Oryza Sativa L.) are obviously lower than those of superior spikelets, especially under abiotic stress. However, different responses to heat stress are seemed to show between the superior and inferior spikelet, and this response is scarcely documented that the intrinsic factors remain elusive. In order to reveal the mechanism underlying, two rice plants with different heat tolerance were subjected to heat stress of 40°C at anthesis. The results indicated that a greater decrease in fertility and kernel weight was observed in superior spikelets compared to inferior spikelets. This decrease was primarily ascribed to their different organ temperatures, in which the temperature of the superior spikelets was significantly higher than that of inferior spikelets. We inferred the differences in canopy temperature, light intensity and panicle types, were the primary reasons for the temperature difference between superior and inferior spikelets. Under heat stress, the fertility and kernel weight of superior and inferior spikelets decreased as the panicle numbers per plant were reduced, which was accompanied by significantly increasing the canopy temperatures. Thus, it was suggested that the rice plant with characteristic features of an upright growth habit and loose panicles might be more susceptible to heat stress resulting from their higher canopy and spikelets temperatures.In general, the fertility and kernel weight of inferior spikelets of rice ( Oryza Sativa L.) can be classified as superior spikelets and inferior spikelets, in which the former are located on apical primary branches, while the latter are located on proximal secondary branches  were carried out at the Fuyang farm of the China National Rice Research Institute, Hangzhou, Zhejiang Province, China, from March to October in 2014. Each pot was filled with 15 kg of paddy soil blended with 20 g of compound fertilizer (N:P:K = 15:10:14). Two rice genotypes differing in heat resistance, i.e., V20 (heat resistance) and Zhong9 (heat susceptible), were directly sown in pots with eight seedlings per pot after germination. The seedlings at the 5.0–5.5 leaf stage were thinned to three leaves and grown outdoors until heat stress was imposed. The rice plants were subjected to heat stress of 40°C for 7 h from 9:30 to 15:30 every day from anthesis for 5, 10, and 15 days. The temperature was monitored and regulated by an automatic temperature control system with night and day temperatures of 30 and 40°C under heat stress, while the temperature was set at 24 and 33°C  in another greenhouse as controls. Both heat stress plants and the controls were exposed to normal sunlight. Six panicles (SP) were kept on each rice plant before heat stress was imposed, and about 1–3 small tillers were removed in each rice plant. However, to reveal the effect of the canopy temperature on the fertility and kernel weight in the superior and inferior spikelets, another two plant densities were subjected to heat stress for 10 days via controlling the panicles for each rice plant, including four panicles per plant (FP), and two panicles per plant (TP). In these two treatments, the main panicle and bigger panicles were kept, and the others were removed before heat stress was imposed. During the temperature treatment, the organ and canopy temperature, pollen viability, pollen numbers and pollen germinating on the stigma, reactive oxygen species (ROS), soluble sugars, starch branching enzyme activity, and phytohormones of superior and inferior spikelets were determined. Superior and inferior spikelets were sampled according to the methods of TM S65 system  with a wide-angle camera lens (18 mm IR-LENS). The spectral range of this camera was 7.5–13 mm, and the detectable temperature range was –40 to 1,500°C. Temperature differences of less than 0.06°C  could be detected with this camera. The camera was set up 0.5 m away from the rice plants. A black cloth was used to minimize the interference from other substances when recording the temperature of superior and inferior spikelets. The data were analyzed with Therma CAM Researcher Pro 2.7 software .The temperatures of superior and inferior spikelets of rice with or without flag leaves were determined from 9:30 am to 10:30 am using an FLIR ThermaCAMA meteorological automatic recorder  was placed in the middle of the rice plant populations to record the canopy temperature, relative humidity, and illumination intensity. For each rice genotype, two different vertical heights based on the canopy height in which superior and inferior spikelets were located were established. The temperature, relative humidity, and illumination intensity were recorded every 3 min.The superior spikelet and inferior spikelet were collected on day 2–4 of heat stress treatment, and their relative water content (RWC) were determined according to the equation RWC = 100(FW – DW)/(TW – DW), where FW was fresh weight, DW was dry weight and TW was turgid weight. Turgid weight was determined after immersing the sample in the distilled water for 24 h at 48°C in darkness; dry weight was measured after oven-drying the sample for 48 h at 85°C.