show inconsistent results (Papers II and V), which may be due to uneven weed stands.
This may be a result of the covered flamers being able to reduce the plants to a certain extent also at very low doses. Moreover, the larger part of the weeds were emerged at the time of treatment, and therefore, there were few additional newly-emerged weeds to be controlled by the second treatment. In some cases, even repeated flaming during the same day may be justified (Lien et al., 1967). In Paper III, considerably lower temperatures were measured 1 cm above the soil surface between the ridges on a rough surface than on a smooth surface. Several types of flamers exist, some with open burners and others with burners underneath an insulated cover.
The reason why such air assisted burners are not used in practice is probably that they would make the flamers even more expensive. When the flamer was steady, however, a burner angle of 67° caused considerable flame deflection upwards. A flamer with a very high propane input of 62 kg h-1 m-1 (800 kw m-1) showed lower energy efficiency than a flamer with a propane input of 34 kg h-1 m-1 (430 kw m-1) (Paper V). Storeheier (1991, 1994), however, found poorer performance of higher burner power, in terms of temperature sum per unit propane, in particular for a cover height below 10 cm. One flamer was relatively more effective on smaller plants, whereas another flamer was better on larger plants. That data on lethal leaf temperatures during flame treatment are few and inconclusive is probably a result of these factors.
For example, for a dynamic open flamer in Paper III, a temperature peak of 960°C was recorded when the rear part of the flame passed the thermocouple (Fig.
However, for the reduction in the number of larger plants, the correlation was generally higher when the base temperature 400°C was used. It is therefore impossible to define a general temperature or temperature sum to be reached in order to achieve sufficient weed control. 2, which acts largely by infrared radiation, gave a lower temperature sum per unit propane than other flamers at a height of 1 cm (Fig. Several measurement errors may also be involved in this type of measurements (Doebelin, 1990). In potato production, a broadcast application of flaming requires about ten times more energy than do mechanical and chemical methods for weed control (Fykse, 1985a) and haulm killing (Jolliet, 1993). Flaming is then often applied once only and further weed control is carried out by mechanical and manual methods.
Annexin-V was localized in cells bearing GFP-LC3 punctuated spots, indicating autophagy in immunofluorescence.
A cluster of 50 cells or more was scored as a colony and the surviving fraction was calculated in relation to the un-irradiated control.
The percentage of cells undergoing autophagy was calculated from the ratio of autophagic cells to normal cells bearing GFP-LC3 fluorescence. After adding one drop of 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) cotaining mounting solution, cells were analyzed by fluorescence microscopy.
Fluorescence and differential interference contrast images were obtained every 30 minutes from 3 to 5 days after irradiation. Taken together, these finding suggest that radiation-induced autophagy is proportional to cell death after irradiation at lethal doses.
However, the knockdown of Beclin1 and Vps34 did not inhibit radiation-induced autophagy significantly (Appendix 3B).
This long-term observation of irradiated cells revealed that multi-nucleated cells (aneuploidy), which had shown no discrete LC3 dots but cleaved PARP staining in the previous section, showed a punctuated pattern of GFP-LC3 in the early stages of their aneuploidy or when they attempted mitosis (Appendix 4).
Although PARP is a substrate of caspase-3, PARP cleavage, which we observed as a late apoptosis marker after irradiation, could occur from cellular damage via either apoptosis or non-apoptotic cell death [21].
However, it is still not clear if this autophagy-inhibition related reduction of apoptosis is autophagy-dependent or the direct effect of Atg5 knockdown. These experiments indicate that autophagy is a cell death pathway after exposure to therapeutic intervention including radiation, especially under apoptosis-defective conditions.
However, it was present at 3 days after irradiation and its expression increased for up to 7 days. For example, flaming has proven to be effective in controlling the pesticide resistant potato Colorado beetle (Moyer, 1991; South, 1993).
Contrary to a commonly held belief, however, fuel costs are generally not the most expensive part of the total cost of flame weeding.
However, the effects are usually evaluated as the relative difference between individual treatments, rather than estimating the effective dose and ground speed required to obtain good weed control. On the other hand, there is no simple way of just replacing one burner type with another on an existing covered flamer, as the whole flamer may then have to be re-designed and re-optimized in terms of burner position, cover height etc.
However, as the aim of this work was to study some principal issues, and to develop experimental methods, rather than give specific recommendations to users on how to use a certain machine, this lack of repetition was considered acceptable.
The ground speeds were determined by repeated measurements of the time taken by the tractor to travel a certain distance.
