When the left and right eyes are simultaneously presented with different images, observers typically report exclusive awareness of only one image. This phenomenon is termed binocular rivalry, reflecting the fact that the dominant image alternates every few seconds in a cycle of perceptual competition that continues indefinitely. Despite the apparent continuity in perceptual switching, we now demonstrate that the initial “onset” period is fundamentally different to all subsequent rivalry epochs. Using brief intermittent presentations, rivalry dominance shows strong biases such that the same target is perceived with each successive stimulus onset. These biases remain consistent within any given location, but vary across the visual field in a distribution that is stable over multiple weeks but highly idiosyncratic across observers. If the presentation exceeds ∼1sec at any location, however, the very different and much more balanced alternations of sustained binocular rivalry become apparent. These powerful onset biases are observed with brief intermittent presentations at a single location or with continual smooth motion of the targets. Periods of adaptation to one of the rivaling targets induced local switches in dominance to the non-adapted target. However, these effects were generally limited to the spatial site of adaptation and had less influence over each subsequent cycle of the target. We conclude that onset rivalry is independent of sustained rivalry and cannot be explained by local regions of monocular dominance or memory of past perceptual history, but rather reflects low-level, spatially localized factors that are stable over periods of weeks. These findings suggest that brief presentation paradigms are inappropriate for their current use in studies of the mechanisms underlying sustained rivalry. However, brief presentations are ideal for investigating early stages of perceptual competition. What happens when the brain is presented with ambiguous visual input? For the most part, the visual system tends to “choose” one valid interpretation at the expense of the alternatives. This winner-takes-all strategy is illustrated by popular illusions such as the Necker cube and binocular rivalry. In the case of binocular rivalry, two different images are presented simultaneously to the corresponding location in each of the two eyes. Under these conditions, the observer will be more likely to see one image rather than a superposition of the two. After a few seconds, the previously suppressed image will become dominant and then continue to cycle between suppression and dominance in a quasi-regular fashion Due to the dramatic switches in perception between complete dominance and total suppression of an image, binocular rivalry has attracted considerable attention. Most notably it has been used as a tool to investigate the neural correlates of conscious experience During the period of “normal” sustained rivalry the frequency of rivalry transitions can be manipulated through changes to stimulus parameters such as contrast, luminance, motion and salience We were interested to investigate whether permanent perceptual biases could ever be observed at rivalry onset. It was reasoned that demonstration of an “onset bias” would require: 1) evidence of a dominant perceptual state-the near exclusive dominance of the same target across each successive stimulus presentation, 2) evidence against perceptual stabilization-any occurrence of a switch in perceptual dominance would have to be temporary, reverting quickly to the originally dominant state . Furthermore, on the rare occasions that the non-dominant target was perceived, the dominance quickly returned to the target with the strongest perceptual bias. However, when the same targets were presented continuously for 60sec these large biases in dominance were no longer seen  . Together they showed that perceptual memory is location specific In line with the foveal results described in experiment 1, during peripheral presentation, rivalry dominance was again reported to have very strong and consistent biases at each location. Surprisingly, however, the bias varied at different locations. For example, one target might dominate seven of the eight locations, but the dominance would be reversed in the eighth location  column 1One possibility was that onset rivalry may depend on the sequence of stimuli rather than their locations such that, for example, dominance begins with one target but switches on the eighth stimulus to the alternative target. To test this, subjects were presented the same rivalry targets used in experiment 2a, however, this time the order of presentation was randomized rather than sequential. Illustrated in column 2 of 2 = 0.25, ß = −0.8; S2: r2 = 0.10, ß = −0.16; S3: r2 = 0.15, ß = 0.07; S4 r2 = 0.42, ß = 0.14, df = 6). Using a two tailed t-test, none of these correlations was found to be significant. In the foveal condition, the correlation approached but did not reach significance . Given the small number of subjects and locations tested, it is difficult to determine the exact relation between the onset and sustained biases. However, given the small slopes of the relation between the two , we conclude that there is a large degree of independence between the dominance properties of rivalry seen at onset and that seen during sustained viewing. More importantly, the lack of any obvious correlation can be taken as further evidence that the “onset” biases observed were not reflecting general inherent biases in monocular dominance.Another potential explanation is that local biases reflect localized regions of monocular dominance. Given that binocular rivalry involves competition between images presented to the two eyes, it is important to rule out the possibility that the local onset biases result from regions of monocular dominance due to some sort of inherent variability in the retina or low-level cortical areas. To test this possibility, subjects from experiment 2a were retested after the eye of presentation was switched for each target . Under these conditions onset rivalry still shows clear location specific biases. The interesting thing to note is that the observed biases are different but not complementary/opposite to those reported under the original conditions  column 3In summary, results from the first 2 experiments show that dominance at rivalry onset is determined by location specific factors that are different from those biasing dominance during sustained rivalry. The biases cannot be explained by local variability in monocular dominance or the build up of rivalrous inhibitory interactions over the target path history.Together the results from experiment 1 and 2 suggest the existence of an onset suppression that is quite distinct from the subsequent state of binocular rivalry. Experiment 3 explored whether similar local biases occur during sustained presentation of a continually moving target. The one study that has previously investigated this question suggests that continuous motion of a rivalry target can slow down and even stabilize rivalry alternations Data from all four subjects showed that the local biases are nonexistent when the targets are moved very slowly around the periphery. This result is consistent with that reported by Blake et al (2003). However, contrary to their claim that no local biases in dominance exist, we show that such biases do exist if the target motion is fast enough  intermittent paradigm of experiment 1 was used. Consistent with experiment 3, the target completed a cycle every 10, 20 and 100sec. Looking at the results from the 100sec condition, it is clear that location specificity is still seen at onset, even at local inter-stimulus intervals of up to 99sec. To further test the influence of perceptual memory and the stability of the onset bias, subjects were also re-tested 1–2 weeks later . The facThe role of stable local factors in onset rivalry is clear, but the question remains whether anything can affect this onset bias. Blake et al., (2003) found that if the targets are made to move over an area that had previously been adapting to one of the rivaling target, it was possible to induce a switch favoring the non-adapted target. In experiment 5, the aim was to determine if it was similarly possible to induce a switch in perceptual dominance when the targets were moving fast enough to show the onset effect (10sec cycles). Two subjects from experiment 3 participated in this final condition. Each trial began with 60sec of adaptation to a stationary version of the non-dominant target (dominance was based on subjects reports in experiment 3–red gratings in both cases). In all other aspects the adaptor and target were identical. At the end of the adaptation period the adaptor was extinguished and the target was presented. The target always appeared in the location 45° preceding the place of adaptation and began moving immediately along the same circular trajectory used in experiment 3 . Subjects were asked to fixate for the entire duration of the trial and to report the perceptual dominance during the 2min binocular rivalry portion of the trial. Each subject participated in four trials with the adaptor being located in a different location for each trial . As shown in This study aimed to demonstrate a single point–onset rivalry represents an early phase of perceptual competition that is distinct from sustained binocular rivalry. It is true that onset and sustained rivalry are both characterized by the exclusive dominance of one rivalry target at the expense of the alternative, temporarily suppressed, image. In this respect, the two states are phenomenologically identical. However, onset and sustained rivalry appear to be quite different in regards to the factors biasing dominance and may be driven, at least partially, by different underlying mechanisms.In experiments 1 and 2, a brief (1sec) rivalry target was presented at 10sec intervals either in the fovea or in one of eight locations around the periphery. In all cases the dominance during the intermittent presentation was uncorrelated with the dominance during sustained rivalry. The most striking observation from these initial experiments was the location specificity and the magnitude of bias during the intermittent presentation. This location specificity could not be explained by regions of monocular dominance (eye-of-origin factors) or path history . In experiment 3 the location specificity remained even when the targets were moved smoothly around the visual periphery. Interestingly, this location specificity broke down when the motion speed was reduced to speeds of 3.6°/sec, suggesting that the onset bias is sensitive to the presentation duration at any location and not “onset” per se. Experiment 4 used the intermittent (1sec) presentation to ensure that the lack of location specificity at slower speeds was, in fact, due to the reduction in speed and not the result of the increased intervals between subsequent target cycles. Using this paradigm, the