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Earlier studies have suggested that the higher locomotive costs of smaller bears could be related to increased stride frequency, because more steps will be needed to maintain the same speed as larger bears (Heglund and Taylor, 1988; Best et al., 1981). Energy cost per gram of body weight per stride is relatively constant across animals of drastically different masses moving at the same speed (Heglund et al., 1982), so although heavier animals require more energy to move per stride, the longer stride length and lower stride frequency could result in increased efficiency over the same distance (Heglund et al., 1982). Incremental rates of energy use during terrestrial locomotion can also change with transitions to different gaits (Chassin et al., 1976; Heglund and Taylor, 1988; Reilly et al., 2007; Watson et al., 2011), although this has not yet been studied in polar bears and warrants further attention because it could affect the shape of oxygen consumption curves at higher speeds.
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study
| 100.0 |
Pursuits (and capture) of flightless snow geese lasting longer than 12 s have been documented (Iles et al., 2013), and we have observed multiple examples of this behaviour in recent years (LJG & RFR our unpublished data). Our analyses here indicate that these observations are to be expected, given that prolonged (i.e. >20 min) pursuits of even distant geese (i.e. farther than 500 m) can be energetically profitable, especially for polar bears in the size range for which there are data (Figs 3 and 4). Of those, smaller bears are capable of profitably engaging in pursuits of more distant geese and at higher pursuit speeds, given their lower overall level of energy expenditure (Fig. 4). In western Hudson Bay, subadult polar bears (those that are included in the studied size range) as well as females with cubs tend to arrive onshore in spring earlier than larger, mature individuals (Rockwell and Gormezano, 2009). Interestingly, our results suggest that these younger and smaller bears, which have recently been shown to have lower survival (Lunn et al., 2016) and which may be disproportionately affected by lost opportunities to hunt seals as a result of climate change (Regehr et al., 2007; Rockwell and Gormezano, 2009), should have an inherently better ability to recover caloric deficits via terrestrial prey.
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study
| 100.0 |
Prolonged chases of flightless snow geese can be energetically profitable over a range of pursuit speeds for polar bears in the 125–235 kg size range. The same is likely to be true for larger bears, those outside the range of available oxygen consumption data, because only postural cost (y-intercept) is mass dependent and it scales at the 0.25 power (Fig. 4; Taylor et al., 1970). Extrapolations past the upper limit of speeds for which there are data assume that the functional basis for the modelled trend remains the same, an assumption that may be violated if polar bears change gait and energy efficiency at higher speeds. Nevertheless, based on our top model, we project that a 320 kg bear running at 20 km/h would expend the calories contained in an adult goose in 33 s, a value that is reasonably comparable to the estimate of 12 s previously suggested by Lunn and Stirling (1985) using a different model. However, we note that our model also predicts that 320 kg bears can more profitably engage in much longer pursuits at slower speeds (e.g. our model predicts that pursuits of geese lasting up to 13.3 min are energetically profitable for a 320 kg polar bear running at 7.9 km/h).
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study
| 100.0 |
Although polar bear locomotion is considered relatively inefficient, they typically walk slowly, with a steady gait of ~5.5 km/h (Stirling, 1988). They average 1–5 km/h over longer distances, periodically interspersed with rest stops, and can sustain these speeds for extended periods while covering large distances (Harrington, 1965; Amstrup et al., 2000; Anderson et al., 2008; Durner et al., 2011; Whiteman et al., 2015). For example, Amstrup et al. (2000) reported many polar bears sustaining average travel on the ice at >4 km/h for up to 20 h, with some maintaining these speeds for >40 h. In a controlled experiment, polar bears trained to walk on treadmills were likewise able to walk for long periods, continuing exercise for up to 90% of 6 h walking sessions (Best, 1982). However, during these trials the polar bears thermoregulated behaviourally by leaving the treadmill temporarily to ingest snow when their core temperatures reached a particular threshold (Best, 1982). Best (1982) suggested that hyperthermia, not fatigue, was more likely to be a limiting factor to continuous locomotion. Polar bears have also been observed sustaining higher speeds (approaching 10 km/h) for shorter periods of time while on the ice (i.e. 1–8 h; Amstrup et al., 2000), where low ambient temperatures and strong winds would be likely to reduce the risk of hyperthermia (Best, 1982).
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study
| 99.94 |
In contrast, while on land during the ice-free season in western Hudson Bay, when ambient temperatures are considerably higher, polar bears limit their daily movements, remaining inactive for long periods (Knudson, 1978; Latour, 1981). However, they have been observed engaging in faster-paced pursuits after caribou and waterfowl (e.g. Brook and Richardson, 2002; Iles et al., 2013; LJG & RFR our unpublished data). In such cases, hyperthermia, rather than lack of profitability, may be a limiting factor to sustained activity for several reasons. Polar bears are typical of non-sprinting mammals in that almost all the heat produced during exercise is immediately dissipated and little is stored (Taylor et al., 1970; Best, 1982), making warmer ambient temperature conditions particularly problematic because they reduce the potential for heat dissipation during exercise. For example, 218–239 kg polar bears walking at 7.9 km/h reached their upper critical temperature (when core body temperature can no longer be regulated) at about −33°C. Furthermore, these captive bears could sustain this activity at temperatures only up to −20°C when allowed to ingest snow before returning to walk (Best, 1982).
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study
| 100.0 |
Interestingly, many pursuits by wild bears have been observed in or near ponds, lakes and rivers (Iles et al., 2013; LJG & RFR our unpublished data), with the bear often lying in shallow streams and ponds immediately after the pursuit (Fig. 5). Immersion in water has been shown to reduce a polar bear's core body temperature substantially both before and after sustained exercise (Øritsland, 1969; Frisch et al., 1974). In general, the thermoregulatory costs of exercise for polar bears can be somewhat dissipated by certain behaviours, but these costs probably often constrain the duration and speed of a wild goose chase, especially during warm summer days. Figure 5:A subadult male polar bear in the Mast River (Wapusk National Park) after killing at least five flightless snow geese in three chases. After the chases, the bear walked into the river, lay down and drank periodically. Photographed on 13 July 2013 by R.F.R.
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study
| 99.94 |
A subadult male polar bear in the Mast River (Wapusk National Park) after killing at least five flightless snow geese in three chases. After the chases, the bear walked into the river, lay down and drank periodically. Photographed on 13 July 2013 by R.F.R.
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other
| 99.94 |
Additional research is clearly needed to gain a full understanding of the thresholds of inefficiency of foraging pursuits associated with polar bear locomotion. This is especially true for larger-sized bears and for all bears travelling near their maximal speeds. Such data are crucial for understanding the potential importance of land-based foraging behaviour. Polar bears currently consume various foods on land (e.g. Gormezano and Rockwell, 2013a,b and references therein), but the profitability of these foods and their contribution towards the persistence of polar bears in the face of climate change remains debatable (e.g. Gormezano and Rockwell, 2015; Rode et al., 2015; Pilfold et al., 2016). To clarify these issues, studies are required either that provide complete data allowing the calculation of energetic and nutritional costs and gains or (preferably) that allow those costs and gains to be measured directly.
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study
| 91.0 |
Visual illusions are fun, but they are also insightful (Carbon, 2014)—the great pedagogic value behind such illusions is that most readers, while being amused, also experience perceptual insights which assist the understanding of rather complex perceptual processing (see Gregory, 2009). Here, I present a very simple illusion which was inspired by a discussion which I involuntarily witnessed in a German photographic shop back in the year 1989, when photoprints in Germany were typically made in sizes of 9 × 13 cm (small purchase option) or 13 × 18 cm (large option). The customer who was currently being served was complaining about the incomprehensible billing policy of the shop—from his perspective, the large purchase option was only marginally larger than the small option, but incomparably more expensive. Indeed, when we looked at the specific setting, with the smaller sized print being centered on the larger one, the larger photo print looked only marginally larger. Ignoring all mathematically based means, the vendor followed this perceptual path, puzzling about the pricing policy himself. After a while, I chimed into the conversation with a simple mathematical argument: to compare the size of a 9 × 13 cm and a 13 × 18 cm print, just “cancel the common 13” and you will get a doubled remaining side as 18 = 2 × 9, so the larger option is 100% larger than the smaller one, the size is doubled! Both attendees looked through me, fully confused, a bit shocked and very doubtful . . . doubled? No way! They did not find basic mathematical rules convincing at all. So I decided to change my persuasion strategy (purely for practical reasons; I was late and wanted to be served quickly!) by visually demonstrating the fractional arithmetic via a very subtle, but extremely insightful change of the configuration: simply by rotating the smaller print by 90° and positioning it in such a way that the large edge was aligned with the small edge of the large print, both attendees exclaimed “wow!” in unison. Instantly, they had understood the size relationship of the two prints—the larger print was really double the small one; exactly double!
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other
| 99.9 |
Twenty-five years later, we can easily replicate the whole setting, simulating this aha!-insight effect (inspect therefore particularly Figure 1(a) and (g)): Just take two sheets of paper (e.g., A4); one original-sized, one halved by folding, and compare them in terms of area size by centering the halved sheet on the center of the original one! We perceive the larger sheet as far less than double the size of the smaller one. For instance, by asking people to assess how much bigger the larger sheet is compared with the smaller one (in percentage; thus, “100%” would be the correct answer1); most people strongly underestimate the size of the larger sheet. When I asked 102 participants (undergraduates of psychology, 76 female, mean age 20.8 years), two main results were retrieved: (a) although the entrance requirements for starting the study of psychology are extremely high in Germany, 14 persons were not able to operate with percentages in a meaningful way, an inability which is not at all uncommon (Siegler & Lortie-Forgues, 2015) besides severe forms of problems with numbers and problems applying mathematical routines such as dyscalculia (with a prevalence rate of about 6%, see Shalev, Manor, Auerbach, & Gross-Tsur, 1998)—consequently these persons were excluded from further analyses; and (b) remaining participants (88 undergraduates of psychology, 68 female, mean age 20.9 years) showed the same aha-effect as was described in the photographic shop after having had rotated the sheets. Before doing this, I confronted them with a series of geometrical settings (see the top row of Figure 1 for the A4 settings), first consisting of an A4 and a halved A4 (i.e., A5) sheet, always starting with the setting shown on the very left side, progressing to the version on the very right side, one after another. After this A4 series, the same general settings were shown for the U.S.-letter size plus the halved U.S. letter (“half letter”), see the bottom row of Figure 1. Participants assessed the configurations of the first “A4” series very similarly to the second “letter” series—exact values plus effect sizes for one-sample t-tests against the true value of 100% can be retrieved from Figure 1. Figure 1.Overview of the employed experimental conditions, already in the experimental order which was realized, starting with a centered version (a/g) and always ending with a fully aligned version (f/h)—the participants were first exposed to the A4 paper size setting (Series #1), then to the U.S. letter size setting (Series #2). Percentage values show the mean estimations of how much bigger the larger sheet is compared with the smaller one (100% would be the correct answer, e.g., 64.0% in the case of Figure 1(a) means that the area of the bigger sheet is strongly underestimated, d = 1.16); *** indicate p-values < .001. Effect sizes are expressed as (Cohen’s) d’s for one-sample t-tests against 100%.