One objection to altering the fuel pressure is that this also changes the propane combustion, the flame length and the resulting penetration of the flame. Based on this relationship, it can be shown that the lethal temperature sum will also be lower as the temperature increases, which was verified by Storeheier (1991, 1994). Moreover, the dose level chosen for such vertical assessment will greatly affect the size of any difference. A related concept with intensity-response curves for mechanical weed control has been developed by Rasmussen (1991). At later stages, these species could not be controlled with one treatment, whatever the dose, because of their regrowth capacity. Moreover, the larger amount of reserve food in the roots increases the plant's ability to regrow. The other type uses an infrared (IR) radiant gas burner, that operates at red brightness temperatures of about 900°C with essentially no visible flame on the combustion surface. However, burners with a short flame require a lower burner height if the hot part of the flame is to reach the ground.
In practice, change of nozzle size, usually by change of burner, has the greatest influence on the burner power. These results agree with thermodynamic modelling by Bertram (1991), which indicates that for a given type of flamer, there is an optimum fuel input.
European commercial flamers usually have a fuel input between 10 to 25 kg h-1 m-1, which allows effective ground speeds in the range of only i to 4 km h-1 (Ascard, 1988, Hoffmann, 1989). At this speed, an individual cotton plant is exposed to the high temperature for about a tenth of a second. D'Hulster (1985) reports that the effective ground speed was on average 4 km h-1 with tandem burners as compared to 3 km h-1 with single burners. The difference between the flamers cannot be attributed to the cover only, as the burners and burner arrangements were also different, but the results still indicate opportunities to lower the energy requirement by improving the design of a flamer.
Storeheier (1991) used a similar method for flame treatment, in which plants were moved at different speeds through a stationary flame. Moreover, the laboratory conditions in the rail track with no plants, no wind and a smooth surface are very different from the field conditions.
Such calculations should, however, not be generalized as the energy input will vary largely both within and between crops, depending on, for example, herbicide and application method (Pimentel, 1992). Some of these punctuated GFP-LC3 bearing cells formed conglomerated spots and died in final phase.
For analysis of the fate ofautophagic cells, cells were analyzed if they showed punctuated GFP-LC3 3 days after irradiation.
Statistical significance was considered to have been reached if the p-value was less than 0.05.
Subsequent experiments measuring both autophagy and apoptosis at subconfluent cell culture density showed no remarkable change compared to the control at 5 Gy. The subG1 population, which was not observed at 1 day after irradiation, became apparent at 3 days after irradiation. 3B) and did not show a dose-dependent response to treatment with up to 30 Gy, whereas staurosporine (500 nM-1 I?M) induced a decrease in caspase-3 (Appendix 2). The observation of GFP-LC3 granule bearing cells suggests that irradiated cells are commonly present with indicators of autophagy and that some of these autophagic cells eventually die via either membrane disruption or post-mitotic catastrophe. These stressed cells activated autophagy to recycle proteins in response to increasing demand for energy. Flaming has also been tested for partial soil disinfection, where very high flaming intensities are used and the upper soil layer is lifted up and dropped through a flame curtain to expose the soil particles to the heat (Kraus, 1973; Hoffmann, 1989). Several attempts have been made to evaluate flamers based on temperature measurements, although the correlation between these measurements and the weed control is poorly understood. As a result, the energy efficiency of the flamer may vary when the fuel pressure varies (Vester, 1990; Paper IV). This can be explained by the higher heat transfer rate to the plants at higher temperatures (e.g. To prevent regrowth from old plants, the flame must penetrate the leaf canopy and also reach the axillary buds at the lower nodes, which may be protected by surrounding leaves, leaf sheets and petioles. These burners heat ceramic and or metal surfaces which then radiate heat towards the target.
The upper level is probably influenced by several factors including burner type and design of the flame cover. However an experimental flamer built by Vester (1987b, 1988, 1990) gave an effective ground speed of 9 km h-1 at a burner power of 45 kg h-1 m-1. However, when speeds were reduced to 3.2 km h-1, serious damage to plants occurred at all fuel rates. Loewer & Mayeux (1970) concluded that tandem burners were superior to a single burner, in terms of the percentage of dead seeds at similar ground speed. Thus, evaluating flamers based on temperature measurements is a questionable method when knowledge about the relationship between temperatures and weed control is lacking. They suggested that continued high level autophagic response may lead to cell death, but they evaluated neither the dose-response relationship nor cross-talk between autophagy and apoptosis [10].
Cells were seeded in 96-well plates at 1,500 cells per well and allowed to attach overnight before treatment with radiation. These results suggest that radiationinduced apoptosis measured by annexin-V is independent of caspase activation.