strong local biases reappeared even when the inter-stimulus intervals were extended to 100sec (matching the slow smooth motion condition in experiment 3). Finally, we showed that periods of adaptation to one of the rivaling targets could induce local switches in dominance to the non-adapted target. However, these effects were generally limited to the spatial site of adaptation and had less influence over each subsequent cycle of the target . Together these results suggest that binocular rivalry at onset is a distinct phenomenon that is influenced by local factors independent of presentation eye and path history. Importantly, these local biases were found to be stable across periods ranging from 10sec to 2 weeks. The localized short term nature of the adaptation results suggests that, while the biases can be temporarily altered, the main factors appear to be stable local endogenous cues. “Perceptual memory” and other direct manipulations of the stimulus may (and likely do) impose some influence on the generation of onset dominance, however, these effects appear to play a lesser role.The current results are particularly relevant to three recent studies that all reported perceptual stabilization with presentation times ranging from 0.5-1.2sec separated by offset durations of between 2-5sec Here we are claiming that onset rivalry is a distinct form of perceptual competition. Onset and sustained rivalry may involve identical mechanisms that differ only in their relative role in biasing dominance or their sensitivity to factors such as attention shown recentlyThe fact that we do not get an inversion of results when the eyes are swapped suggests that the bias is not simply one of local regions of ocular dominance or retinal variations in color sensitivity. However, the location specificity observed suggests that the biases are likely to occur at a level at which retinotopic areas are clearly defined and stable over weeks, which is likely to be at a level “lower” than the frontal parietal networks that have been implicated in some rivalry models. It is worth noting that the location specificity observed in the onset biases did not show any systematic distribution across the visual hemi-fields. This result differs from the clear hemi-field asymmetries in switch rate and dominance durations found in normals From the current set of experiments it is clear that binocular rivalry dominance is initially determined by low-level, spatially localized factors that are stable over periods of weeks. However, further experiments are needed to pinpoint, more exactly, the structures and mechanisms involved in onset rivalry. The implications of the proposed existence of onset rivalry raises questions about intermediate processes like flash suppression A total of 13 subjects participated in these experiments  aged between 20 and 33 participated in this study. An additional 4 subjects recruited for the study, were unable to participated because of their inability to experience definable perceptual dominance . All data collected from the 13 eventual participants is presented.Apart from one of the authors (OC), all subjects were trained psychophysical observers but naïve to the aims of the experiment. All subjects reported normal or corrected to normal vision. These experiments were undertaken with the understanding and written consent of each subject, the review of the Federally mandated Harvard University Committee on the Use of Human Subjects in Research, and in conformation with The Code of Ethics of the World Medical Association (Declaration of Helsinki).™TM software . The stimulus background consisted of a uniform gray background (35cd/m2). During the foveal presentation used in experiment 1, the patches were either red  and black or green  and black gratings of 100% contrast  monitor viewed through a mirror stereoscope. Stimulus were programmed and presented using VisionShellrast see .Responses were reported by key press on a standard computer keyboard with depression of the key signaling the dominance of the corresponding target. During intermittent conditions, subjects were asked to report the dominant percept after each 1sec presentation. In the case of sustained stimulus presentation, either in a single location for 60sec or during the smooth target motion, subjects were instructed to continually report the relative dominance of each of the two percepts. Subjects were instructed to focus on the target color and they were not given the opportunity to report mixed dominance states but were forced to indicate the dominance of one of the two targets (red or green–see below for more details).Eye of presentation was counter balanced for color and orientation across subjects but was kept constant within each subject. The experimental order and motion direction (clockwise and counter-clockwise) was also counterbalanced. Every trial was repeated once before the subsequent condition was tested. However, due to space limitation, here we present only responses for the initial trials.Because one aim of the study was to consider the role perceptual memory or adaptation, we did not allow subjects to have a practice. Instead, responses were recorded from the initial stimulus presentation. After the completion of this initial trial , subjects were questioned in detail about their perceptual experience and their understanding of task. The majority of participants reported that the dominance during each presentation was very clear with either total or near-total dominance through the trial. The strength of dominance experienced was more in line with that achieved during flash suppression rather than the dynamic mixed states that are often experienced during rivalry transitions. A minority of 4 participants, however, reported experiencing a mixed state in which the relative dominance of the two targets ranged from 50:50 to approximately 30:70 on each presentation. These individuals described it as an indecipherable mixed state that was the dominant percept at nearly all locations for the entire trial. In an attempt to induce reportable dominance, people in this final group were presented with a variety of different stimulus conditions involving changes in relative stimulus strength and eye of presentation, multiple readjustments of the mirror stereoscope were also made. In all cases, these subjects were rejected from further part in the study after approximately 20minutes of unsuccessfully attempting to induce reportable dominance of either of the two stimuli. Because these 4 participants were unable to complete any of the experimental conditions they were not included in the participant description above.Saturation of both targets was always kept at 100% for experiment 1. However, some minor changes to the target saturations were needed for some participants in the periphery intermittent presentation experiments (2&4). After the initial presentation of the experimental trial the phenomenological reports fell into two types 1) clear dominance during each one second presentation but alternating dominance between location–some in this final group spontaneously reported the location specificity of the relative dominance of the two rivaling colors , 2) exclusive dominance of one of two colors in every location on multiple cycles through the 8 locations. In this second case, the total dominance of one target for an entire trial was considered uninformative in respect to questions of both perceptual memory and location specificity. Therefore, to bring the stimulus closer to a point of equivalence, the saturation of the dominant target was successively reduced (e.g. the red of the red black patch was desaturated progressively toward gray) until the subject verbally reported experiencing dominance of the non-dominant target in at least one or two locations during each cycle of the stimulus (∼20% dominance for the non-predominant target). This saturation value was then used for all subsequent experiments that the individual participated in.It should be noted that, despite the clear dominance, some participants reported a small but perceptual crescent of the non-dominant target around the target periphery. However, because the crescent was small and consistently present, participants were able to ignore it and focus on the central portion of the target while determining their responses.


Arabidopsis seedlings in a 96-well format and rapidly collecting tissue for subsequent DNA extraction and genotyping. While the originally described Ice-Cap method is an effective tool for high-throughput genotyping, one shortcoming of the first version of Ice-Cap is that optimal seedling growth is highly dependent on specific environmental conditions. Here we describe several technical improvements to the Ice-Cap method that make it much more robust and provide a detailed protocol for implementing the method.We previously developed a high-throughput system called 'Ice-Cap' for growing The key innovation underlying Ice-Cap 2.0 is the development of a continuous watering system. The addition of the watering system allows the seedling growth plates to be incubated without a lid for the duration of the growth period, which in turn allows for much more uniform and robust seedling growth than was observed using the original method. We also determined that inserting wooden skewers between the upper and lower plates prior to tissue harvest made it easier to separate the plates following freezing. Seedlings grown using the Ice-Cap 2.0 method remain viable in the Ice-Cap plates twice as long as seedlings grown using the original method.The continuous watering system that we have developed provides an effective solution to the problem of sub-optimal seedling growth that can be encountered when using the originally described Ice-Cap system. This novel watering system and several additional modifications to the Ice-Cap procedure have improved the robustness and utility of the method. Plant scientists increasingly need to genotype large numbers of individual plants as a part of their research programmes. For example, when performing map-based cloning to locate the genetic lesion responsible for a specific phenotype, it is often necessary to genotype thousands of individual plants from segregating populations. For crop species, the process of marker-assisted selection can involve genotyping several thousand individuals in a large population. To optimize the efficiency with which these types of experiments can be performed, it is necessary to develop a streamlined plant genotyping pipeline. We previously described a method named "Ice-Cap" to address these needs . Ice-CapMurashige and Skoog (MS) basal salt mixture e.g. Sigma-Aldrich, St. Louis, .4-Morpholinoethanesulfonic acid (MES) e.g. Sigma-Aldrich, .Plant cell culture tested agar e.g. Sigma-Aldrich .Sterile pipette tips, 20–200 μL size.Multi-channel pipette trough e.g. Fisher Scientific 50 ml pipette basin .Surgical tape e.g. 3 M Micropore surgical tape .96-well deep well plates (referred to as 'upper seedling