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study
| 100.0 |
Overview of the employed experimental conditions, already in the experimental order which was realized, starting with a centered version (a/g) and always ending with a fully aligned version (f/h)—the participants were first exposed to the A4 paper size setting (Series #1), then to the U.S. letter size setting (Series #2). Percentage values show the mean estimations of how much bigger the larger sheet is compared with the smaller one (100% would be the correct answer, e.g., 64.0% in the case of Figure 1(a) means that the area of the bigger sheet is strongly underestimated, d = 1.16); *** indicate p-values < .001. Effect sizes are expressed as (Cohen’s) d’s for one-sample t-tests against 100%.
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study
| 99.94 |
The main result was that for all conditions but the last one of both series (Figure 1(f) and (l)), we obtained medium-to-large effects (Cohen’s d ’s > 0.68; for A4-settings always d ’s > 0.93 indicating large effects through-out) in terms of deviations from the true value of 100%, meaning that the area size of the large rectangle was strongly underestimated. This effect was strongest when the small rectangle was centered to the large one (Cohen’s d ’s > 0.95)—and even rotating and aligning it to one single side did not dismiss this very large perceptual effect. The only way of escaping this strong visual illusion was to align two sides of both sheets at once in such a way that the small rectangle halved the large one (Figure 1(f) and (l)). This provides some indication of how the effect emerges: We seem to face a general incapability of accurately comparing more than one geometrical dimension at once. Such effects are already known from other illusions and perceptual phenomena. For instance, Piaget, Inhelder, and Szeminska (1960) already showed that (primary school) children predominantly used just one single dimension (height) to estimate volume, the so-called centration hypothesis—but see counter-evidence in later work where the integration of all three dimensions have been documented (Ebersbach, 2009). That adults are also susceptible to such volume illusions has been extensively documented, even for everyday-life objects such as typical food and drink packages (Raghubir & Krishna, 1999)—again, it seems that simple measures or single dimensions are predominantly used to estimate more complex measures or to predict the outcomes of actions by analyzing two concurrent movements (Hergovich, Grobl, & Carbon, 2011). Perceptual research, however, also offers some alternative explanations for the revealed effect. First of all, the general tendency to underestimate areas documented by psychophysical power functions with exponents below one (e.g., Ekman & Junge, 1961) cannot account for the effect seen here as the relationships between the two physical areas remained constant at 2:1 across all conditions. Assimilation theory (Pressey, 1967, 1971) appears to offer a more promising account. According to this theory, in a group of objects the ones with the extreme values, for example, lengths, will be misperceived toward the average values (the theory has many names in science, for example, “regression to the mean” in statistics or “central tendency error” in applied fields). In the given case, people would adapt their length or area assessments toward the mean of the given context lines or areas, respectively, so would underestimate the size of the bigger rectangular area. However, assimilation theory will be quite ambiguous in predicting the specific outcome for the cases of 45° rotated configurations depicted in Figure 1(e) and (k), as it is not clear how the edges will assimilate—with the smaller or longer ones? Another alternative explanation arises by comparing the area of the smaller object and the non-covered, residual of the larger rectangle. As is seen particularly clearly in the centered cases in Figure 1(a) and (g), the residual area is quite narrow, in fact much narrower than the smaller object. Indeed, we observe particularly large effects in Figure 1(a) and (b) as well as in Figure 1(g) and (h). Further qualification of such an explanation, however, reveals that the resulting underestimation of the summed up narrow areas is again an indication of problems in integrating different dimensions; we could also reconcile it with assimilation theory operating with areas instead of lengths.
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study
| 99.94 |
In everyday life, we solve such perceptual-cognitive bottlenecks by reducing the complexity of such a task via aligning parts with same lengths; actually, what Figure 1(f) and (l) provides is a kind of geometrical fractional arithmetic as one side of the first object fully cancels out one side of the second—to be compared—object. This reduces the degrees of freedom to just one—now only one remaining side has to be compared with the other side, and this works out brilliantly as shown by data provided in Figure 1 (for versions 1(f) and (l)). If we follow such a strategy to reduce the complexity of this perceptual task, we are able to validly estimate “area sizes”—in fact we are then only estimating unidimensional information—if we do not follow such a strategy, we evidently and substantially fail. The most important insight from the entire story seems to be: sometimes, insights from perceptual illusions are much more striking than even mathematical proof, particularly if such illusions show a high degree of Prägnanz! Finally, the enjoyment of perceiving such an emerging Gestalt and the insight it creates gives us the pleasure needed to continue in our path of perceptual learning (Muth & Carbon, 2013).
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other
| 99.6 |
Dental caries is the localized destruction of susceptible dental hard tissues by acidic by-products from the bacterial fermentation of dietary carbohydrates . The disease process is initiated within the bacterial biofilm (dental plaque) that covers the tooth surface . The development of dental plaque involves the adhesion of bacteria and subsequent colonization. The influence of the adsorbed proteins on bacteria adhesion has been suggested as playing a major role in bacteria–tooth interactions . Compared to coronal caries (dental caries development in the tooth crown), the prevalence of root caries (dental caries development in the tooth root) is increasing. This can be due to extended root exposure time by increased life expectancy and the special anatomical location of the root in the oral cavity . A systematic review concluded that about 40% of elderly people aged 70 suffered from untreated root caries . Dentine on the root surface is soft and porous. Bacteria penetrate further into the tissue at an earlier stage of lesion development in root caries . Thus, control of bacterial initial adhesion to the root surface is critical for the prevention of root caries .
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review
| 99.9 |
Polyethylene glycol (PEG) was approved by the US Food and Drug Administration for internalization in the human body . It is used as an anti-biofouling material to provide a hydrophilic environment on a substrate surface. Although the mechanism is not fully understood, it has been suggested that the hydrophilic surfaces could act as a good anti-biofouling barrier and that the protein and cell resistance of surface-immobilized PEG could be attributed to the large exclusion volume, high mobility, and steric hindrance effects of the highly hydrophilic layer . PEG was shown to reduce protein adsorption and platelet adhesion in a blood-material interface . A study synthesized a methacryloyloxydecyl phosphate PEG for the prevention of Streptococcus mutans adhesion on hydroxyapatite and found that its inhibitory effect on bacterial binding was diminished by saliva protein .
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study
| 99.94 |
Mechanical plaque removal methods such as tooth brushing and dental flossing have been advocated as reducing the biofilm formation. However, these mechanical plaque removal methods require good manual dexterity and can be difficult to implement in certain circumstances, such as in older patients . Exposed dentine on the root surface is more susceptible to caries than enamel. PEG can be used as an anti-biofouling material in inhibiting dental biofilm formation on the root dentine surface. Polydopamine is a bio-inspired polymer that can form a strong adhesive interaction with various substrates . It also provides a versatile platform for secondary reactions for diverse functional applications . Studies have indicated that polydopamine was effective in surface functionalization and biomolecule covalent immobilization . The covalent immobilization of biomolecules maintained stable and long-term performance. The objectives of this study were to develop a polydopamine-induced-PEG coating method for the dentine surface and to investigate its anti-biofouling effect against a multi-species cariogenic biofilm.
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study
| 99.94 |
The contact angles for Groups 1 to 4 were 33.8° ± 2.5°, 77.8° ± 5.6°, 43.8° ± 2.8°, and 56.7° ± 5.9°, respectively (p < 0.001; Figure 1). The multiple-comparison result is shown in Figure 1. The small contact angle of the hydroxyapatite disk treated with polydopamine and PEG indicated a strong hydrophilic property of the surface.
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study
| 100.0 |
The change in the surface density of quartz crystal over time in Group 1 to Group 4 is shown in Figure 2. Results of the FTIR showed peaks at 1600 and 1353 cm−1 in the polydopamine spectrum, indicating aromatic ring chains and bonds between the phenyl groups in polydopamine, respectively (Figure 3, PDA group). For the PEG spectrum, the peaks at 1061 and 1123 cm−1 represented the C–O stretching vibration and the O–H bending vibration . The peaks at 3172, 3278, and 3345 cm−1 represented free –OH and –NH3. These peaks were observed in the PEG spectrum but not in the dopamine + PEG spectrum. This suggested that no free –OH and –NH2 could be found in the dopamine + PEG treatment and that PEG was grafted chemically to polydopamine .
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study
| 100.0 |
The optical density from Groups 1 to 4 were 0.058 ± 0.007 ng/cm2, 0.115 ± 0.014 ng/cm2, 0.093 ± 0.01 ng/cm2, and 0.1 ± 0.015 ng/cm2, respectively (p < 0.01). The mucin absorbed by the hydroxyapatite disks of Group 1 was significantly less than that of Groups 2, 3, and 4.
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study
| 100.0 |
The surface morphology of the cariogenic biofilm on the dentine surface after 48 h under SEM is shown in Figure 4. A monolayer of biofilm was found in Group 1. The bacteria were sparsely distributed on the dentine surface. The openings of dentinal tubules were visible. A dense biofilm with confluent bacteria covering the dentine surface was observed in Groups 2 to 4.
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study
| 100.0 |
Figure 5 showed CLSM images of the live/dead staining of different groups on the dentine surface after 48 h. The red-to-green ratio of Groups 1 to 4 were 0.12 ± 0.05, 0.19 ± 0.05, 0.11 ± 0.04 and 0.04 ± 0.02, respectively (p = 0.001). Multiple comparisons showed that the red-to-green ratio of Group 4 was smaller than that of the other groups, and the largest red-to-green ratio was shown in Group 2.
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study
| 100.0 |
The log (CFU/mL) of Groups 1 to 4 were 4.9 ± 0.4, 5.8 ± 0.1, 5.7 ± 0.1, and 5.8 ± 0.2, respectively (p < 0.05). Multiple comparisons showed the log (CFU/mL) of Group 1 was smaller than that of the other groups, and there was no significant difference in log (CFU/mL) among Groups 2, 3, and 4.
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study
| 100.0 |
The results of the experiments on contact angle and QCM suggested that a novel polydopamine-induced-PEG coating on the dentine surface was developed in this study. This polydopamine-induced-PEG coating had the anti-biofouling effect in inhibiting salivary mucin absorption and cariogenic biofilm formation. The substrates used in this study are hydroxyapatite disks and human dentine blocks. The hydroxyapatite disks were standardized and made from hydroxyapatite powder. They were used for the assessment of the contact angle and mucin absorption because these two assessments require high test–retest variability. Autoclaving was used to sterilize both hydroxyapatite disks and dentine blocks because this would render the teeth free of viable microorganisms . Although autoclaving of teeth may reduce dentine microhardness, an in vitro study has shown that it did not significantly alter the physical properties . Contact angle quantifies the wettability of the polydopamine-induced-PEG-coated hydroxyapatite by water via the Young equation. Spectrophotometry is widely used for the study of chemical substances and can determine the amount of mucin absorbed on the hydroxyapatite disk through calculations of observed wavelengths. The early stage of bacterial invasion in the caries process involves Streptococci, Lactobacilli, and Actinomycetes. Streptococcus mutans is the most important odontopathogens involved in the initiation and progression of caries. Actinomyces israelii has the potential to invade dentinal tubules and is associated with root surface caries. Lactobacillus acidophilus is one of the most abundant species frequently found in both superficial deep carious lesions . For the foregoing reasons, these three cariogenic species were chosen to form a multi-species cariogenic biofilm. It is noteworthy that dental caries is a polymicrobial infection process caused by over 700 species of oral bacteria . In addition, this study incubated the bacteria on the root dentine surface with no prior saliva contamination. The results of this in vitro study need to be interpreted with caution.