–2s–1; ambient CO2 (450 μmol mol–1); 500 μmol s–1 flow speed; and temperatures according to the treatment. The spikelets were kept in the leaf chamber of the instrument for 10 min. The spikelet areas were estimated by SC-G rice grain appearance quality image analysis system developed by Hangzhou WSeen Detection Technology Co., Ltd, China  under the following conditions: photosynthetic photon flux density of 1200 μmol md, China .2). Pollen grains were removed from the anthers of the florets, placed into a drop of KI/I2 on a glass slide, and observed and photographed under a light microscope .Pollen viability was determined using the method of –1NaOH for 6–10 min at 56°C, and then stained in 0.1% (w/v) aniline blue solution for 24 h. The pollen numbers and pollen germinating on the stigma were observed and photographed at 350 nm with a fluorescence microscope .More than 50 flowering spikelets were collected at 10:00–11:00 am on day 4 of the heat stress treatment. The pollinated stigmas were fixed in Carnoy’s fixing reagent . The samples were then washed with water, incubated in 10 mol LFor visualization and analysis of stigma ROS, the oxidation-sensitive probe DCFH-DA was used, as previously described by 3, and ZR (zeatin riboside), were provided by The superior and inferior spikelets were collected on day 14 after heat stress to determine the soluble sugar concentrations, starch branching enzyme activities, and phytohormones. The concentration of soluble sugar was measured using the phenolsulfuric acid method . Frozen At maturity, heat stressed and control rice plants were harvested, dried at 50°C for 48 h, and then the fertility and kernel weight of superior and inferior spikelets were determined.Data were processed with SPSS 11.5 for Windows, and each value of mean and standard error in the figures represent four replications (unless otherwise stated). Tukey’s least significant difference (LSD) at a probability level of 5% was used to compare the differences between treatments and genotypes.Figure 2). The fertility of inferior spikelets were significantly higher than those of superiors in both rice genotypes when the rice plants were subjected to heat stress from anthesis for 5, 10, and 15 days. It is worth noting that a remarkable reduction in spikelet fertility was attained as the panicle numbers were reduced under heat stress for 10 days, especially for the inferior spikelets. The highest fertility of inferior spikelets was observed in the rice plants with SP; followed by those with FP (four panicles) and TP (two panicles).Undoubtedly, the superior spikelets of rice exhibited greater fertility than inferiors at normal temperature, however, the results were reversed under heat stress at anthesis . Nevertheless, for those rice plants subjected to heat stress for 5, 10, and 15 days, a greater reduction was also observed in the superior spikelets than inferiors compared with their respective controls. This effect diminished as the panicle numbers per plant decreased, especially for inferior spikelets. Compared with controls, higher decreases were observed in those rice plants with FP or TP than those with SP under heat stress.Similar to the changed pattern of spikelet fertility, the kernel weight of superior spikelets was also significantly higher than that of inferior spikelets in both control group genotypes . Approximately 2.40 and 2.36°C temperature differences were shown between the superior and inferior spikelets of V20 and Zhong9, respectively, under control conditions , while they increased to 4.22 and 4.28°C, respectively, under stress . Surprisingly, temperature differences over 7.0°C between the superior and inferior spikelets was also observed in both genotypes (data not shown).Regardless of natural conditions or heat stress, the temperatures of superior spikelets were always significantly higher than those of inferiors for both rice genotypes , while they only increased to 2.03 and 2.54°C under heat stress . Generally, the spikelet temperatures increased after removing the flag leaves, especially for inferior spikelets.The temperature differences between superior and inferior spikelets were reduced if the flag leaves were removed. Temperature differences between the superior and inferior spikelets in V20 and Zhong9 under control conditions were approximately 1.75 and 1.27°C, respectively . When the rice plants were subjected to heat stress of 43–44°C , obvious temperature differences were shown between those spikelets located in A and B, which were at the same canopy height, in both genotypes. However, no remarkable difference was observed among those spikelets located in A, C, and D with different canopy heights. These results indicated that, in this case, the spikelet temperature was determined by the panicle types rather than the canopy heights or positions.Interestingly, the temperature differences existed between the superior and inferior spikelets with or without the flag leaves under heat stress, although the difference in the former was higher than the latter . When the rice plants were subjected to the heat stress of 40 to 41°C, the mean air temperature differences between the canopies where the superior and inferior spikelets