plates' in the Ice-Cap protocol) e.g. Fisher Scientific Nunc brand, 1-mL filter plates without frit . These plates are manufactured with a hole in the bottom of each well to allow roots to grow down into lower root plates.96-well PCR plates (referred to as 'lower root plates' in the Ice-Cap protocol) e.g. Fisher thin-wall PCR plates with raised rims . These PCR plates have a raised rim to fit securely onto the upper seedling plates.Clear plastic lids e.g. lids taken from Falcon Microtest flat bottom 96-well polystyrene plates . Lids must be correct size to fit onto tops of upper seedling plates.Metal ball bearings e.g. 3/32-inch diameter stainless steel balls .Adhesive sealing film e.g. Fisher Scientific .Elastic bands e.g. 7.5 cm (3") elastic hair bands purchased from grocery store.4 mm diameter wooden skewers. Purchased from a grocery store. These skewers are the type that is typically used to prepare kabobs.Dry ice.95% (v/v) ethanolThermal adhesion foil e.g. Easy Peel heat sealing foil A pH meterMulti-channel pipettors with volume ranges from 20 μL to 180 μLArabidopsis seed-loading device A 96-well Large, shallow metal pan with a rim e.g. 43.8 cm × 30.8 cm × 2.5 cm (17.25" × 12.15" × 1") cookie sheet. We use a rimmed metal cookie sheet , available from a cooking supply store. The pan should be just deep enough so that when the pan is filled with water the water will just cover the tops of wells of the lower root plate without submerging upper seedling plate. To achieve the desired water depth it may be necessary to place a thin sheet of glass on the bottom of the cookie sheet.A small submersible water pump e.g. small tabletop fountain pump , available from a garden supply store.A 26-L polypropylene storage box e.g. 53.3 cm × 38.1 cm × 14 cm (21" × 15" × 5.5")  or other similar watertight container. Container must be longer and wider than the metal pan to allow water to overflow from the pan down into the container. Container must be deep enough to completely submerge fountain pump.A metal rack. We placed the metal pan on a custom-made rack constructed of 2.5 cm (1") metal bars with adjustable screws for levelling the metal pan. This metal rack was held above the bottom of the 26-L storage box by the plastic ledges present in the sides of the storage box.Some 12.7 mm (1/2") diameter plastic tubing or size appropriate for the fountain pump.Small water-resistant clamps which are used to secure the plastic tubing to the side of the metal pan.A hand-held microseal roller e.g. Roller for Microseal Film MJ Research, Inc. .A 1–2 cm wide soft bristle artist's paintbrush, purchased from a craft store.A manual heat-sealer e.g. Thermo-Sealer from ABgene USA .A 96-well metal thermal block e.g. Cole-Parmer 96-well microplate heating block module .A glass dish e.g. Pyrex 2-L baking dish, 11 cm × 7.5 cm × 4 cm (11" × 7.5" × 1.75"), purchased at a cooking supply store. Dish is used for dry ice/ethanol bath and must be freezing tolerant.A Genogrinder or paint shaker modified to hold 96-well plates e.g. 2000 Geno/Grinder machine .A centrifuge equipped with microplate carriers e.g. Beckman Coulter Allegra™ 25R Centrifuge equipped with S5700 rotor and microplate swing buckets.Some 1 cm diameter rubber tubing or appropriate size to attach to compressed air supply for use in recovering individual seedlings from Ice-Cap plates after genotyping has been completed.The key innovation in Ice-Cap 2.0 is a novel automatic watering system Fig. . BrieflyAs described above, the addition of this type of metal tray alone does not solve the problem of keeping the lower plate constantly hydrated because the water level in this tray would rapidly drop over the course of a single day due to evaporation. To address this problem we suspended the pan above a larger reservoir filled with several liters of water. Adjustable screws allow the upper pan to be leveled to achieve uniform water depth. A submerged pump constantly pumps water from the high-capacity lower reservoir to the upper pan. The continual overflow from the upper pan spills back into the lower reservoir and ensures that the water level in the upper pan is constantly at the correct level to keep the lower root plates filled with water while avoiding submerging the seedlings. Water is added to the lower reservoir as needed so that the level does not drop below that of the recirculating pump, typically once per week. The system is large enough to accommodate eight Ice-Cap plates in a single fountain, allowing 768 seedlings to be grown in a 60 cm by 40 cm space.Arabidopsis thaliana or rice seedlings are grown in a 96-well format by placing a single seed in each well of a 96-well upper seedling plate that contains nutrient agar. As the seedlings develop, the roots penetrate the agar and grow down through a hole in the bottom of the upper seeding plate into a lower 96-well plate containing water and a metal ball. The upper and lower plates are held together by elastic bands during the growth period. After ca. two weeks of growth the roots reach the bottom of the lower root plate. Tissue harvest is then performed by transferring the stacked plates to a dry ice/ethanol bath to flash freeze the water in the lower root plate. The resulting ice captures the root tissue, but does not freeze the seedlings in the upper plate. The upper and lower plates are then manually separated, yielding an upper plate containing 96 viable seedlings that can be transplanted to soil and a lower plate containing 96 root tissue samples. Genomic DNA is extracted from the root tissue by adding extraction buffer to the lower root plate, sealing it with foil, shaking it to pulverize the root tissue and centrifuging to pellet the cell debris. The resulting aqueous solution contains genomic DNA of suitable purity to be added directly to a PCR reaction for genotype analysis. When Arabidopsis seedlings are processed using this protocol the average yield of genomic DNA is 400 ng per sample, which is sufficient to perform hundreds of PCR reactions [In the original Ice-Cap system , Arabidoeactions .One shortcoming of the original Ice-Cap system was that seedling growth was sensitive to subtle changes in environmental conditions. The humidity level and light intensity of the growth chamber affected the performance of the original Ice-Cap system such that optimal growth could only be achieved within a narrow window of conditions. If the humidity was too low, the water level in the lower root plate dropped quickly and exposed the roots of the growing seedlings. If the water level dropped below the tip of the root, the seedling would die. Manually adding water to the lower root plate is not an option because the process of separating the upper and lower plates to add water would damage the roots and risk cross-contamination between adjacent wells.To address this problem, we developed an automatic watering system that maintains a constant water level in the lower root plate for the duration of the growth period. Here we present a protocol detailing this novel watering system as well as other improvements to the Ice-Cap system.NOTE: We found that a mixture of one part tap water to three parts distilled water provides better growth performance than pure distilled water. The reservoir is typically filled with a total volume of 20 L of water. Subsequent re-filling of the reservoir to replenish water lost to evaporation should be performed using pure distilled water.1. Place the small submersible pump in the bottom of the large plastic reservoir. Place the metal support rack in the reservoir container. Place the metal tray on top of rack and level by adjusting the levelling nuts on the support rack. Clamp the outflow hose from the pump onto the rim of the metal pan and fill the reservoir with water. NOTE: If the water level is too deep then it will be necessary to place a sheet of glass on the bottom of the metal tray. Choose a sheet of glass of a thickness that brings the lower root plate up to the desired depth relative to the water level.2. Turn on the fountain pump and allow the metal tray to fill with water. Check the water level in the metal tray as follows. Fill each well in a 96-well lower root plate to capacity with water using a multichannel pipette. Place an empty 96-well upper seedling plate on top of the lower root plate and place the stacked plates in the water filled tray. Visually inspect the plates to make sure that the water level is just above the rims of the wells of the lower root plate. We experimented with several different volumes of growth medium for the upper seedling plate and determined that seedlings grown in 525 μL of agar medium were larger, greener and grew more quickly than seedlings grown in the 125 μL of medium described in the original version of Ice-Cap (data not shown).1. Prepare sterile agar growth medium composed of 0.5× Murashige and Skoog basal salt mixture, 2 mM MES, 0.6% agar, pH 5.7. Place the autoclaved medium in an incubator set at 65°C, which will prevent the medium from solidifying. (One liter of medium is sufficient for the preparation of 13 Ice-Cap seedling growth plates).NOTE: It is very important that the 96-well plates are completely dry and at room temperature before applying the sealing film, otherwise leakage will result when the agar medium is added in the next step.2. Perform the following operation in a sterile laminar flow hood. Seal the bottom holes of autoclave-sterilized 96-well upper seedling plates with removable adhesive sealing film using the hand roller. 