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study
| 99.94 |
After we confirmed that PEG connected to hydroxyapatite via dopamine, human dentine blocks were used to study the inhibition effect of this polydopamine-induced-PEG coating on dentine against a cariogenic biofilm formation. The CFU and SEM results corroborated that dopamine-induced-PEG had inhibited the growth of a cariogenic biofilm on the dentine surface. Rinsing with distilled water did not notably reduce the surface density after the polydopamine-induced-PEG treatment. This suggested covalent binding between the polydopamine and PEG, which made the coating surface more stable.
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study
| 100.0 |
QCM is a well-suited technique for monitoring mass attached to coating equipment in a vacuum. It can be used for the investigation of the adsorption and surface reaction in the monolayer range via changes in the resonant frequency . The substrate of QCM is standard quartz crystal, and this is used to detect minute changes on the surface at the nano-gram level. Since no hydroxyapatite substrate was used, the results of QCM could only indirectly indicate the binding between polydopamine and PEG based on changes in the surface density. A FTIR study is necessary to determine whether PEG reacted with polydopamine. In addition, the results of the contact angle assessment demonstrated that the polydopamine-induced-PEG modified the hydroxyapatite surface by making it more hydrophilic.
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study
| 100.0 |
PEG is a water-swellable, non-toxic, and biocompatible polymer. Its use in biotechnology has been reported in the literature . Polymer brushes consisting of PEG opened a wide door in biomaterials research due to the suitable properties of the polymer . Polymer brushes are linear polymer chains terminally anchored to solid surfaces. If the distance between the anchoring points of the surface-grafted chains is small, interchain correlations occur. The tethered chains are stretched away from the surface leading to a “brush”-like conformation. Such polymeric monolayers play an important role in a wide range of colloid stabilization, tribology, lubrication, and rheology . PEG-modified surfaces have a high degree of hydration. The modified surfaces are effective in reducing diatom adhesion and weakening protein adsorption . This study showed that the tooth surface became more hydrophilic after coating it with polydopamine-induced-PEG. The resistance to adhesion of protein and bacteria to the PEG-modified surface can be attributed to the large exclusion volume, high mobility, and steric hindrance effects of this hydrophilic layer (Figure 6) .
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study
| 100.0 |
The bonding of adhesives to enamel is reliable and strong because enamel is highly mineralized. However, the bonding of adhesives to dentine is less satisfactory because dentine contains, by weight, 20% organic material (mainly collagens) and 10% water. Mussels can attach to various surfaces in aqueous conditions, ranging from natural inorganic materials and organic materials to synthetic materials . Such adhesive properties rely on exhaustively repeated 3,4-dihydroxy-L-phenylalanine (DOPA) and amine . Dopamine was identified as a small-molecule compound that contains both DOPA and amine (Figure 7). It has strong and good biodegradability and low cytotoxicity . The oxidative polymerization of dopamine in aqueous solutions spontaneously forms polydopamine . It mimics repeated DOPA and amine and therefore exhibits a strong adhesive property in relation to various substrates under wet conditions . Polydopamine is used for biomaterial surface modification because it is easy to obtain abundant active groups. These active groups are mostly phenolic hydroxyl/o-quinone and amino/imino for bimolecular immobilization on the material surface with minimal change in the chemical structure of biomaterials . The plausible mechanism of polydopamine-induced-PEG coating is shown in Figure 7. Polydopamine connects PEG with an active group of o-quinone. It can also attach to hydroxyapatite with phenolic hydroxyl (Figure 7c). This mechanism could be the reason for the promising anti-biofouling effect of polydopamine-induced-PEG coating on dentine.
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study
| 99.94 |
Mucins are the fundamental organic components of mucus in human saliva. It remains controversial whether mucin reduces or enhances cariogenic bacterial adhesion . However, mucin may act as a receptor for accumulation and biofilm formation, and it plays an important role in the agglutination/aggregation of a number of microorganisms . The mucin absorption measured by a spectrophotometer in this study demonstrated that polydopamine-induced-PEG coating inhibited mucin adhesion to hydroxyapatite. This can be one of the main reasons for the biofilm-inhibitory effect of polydopamine-induced-PEG. Likewise, polydopamine-induced-PEG showed an inhibitory effect on cariogenic biofilm formation on dentine. The total bacterial amount of the polydopamine-induced-PEG group was significantly lower than that of the other three groups, and the quantity of the biofilm does increase with time. The reason might be that the “micro-brush” effect of polydopamine-induced-PEG coating is a mechanical effect rather than a chemically antimicrobial function. It is plausible that the accumulation of bacteria on the dentine surface weakened the “micro-brush” effect by occupying the space for PEG movement, which in turn caused more bacteria to settle on the dentine surface. Thus, it is difficult to rely completely on this function to protect the tooth surface due to the complex environment of oral biofilm. The polydopamine-induced-PEG should be applied more frequently to sustain the anti-biofouling effect. Polydopamine-induced-PEG may be used as an anti-biofouling agent in mouth rinse and to prevent cariogenic biofilm formation after teeth cleaning. Another clinical application involves adding it to a varnish to protect the tooth from biofilm adhesion. This in vitro study demonstrated the promising results of a novel anti-biofouling method to help control cariogenic biofilm formation. Further studies aiming to simplify the coating process and to sustain the coating are needed.
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study
| 99.94 |
This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (IRB UW13-555). Extracted sound human third molars were collected with patient consent. They were stored in distilled water at 4 °C and were used within one month of extraction. Thirty dentine blocks of 2 × 2 × 4 mm3 were prepared from a tooth root modified from our previous protocol . The blocks were treated with 1% acetic acid for 5 s and ultrasonically washed with deionized water to remove the smear layer . In addition, 36 hydroxyapatite disks were prepared by compressing hydroxyapatite powder (Sigma-Aldrich Co., St. Louis, MO, USA) into a circular mold (12 mm in diameter, 1 mm in thickness). They were sintered at 900 °C for 2 h. The surfaces of the hydroxyapatite disks were polished using micro-fine 4000 grid sanding paper. Commercially available methyl-PEG-amine (mPEG-NH2 Mw = 2000) (Shanghai Science Peptide Biological Technology Co., Ltd., Shanghai, China) and polydopamine (Sigma-Aldrich, St. Louis, MO, USA) were used in this study.
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study
| 100.0 |
The dentine blocks (2 × 2 × 4 mm3) and hydroxyapatite disks (diameter: 10 mm) were autoclaved for sterilization. They were randomly and equally divided into 4 experimental groups. In Group 1, dentine blocks and hydroxyapatite disks were immersed in 2 mg/mL of freshly prepared polydopamine solution (in 10 mM Tris buffer, pH = 8.5) at 23 °C for 12 h in the dark . After rinsing for 10 min with Tris buffer to remove non-attached polydopamine and drying under nitrogen, the blocks and the disks were then immersed in a freshly prepared solution of 1 mg/mL of mPEG-NH2 (in 10 mM Tris buffer, pH = 8.5) at 23 °C for 4 h. They were then rinsed with Tris buffer for another 10 min to remove any extra solution . In Group 2, dentine blocks and hydroxyapatite disks were immersed in a freshly prepared solution of 1 mg/mL of mPEG-NH2 at 23 °C temperature for 4 h. They were rinsed with Tris buffer for 10 min to remove residual solution. In Group 3, dentine blocks and hydroxyapatite disks were immersed in 2 mg/mL of freshly prepared polydopamine solution at 23 °C for 12 h in the dark. They were rinsed with Tris buffer for 10 min to remove nonattached polydopamine. In Group 4, dentine blocks and hydroxyapatite disks were immersed in distilled water.
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study
| 100.0 |
The degree of wetting (wettability) of the hydroxyapatite disks with coating was evaluated by measuring the contact angle using a drop-shape analyzer (DSA100. Krüss GmbH, Hamburg, Germany) equipped with a pendant drop module. It quantifies the wettability of the hydroxyapatite samples by water via the Young equation. Three replicates were measured for each group, and three samples per group were assessed. Each water drop (5 µL) was deposited onto the disk surface and kept for 15 s. Then, an image of the drop was taken by a built-in camera and analyzed using the software (Image J 1.51a, National Institutes of Health, Bethesda, MD, USA) supplied by the manufacturer. All measurements were conducted at room temperature.
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study
| 100.0 |
The affinity of the polydopamine-induced-PEG to the surface was studied by quartz crystal microbalance (QCM; Seiko QCM 922, Princeton Applied Research Inc., Oak Ridge, TN, USA). Mass variation before and after coating was measured. The change of the effective surface mass on the quartz crystal altered the gold resonant frequency. The differences in the resonant frequency were recorded as a function of the potential, and the corresponding mass changes were calculated. The experimentally determined Sauerbrey constant for the gold resonator was 176.0 Hz cm2·µg−1 .
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study
| 100.0 |
QCM study was carried out with 0.198-cm2 standard-finished 9-MHz AT cut gold resonators (Princeton Applied Research Inc., Oak Ridge, TN, USA) sputtered on quartz. All the experiments were conducted by dipping the quartz crystal into 4 mL of experimental solutions in a 16-well plate at 23 °C. The quartz crystal was dipped into a sequence of reagents, which is described in Section 2.2 for Groups 1–4. Distilled water (pH = 7.0) was used to remove the unstable reagent from the quartz crystal surface. The real-time frequency changes during the experiment were recorded using WinQCM computer software (Princeton Applied Research Inc., Oak Ridge, TN, USA). The surface density of the quartz crystal was calculated.
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study
| 100.0 |
Absorption of mucin on the surface of the hydroxyapatite disks with coating was evaluated. The disks were immersed in 10 mg/mL of mucin obtained from bovine submaxillary glands (Sigma–Aldrich, Co., St. Louis, MO, USA) for 90 min. They were rinsed with phosphate buffer solution (PBS) to remove the residual mucin before they were stained with Alcian blue solution (pH = 2.5). The absorbed mucin was then extracted using 30% hydrogen peroxide. The optical density of the supernatant (200 mL) was measured spectrophotometrically at 595 nm . The expected difference is at least 10°. Six hydroxyapatite disks per group were studied.