located were increased to 3.2 and 2.4°C in V20 and Zhong9, respectively . Nevertheless, this difference was reducing as the panicle numbers per plant were decreased. There were only 1.5 and 1.1°C temperature differences shown in those rice plants with FP and TP, respectively . Additionally, the illumination intensity above the canopy was also obviously higher than that below under heat stress . In contrast, lower relative humidity was shown in the former than the latter .The air temperature within the canopy decreased as the canopy height was reduced either under natural conditions or heat stress (data not shown). Under a natural condition with a 33–34°C air temperature, the mean temperatures of the canopy in which the superior spikelets located were approximately 32.7 and 33.2°C, respectively, in the rice genotypes V20 and Zhong9, while those of the inferiors were approximately 30.4 and 30.9°C . Nevertheless, little difference in transpiration rates was observed between the superior and inferior spikelets whether under natural condition or heat stress except for the Zhong9 under heat stress . The transpiration rate of the superior spikelets was obviously higher than that of inferiors of Zhong9 under stress condition. Surprisingly, the RWC of the spikelets was not determined by their positions or temperature settings because no difference was shown between the superior and inferior spikelets either under control condition or heat stresses in both genotypes .The spikelet transpiration rates were significantly increased in both genotypes under heat stress compared to their respective control, particularly in Zhong9 . Heat stress caused little effect on the pollen viability of superior spikelets, but significantly decreased that of inferior spikelets of V20. The pollen viability of Zhong 9 was not affected by heat stress that no obvious difference showed between the heat stress treatment and normal temperature treatment . Furthermore, there was no significant difference in pollen viability shown between the superior and inferior spikelets under either natural condition or heat stress.For the genotype V20, higher pollen viability was observed in superior spikelets than inferiors under both natural condition and heat stress, in particular the latter . Surprisingly, pollen germination was found on the stigma of superior and inferior spikelets under both natural and stress conditions.Heat stress obviously decreased the pollen number on the stigma of superior and inferior spikelets in both rice genotypes, especially in the superior spikelets. We found that a remarkable decrease was shown in superior spikelets compared to inferiors under heat stress . Nevertheless, ROS were induced by heat stress and were significantly increased in both superior and inferior spikelets compared to their respective controls. In sum, the relative value of ROS in superior spikelets was obviously higher than that of inferior spikelets under heat stress.In terms of stigma ROS values, no obvious difference was observed between the superior and inferior spikelets under natural conditions in both rice genotypes . Under natural condition, higher starch branching enzyme activity was observed in superior spikelets than inferiors, whereas that of the former was significantly lower than the latter when encountering heat stress in both genotypes.Heat stress at anthesis significantly decreased the starch branching enzyme (Q enzyme) activity of superior and inferior spikelets in both genotypes, especially in the former . When rice plants were subjected to heat stress, the soluble sugar of inferior spikelets was obviously higher than that of the superior spikelets.There was no obvious difference in soluble sugar between the superior and inferior spikelets in both genotypes under natural condition (Table 1). Under natural condition, the IAA concentration of superior spikelets was 66.1 and 39.5% higher than those of inferiors in V20 and Zhong9, respectively, while the difference was reduced under heat stress. Only a 14.7% higher concentration of IAA was found in superior spikelets than inferior spikelets in Zhong9 under heat stress, while it changed to a higher IAA concentration in the inferior spikelets than superiors in V20. Heat stress significantly decreased the ABA concentration at anthesis, in which a higher decrease was shown in the inferior spikelets of V20, whereas for Zhong9 it was shown in the superior spikelets. The GA3 concentration of spikelets in V20 was not affected by heat stress, and no obvious difference was demonstrated between the superior and inferior spikelets. Heat stress significantly decreased the GA3 concentration of superior spikelets in Zhong9, whereas a great increase was shown in the inferior spikelets under heat stress compared with that under natural condition.The phytohormones, including ZR, IAA, ABA, and GA3, were determined on day 14 of the heat stress treatment. Heat stress caused little effect on ZR, and there was no obvious difference between superior and inferior spikelets under either natural condition or heat stress . This suggested that heat stress at anthesis was more damaging to the superior spikelets