3. While the growth medium is still molten, use a multi-channel pipette to dispense 525 μL of growth medium into the bottom of each well. (Molten medium can be poured into disposable plastic troughs to facilitate pipetting). Leave the 96-well plates in the sterile hood while the medium solidifies. Once the medium has solidified and cooled to room temperature, cover the plates with a clear plastic lid, wrap in Al foil and store at 4°C for future use.Arabidopsis seeds by soaking them in 95% (v/v) ethanol for 5 min, remove excess ethanol and allow them to air dry. NOTE: It is very important that the seeds are free from any small particles of soil, plant tissue, or other debris that could fall into the holes in the seed loader.1. Surface sterilize quickly invert the plate and loader to allow the seeds to fall into the wells in the upper seeding plate. Firmly tap the back of the metal plate to dislodge any seeds that may be stuck in the seed loader. Remove the seed loader from the top of the seedling growth plate. There should now be a single seed on the surface of the agar in each well of the upper seedling plate.2. Use the 96-well seed-loading device to place one seed on the surface of the agar in each well. This is done by sprinkling the surface of the seed loader with several hundred seeds and using a soft bristle paintbrush to spread seeds evenly across the surface of the loader. A single seed should fall into each well in the seed loader. Use the paintbrush to carefully brush off excess seeds so that there is a single seed in each hole and no loose seeds on the surface of the seed loader. Place an upper seedling plate upside down on the seed loader and use the metal notches on the seed loading device to position the plate so that each well in the seedling growth plate is aligned over a single seed in the seed loader. Firmly hold the upper seedling plate against the seed loader and NOTE: Seeds can also be plated individually. For example, we found that individual seeds can be transferred using a small (20–200 μL) sterile pipette tip attached to the end of a disposable 2 mL glass pipette . The end of the pipette tip is first moistened by dabbing the agar in the upper seedling plate and pick up a single seed. The glass pipette is used as a handle to allow you to gently place a single seed on the surface of the agar in each well of the upper seedling plate.3. Place a clear plastic lid on each plate and seal the gap between the lid and the upper seedling plate with surgical tape.4. Wrap the plates in foil and place them at 4°C for 2–7 d to stratify the seeds. This treatment improves germination rate and synchrony.In the original Ice-Cap method the clear plastic lids were left on the upper seedling plates for the duration of the growth period. We have now determined that removing the clear plastic lids from the upper seedling plates after 2 d of growth and leaving them off for the remainder of the growth period allows for much more vigorous seedling growth .2. After 2–3 d incubation under constant light, remove the lids from the upper seedling plates and place the upper seedling plates on top of lower root plates as follows. To begin with, prepare the lower root plates by placing a single metal ball bearing into each well of the lower root plates. A simple home-made device can be used to dispense single metal balls into the lower root plates  ethanol mixture. Place the metal thermal block in freeze-resistant glass dish. Place dry ice in dish around the metal thermal block. For a 2-L (28 cm × 18 cm × 4 cm) dish we use about 1 kg dry ice. Carefully pour 95% (v/v) ethanol into the glass dish until the level of the dry ice and ethanol is just below the surface of the metal thermal block. Allow the metal thermal block to chill for about 20 min to equilibrate.NOTE: The best way to separate the two plates is to peel the upper seedling plate away from the lower root plate in the same manner you would peel the backing off a sheet of adhesive film. During this process the roots that are frozen in the lower root plate break off at the level of the ice, leaving a sample of frozen root tissue in the lower plate and a viable seedling in the upper plate. Leaving the skewers in place during the freezing of the lower root plate facilitates the separation of the upper and lower plates after the water in the lower root plate had frozen solid. NOTE: Following tissue harvest, the upper seedling plates should be transferred to fresh lower root plates filled with water. The stacked plates can then be returned to the continuous watering system to allow the seedlings to continue growing (see below).3. Place the wells of the lower 96-well root plate into the holes in the thermal block with the upper seedling plate still held in place by the elastic bands and the skewers still in place between the plates. Leave the stacked plates on the thermal block for 5 min, during which time the water in the lower root plate will freeze solid. Take the stacked plates from the thermal block, remove the elastic bands, and remove the skewers from between the plates. Carefully separate the upper and lower plates. 4. Allow the water in the lower root plates to thaw at room temperature. Add 25 μL of Tris-EDTA buffer  to each well. Seal the lower root plates using thermal adhesion foil and a manual heat sealer.5. Load the sealed plates containing water, root tissue, Tris-EDTA buffer and the metal ball bearings into the Genogrinder machine. Shake for 5 min at 1,500 strokes/min. Remove plates from the Genogrinder and spin them in a centrifuge equipped with microplate carriers for 10 min at 3,500 rpm at room temperature to pellet cell debris. Carefully peel off the sealing foil and transfer 20 μL of the supernatant to a clean 96-well plate containing 180 μL of distilled water. This diluted sample can be used as template for PCR genotyping reactions and any unused samples can be placed at -20°C for long-term storage.Following tissue harvest, the upper seedling growth plates should be placed in fresh lower root plates and returned to the continuous water system. The seedlings can be maintained in these 96-well seedling growth plates for up to two additional weeks of growth. During this time period genotype analysis can be performed on the DNA samples collected from each plate of seedlings. When individual plants of the desired genotype are identified, those seedlings can be easily transferred out of the 96-well growth plate and into soil using the procedure described below.1. Remove the lower root plate from the upper seedling plate. Any root tissue that has grown down into the new lower root plate must be snipped off to allow the seedling to be easily removed from the upper seedling plate. This second removal of root tissue does not affect seedling survival (data not shown).2. Two different approaches can be used to remove individual seedlings from the seedling growth plate:NOTE: Care must be taken when performing this operation so that the seedling of interest does not inadvertently shoot across the lab. It is advisable to practice this technique using control seedlings before attempting to collect rare individuals with a desired genotype. Transfer the entire agar plug containing the seedling to soil, making no effort to remove the agar from the root system. The agar plug containing roots is buried in the moist soil, leaving the hypocotyl and leaves exposed to the air.Option 1. Place a rubber hose over the opening in the bottom of the well containing the seedling of interest and connect the other end of the hose to a compressed air source. Carefully increase the air pressure until the plug of agar containing the seedling slides out of the well onto the lab bench. NOTE: The blunt end of the skewer usually works better for this than the sharp end. It is also possible to use a combination of the pressurized air and wooden skewer approaches to remove desired seedlings from the seedling growth plates.Option 2. Use a wooden skewer to carefully push the agar plug out the top of the upper seedling plate. 3. Cover the transplanted seedlings with a plastic dome to maintain high humidity for the first 2 d following transfer to soil.Arabidopsis thaliana plants that are segregating T-DNA insertions at multiple loci. This application is one of several possible uses for the Ice-Cap system, which include map-based cloning and screening for rare recombinants between tightly linked mutant loci. We have previously shown that the Ice-Cap method can be used with rice seedlings [The continuous watering system described in this paper provides an effective solution to the problem of sub-optimal seedling growth that can be encountered when using the originally described Ice-Cap system. The continuous watering system and other modifications to the Ice-Cap method have improved the robustness and utility of the system. The optimized protocol provides a cheap, simple and effective method for extracting genomic DNA samples from thousands of viable plant seedlings. We are currently using the Ice-Cap system to analyze the progeny of eedlings  and it iThe author(s) declare that they have no competing interests.KAC participated in the design of the study, tested the continuous watering system and other improvements and drafted the manuscript. PJK conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Vaccines that protect against infection with the types of human papillomavirus (HPV) commonly associated with cervical cancer(HPV 16 and 18) and genital warts (HPV 6 and 11) are expected to become available in the near future. Because HPV vaccines areprophylactic, they must be administered prior to exposure to the virus, ideally during preadolescence or adolescence. The youngage of the target vaccination population means that physicians, parents, and patients will all be involved in the decision-makingprocess. Research has shown that parents and patients are more likely to accept a vaccine if it is efficacious, safe, reasonably priced,and recommended by a physician. Widespread education of physicians, patients, and parents about the risks and consequences ofHPV infection and the benefits of vaccination will be instrumental for fostering vaccine acceptance. Human papillomavirus (HPV) is the most common sexuallytransmitted infection (STI) and a known risk factor for cervicalcancer , 2. HPV The average age of first sexual intercourse inNorth Americais approximately 16 years for both men and women .To provPopulation awareness of the risks associated with acquiring HPV and the consequences associated with infection is low.Numerous studies have demonstrated that most patients areunaware of HPV and its association with genital warts andcervical cancer , 8–14. Vaccines designed to reduce the incidence of HPV infectionare in late stages of clinical development and studies to datehave demonstrated that these vaccines are safe and highly efficacious.Widespread acceptance of HPV vaccines are likelyto lend enormous health benefits by decreasing morbidityand mortality associated with cervical cancer and by reducingthe psychosocial burden of both genital warts and abnormalPapanicolaou (Pap) test results. Savings in health careexpenditures, including treatments for genital warts, preinvasivecervical lesions, and cervical cancer would also be considerable.Historically, however, vaccine availability has not alwaystranslated to widespread use. Underestimates of both thelevel of risk and the severity of HPV-associated disease may become barriers to vaccine acceptance . Hence, n = 20) and adult women (n = 20) attending two urban STI clinics . In. In30]. Educational efforts aimed at health care professionalsshould include physician assistants and nurse practitioners,as well as physicians. Educating patients about HPV andthe HPV vaccine will be time consuming, and the assistancethese groups can provide will prove to be invaluable.Clinicians should provide information in an individualizedfashion, tailoring educational sessions according to thepatient's background, age, and literacy level . An effoProphylactic HPV vaccines may soon become available forpublic distribution; however, general knowledge about HPVand HPV-associated disease is limited and may affect vaccineacceptability. Research suggests that vaccine acceptance willbe maximized by effectively communicating the risks associatedwith HPV infection and the benefits of vaccination. Educationalinitiatives targeted towards patients, parents, andhealth care providers will play key roles in fostering positiveattitudes towards vaccination.