Independent of its function in autophagy, Atg5 can be cleaved following death stimuli, and the cleaved product appears to promote mitochondria-mediated apoptosis [23]. An increase in the lower band accompanied by an increase in the ratio of the lower to the upper band is indicative of autophagy. However, the thinner thermocouples were very fragile and broke easily, especially when the stronger flamers were used at low ground speeds.
The first real attempt to establish dose-response curves for flaming was made by Vester (1990), who over a limited dose range fitted a straight line between the logarithm of the plant response and the propane dose per unit area.
Fenwick & Lien (1968) also showed that flaming may either increase or decrease germination of different species, and suggested that flaming may have increased germination by breaking the dormancy of seeds on the soil surface. When flamed at moderate doses, the plant parts above the ground will only partly desiccate and the sensitive parts may be only partly damaged.
This kind of IR radiator was tested for thermal defoliation of cotton in the USA during the 1960s (e.g.
Thus, major changes to the fuel input of a flamer may have to be followed by change of burner type as well as modifications of the burner setting and cover design (Storeheier, 1991, 1994). The great importance of the exposure time for the cotton stem can be explained by a low heat transfer coefficient for this relatively thick and corky cotton stem. However, none of the five above mentioned studies showed whether two-stage flaming was more or less fuel efficient than ordinary one-stage flaming. In contrast to this concept of autophagy-mediated cell death, some researchers showed that inhibition of autophagy could sensitize glioma cells to radiation [11].
Therefore, knockdown of the pro-apoptotic molecule Atg5 could cause the inhibition of apoptosis directly. Flamers with covered burners were generally more effective than an open flamer, especially on larger plants and tolerant species. In addition, the thermal parameters obtained from the 0.13 mm thermocouples did not give higher correlation with the weed control than those from the thicker ones. Contrary to the findings mentioned above, Hoffmann (1990) argues that a certain amount of heat must act on the leaf surface, and that it does not matter whether this amount of heat is achieved by a high temperature during a relatively short time or by a lower temperature during a longer time. The ability of the damaged plants to regrow will then depend on the energy reserves of the plants, environmental conditions such as soil moisture, and competition from neighbouring plants.
Thus, temperatures recorded by the thermocouples represent neither the temperature in the plant nor in the air but in the thermocouple itself. It is generally accepted that autophagy-mediated cell death occurs in proportion to the degree of intracellular damage, and that autophagy occurs more under apoptosis-defective conditions [12,13]. The fixed cells were then washed with distilled water five times and air-dried, after which the dried cells were stained with 0.4% SRB in 1% acetic acid solution for 30 minutes, washed with 1% acetic acid solution and again air-dried.
Apoptosis was then observed and compared to that of irradiated parent cells without the inhibitor at 5 days after the irradiation. The effective ground speed was generally higher for flamers with a higher fuel input, but only up to a certain level.
Thus, when an open burner is used perpendicular to the row, the higher energy input cannot be utilized as it is in the "oven" under a covered flamer. Thin thermocouples will, however, measure temperatures from a dynamic flame that are reasonably close to the air temperature and thereby related to the convective heat transfer rate to the plants. Thus, we could postulate that autophagy after irradiation plays different roles according to the dose of radiation and propensity of cells to undergo apoptosis after lethal damage. Stained SRB, which is proportional to cellular protein mass, was resolved with 10 mM Tris-base solution (pH 10.5) and its absorption was measured at 510 nm.
Cells bearing GFP-LC3 puncta, which were quantified and averaged from 10 high-power fields, showed a dose-dependent increase at 3 days after irradiation (D) and an increase from 3 to 5 days after irradiation at 10 Gy (E). A flamer with a relatively high propane consumption of 34 kg h-1 per metre working width (440 kw m-1) allowed an effective ground speed of 8 km h-1 when smaller plants were treated, whereas the effective ground speed was 2.6 km h-1 for a flamer with the more usual burner power of 12 kg h-1 m-1 (150 kw m-1).
Truncated Atg5 (24-kDa) translocated from the cytosol to mitochondria, triggering cytochrome c release and caspase activation.
Temperatures from the flamers were measured above the ground under weed-free conditions in the laboratory. Several investigations and practical experience indicate that pure infrared radiation is inferior to flaming in terms of weed control, energy efficiency, capacity and costs (e.g. However, the role of Atg5 provoking apoptotic cell death according to the different kinds of death stimuli remains elusive [12]. These results are in accordance with relative radio-sensitivity of LN229 compared to U87 and U373 at 5 Gy. For example, the performance of IR and flame weeders seems to interact with the plant species. Parish (1989a) achieved similar control of Sinapis alba with electrical infrared emitters and propane flamers at similar energy inputs, but the energy requirement to control ryegrass (Lolium italicum) was considerably higher for IR radiation than for the flamer.

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