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study
| 100.0 |
A multi-species cariogenic biofilm was developed using a method modified from our previous study . Briefly, Streptococcus mutans from the American Type Culture Collection (ATCC) 35668, Lactobacillus acidophilus ATCC 9224, and Actinomyces israelii were anaerobically cultured on blood agar plates at 37 °C for 48 h. A single colony was picked from each plate to prepare 24-h broth cultures in brain heart infusion (BHI) supplemented with 5% sucrose at 37 °C under anaerobic conditions (95% nitrogen and 5% carbon dioxide). Subsequently, bacterial cell pellets were harvested and resuspended in the BHI to a cell density of McFarland 2 (106 cells/mL). A 500-µL aliquot of each bacteria culture was mixed and inoculated on each dentine block sitting in a well of a 24-well plate with BHI. The dentine blocks were aerobically incubated in 1 mL of mixed BHI suspension (adhesion phase) at 37 °C for 90 min. After rinsing with PBS to remove non-adherent cells, the dentine blocks were aerobically incubated in the BHI at 37 °C (biofilm phase). The dentine blocks were taken out after 48 h for analysis; the BHI was refreshed after 24 h .
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study
| 100.0 |
Scanning electron microscopy (SEM) was used to monitor the topographical features of the biofilm. In preparation for SEM, the dentine blocks with biofilm were rinsed in 4% formaldehyde followed by 1% PBS; they were then placed in 1% osmium tetroxide solution for 60 min. The blocks were washed in distilled water and dehydrated in a series of ethanol solutions at increasing concentrations (70% for 10 min, 95% for 10 min, and 100% for 20 min). The blocks were dried in a desiccator and sputter-coated with gold. The surface topographies of biofilms were studied under SEM (Hitachi S-4800 FEG Scanning Electron Microscope, Hitachi Ltd., Tokyo, Japan) at 12 kV in high-vacuum mode . Two dentine blocks per group were used in this qualitative experiment.
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study
| 100.0 |
Confocal laser scanning microscopy (CLSM) (Fluoview FV 1000, Olympus, Tokyo, Japan) was employed to assess the viability of the biofilms. The bacteria on the dentine surfaces were labeled in situ using two fluorescent probes: propodium iodide and SYTO-9 dye (LIVE/DEAD BacLight Bacterial viability kit, Molecular Probes, Eugene, OR, USA). Five images from the middle layer of each biofilm were obtained using CLSM and were analyzed using Image J (Version 1.51a, National Institutes of Health, Bethesda, MD, USA). The red-to-green ratio was calculated to denote the ratio of dead-to-live bacteria, respectively . Two dentine blocks per group were used in this qualitative experiment.
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study
| 100.0 |
Serial 10-fold dilutions of homogenized biofilm samples in 1% PBS were collected from dentine blocks and then plated in duplicate with a spiral plater (Autoplate 4000; Spiral Biotech Inc., Norwood, MA, USA) onto horse blood agar (Defib Horse Blood; Hemostat Laboratories, Dixon, CA, USA). The plates were incubated aerobically for 72 h to assess the bacterial colony-forming unit (CFU) per mL. Biofilms on six dentine blocks per group were studied.
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study
| 100.0 |
A Shapiro–Wilk test was used to assess the data for normality. One-way ANOVA was performed to compare the contact angle, mucin absorption, and log CFU among the four experimental groups. A Bonferroni test was carried out for multiple comparisons of the result. All the analyses were conducted using IBM SPSS version 2.0 software (IBM Corporation, Armonk, New York, NY, USA). The cut-off level for significance was taken as 5% for all the analyses.
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study
| 100.0 |
As one of the potent and efficient cancer therapies, radiation is widely used to treat a variety of cancer types. Ionizing radiation (IR) works by damaging the DNA of normal and cancerous tissue leading to cellular death . The goal of radiation therapy (RT) is to maximize damage to cancer cells while keeping collateral damage to a minimum [1, 2]. Studies in this field revealed that IR-induced DNA damage repair is more efficient in normal cells than in tumor cells, which display dysregulated repair . Standard RT protocols for cancer patients use a low dose (1.8–2.0 Gy) exposure repeated daily until the maximal tolerable normal tissue doses are reached. Given that the total dose delivered to tumors is generally determined by normal tissue toxicity rather than by the curable dose required for tumor eradication, the overall local cure using fractionated approach is ~65% of all tumors, and curability depends on tumor type and size. Following the development of intensity modulation RT (IMRT) and image guidance RT (IGRT), which can improve precision in tumor targeting and reduce normal tissue exposure, SDRT is gradually becoming an alternative therapeutic strategy. SDRT has shown clinical benefit with local control rates at over 90% in a variety of cancer types, including tumors considered resistant to conventional fractionated RT [3, 4]. Although SDRT presents a practical alternative approach for fractionated radiotherapy, its biological mechanism remains elusive. We recently reported that SDRT induces a rapid wave of endothelial cell apoptosis via ceramide generation in both normal gastrointestinal tract and tumors [5–7]. In this regard, SDRT launches a rapid translocation of acid sphingomyelinase (ASMase) to the endothelial plasma membrane, where ASMase converts sphingomyelin to the second messenger ceramide and the latter mediates apoptotic signaling [8, 9]. The 20-fold enrichment of a non-lysosomal secretory form of ASMase results in endothelial cells being particularly vulnerable to radiation-induced ASMase-mediated generation of the pro-apoptotic second messenger ceramide [10–12]. Importantly, our previous studies showed that the angiogenic factors bFGF or VEGF restrain radiation-induced ASMase activation, ceramide generation, and consequent endothelial apoptosis [12, 13]. Conversely, anti-angiogenic agents, such as anti-VEGFR2 antibody, antagonize these effects, synergistically increasing radiation-induced ceramide elevation and enhancing apoptosis . Furthermore, in vivo these agents cause a synergistic increase in radiation-induced tumor endothelial apoptosis and enhanced tumor response to SDRT . These studies therefore define that an ASMase/ceramide pathway-dependent endothelial response plays a crucial role in tumor cure by SDRT and is modulated by angiogenic factors.
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study
| 99.94 |
Tumor angiogenesis, the recruitment of new blood vessels, is essential for tumor growth and metastasis, and is driven by a balance between anti-angiogenic and pro-angiogenic factors . Anti-angiogenic therapy is emerging as an effective treatment for various tumor types through direct targeting of VEGF (such as the antibody bevacizumab) or the inhibition of VEGFRs by multi-target tyrosine kinase inhibitors (TKIs) [16–18]. These anti-angiogenesis strategies interfere with either the development or functionality of the tumor-associated vasculature, and thereafter lead to suppression of oxygen and nutrition supply to cancer cells . Recently, two different concepts have proposed that anti-angiogenic tumor therapy may either ‘‘normalize’’ dysfunctional tumor vasculature, which therefore facilitates drug delivery, or prevent recruitment of circulating endothelial precursors into the tumor [18, 19]. Although the outcomes of some clinical studies support either of these hypotheses, to date anti-angiogenesis therapy has yielded only modest therapeutic gains. The accurate mechanisms remain to a large extent unknown and the lack of an optimized mode of application limits the utility of this approach.
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review
| 99.9 |
Pazopanib, (GW786034B, 5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methyl-benzenesulfonamide), a novel and potent vascular endothelial growth factor receptor inhibitor , is a small-molecule inhibitor shown to target both tumor and endothelial cells in multiple myeloma . Pazopanib targets the TKRs including VEGFR-1/2/3, PDGFRα/β, and c-KIT . Pre-clinical studies have shown that Pazopanib can inhibit tumor angiogenesis and the growth of several human tumor xenografts (multiple myeloma, colon, melanoma, prostate, kidney) in mice . In addition, in 2009 Pazopanib was approved by the US FDA for the treatment of patients with advanced renal cell carcinoma (RCC). Additionally, several recent phase II and III studies have shown a significant clinical benefit of Pazopanib in a variety of malignancies, including soft tissue sarcoma, thyroid cancer, and ovarian cancer [21–23]. In the current study we tested the curative potential of a combination of SDRT with Pazopanib on xenografts of human sarcoma tumors, a chondrosarcoma (JJ012) and a neurofibrosarcoma (MPNST3). Our results revealed that a single dose of Pazopanib mimics the anti-VEGF/VEGFR impact on tumors subsequently exposed to SDRT, increasing ASMase activity in the serum and tumor endothelial dysfunction, enhancing tumor response, and exhibiting critical dependence on timing relative to SDRT exposure. These results suggest that Pazopanib has a similar mechanism of action to the one we previously demonstrated for anti-VEGF/VEGFR2 antibodies. As a short-acting anti-angiogenic, Pazopanib might be optimal for endothelial-mediated radiosensitization, and in combination with SDRT it might allow dose de-escalation, thus significantly expanding the range of clinical indications for SDRT.
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study
| 99.94 |
Our previous studies have shown that angiogenic factors protect endothelial cells from radiation-induced apoptotic death, and anti-angiogenics antagonized this effect and increased tumor response [14, 23]. Here we tested the effect of radiation therapy in combination with Pazopanib, a VEGFR inhibitor and a short-acting anti-angiogenic agent, on two mouse models of human sarcoma. Athymic or ICR/SCID mice were transplanted with JJ012 or MPNST3 sarcoma tumors respectively. When tumor volume reached 150 mm3 the tumors were treated with IR and/or Pazopanib, and their volumes were measured. As shown in Figure 1A and 1B, Pazopanib alone (single-dose or two-doses) administration resulted in a slight tumor growth delay relative to non-treated control mice in both sarcomas, whereas no significant difference between a single dose (−1 h) or two-doses (−8 h and −1 h) pre-administration cohorts was observed. SDRT (a single dose of 30 Gy) yielded a significant tumor response (p < 0.05 vs control) in MPNST3 tumors. Pre-treatment with single-dose or two-doses of Pazopanib prior to SDRT, radiosensitized MPNST3 response and led to enhanced tumor growth delay as compared to SDRT alone (Figure 1A). Notably, single-dose Pazopanib administration resulted in a greater tumor growth delay than in the two-doses Panzopanib administration cohort. A similar result was obtained in JJ012 tumors, SDRT alone (15 Gy) robustly reduced tumor growth, while pre-treatment with Pazopanib (single- or two-dose/s) significantly increased the radiation effect on tumor growth inhibition (Figure 1B, p < 0.05 vs SDRT alone). Although sarcoma tumors are modestly radio-responsive, pre-treatment with Pazopanib radiosensitized both JJ012 and MNPST3 sarcoma tumors and improved their response significantly.
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study
| 99.94 |
MPNST3 tumor tissue was transplanted subcutaneously into the right flank of ICR-SCID mice (A) and JJ012 cells were implanted subcutaneously into the right flank of athymic mice (15 × 106cells/mouse) (B). When the tumors reached the volume of ~150 mm3, the mice were administered a single p.o. dose of Pazopanib (100 mg/kg) at 1 h or two p.o. doses at 8 h and 1 h prior to 30 Gy or 15 Gy SDRT. The tumor volumes were measured for the indicated days. Volumes are mean ± SEM. Arrows indicate the day of SDRT. (*p < 0.05; 30 Gy vs 30 Gy + Paz and 15 Gy vs15 Gy + Paz).