Furthermore, we evaluated the efficiency of the fixative liquid, Easyfix®, to preserve HPV DNA in the collected samples.Although liquid-based cytology (LBC) is now recommended for cervical cancer screening, it requires expensive automated devices and materials. To evaluate the efficiency of inexpensive LBC methods relying on an inexpensive fixative liquid, Easyfix® system and 215 were performed using the Turbitec® system. To control the quality of the preservation liquid, Easyfix®, we correlated the results of HCII assays with those of HPV PCR.266 LBC were compared with 174 colposcopies and 91 Loop Electrosurgical Excision Procedure (LEEP). Among the LBC, 51 were performed using the Papspin® and Turbitec® systems, the sensitivities were respectively 82.6%  and 75.0%  and the specificities were 92.6%  and 96.2% . We find no statistical difference between the results of the both systems (p = ns). The sensitivity of the HCII was 86.4%  and the specificity was 39.4% . The comparison between HCII and HPV-PCR shows a good correlation: the kappa was 0.89.For PapspinLBC performed by cytocentrifugations are inexpensive, reduce inadequate smears, show excellent efficiency and allow HPV detection by molecular biology. For more than ten years, liquid-based cytology (LBC) has been developed for cervical cancer screening. Unlike to Conventional cervical Smears (CS), cells are scattered in a fixative liquid to produce a thin layer of cells on slides. The main advantages of this technique are to reduce the number of inadequate smears and to provide enough cells for the detection of infectious agents such as human papillomavirus (HPV) through molecular biology techniques -3. At th® system , in the CytoSCREEN® system  and in the Turbitec® system . These last two techniques use the Hettich cytocentrifuge .The LBC performed by cytocentrifugations exist since the 1970s and have been developed for the automated reading of cervical cancer cytology. Preliminary studies show that they significantly reduce the cost of LBC -13. In C® and the Turbitec® systems. In addition, we evaluated the efficiency of the fixative liquid, Easyfix®  to preserve HPV DNA.The first purpose of this study was to evaluate the performance of two LBC performed by centrifugations: the PapsinA total of 268 LBC were collected from 177 women (mean of age: 37.3 +/-10.8 years) from January 2002 to December 2004 in a routine gynecologic setting at the Department of Gynecology . Indications for gynecological consultation were based on previous abnormal conventional cervical smears in a population-based screening. According to the histological diagnosis, the age groups were quite homogeneous, except for the women in the CIN3 group who were slightly younger than those of the other groups (Table ® and 144 for Turbitec®) and 92 Loop Electrosurgical Excision Procedure (LEEP) (21 for Papspin® and 71 for Turbitec®) were correlated with all LBC. All the cells were collected using a Cervexbrush® , immersed in 15 ml of a non-buffered alcoholic fixative liquid containing 30% ethanol . 2 LBC corresponding to a cytological and histological diagnosis of cervical adenocarcinoma were discarded. All the patients were informed of the purpose of this study and agreed voluntarily to take part in it.174 colposcopies associated with biopsy  for at least 30 seconds.The EasyfixTurbitec® technique, 4 drops albumin  were put on a polylysined slide placed in the centrifuge chamber (Hettich Centrifuge®). The centrifuge chambers had a final volume of 6 ml; the surface of cells projection on the slide was 240 mm2. The cellularity was estimated using a photoelectric cell analyzer (Labonord) according to the dilution of the Easyfix® sample: 200 μl, 500 μl, 1 ml, 3 ml or 5 ml of Easyfix® solution. This volume was placed in the centrifuge chamber and diluted with an alcoholic fixative liquid containing polyethylene glycol , in order to obtain a finale volume of 6 ml. After 10 minutes centrifugation at 2000 revolutions per minute (rpm), the liquid was discarded.For the Papspin® technique was performed as described previously in the literature with the exception of the use of the Easyfix® and the Cytofix® fixative liquids [2. The volume of dilution was respectively 500 μl, 1 ml and 2 ml and the finale volume was 3 ml, according to the turbidity of the sample. The chambers (Megafunnel® – ThermoShandon) were centrifuged 5 minutes at 1250 rpm (CytoSpin® – ThermoShandon) [The  liquids . The surShandon) -12.For cytology diagnosis, we used the Bethesda system 2001: within the normal limits (WNL), atypical squamous cells cannot exclude high grade squamous intraepithelial lesion (ASC-H), atypical squamous cells of undetermined significance (ASC-US), low and high grade squamous intraepithelial lesions (Lg-SIL and Hg-SIL) . The hisHybrid Capture II®  were also realized (38 for Papspin® and 187 for Turbitec®). The HCII assays were performed on 5 ml of the residual liquid-based samples. After 5 min. centrifugation at 2000 rpm, the supernatant was discarded. The cellular pellet was washed once with 1 ml PBS, resuspended in 100 μl of Cervical Sample ®  and denatured in an alkaline solution. Classical hybridization, detection and calibration were made according to the HCII kit's instructions [225 HPV tests performed by the ructions ,16. We o® solutions for 3.2 +/- 0.9 months. Total cellular DNA were extracted by a freeze/defreeze method as previously described [We also performed PCR techniques to detect HPV DNA in 72 samples fixed in Easyfixescribed  or were escribed . HPV-speescribed .2 or a Fisher exact, ANOVA and kappa of Cohen tests were performed. A p-value less than 0.05 was considered statistically significant. The results are expressed as a mean +/- standard deviation or as a percentage.A Chi ® 1.7  programs.We use the Epi Info 2002 revised 2003  and the Analyse-it® system, although a higher background  was observed but did not influenced the lecture  vs 24/215 for Turbitec® (11.1%), low cellularity for 1/51 (1.9%) vs 4/215 (1.8%), hemorrhage for 2/51 (3.8%) vs 6/215 (2.7%), severe inflammation for 1/51 (1.9%) vs 1/215 (0.4%) and severe cytolysis for 2/51 (3.8%) vs 2/215 (0.9%) of LBC. However, we can not draw any statistical conclusion because of the too low number of LBC.According to Bethesda 2001, only 2 smears (0.9%) were classified as inadequate for cytology interpretation because of too low cellularity. These two smears were performed by Turbitec® liquid, we compared the results of 2 methods of LBC centrifugations with the histological diagnosis.In order to evaluate the performance of the LBC performed by cytocentrifugations from ours clinical samples preserved in the Easyfix® and the Papspin® cytocentrifugation systems . This is probably due to the fact that Papspin® LBC were performed after Turbitec® LBC when ours cytologists and pathologists were more used to the Bethesda system 2001.The comparison between the results obtained by the two cytocentrifugation LBC and those obtained by histology is shown in Table ® and 187 from the Turbitec® methods were also analyzed in the HCII assay. The frequency of HPV performed by HCII was respectively for Papspin® and Turbitec® of 50.0% vs 30.0% for WNL, 60.0% and 51.8% for ASC-US, 100% vs 100% for ASC-H, 87.5% vs 71.4% for Lg-SIL and 100% vs 85.0% for Hg-SIL. Those results suggested that HPV DNA was more detected among the ASC-US than among the WNL and that the HPV DNA is more frequently detected for ASC-H, Lg-SIL and Hg-SIL. However, because of the small cases in each group, no conclusion can be made.38 samples from the Papspin® and Turbitec® methods were calculated using the CIN2+ histology detection as the reference standard for the cytological diagnosis of Hg-SIL. Respectively for Papspin® and Turbitec®, the sensitivity was 82.6%  and 75.0%  and the specificity was 92.6%  and 96.2% . There was no statistical difference between the 2 LBC systems (p = ns).The sensitivity and the specificity of Papspin®, all Lg-SIL and all positive for HPV/HCII (4/4) and of 22 Turbitec®, 11 Lg-SIL (8/11 positive for HCII), 5 ASC-US (4/5 positive for HCII) and 6 ASC-H (6/6 positive for HCII). The false positives LBC consist of 2 for Papspin® and 4 for Turbitec®, all positive for HCII (2/2 and 4/4) and all with a colposcopy with only one small biopsy which is no representative of the whole cervix.The false negatives LBC (with CIN2+ as reference standard) consist of 4 Papspin® fixative liquid to preserve HPV DNA in the residual materials of 72 LBC performed by cytocentrifugations. Even after 3.2+/-0.9 months, at room temperature, beta-globin or HPV DNA could still be amplified by PCR in those samples. The comparison between HPV detection using either general primer PCR or HCII tests showed a Kappa test of 0.89 (p < 0.001). Three cases were positive for the HPV PCR and negative for the HCII and one case was negative for the HPV PCR and positive for the HCII. This last case was also negative for the beta-globin PCR explaining that HPV could not be detected because of the poor quality of the extracted cellular DNA. The stability of DNA in the Easyfix® medium after 3 months was in general excellent.We also evaluated the quality of the Easyfix® and the Turbitec® systems. We use colposcopy with biopsy or LEEP as reference standard. The diagnosis threshold was set up to the high-grade cervical intraepithelial neoplasia (CIN2+). This could have led to a selection bias of a high-risk patient population. Therefore, ours results are only acceptable in the framework of diagnosis but must be interpreted with some caution in the framework of cervical cancer screening in which the number of healthy patients should usually be as high as possible.In this study, we demonstrated the feasibility and the efficiency of inexpensive LBC performed by cytocentrifugations: the Papspin® system and Bergeron et al. (2003) [® system). These authors also demonstrated the superiority of the quality of LBC in comparison with those of CS. With cautions of small number of our samples and using the norms of the Bethesda system 2001, we also found only 0.9% of inadequate cytology [®, Autocyte PREP® and the new manual LBC of Digene , Alves et al. (2004) also concluded that in spite of the different methodologies, the 3 methods adequately preserved cellular structure for morphologic evaluation [® and a manual LBC, called MonoPrep2®, drew similar conclusions [The adequacy of the LBC has been already described in the literature: Weynand et al. 2003)  describe. (2003)  found 0.03  descrcytology . Chhiengaluation . Nam et clusions  All of t® and Turbitec®, are quite similar. Indeed, their sensitivity are respectively 82.6 % and 75% (p = ns) and their specificity are respectively 92.6 % and 96.2% (P = ns). The small number of Papspin® LBC in our study is due to the subsequent choice for the Turbitec® method which is more cost-effective. By comparison with published results our LBC efficiency performed by cytocentrifugation seems better than CS. A sensitivity of 68% and the specificity of 79% has been indeed reported for the CS and similarly a sensitivity of 76% and a specificity of 86% was reported by others for the Thinprep® [Nevertheless, rare are the studies describing the accuracy, in terms of sensibility and specificity, of these manual LBC and particularly for cytocentifugation methods. In our study, to detect the CIN2+, the efficiency of ours LBC, performed by cytocentrifugations, Papspinhinprep® -26. Recehinprep® .One main advantage of LBC is that they allow ancillary techniques such as those used in immunocytochemistry or molecular biology -32.® fixative fluid, used in this study for both LBC, is not yet accredited for the use of HCII. With a Kappa test of 0.89 between DNA/HPV PCR and HCII, and with the comparison with the cytological or histological diagnosis, we can conclude that this liquid is efficient for molecular biology and for the HCII technique. Similar conclusions have recently been reached by Leduc et al.(2004) who compared 250 cervical samples which have been fixated with both Easyfix® and Cervical Sampler liquid® (Digene Corp). They found a Kappa of 0.74 [® (Digene) and found a sensitivity of 94.2% and a specificity of 52.4%[The Easyfix of 0.74 . In our  of 52.4%. Howard  of 52.4%. In our Actually, it is generally accepted that the HCII test is more sensitive and less specific than cytology to identify Hg or Lg-SIL. However, in the case of ASC-US, a combination of HPV DNA and Papanicolaou smears can certainly increase the sensibility of the cervical cancer screening -39. Like® as no-buffered alcoholic fixative liquid.We demonstrated by this study that LBC performed by cytocentrifugation are efficient and also allow the HPV DNA preservation in the EasyfixInexpensive LBC performed by cytocentrifugations can be performed by small laboratories which cannot invest in expensive automated equipments.


Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer.K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact testsHPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis.Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype. Colorectal carcinogenesis is a complex, multistep process involving environmental and lifestyle factors, sequential genetic changes and possibly viral components in discrete geographical areas. Genetic changes inactivating tumor suppressor and mismatch repair genes or activation of oncogenes which are involved in cell growth, proliferation and differentiation are implicated in the development of colon carcinoma. The target genes for these alterations are APC (Adenomatous Polyposis Coli), the ras family and p53 . The rasThe association between human papillomavirus (HPV) infection and the development of cervical and anogenital tumors is widely accepted. However, the relationship between human papilloma viruses and malignant diesases at various body sites, including the upper respiratory and digestive tracts and the breast is still not clear -17. HPV Although the role and distribution of K-ras mutations in colon cancer has been studied extensively there are no reports in the literature investigating the K-ras mutation status in HPV-associated colorectal cancer. The present study was undertaken to investigate the role of K-ras codon 12 and codon 13 mutations in HPV-associated colon tumors.2, 10 pmol each of the forward and reverse primers, 1U Taq polymerase , 200 μM dNTP mix and 20 mM Tris-HCl, pH 8.3. The reaction mixture was heated to 95°C for 5 min. for initial denaturation, followed by 35 cycles of denaturation at 95°C for 1 min., annealing at 60°C for 1 min. and extension at 72°C for 1 min. Final extension was allowed to proceed for 6 min. at 72°C.Tumor samples were obtained at the time of surgery from 53 patients with colon cancer. The corresponding normal tissues surrounding the tumors were also analyzed. The study was approved by the Institutional Ethics Committee. Genomic DNA was extracted from the tumors and corresponding normal colon tissue samples by phenol/chloroform extraction. To detect K-ras codon 12 and 13 mutations, DNA was amplified by a mutagenic PCR assay. A mismatched upstream primer for codon 12 and a mismatched downstream primer for codon 13 which introduced a Bst N1 (codon 12) and a HaeIII (codon 13) restriction site in the wild type allele, respectively, were used for amplification as described previously . The PCRDigestion was performed in a total volume of 25 μl containing 10 μl PCR product, 2.5 μl 10x digestion buffer, 5 μg BSA and 10 U of Bst N1 or HaeIII by overnight incubation at 60°C or 37°C, respectively. The digested products were separated by 8% non-denaturing polyacrylamide gel electrophoresis at 120 V for 3 h, the gel was stained with ethidium bromide and the genotypes were determined using a video gel documentation system .2, 200 μM of each dNTP, 150 ng sample DNA, 25 pmol of each primer and 2.5 U Taq polymerase. The reaction was begun by incubation at 95°C for five minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute.To investigate the HPV infection specific regions from the HPV genome were amplified by the MY 09 and MY 11 consensus primers which amplify a region of about 450 bp from the L1 open reading frame. DNA from a HPV-positive patient with cervix cancer was used as the positive control and DNA from lymphocytes were used as the negative control. The PCR reaction mix contained 500 mM KCl, 100 mM Tris-HCl, pH 9.0, 3 mM MgCl30 μl of the PCR product was separated on a 1.5 % agarose gel and the DNA samples were blotted onto positive-charged nylon membranes . The probes used in the study were cloned into the pBR322 plasmid. They were separated from the vector by digestion with Bam H1 and Eco RI for HPV16 and HPV18, respectively. The probes were non-radioactively labeled at their 3'-end by incorporation of digoxigenin-labeled dideoxyuridine-triphosphate (DIG-ddUTP). Hybridization was performed at 68°C for 18 hours. The hybridized probes were visualized by an anti-DIG-alkaline phosphatase conjugate-catalyzed color reaction using BCIP (5-bromo-4-chloro-3-indolylphosphate) and nitroblue tetrazolium salt .Statistical analyses were performed by using the chi-square and Fisher's exact tests with a significance level of p < 0.05.To analyze the coexistence of human papillomavirus infection and K-ras gene activation in colon carcinoma, we investigated 53 tumor samples for the presence of HPV infection  and K-ras codon 12 and codon 13 mutations. Codon 12 mutations were detected in 31 cases (58.5%) while fragments of the HPV genome were found in 43 (81.2%) cases. K-ras gene codon 13 mutations were not observed in the tumor samples of colon cancer patients. In 17 samples (32 %) genomic HPV fragments were also detected in the corresponding adjacent normal tissue. K-ras codon 12 or codon 13 mutations were not present in any of the normal tissue samples.24 of 43 (55.8 %) HPV-positive samples and 7 of 10 (70%) HPV-negative samples analysed harbored a mutation in codon 12 of the K-ras gene. HPV 18 and HPV 33 were the most frequent types . HPV 6, HPV 16, and HPV 11 were detected in 8, 5, and 2 cases, respectively. HPV 18 was not detected in the adjacent normal tissue samples. The most prevalent HPV types in normal tissue were HPV 16 (15%) and HPV 11 (13.2%). The distribution of HPV types between the normal and tumor tissue was not statistically significant except for HPV types 18 and 33 (p < 0.001).Statistical analysis did not reveal an association between the HPV infection and K-ras mutation Table . When weThe current genetic model for colon carcinogenesis depicts sequential accumulation of mutations in specific cancer-related genes, including APC, K-ras, and p53 that drive the transition from normal epithelium to increasing adenomatous dysplasia and finally to cancer ,25. DuriNumerous studies in the literature support the role of the ras protein and HPV viruses and indicate their probable cooperation in the pathogenesis of cervical neoplasias -28. MazuThe role of the ras oncogene ,30-35 anIn this study we observed mutations in the K-ras gene in 58.5 % of the tumor samples. This mutation frequency is slightly higher than the frequencies reported for the K-ras gene by other investigators in colon cancer -35,39. KBodaghi et al  reportedOur results suggest that mutational activation of the K-ras gene is common in colon carcinogenesis and infection with HPV may represent an additional important early step in the development of premalignant transformation. Further studies incorporating larger sample sizes are necessary to provide more definitive data on the potential role of these factors in colon carcinogenesis.The author(s) declare that they have no competing interests.NB conceived and participated in the design of the study, performed the experiments and drafted the manuscript. AT carried out the experiments. ND participated in the design and coordination of the study and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


We have previously shown the high prevalence of oral anti-human papillomavirus type 16 (HPV-16) antibodies in women with HPV-associated cervical neoplasia. It was postulated that the HPV antibodies were initiated after HPV antigenic stimulation at the cervix via the common mucosal immune system. The present study aimed to further evaluate the effectiveness of oral fluid testing for detecting the mucosal humoral response to HPV infection and to advance our limited understanding of the immune response to HPV.The prevalence of oral HPV infection and oral antibodies to HPV types 16, 18 and 11 was determined in a normal, healthy population of children, adolescents and adults, both male and female, attending a dental clinic. HPV types in buccal cells were determined by DNA sequencing. Oral fluid was collected from the gingival crevice of the mouth by the OraSure method. HPV-16, HPV-18 and HPV-11 antibodies in oral fluid were detected by virus-like particle-based enzyme-linked immunosorbent assay. As a reference group 44 women with cervical neoplasia were included in the study.Oral HPV infection was highest in children , followed by adolescents , and lowest in normal adults . The predominant HPV type found was HPV-13  followed by HPV-32 . The prevalence of oral antibodies to HPV-16, HPV-18 and HPV-11 was low in children and increased substantially in adolescents and normal adults. Oral HPV-16 IgA was significantly more prevalent in women with cervical neoplasia  than the women from the dental clinic . Significantly more adult men than women displayed oral HPV-16 IgA  and HPV-18 IgA .The increased prevalence of oral HPV antibodies in adolescent individuals compared with children was attributed to the onset of sexual activity. The increased prevalence of oral anti-HPV IgA in men compared with women was noteworthy considering reportedly fewer men than women make serum antibodies, and warrants further investigation. The involvement of human papillomaviruses (HPV) in squamous cell carcinomas of the anogenital region is widely accepted. HPV infection has also been demonstrated in several disorders of the oral and tonsillar regions  but unliVaccines for the control of HPV infection are presently in the process of being released for general use. In Africa with its huge burden of HPV-associated cancers, novel vaccines against HPV are under development that could enable the vaccination of large sectors of the population . The intThe testing of oral fluid for antibodies has proved most useful as an HIV-1 screening tool as oral HIV-1 IgG antibodies closely reflect HIV-1 serostatus . The oraHPV seropositivity has been shown to be a biomarker of past and present HPV infection and lifetime number of sexual partners . The preThe present study aimed to evaluate the extent of oral HPV infection and the prevalence of oral IgA and IgG antibodies to HPV-16, HPV-18 and HPV-11 in members of a normal