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study
| 100.0 |
Recent studies from our group and others demonstrated that the in vivo apoptotic damage delivered by SDRT to endothelial compartment is critical for SDRT-induced tumor cure, whereas angiogenesis factors (VEGF, FGF etc.) partially reversed this effect [6, 13, 14]. We showed above that Pazopanib enhanced SDRT effect in two human sarcoma tumor models. Next, we determined the effect of the combined therapies on the vasculature. First, we evaluated TEC apoptosis at 6 h post-radiation. JJ012 and MNPST3 tumors were pre-treated with Pazopanib (−1 h, 100 mg/kg p.o.) and irradiated with 15 Gy or 30 Gy accordingly. As shown in Figure 2A and 2B, compared to a baseline TEC apoptosis in non-treated control JJ012 tumors (8.8 ± 2.1%) and MNPST3 tumors (6.1 ± 1.2%), Pazopanib alone generated a modest increase of TEC apoptosis in JJ012 (13 ± 5.6%) and in MPNST3 (12.2 ± 3.2%). While SDRT alone triggered a significant increase in TEC apoptosis (18 ± 3% in JJ012 tumors and 15.7 ± 2.6% in MPNST3 tumors), in the combination treatment with Pazopanib and SDRT there was a further increase in the TEC apoptosis (26.7 ± 2.7% in JJ012 tumors and 21.3 ± 2.3% in MPNST3 tumors), which was significantly higher than each treatment alone. These observations indicated that the combination of Pazopanib and SDRT significantly increased TEC apoptosis in human sarcoma animal models when Pazopanib is administered 1 h before radiation.
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study
| 100.0 |
Pazopanib (100 mg/kg p.o.) was administered to MPNST3 bearing ICR-SCID mice (A, C) and JJ012 sarcoma bearing SCID mice (B, D) and after 1 h tumors were irradiated with 30 Gy and 15 Gy, respectively. Tumors were removed 6 h after irradiation, fixed in 4% paraformaldehyde, and embedded in paraffin. 5-mm sections of tumor specimens were double stained with MECA-32 (red), to detect TEC, and cleaved caspase-3 , to identify apoptotic cells. Data (mean ± SD) are collected from 5 mice per group with 1000 endothelial cells evaluated. Scale bar, 50 μm. (*p < 0.05).
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study
| 100.0 |
Tumor angiogenesis is a critical factor for tumor growth, and MVD is an important parameter for assessing angiogenesis in tumors (14, 15). Subsequently, we quantified the effect of combination of SDRT and Pazopanib on tumor MVD. Figure 2C and 2D show that Pazopanib alone reduced MVD to 56.2% ± 14.5% (vs control) in JJ012 tumors and to 79.5% ± 10.8% (vs control) in MPNST3 tumors. Whereas SDRT alone caused a decrease in MVD in JJ012 to 68.8 ± 23.3% (vs control) and to 84.6 ± 16.8% (vs control) in MPNST3, the combination of SDRT and Pazopanib showed an enhanced decrease in MVD in JJ012 (34 ± 11.8%, vs control) and MPNST3 (62.3 ± 11.8%, vs control). These results indicate that Pazopanib enhances SDRT effect on tumor growth delay by significantly increasing SDRT-induced TEC apoptosis and reducing angiogenesis in these tumors.
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study
| 100.0 |
Subsequently, irradiation with 30 Gy combined with Pazopanib pretreatment of MPNST3 tumors resulted in 50% decrease in perfusion in the tumors 30 minutes post SDRT, compared to untreated tumors (Figure 3, p < 0.01). Treatment with 15 Gy or 30 Gy with Pazopanib and Pazopanib alone caused a 30% decrease. Radiation alone did not cause a decrease in perfusion in this tumor model. These results indicate that the improved tumor growth delay in response to combination of Pazopanib and SDRT treatment are due to enhanced microvascular dysfunction generated by this combination treatment in these sarcoma tumor models. Similar results were obtained in pre-clinical mouse tumor models using SDRT in which the perfusion reduction was accompanied by secretion of ASMase into the systemic circulation within the first hour of radiation (Campagne and Fuks submitted publication).
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study
| 100.0 |
Perfusion was measured by injection of Hoechst 33342 fluorescent dye. Irradiation with 30 Gy combined with Pazopanib pre-treatment (100 mg/kg p.o.) resulted in 50% decrease in perfusion in the tumor 30 minutes post RT, compared to the untreated tumors (*p < 0.01).
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study
| 100.0 |
Accumulated evidence establishes that SDRT-induced TEC apoptosis depends on ASMase/Ceramide-mediated signaling in vivo [6, 14, 23]. SDRT induces rapid translocation of ASMase to endothelial plasma membrane where it generates ceramide and initiates the apoptotic signal. Next, we evaluated whether the effects of combination of Pazopanib and SDRT on the TEC in vivo are mediated via the ASMase activity. JJ012 and MPNST3 bearing mice were treated as described above, and mouse serum was collected at 1 h and 6 h after SDRT. Both Pazopanib treatment and SDRT increased serum ASMase activity at 1 h and 6 h post radiation in both human sarcoma models (Figure 4A and 4B) as compared to baseline control cohorts. Combination of Pazopanib and SDRT induced ASMase activity up to 3.91 ± 0.55 mol/ul/h at 1 h and 2.61 ± 0.13 mol/ul/h at 6 h after radiation, respectively, significantly higher than Pazopanib alone (2.69 ± 0.32 mol/ul/h at 1 h and 1.9 ± 0.24 mol/ul/h at 6 h) or SDRT alone (2.78 ± 0.35 mol/ul/h at 1 h and 2.3 ± 0.11 mol/ul/h at 6 h) in MPNST3 tumors. Similar results were achieved in JJ012 sarcoma model. Combined therapy increased ASMase activity to 3.91 ± 0.45 mol/ul/h at 1 h and 2.6 ± 0.32mol/ul/h at 6 h post-SDRT, significantly higher than Pazopanib alone (2.69 ± 0.45 mol/ul/h at 1 h and 2.77 ± 0.56 mol/ul/h at 6 h) or SDRT alone (1.8 ± 0.6 mol/ul/h at 1 h and 2.29 ± 0.67 mol/ul/h at 6 h). Altogether these data indicate that the combination of SDRT with the short-acting anti-angiogenic agent, Pazopanib, generates a ceramide-driven vascular dysfunction resulting in significant tumor growth delay in two human sarcoma models.
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study
| 99.94 |
MPSNT3 (A) and JJ012 (B) tumors were irradiated with 30 Gy or 15 Gy after 1 h pre-treatment with Pazopanib (100 mg/kg p.o.). Mouse serum was collected at 1 h and 6 h after radiation and ASMase activity was measured by quantifying conversion of (14C)sphingomyelin to the product (14C)phosphocholine. Data is mean ± SD. *p < 0.05 and **p < 0.01.
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study
| 100.0 |
Angiogenic factors (bFGF and VEGF, etc) inhibited SDRT-induced EC apoptosis, although the underlying mechanism remains unclear [13, 14]. In order to demonstrate that the synergistic effect of Pazopanib results from inhibition of VEGF signaling we tested these effects on BAEC in vitro, which has been demonstrated to be an ideal model for radiosensitization to SDRT . As shown in Figure 5A, 100 ng/ml VEGF elicited a rapid activation of VEGFR2 and 2 crucial downstream proteins, ERK and Akt in BAECs. Pazopanib at a low concentration (10 ng/ml) did not repress VEGF-induced phosphorylation of VEGFR2, Akt and Erk, whereas high concentration of Pazopanib (>100 ng/ml) showed a dramatic inhibition effect of all three, and 500 ng/ml Pazopanib led to a complete repression of phosphorylation of these targets. These results indicated that Pazopanib is a potent inhibitor of VEGF signaling in BAECs. Next, we assessed whether Pazopanib administration could induce BAEC apoptosis. Similar to our results with DC101 (antibody against VEGF - R2) , Pazopanib administration showed the best induction of apoptosis in BAEC at 24 h (Figure 5B). Based on these results we pre-incubated the cells with Pazopanib for 16 h and examined a dose response radiosensitization in BAECs. As shown in Figure 5C, a massive increase in BAEC apoptosis (15.8% ± 1.5% at 5 Gy and 28.9% ± 2.2% at 10 Gy) was achieved at 8 h post SDRT, whereas Pazopanib alone (100 ng/ml) induced a very minimal apoptotic effect. Importantly, substantially enhanced EC apoptosis (22.9% ± 2.5% at 5 Gy and 40.8% ± 1.9% at 10 Gy) was generated in the combined condition (Figure 5C), which was significantly higher than with each treatment alone. These results provided strong evidence that Pazopanib has a direct effect on the endothelial cells and synergizes with SDRT in their dysfunction.
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study
| 100.0 |
(A) BAEC were pre-treated with Pazopanib at the indicated doses for 1 h, and stimulated with VEGF (100 ng/ml) for 10 min. Cell lysate was analyzed by western blotting. Endogenous VEGFR2 were immunoprecipitated and analyzed by western blotting against anti-phosphotyrosine antibody. (B) BAECs were exposed to Pazopanib at the indicated dose and time. Cells were fixed, and apoptosis was assessed using bis-benzimide staining. (C) After pre-treatment with Pazopanib (100 nM) for 16 h, BAECs were irradiated at the indicated doses. Cells were fixed at 8 h post-radiation, and apoptosis was assessed using bis-benzimide staining. Each value represents the mean ± SD of duplicate determinations from three separate studies. * p < 0.05.