healthy population, male and female, children to adult, and to assess the efficacy and acceptability of oral fluid sampling for HPV testing in young children and older individuals. To our knowledge the presence of HPV antibodies in oral fluid have not previously been reported in such populations. The study also hoped to further our limited basic understanding of the immune response to HPV infection in especially men who are being considered for inclusion in HPV vaccination strategies as men sexually transmit HPV to their female partners. SignifiParticipants in the present study were visitors to dental clinics in the Cape Town metropolitan area for normal dental treatment during 2003. Informed consent or parental consent for the children was obtained from all individuals and approval for the study obtained from the Ethics Committee for Human Research of the University of the Western Cape. There were 114 children , 78 adolescents  and 116 adults  with adequate buccal samples who were included in the study. Further participants were 44 women who formed part of study investigating cervical immune responses to HPV-16 . The latter women aged between 18 and 40 years were recruited during 2004 and 2005 from amongst first time attendees of the Colposcopy Clinic at Groote Schuur Hospital, Cape Town, South Africa. Of the 44 women, 19 were diagnosed by histology and/or colposcopy with high grade cervical intraepithelial neoplasia (CIN2/3), 13 with low grade cervical disease (CIN1) and 12 had cleared their CIN (CIN0) . The latter (CIN0) had been referred to the colposcopy clinic with high grade cervical disease as diagnosed by Pap smear but on subsequent histology or colposcopy or both showed no CIN.Trained dentists collected buccal samples for HPV DNA determinations by brushing the entire mucosa of both inner cheeks with a CerviBrush (Digene). Brushes were placed in sterile Digene tubes containing transport buffer. Oral fluid samples were collected with an OraSure pad  as described previously , samplinDNA was extracted and purified from buccal cells using QIAamp DNA minikit (Qiagen) and buccal swab protocol. The quality of the DNA and thus the adequacy of the buccal sample, was evaluated by amplifying a section of the CCR-5 gene as described by Michael et al, 1997) [97 [19]. 2 tissue culture flasks, at an MOI of ~10. Cells were harvested by centrifugation at 5000 × g, 6 days post infection [All virus-like particles (VLPs) for antibody enzyme-linked immunosorbent assay (ELISA) were composed of major capsid protein L1 only. HPV-18 VLPs were obtained from MedImmune Inc . HPV-16 and HPV-11 VLPs were prepared using recombinant baculoviruses expressing either HPV-11 or HPV-16 L1 protein to infect monolayer cultures of Sf21 insect cells in twelve 75 cmA capture ELISA was used to detect oral HPV IgA and IgG antibodies to HPV-16, HPV-18 and HPV-11. Oral fluid and serum from CC patients were tested for HPV-16 antibodies only. Serum antibody detection was by direct VLP-16-based ELISA. ELISAs were a modification of that described by Studentsov et al., 2002 , using p2 test of independent proportions was used to determine the Odds Ratios (OR) and 95% confidence intervals (95% CI) for the association of antibodies between different groups. Agreement between IgA and IgG within the same sample and between serum and oral fluid antibodies within the same individual was evaluated by Kappa statistic. If Kappa = 1, then there is perfect agreement and if Kappa = 0 there is no agreement. A kappa <0.4 indicates that there is poor agreement, a kappa 0.4–0.75 indicates fair to good agreement and a kappa > 0.75 indicates excellent agreement. [The Xreement. .All CerviBrush-collected buccal samples were CCR-5 positive. Extraction controls  were included during all extractions and all were negative for both CCR-5 and HPV DNA. Oral HPV DNA was detected in 17/308 buccal specimens from the dental population (5.5%) with the prevalence of oral HPV highest amongst the children  then the adolescents  and lowest amongst adults . The most prevalent type was HPV-13 found in 41.2% (7/17) of the HPV positive samples or in 2.3% of the participants. The demographics, oral HPV types and oral HPV-16, HPV-18 and HPV-11 antibody prevalence of the 17 people infected with oral HPV are shown in Table In contrast to the oral HPV prevalence, the prevalence of oral HPV antibodies was lowest in the children, increased in adolescents and was highest in adults Fig . Of the There was low concordance between the prevalence of oral HPV-16, HPV-18 or HPV-11 IgA and IgG antibodies within the same sample as determined by kappa . There were 15/116 (12.9%) adults with corresponding IgA and IgG to HPV-16, three of these adults also had corresponding IgA and IgG to HPV-18 and to HPV-11 and 3 had corresponding IgA to HPV-11 as well as HPV-16. No adult had corresponding IgA and IgG to HPV-18 or HPV-11 unless they had corresponding HPV-16 IgA and IgG antibodies (data not shown).Antibody prevalence in oral fluid was assessed according to gender in the three age categories of dental clinic participants Fig . AmongstAmongst the women from the colposcopy clinic, HPV-16 oral IgA was found in 30/44 women (68.2%) and HPV-16 oral IgG in 14/44 (31.8%) women. The prevalence of oral HPV-16 IgA was significantly higher in these women than the women from the dental clinic  but not for oral HPV-16 IgG . The prevalence of HPV-16 serum IgA in the colposcopy clinic patients was 50%, serum IgG 34.1%. There was low agreement between the presence of oral or serum HPV-16 antibodies in the same individual. There was also low agreement between the presence of HPV-16 IgA or IgG within the same individual .The prevalence of HPV-16 antibodies in oral and serum samples from these women stratified according to the grade of CIN and the presence of cervical HPV-16 are shown in Table Alpha-papillomavirus genus which includes mucosal and cutaneous HPV types [The oral HPV types found in this study belong to the PV types . The prePV types . HPV-13 PV types ,28. The PV types  and was PV types  and possThe oral sampling of oral fluid for this study was favourably accepted by the children, adolescents and adults, even in children as young as one year old. Three oral fluid samples were discarded because they showed a lower than normal total IgA level (<0.5 μg/ml). This occurred in children < 2 years and was probably a consequence of the sampling device not remaining in the mouth for the correct length of time. A significant finding was the increased prevalence of oral HPV IgA in adult men compared with women. Gender differences in seropositivity to HPV have been reported. The prevalence of HPV serum antibodies is apparently consistently lower in men than in women, even in men with high risk sexual activity ,18. The We postulate the site of the HPV infection in the men (and women) with oral antibodies to be anogenital, based on our findings in women with cervical neoplasia. In support of this, almost no HPV-16, HPV-18 and HPV-11 types were found in the mouth. The role of HPV infection in cancers at male and female anogenital sites has been clearly demonstrated . TonsillIn the present study, the high prevalence of oral HPV antibodies in adolescents was notable. HPV seropositivity is related to sexual activity  and oralWe believe that sampling oral fluid for HPV antibodies shows promise as a simple, non-invasive method to test for the presence of antibody responses to HPV infection. However larger studies are required to validate oral fluid antibody sampling as either a surrogate for serum testing or as a biomarker of anogenital HPV infection. We postulate the site of infection eliciting oral HPV antibodies to be mainly anogenital. As an adjunct to serology, oral testing could increase the sensitivity of HPV analysis, covering a broader range of humoral responses and contribute to our understanding of the immune response to HPV. It is well known that HPV serology underestimates genital HPV infection even when restricted to the same type HPV DNA as detected in the lesion. Conceivably certain individuals could mount more effective mucosal responses to HPV infection than systemic responses and oral IgA testing could serve as an adjunct to or even replace serum testing for genital HPV infection in children. A large study is underway to further investigate oral HPV IgA as a biomarker of genital HPV infection in women.Most significant was the finding that more men than women display oral antibodies, considering the opposite is found for serum responses. Studies are planned to assist in the further understanding of the mucosal immune response to HPV in men and the site of the HPV infection eliciting these responses. The possible association of oral HPV antibodies with genital HPV infection especially in adolescents requires further clarification.The author(s) declare that they have no competing interests.DM: manuscript writing, antibody determinations, data analysis and data interpretation; CS: Buccal HPV type determination; AJ and DD: acquired dental patient data and collected the buccal samples; LS: participated in study design and coordination; JP designed and co-ordinated the study of the immune responses in women with CIN; LD provided the samples from the women attending the colposcopy clinic; EPR and EvdW: manuscript review and development and production of HPV-16 and HPV-11 virus-like particles; A-LW: Principal Investigator on the study – participated in the study design, manuscript review. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Treatment options for comminuted tibial shaft fractures include plating, intramedullary nailing, and external fixation. No biomechanical comparison between an interlocking tibia nail with external fixation by an Ilizarov frame has been reported to date. In the present study, we compared the fatigue behaviour of Ilizarov frames to interlocking intramedullary nails in a comminuted tibial fracture model under a combined loading of axial compression, bending and torsion. Our goal was to determine the biomechanical characteristics, stability and durability for each device over a clinically relevant three month testing period. The study hypothesis was that differences in the mechanical properties may account for differing clinical results and provide information applicable to clinical decision making for comminuted tibia shaft fractures.In this biomechanical study, 12 composite tibial bone models with a comminuted fracture and a 25 mm diaphyseal gap were investigated. Of these, six models were stabilized with a 180-mm four-ring Ilizarov frame, and six models were minimally reamed and stabilized with a 10 mm statically locked Russell-Taylor Delta™ tibial nail. After measuring the pre-fatigue axial compression bending and torsion stiffness, each model was loaded under a sinusoidal cyclic combined loading of axial compression  and torque  at a frequency of 3 Hz. The test was performed until failure (implant breakage or ≥ 5° angulations and/or 2 cm shortening) occurred or until 252,000 cycles were completed, which corresponds to approximately three months testing period.In all 12 models, both the Ilizarov frame and the interlocking tibia nail were able to maintain fracture stability of the tibial defect and to complete the full 252,000 cycles during the entire study period of three months. A significantly higher stiffness to axial compression and torsion was demonstrated by the tibial interlocking nail model, while the Ilizarov frame provided a significantly increased range of axial micromotion.This is the first study, to our knowledge, which compares the biomechanical properties of an intramedullary nail to an external Ilizarov frame to cyclic axial loading and torsion in a comminuted tibia shaft fracture model. Prospective, randomized trials comparing Ilizarov frames and interlocked tibial nails are needed to clarify the clinical impact of these biomechanical findings. Open fractures of the tibia with bone loss or extensive comminution can be treated by a variety of techniques -4. A comThe present study on a biomechanical model was designed to investigate the fatigue behaviors of an interlocking tibial nail and the Ilizarov frame under a combined load of axial compression, bending and torsion. We believe that the understanding of the mechanical differences of both devices may provide new information applicable to clinical decision making in the treatment of comminuted tibial shaft fractures.A) and of the IMN construct (B).Twelve composite tibia bone models with a 25 mm diaphyseal gap were used for this biomechanical study to model a comminuted tibial fracture . Six mod®, and mounted on an 858 Bionix™ material-testing machine. To eliminate the potential for testing machine related data scatter, both the Ilizarov and IMN constructs were tested alternately on the two 858 Bionix™ systems. Custom-made loading fixtures were used to facilitate a clinically relevant combined loading of torque and axial compression bending with different proximal (23 mm) and distal (9 mm) offsets from the tibia's mechanical axis.Each construct was potted proximally and distally in a pair of loading fixtures, using Fast Cast® system). The stiffness was calculated from the slope of the load-deflection curve. A ramp compressive load at a rate of 0.2 in/min and a maximum of 178 N (40 lbf) was applied to observe bending stiffness in axial stress. For the torsion stiffness, a ramp torsion load at a rate of 5°/min and a maximum of 17 lbf-in (1.92 Nm) was applied.A linear variable differential transformer (LVDT) was mounted at the simulated fracture site to measure the fracture gap distance. The initial gap distance and pre-fatigue axial compression bending and torsion stiffness of both models were measured and documented prior to the start of the experiments. Axial deflection, torque and rotation were recorded by the LVDT  and torque of 1.7/17 lbf-in (0.19/1.92 Nm) at a frequency of 3 Hz. Load was applied until either failure occurred, as defined by an implant breakage or ≥ 5° angulation and/or 2 cm shortening, or when the three cycle periods of 252,000 cycles were completed, which corresponds to a simulated clinical loading time of approximately 3 months. Every 84,000 cycles the test was interrupted to re-measure the stiffness and the gap distance under zero load. The applied loading stress which was estimated to be clinically relevant has previously been determined in a different biomechanical study using unilateral external fixators ,17.® software and statistical analysis was performed by ANOVA with a P-value < 0.05 being considered statistically significant.All the data were collected and analyzed by Lab ViewP > 0.05). Similarly, no significant difference of the gap distance change over time/cycles was observed within the individual groups . However, a significant reduction in axial compression bending stiffness .All our 12 model systems could successfully conclude the 252,000 cycles without any implant breakage or deformity equivalent to clinical complications, such as ≥ 5° angulation and/or ≥ 2 cm shortening. Neither the axial compression bending nor the torsion stiffness was shown to change statistically over time within the individual groups  throughout the entire testing period, implicating that both constructs were able to maintain fracture stability. Likewise, neither model lead to a permanent deformity in terms of a malalignement. A composite tibia was chosen over a cadaveric model due to the more standardized features under different loading stresses . The comOur results indicate that both the Ilizarov frame and a statically locked intramedullary nail are able to maintain fracture stability over three months of normal clinical use in a comminuted tibial defect model. This model reflects a "worst case scenario", since under normal clinical conditions bone formation would typically occur enabling the bone to increasingly bear more load with time."in vitro" study. This model does not account for the potentially important biomechanical influence of the continuously changing stiffness due to the kinetics of fracture healing. However, as mentioned above, the composite tibia model offers the unique advantage of highly standardized biomechanical properties with regard to the reproducibility of different loading stresses, as opposed to the interspecimen variability in cadaveric or "in vivo" studies [On other hand, since our model does not provide increasing stability at the fracture site due to callus formation over time, it must be considered a pure  studies .In this test design, the implants bore the full load throughout the duration of the test and healing callus did not influence biomechanics of fixation. Neither the intramedullary nail nor the Ilizarov frame failed in simulated weightbearing conditions over three months. This validates the immediate weightbearing concept of Illizarov and implies a similar potential for locked intramedullary tibial nails.We utilized a simple four-ring, eight olive wire Ilizarov fixator construct for this study. Unilateral external fixators may demonstrate plastic or slip failure of frames during weightbearing with unstable fractures and frame fatigue may affect long-term interfragmentary stability . The oveThis biomechanical study on a comminuted tibia shaft fracture model demonstrates a significantly higher stiffness for axial compression and torsion by an interlocked tibia nail, as compared to an external Ilizarov frame. The Ilizarov construct, however, provided an increased axial micromotion. Prospective, randomized trials comparing Ilizarov frames and interlocked tibial nails are needed to clarify the clinical impact of these biomechanical findings.There are no financial interests by any of the authors regarding the present project.LL performed the biomechanical testing experiments and assisted with analysis of the data and writing of the manuscript. EH and GCP analyzed the data and wrote the final version of the manuscript. WRS, PFS, and SJM were responsible for conception and supervision of the study, planning of the experiments, and writing the manuscript.


It is now clearly established that proteolytic enzymes, including plasminogen activator (uPA), play an important role in breaking down the extracellular matrix, which is considered to be a step in metastasis formation. Plasminogen activators are controlled at various levels. Two inhibitors, PAI-1 and PAI-2, have been identified, the latter being more specific for uPA. In attempts to determine their prognostic value, it is essential to investigate the relative importance of these parameters and their interactions. We used an immunoenzymatic method to assay uPA, PAI-1 and PAI-2 antigens in cytosols prepared from 314 primary breast tumours. The patients were followed up for a minimum of 6 years and all relevant clinical and laboratory findings were recorded. Univariate analysis confirmed the poor outcome of patients whose tumours contained large amounts of uPA and PAI-1. In addition, low levels of PAI-2 correlated with shorter disease-free survival in the overall population (P = 0.02), post-menopausal women (P = 0.02) and women without lymph node involvement (P = 0.02). Multivariate analysis in the 'main effects' Cox model identified node involvement, macroscopic tumour size and PAI-2 as significant variables. The 'interactive' model, taking into account interactions between uPA and its two inhibitors, identified a first subgroup with a very poor prognosis associating either high levels of PAI-1 with low levels of PAI-2 in the overall population and the women with no node involvement or high levels of uPA with low levels of PAI-2 in the group of menopausal women. We conclude that PAI-1 provides the same prognostic information as uPA, and does not appear to play a role as an inhibitor. In contrast, PAI-2 increases the prognostic value of uPA, particularly in post-menopausal women, and PAI-1 in patients with no node involvement.