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study
| 100.0 |
In order to establish that the Pazopanib-induced radiosensitization is mediated via ASMase/ceramide pathway activation, we assessed ASMase activity and ceramide levels in BAEC after Pazopanib treatment in combination with SDRT. As shown in Figure 6A, 10 Gy radiation rapidly triggered an increase in ASMase activity in BAEC from a baseline of 68.34 ± 2.41 mol/mg/h to peak at 90.42 ± 1.84 mol/mg/h at 2 min after radiation, similar to our previously reported results in these cells. Pre-treatment of Pazopanib further elevated ASMase activity up to 101.78 ± 2.6 mol/mg/h at 2 min, which was significantly higher than radiation alone. Combination of Pazopanib and SDRT also enhanced the total ceramide content compared to Pazopanib or SDRT alone treatment (Table 1). Recent studies revealed that C16-ceramide was preferentially associated with stress-induced apoptosis in a variety of cell types [25–27]. In this study, C16-ceramide, the apoptogenic ceramide, level was immediately enhanced after radiation treatment, and increased from a baseline of 72.71 ± 0.47 pmol/106 cells to 112.25 ± 5.88 pmol/106 cells at 2 min and to 127.36 ± 3.04 pmol/106 cells at 10 min (Figure 6B and Table 1). Pazopanib induced a significant increase in the generation of ceramide (243.03 ± 3.65 pmol/106 cells) at 2 min, which was maintained over 10 min (249.48 ± 1.8 pmol/106 cells, p < 0.05% vs SDRT alone). There were no significant differences in the other ceramide species levels in response to Pazopanib or SDRT alone or in their combination. These results indicate that Pazopanib potentiates radiation-induced apoptosis in endothelial cells via modulation of ASMase/ceramide signaling.
|
study
| 100.0 |
BAECs were irradiated at 10 Gy, followed by pre-treatment with Pazopanib for 16 h. Cells were collected at indicated time after radiation and ASMase activity was measured by quantifying conversion of (14C)sphingomyelin to the product (14C)phosphocholine. The result represents the duplicate determinations from 2 experiments. Data is the mean ± SD. * indicated p < 0.05 (10 Gy vs 10 Gy + Paz) (A). After pre-treatment with Pazopanib for 16 h, BAECs were irradiated at 10 Gy. Total lipids were extracted at the indicated time after radiation, and C16-ceramide level was measured by liquid chromatography, electrospray ionization-tandem mass spectrometry. Data is the representative determination from 2 experiments. Data is the mean ± SD. *p < 0.05 and **p < 0.01; 10 Gy vs 10 Gy + Paz (B).
|
study
| 100.0 |
BAEC were irradiated at 10 Gy after pre-treatment with Pazopanib for 16 h. Total lipids were extracted at the indicated time post radiation, and ceramide content was measured by liquid chromatography, electrospray ionization-tandem mass spectrometry. Value is the mean ± SD. Paz: pazopanib; RT: radiation treatment.
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study
| 99.94 |
In this study, the molecular mechanism of combination of anti-VEGF reagent and SDRT on tumor response via amplification of ceramide-driven endothelial apoptosis was investigated in two human sarcoma animal models. Our findings revealed that pre-treatment with Pazopanib prior to SDRT enhances tumor endothelial dysfunction and tumor growth delay in these tumors. The results also confirm that the tissue VEGF confers resistance to radiotherapy, and provide a pre-clinical basis to the use of Pazopanib as an SDRT sensitizer to synergistically improve local tumor response.
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study
| 100.0 |
Emerging evidence indicates that SDRT, such as used in this study, may provide therapeutic benefit to some human tumors, even to those considered resistant to conventional fractionated radiotherapy schemes and chemotherapy. Early clinical studies demonstrated that 24 Gy SDRT alone can locally cure >90% of human cancer regardless of tumor type or size, however this curative effect can be acquired only in cases where normal tissues could be completely avoided [4, 28, 29]. Thus, SDRT use is limited in many clinical settings by close proximity of the tumor to critical normal tissue. According to this principle, the critical dose limitation toxicity for SDRT in multiple clinical settings appears to generally be within the range of 14–16 Gy. Therefore, the challenge is to improve tumor response to lower doses of SDRT, and radiosensitization via temporally constrained use of anti-VEGFR agents may be the answer. Prior work demonstrated that bursts of free radical species, generated by waves of hypoxia/reoxygenation occurring after each radiation exposure, lead to induction of hypoxia-inducible factor-1α (HIF-1α) activity . The up-regulation of HIF-1α elevates overexpression of VEGF and other pro-angiogenic factors, which protect tumor endothelium and confer radioresistance . A recent report from a group at MSKCC showed that a hypoxia-activated chemotherapeutic TH-302 combined with VEGF-A inhibition and RT, an exploratory tri-modality therapy, dramatically enhanced tumor response in preclinical models of sarcoma via increasing DNA damage and apoptosis in endothelial cells and decreasing HIF-1α activity . These results indicate that an anti-VEGF approach may improve the radiosensitization and increase local tumor response. Consistent with our previous publications, our current results further provide direct and clear evidence that anti-VEGF treatment synergistically improved tumor response through radiosensitization.
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study
| 99.94 |
SDRT induced in vivo and in vitro endothelial cell apoptosis via activation of ASMase/ceramide signaling, which contributes to tumor growth delay or cure [6, 9, 14]. Within these studies, SDRT triggered TEC apoptosis as early as 2 h and peaked at 6–8 h post radiation treatment [6, 14]. Our results showed that SDRT drove an increase of in vivo TEC and in vitro BAEC apoptosis at 6–8 h post RT, which supported these findings. Tumor vasculature has been shown to be largely affected by RT depending on the number of fractions, dose rate, and total radiation dose . SDRT can induce rapid apoptosis of TEC, whereas the importance of vascular damage in tumors receiving conventionally fractionated radiation therapy (CFRT: 1.8–2 Gy per fraction) is more controversial [6, 7, 30]. A recent study suggested that 2 Gy irradiation dose induced minimal endothelial cell apoptosis followed by a later increase in vessel diameter, microvascular density and vessel leakiness in normal brain blood vessels . However, few reports so far provided clear proof that CRFT induced TEC apoptosis. Thus, the biological mechanism underlying fractionated radiotherapy may greatly differ from SDRT used in this study, which mainly functions via ASMase/ceramide-mediated vascular dysfunction.
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study
| 99.94 |
As a generic mediator of stress, the ASMase/ceramide pathway has been shown to function on trans-activating pathogenesis of tissue damage in multiple models of human disease . Endothelial cells are particularly sensitive to SDRT-induced apoptosis in vitro and in vivo because they have 20-fold higher levels of secretory ASMase relative to other mammalian cells [5, 10, 35]. Our results showed that SDRT launched ASMase activity in vivo and in vitro, which resulted in a significant increase in endothelial cell apoptosis via enhanced ceramide generation. Although the detailed mechanism by which radiation-induced translocation/activation of ASMase was not addressed here, the accumulated information over the last decade sheds light on this event. Diverse stresses (UV, IR etc) induce ASMase trafficking to the outer leaflet of the plasma membrane, where it converts sphingomyelin to ceramide [9, 36]. This process requires intact microtubules, phosphorylation of Ser508 of ASMase, as well as functional lipid rafts [37–39]. However, the mechanism by which VEGF may repress ASMase activity remains unclear. What is becoming clear from this and our other most recent studies (14) is that anti-VEGF pathway inhibitors synergistically increased SDRT-induced ASMase activity by reversing the effect of VEGF. In addition, several publications revealed that upregulation of ASMase occurred in diverse pathological conditions, and proposed that a significant change of serum ASMase may appear as a novel biomarker in disease [40, 41]. Sathishkumar et al. showed that serum ASMase activity and ceramide content increase following Spatially Fractionated high dose Radiation Treatment (SFGRT) and correlate with the clinical response . Here we also showed that SDRT promoted an increase of ASMase activity in mouse serum at 1 h and maintained high level at 6 h post-SDRT, and an anti-VEGF agent synergistically increased this activity. These results indicate that the changes of ASMase activity can serve as a biomarker when monitoring the delivery of SDRT to cancer patients. We have already confirmed this concept in patients in ongoing clinical trials at MSKCC (Campagne and Fuks, submitted).
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study
| 99.94 |
Activated ASMase converts sphingomyelin to ceramide in the outer plasma membrane and leads to formation of ceramide-rich macrodomains (CRMs), which serve as sites of stress-related specific multiprotein complexes and relay downstream apoptotic signaling [9, 36]. We showed that SDRT induced a rapid increase of the C16-ceramide (Table 1), which is the apoptogenic ceramide species, and Pazopanib synergistically increased the C16-ceramide level. The increase of C16-ceramide contributes to the reorganization of membrane rafts into large signaling platforms, CRMs, which affords a mechanism by which SDRT induces endothelial apoptosis and generates microvascular dysfunction. Recently, increasing studies using LC-MS to determine the changes in specific ceramide species revealed that C16-ceramide was preferentially associated with stress-induced apoptosis in a variety of cell types. C16-ceramide has also been shown to increase the sensitivity of Jurkat T cells and hepatocytes , and human chronic myelogenous leukemia (CML) to Fas-mediated apoptosis. Increases in C16-ceramide levels in Jurkat cells occurred 2 h after initiation of apoptosis by ionizing radiation, whereas no change of C16-ceramide level was observed in RT-resistant cells. In the current study, C16-ceramide levels rapidly increased 2 min after SDRT and remained elevated for more than 10 min. The high radiosensitivity of BAEC may result from the 20–fold higher ASMase level in these cells as compared with other cells in the body. In addition to its crucial role in the formation of CRMs to mediate apoptotic signaling, C16-ceramide may also induce apoptosis by repression of pro-survival pathways. C16-Ceramide has been shown to activate ceramide-activated protein phosphatase leading to the dephosphorylation of p38 and Akt, which may negatively regulate the activities of downstream factors implicated in the regulation of apoptosis [27, 44].
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study
| 100.0 |
Recently, Booth et al. reported that Pazopanib combined with PDE5i or ERBB1/2/4 inhibitor (afatinib) induced tumor cells (fresh PDX isolate of NSCLC) death via ER-induced stress by toxic autophagy and by affecting the chaperone activity in these cells an induction of ER stress-induced autophagy death . The ER stress-induced autophagy death is mediated via the pERK/eIF2α pathway. Furthermore, they also showed that pazopanib and BYL719 combination killed HCT116 cells that express a mutated active K-RAS protein and an activated PI3K-AKT-mTOR signaling pathway indicating that pazopanib-dependent activation of the pERK/eIF2α pathway might be stress- and cell type-specific. Relative to these findings, our study focused on optimizing the effects of RT that induce microvascular dysfunction. Microvascular cells do not overexpress chaperones, a characteristic specific to tumor cells, or other mutation such as K-RAS, nor do they have constitutively active signaling pathways that drive proliferation. Therefore, the pERK/eIF2α pathway-mediated autophagy death may not occur in tumor-associated endothelial cells. The direct impact of SDRT and/or pazopanib on tumor cells, will be investigated in a future study.
|
study
| 100.0 |
The data in this study also indicated that pre-treatment with anti-VEGF agent 1 h prior to SDRT is required for anti-angiogenic de-repression of endothelial ASMase and consequent tumor response. This result supported our previous findings that 1 h prior to RT is the optimal time window for treating tumors using anti-angiogenic agents in combination with SDRT [14, 23]. Notably, this temporal relationship between anti-angiogenic agents and radiation differs from the tumor microvessel normalization hypothesis, which requires at least 24 h to manifest, or constraint of endothelial progenitor recruitment into the damaged site, usually delivered at 24 h prior to irradiation [18, 19]. Although the underlying mechanism remains elusive, given that SDRT increased ASMase activity and ceramide generation just within minutes, it is reasonable to assume that anti-angiogenic agents should be delivered immediately prior to irradiation to de-repress ASMase.
|
study
| 100.0 |
In summary, recent understanding of the effects of the synergistic inhibition of short-acting anti-angiogenic agents and SDRT on tumor endothelium provides new targets for improving local cure of human cancer with radiation. The conclusion from this study, that a single dose of Pazopanib substantially sensitizes tumors to the effects SDRT, indicates this as a highly promising treatment strategy for sarcomas resistant to conventional RT and chemotherapy.
|
study
| 99.44 |
Pazopanib was purchased from GlaxoSmithKline (GSK). Bis-benzimide was purchased from Life Technology (Carlsbad, CA). Anti-VEGFR2 (FLK) was purchased from Santa Cruz Biotech. (Santa Cruz, CA). Anti-Cleaved Caspase3, anti-Meca32, Anti-Akt, anti-Phospho-Akt (Ser473), anti-Erk, anti-Phospho-Erk and anti-GAPDH were purchased from Cell Signaling Technology (Beverly, MA). Protein G Sepharose beads were purchased from GE Health Biosciences GE Healthcare (Piscataway, NJ). [14C-methylcholine] sphingomyelin was purchased from Amersham Biosciences (Piscataway, NJ).
|
other
| 99.94 |
Bovine aortic endothelial cells (BAEC), established from the intima of bovine aorta as described , were maintained in DMEM supplemented with 5% calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine at 37° C in a humidified 10% CO2 chamber. JJ012 cell line and MPNST3 tumors given to us by the Singer lab at Memorial Sloan Kettering Cancer Center cells were maintained in DMEM containing glucose (4.5 g/L), glutamine (2 mM), penicillin (50 U/ml), and streptomycin (100 mg/ml) supplemented with 10% fetal bovine serum in a humidified 5% CO2 chamber.
|
study
| 99.94 |
All the animal experiments were performed according to the guidelines, following a protocol approved by the Institutional Animal Care and Use Committee (IACUC). ICR-SCID mice, 6–8 week-old, were purchased from Jackson laboratory (Bar Harbor, ME); athymic nu/nu mice, 6–8 week-old, were purchased from Envigo (Indianapolis, IN) and housed at the Research Animal Resource Center (RARC) of Memorial Sloan-Kettering Cancer Center. The facility is approved by the American Association for Accreditation of Laboratory Animal Care and is maintained in accordance with the regulations and standards of the United States Department of Agriculture and the Department of Health and Human Services, NIH.
|
other
| 99.94 |
Neurofibrosarcoma (MPNST3) tumor tissue was transplanted subcutaneously into the right flank of ICR-SCID mice and chondrosarcoma (JJ012) cells were implanted subcutaneously into the right flank of athymic mice (15 million cells/mouse). Once tumors reached a size of 100–150 mm3, mice were either treated with SDRT and/or Pazopanib (100 mg/kg, orally). Radiation was delivered using a Philips MG-324 X-ray at 117 cGy/min (50 cm source to skin distance). Mice were lightly sedated with ketamine (0.1 mg/g) and xylazine (0.02 mg/g) and only tumor, surrounding skin and subcutaneous tissues were exposed using a specialized lead jig. Tumor volumes, based on caliper measurements, were calculated daily using the formula of V = (W2 × L)/2 .
|
study
| 99.94 |
ASMase activity was measured by radioenzymatic assay using [14C-methylcholine] sphingomyelin as substrate, as described with minor modifications . Briefly, following treatment with different conditions, cells were washed with ice cold PBS and lysed in PBS containing 0.2% Triton X-100. 2.5 ug cell lysate were incubated with a total 9.5 nmol sphingomyelin substrate mixture including 0.026 μCi[14C-methylcholine] sphingomyelin in reaction buffer (250 mM sodium acetate, pH 5.0 supplemented with 0.1% Triton X-100 and 1 mM EDTA) at 37° C for 2 h. Reactions were terminated by adding 125 ul of mixture of CHCl3:MeOH:HCl, 100:100:1 v/v/v, and the upper phase was removed and quantified by a Beckman Packard 2200 CA Tricarb scintillation counter.
|
study
| 100.0 |
Fluorochrome bis-benzimide trihydrochloride (Hoechst-33258) was used to visualize the morphologic changes of nuclear chromatin in cells undergoing apoptosis as described . Briefly, following treatment BAECs including floating cells were harvested and fixed with 4% paraformaldehyde, washed with phosphate buffered saline (PBS) and stained with 50 μl of 24 μg/ml bis-benzimide trihydrochloride solution for 10 minutes. Apoptotic cells were quantified using an Axiovert S-100 Zeiss epifluorescent wide-field microscope equipped with a DAPI filter set. A minimum of 500 cells was examined per point.
|
study
| 99.94 |
In vivo tumor endothelial cell (TEC) apoptosis was measured by double staining with anti-cleaved caspase 3, to detect apoptotic cells, and the endothelial cell surface marker MECA-32, to identify tumor endothelium. Briefly, tumor specimens were removed after SDRT and/or Pazopanib treatment at the indicated time points, fixed in 4% paraformaldehyde, embedded in paraffin, and 5-μm sections were stained. Red-Green–Blue–merged endothelial cell was counted as an apoptotic cell, and a minimum of 500 endothelial cells were evaluated per point.
|
study
| 99.94 |
After appropriate treatment, cells were incubated at 37° C for the indicated time and reaction was stopped by placing the cells on ice. Floating cells were collected in chilled 13 × 100 mm glass tubes by centrifugation at 500 g for 5 min. Cells were washed once with cold PBS, and lipids were extracted by incubating cells in 0.5 ml of CH3OH for 10 minutes. Cell lysates were then added to the pelleted floating cells. Ceramide levels were analyzed using liquid chromatography, electrospray ionization-tandem mass spectrometry as described previously .
|
study
| 99.94 |
Removed tumor tissues were fixed in 4% paraformaldehyde, and embedded in paraffin. 5-mm sections of tumor specimens were stained with MECA-32 to detect TECs. Microvessel density was quantified using MetaMorph image analysis software and MECA-32+ area was calculated in each section.
|
study
| 99.94 |
Mice were injected with the fluorescent dye Hoechst 33342 (Tocris Bioscience), 20 mg/kg, 30 minutes after irradiation. Mice were sacrificed 2 minutes after injection and tumors were frozen on dry ice in O.C.T. compound (Scigen), cryosectioned and fixed with 4% paraformaldehyde. Tumor sections were imaged using Pannoramic 250 Flash digital slide scanner (3D Histech, Hungary) and fluorescence was quantified using ImageJ software .
|
study
| 99.9 |
Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer-related death with a 5-year survival rate of <5% (Siegel et al. 2016). In contrary to many other forms of cancer the incidence of PDAC is rising and predicted to become the 2nd most common cause of cancer-related death by 2030 (Rahib et al. 2014). Its lethality can be attributed to late diagnosis, almost complete resistance to conventional chemo- and radiotherapy, and a lack of diagnostic biomarkers. Unlike breast or colorectal cancer, the only treatment with curative intent is surgery, for which < 20% of patients qualify; but even then the chance of survival is very low (Lohr 2006). Therefore, PDAC should be treated as a medical emergency (Lohr 2014). Risk factors for PDAC include chronic pancreatitis (CP) and around 5% of CP patients develop PDAC over time (Pinho et al. 2014; Garrido-Laguna and Hidalgo 2015). Both PDAC and CP are characterized by an excessive and reactive stroma, or desmoplasia, which makes it difficult to distinguish between the two conditions (Kloppel and Adsay 2009). The one FDA-approved biomarker for PDAC, carbohydrate antigen 19-9 (CA 19-9), is only useful for prognosis and detection of disease recurrence (Fong and Winter 2012). Therefore there is an urgent clinical need of a non-invasive diagnostic biomarker for PDAC.
|
review
| 99.9 |
Metabolomics is the untargeted analysis of low molecular weight endogenous compounds, metabolites, present in e.g. a defined biological sample such as tissue or a bodily fluid as blood (Fiehn et al. 2000; Fiehn 2002; Spratlin et al. 2009; Dunn et al. 2011). The most widely used analytical platform for metabolomics providing the largest metabolome coverage is liquid chromatography-mass spectrometry (LC-MS) (Want et al. 2007; Patti et al. 2012; Yin and Xu 2014). Given the established connection between cell metabolism and cancer (Hanahan and Weinberg 2011), LC-MS metabolomics has been applied to identify metabolite markers for several cancers (Spratlin et al. 2009; Nordstrom and Lewensohn 2010; Nicholson et al. 2012), including PDAC (Daemen et al. 2015; Rios Peces et al. 2016). Specifically, there have been previous LC-MS based metabolomics efforts to compare PDAC and CP blood samples (Urayama et al. 2010; Fukutake et al. 2015). There is however still no screening test for PDAC available.
|
review
| 99.9 |
In the present study, we have performed LC-MS based metabolomics on blood samples to compare the metabolic profiles of PDAC and CP in two independent cohorts. Both single discriminative metabolites and a panel of metabolites discriminating PDAC and CP were identified. Our findings have potential clinical relevance for early diagnosis of PDAC among CP patients to improve patient survival.
|
study
| 100.0 |
The validation cohort included Swedish PDAC patients (n = 20) and CP patients (n = 31). Plasma samples were supplied by the Karolinska Institutet biobank and had been prepared at different sites following a standardized procedure: whole blood samples were obtained at fasting early in the morning, collected in sodium citrate tubes and immediately placed in −80 °C.
|
study
| 99.94 |
Plasma and serum samples were thawed on ice and 50 µl aliquots were mixed with 150 µl MeOH for protein precipitation. Samples were then centrifuged for 15 min at 15800×g. The supernatant was transferred to a new tube and then evaporated to complete dryness in a vacuum concentrator. Prior to LC-MS analysis, samples were reconstituted in 50 µl MeOH:H2O 1:1 and the two isotopically labelled internal standards phenylalanine (D5) and palmitic acid (D4) (Cambridge Isotope Laboratories) were added at a final concentration of 5 µM.
|
study
| 99.94 |
Metabolites were separated by reversed phase liquid chromatography and detected by electrospray ionization (ESI) mass spectrometry in positive mode. All samples were run in randomized order, randomized by the random number function in Excel (Microsoft, USA).
|
study
| 99.8 |
For the discovery cohort, the analytical platform consisted of a 1200 HPLC-system (Agilent) connected on-line to an LTQ Orbitrap Velos equipped with a HESI probe (Thermo Scientific). 5 µl of each sample were injected onto a Kinetex C18 150 × 2.1 mm column, 2.6 µ, 100 Å (Phenomenex) and the following mobile phases were used at a flow rate of 0.4 ml/min: H2O with 0.1% formic acid (A) and 3:1 acetonitrile:isopropanol with 0.1% formic acid (B). The linear gradient was 0 min, 5% B; 1 min, 5% B; 21 min, 95% B; 26 min, 95% B; 26.5 min, 5% B; 33 min, 5% B. MS data was collected in centroid mode between m/z 80-1200 at an Orbitrap resolution of 60,000 using the following ESI settings: Cap Temp, 350; Sheath Gas Flow, 45; Aux Gas Flow, 15; Source Voltage, 3 kV; S-Lens RF Level, 60%.
|
study
| 99.94 |
For the validation cohort, the analytical platform consisted of a 1290 UHPLC-system (Agilent) connected on-line to a 6550 Q-ToF equipped with a JetStream source (Agilent). 5 µl of each sample were injected onto a Kinetex C18 100 × 2.1 mm column, 2.6 µ, 100 Å (Phenomenex) and the following mobile phases were used at a flow rate of 0.5 ml/min: H2O with 0.1% formic acid (A) and 3:1 acetonitrile:isopropanol with 0.1% formic acid (B). The linear gradient was 0 min, 5% B; 7 min, 95% B; 9 min, 95% B; 9.2 min, 5% B; 11 min, 5% B. MS data was collected in centroid mode between m/z 70-1700 using the following ESI settings: Gas Temp, 300; Gas Flow, 8; Nebulizer Pressure, 40; Sheet Gas Temp, 350; Sheet Gas Flow, 11; Vcap, 4000; Fragmentor, 100; Skimmer1, 45; OctapoleRFPeak, 750. The analysis procedure has also been described previously (Staubert et al. 2015; Lindahl et al. 2016).
|
study
| 99.94 |
The peak heights for the internal standards were continuously monitored during analysis to ensure MS signal stability (Supplementary table 1). In the validation cohort the signal stability was monitored by injection of a pooled QC sample for every sixth sample during the LC-MS analysis. Examples of extracted ion chromatograms are illustrated in Supplementary Fig. 1.
|
study
| 99.94 |
Targeted MS/MS data of a pooled sample was also collected for identification purposes of discriminative features in both cohorts using the ESI settings described above (Supplementary Fig. 2). Samples were analyzed on two different instruments for reasons of instrument availability at the time of receiving the different cohorts to the laboratory.
|
study
| 100.0 |
Discovery raw data files were converted to .cdf format using the file converter tool in Xcalibur version 2.2 (Thermo Scientific). Peak detection, integration and alignment were performed with the open-source software XCMS version 1.30.3 (Smith et al. 2006). The following XCMS settings were used: For peak detection and integration with the centWave algorithm, ppm = 10, snthr = 5, peakwidth = c(5.15), mzdiff = −0.01, prefilter = c(6,100), fitgauss = TRUE; for alignment with Obiwarp, distFunc = “cor”, profStep = 1, grouping parameters bw = 1, mzwid = 0.005, minfrac = 0.5. Isotopes and adducts were not removed.
|
study
| 98.7 |
Sample-wise median normalization (Trezzi et al. 2015) was used to correct for technical variation in e.g. sample preparation and MS signal intensity. Next, a two-step peak filtering approach was applied. First, peaks with an intensity level ≥ 60,000 were retained. Second, only peaks present in at least 75% of samples in at least one group were used for the following statistical analyses.
|
study
| 100.0 |
Fig. 1Schematic overview of data analysis workflow. Univariate (left) and multivariate (right) analyses comparing PDAC and CP were performed in parallel, starting with the abundance filtered discovery cohort data set (step 1). The large number of metabolite features (n = 4578) is due to superfluous features e.g. adducts and isotopes. Following statistical analysis of the discovery cohort (step 2a and b), selected metabolite features were examined manually in the raw data and low quality features (e.g. adducts, isotopes, low intensity peaks) were excluded (step 3). High quality features that were not present in the validation cohort were excluded as well, together with features that could not be identified. The discriminative capacity of the remaining identified features, now referred to as metabolites, was confirmed in the validation cohort by statistical analysis (step 4a and b). Finally, metabolites that were regulated in opposite directions in the two cohorts were excluded (step 5), resulting in three single metabolite markers and a panel of five metabolite markers
|
study
| 100.0 |
Schematic overview of data analysis workflow. Univariate (left) and multivariate (right) analyses comparing PDAC and CP were performed in parallel, starting with the abundance filtered discovery cohort data set (step 1). The large number of metabolite features (n = 4578) is due to superfluous features e.g. adducts and isotopes. Following statistical analysis of the discovery cohort (step 2a and b), selected metabolite features were examined manually in the raw data and low quality features (e.g. adducts, isotopes, low intensity peaks) were excluded (step 3). High quality features that were not present in the validation cohort were excluded as well, together with features that could not be identified. The discriminative capacity of the remaining identified features, now referred to as metabolites, was confirmed in the validation cohort by statistical analysis (step 4a and b). Finally, metabolites that were regulated in opposite directions in the two cohorts were excluded (step 5), resulting in three single metabolite markers and a panel of five metabolite markers
|
study
| 99.9 |
Univariate analysis was performed in the software GraphPad Prism version 6.07 (GraphPad), where PDAC and CP were compared using a Student’s t test with Welch’s correction. In the discovery cohort, metabolite features with p-value > 0.05 were excluded in order to limit the number of features to be examined in the raw data.
|
study
| 99.94 |
For multivariate analysis, data sets were imported into the software SIMCA version 14 (Umetrics). Unit variance scaling was applied to give metabolite features with low and high variation between samples equal importance, and log transformation was applied to shift data towards Gaussian distribution. Principal component analysis (PCA) was initially applied to all variables extracted using XCMS (Supplementary Fig. 3). Subsequently was orthogonal partial least squares-discriminant analysis (OPLS-DA) (Bylesjo et al. 2006) with default SIMCA settings was used to identify metabolite feature patterns discriminating PDAC and CP. The features with the highest discriminatory power between classes were selected based on the variable importance for the projection (VIP) plot. Features significant with a 95% confidence interval, based on the cross- validation, were selected for further analysis. For the discovery cohort, the feature selection was done in the same way with the additional criterion that the VIP-values should be >1.5.
|
study
| 100.0 |
For refined OPLS-DA models, i.e. those built on selected metabolite features only, cross-validation settings were changed from the default 1/7 to 1/5 of samples constituting the internal prediction set. The purpose was to minimize the bias of the refined model towards the selected metabolite features.
|
study
| 99.94 |
The discriminative metabolite features selected in the discovery cohort had to meet several criteria to ensure feature quality, regardless of the method of statistical analysis. First, features were manually examined in the raw data. Isotopes, adducts and poor quality features, e.g. low intensity peaks were excluded following manual inspection of peak shape. This removed features judged by the software to be chromatographic peaks, but which they human eye rapidly can assign to be column bleed or similar artefacts. Second, for the univariate part of the analysis workflow, features were re-integrated in the raw data using the Qual and Quan Browsers in Xcalibur version 2.2 (Thermo Scientific) and their statistical significance was confirmed with a Student´s t test (p < 0.05). Third, ion chromatograms of the selected features were extracted from the validation cohort raw data files using the software MassHunter Qual version B.06.00 (Agilent); discovery cohort features that were either not detected or of low quality in the validation cohort were excluded. Fourth, unidentifiable metabolite features were excluded.
|
study
| 100.0 |
Peak areas of the identified metabolites that met quality control criteria were integrated in the validation cohort raw data files using the “Agile” setting in MassHunter Qual. The median normalized peak area values were used for further uni- and multivariate analyses to confirm the results from the discovery cohort.
|
study
| 99.94 |
Accurate mass measurements were subject to database searches in the public databases METLIN (Smith et al. 2005) and Human Metabolome Database (Wishart et al. 2013) as well as an in-house library comprising 384 synthetic standards. Database hits were then confirmed by retention time match (in-house library only) and MS/MS spectral match from pooled samples (Supplementary Fig. 2). In a few cases additional synthetic standard compounds were acquired and analyzed on the same platform to confirm compound identity.
|
study
| 100.0 |
After abundance peak filtering, the discovery cohort data set contained 4578 metabolite features including isotopes, adducts and low quality features (Fig. 1). 254 of these features had a p-value < 0.05 as determined by Student’s t test with Welch’s correction. After (i) raw data examination, (ii) re-integration and confirmation of statistical significance, (iii) detection in validation cohort and (iv) feature identification, 17 metabolites remained. 11 of these metabolites had a p-value < 0.05 (Student’s t test) in the validation cohort as well. Fold-change calculations showed that the eight metabolites with the lowest intensity were down-regulated in PDAC compared to CP in the discovery cohort, as opposed to up-regulated in PDAC compared to CP in the validation cohort (Fig. 2). These eight metabolites were phospholipids. After exclusion of the phospholipids, the three metabolites glycocholic acid, hexanoylcarnitine and N-palmitoyl glutamic acid remained as single discriminative markers for PDAC compared to CP (Fig. 3). In the discovery cohort, applying an FDR cut-off of 5% to the 254 metabolites did not yield any significant metabolites. However the manual inspection and re-integration together with the validation in an independent patient cohort strengthens the findings.
|
study
| 100.0 |
Fig. 2Fold-change comparison between cohorts. Following confirmation of metabolite significance in the validation cohort (Fig. 1, step 4a and b), fold-change calculations revealed that eight metabolites were regulated in opposite directions in the discovery- and validation cohorts. All eight metabolites were phospholipids that were down-regulated in PDAC compared to CP in the discovery cohort, but up-regulated in PDAC compared to CP in the validation cohort. Consequently all phospholipids were excluded as potential single metabolite markers
|
study
| 100.0 |
Fold-change comparison between cohorts. Following confirmation of metabolite significance in the validation cohort (Fig. 1, step 4a and b), fold-change calculations revealed that eight metabolites were regulated in opposite directions in the discovery- and validation cohorts. All eight metabolites were phospholipids that were down-regulated in PDAC compared to CP in the discovery cohort, but up-regulated in PDAC compared to CP in the validation cohort. Consequently all phospholipids were excluded as potential single metabolite markers
|
study
| 100.0 |
Fig. 3Single metabolite markers discriminating PDAC and CP. After identification of significant features in the discovery cohort (n = 67), feature quality control and confirmation of significance in the validation cohort (n = 51), three single metabolite markers for PDAC compared to CP remained. All three were up-regulated in PDAC. Statistical test: Welch’s unequal variances t test. Box plot settings: Range, minimum to maximum value; line at median
|
study
| 100.0 |
Single metabolite markers discriminating PDAC and CP. After identification of significant features in the discovery cohort (n = 67), feature quality control and confirmation of significance in the validation cohort (n = 51), three single metabolite markers for PDAC compared to CP remained. All three were up-regulated in PDAC. Statistical test: Welch’s unequal variances t test. Box plot settings: Range, minimum to maximum value; line at median
|
study
| 100.0 |
259 of the 4578 metabolite features in the discovery cohort (Fig. 1, step 2b) were selected as potential markers for PDAC as determined by the VIP-plot in the initial OPLS-DA model (Fig. 4a). 19 metabolites remained after feature quality control, detection in validation cohort and metabolite identification. These 19 metabolites were significant in the validation cohort OPLS-DA model (Fig. 4b). A comparison of the loading scatter plots for the discovery- and validation cohort OPLS-DA models showed that a group of 14 metabolites was regulated in opposite directions in the two cohorts (Fig. 4c, d). As in the univariate analysis, all 14 were phospholipids that were down-regulated in PDAC compared to CP in the discovery cohort but up-regulated in PDAC in the validation cohort.
|
study
| 100.0 |
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