than the inferior spikelets. Obviously, this finding was different from those previous results that abiotic stress, including drought, heat and low temperature stress, caused more injury to the inferior spikelets than the superiors . The temperatures of superior spikelet were 2.40 and 2.36°C higher than those of inferior spikelet in V20 and Zhong9 under natural condition, respectively, while the temperature difference were increased to 4.22 and 4.28°C under heat stress . This means that when the temperatures of superior spikelets reached 36.0°C, the inferior spikelet temperature might be less than 33°C. In this case, there is no doubt that superior spikelets might suffer more stress damage than the inferior spikelets.In the present study, a higher decrease in fertility and kernel weight were observed in superior spikelets than inferiors under heat stress . The results agreed with Figures 6G,H), which can exacerbate the temperature differences between the superior and inferior spikelets. We observed that the spikelet temperature could obviously increase by approximately 3–4°C when exposed to direct sunlight (data not shown). This finding was supported by the results of Many factors, such as the organ size, shape, location within the canopy and cooling ability as well as the environment, i.e., the air temperature, relative humidity, wind speed and radiation, are involved in mediating the spikelet temperature . These fFigure 4). Because there was no obvious difference in the transpiration rates between the superior and inferior spikelets under both natural and stress conditions , which played an important role in reducing the plant organ temperatures . Interestingly, this stress caused little effect on the pollen viability and pollen germination on the stigma, which was obviously different from previous results that required further study. Thus, stigma ROS might be the main factor responsible for the higher sterility in the superior spikelets than inferiors, possibly because higher ROS were shown in the pistils of superior spikelets , which could disturb the pollen tube entering into the ovule. In addition, the flowering time might be another important factor. It is well-known that the superior spikelets flower earlier, thus suffering heat stress first. These sterile spikelet increases would benefit those spikelets that flowered later, including the inferior spikelets, because the competition for assimilation exists between the superior and inferior spikelets . Accordingly, the spikelet fertility and kernel weight decreased as the canopy temperature increased under heat stress, especially the inferior spikelets . These findings provide a reference for the ideotype breeding of rice with heat resistance, which has been considered to be effective for breaking the yield ceiling of the irrigated rice crop (We observed that the canopy temperatures increased when reducing the panicle numbers per pot, confirming an obvious relationship existed between the canopy temperatures and the plant architecture (ice crop . The ricice crop , while sice crop , the difice crop . Furtherice crop .Heat stress occurring at anthesis caused more damage to the superior spikelets than the inferiors of rice, which was mainly ascribed to their different organ temperatures. Three factors, including canopy temperature, illumination intensity, and plant type, were involved in mediating the organ temperatures of superior and inferior spikelets. The temperature and illumination intensity within the canopy where the superior spikelets located were significantly higher than that where the inferior spikelets were located. Additionally, the three apical primary branches the superior spikelets located on were always loose, while the three base primary branches the inferior spikelets located on were compacted. Accordingly, the contact area of the superior spikelets to air was much greater than those of inferior spikelets, suggesting the former absorbed more heat energy than the latter. Heat stress occurring at anthesis significantly increased spikelet sterility, and higher reduce were shown in superior spikelet than inferior, which was mainly ascribed to the remarkable decrease in pollen numbers on the stigma, as well as an increase in ROS, especially for the superior spikelets. The obvious decrease in kernel weight of superior spikelets induced by heat stress resulted from their lower Q enzyme activity and IAA content. Additionally, the spikelet fertility and kernel weight decreased as the panicle numbers per plant were reduced under stress, especially for inferior spikelets, which was mainly due to their higher canopy temperature. This finding suggested that those rice plants with characteristic features of upright growth habits and maintenance of an erect position were more susceptible to heat stress due to their higher canopy temperatures.Conceived and designed the experiments: GF, QJ, and LT. Performed the experiments: GF, BF, CZ, and XiaZ. Analyzed the data: YY and TC. Contributed reagents/materials/analysis tools: XY, XiuZ, and GF. Wrote the paper: GF and BF.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The application of microbial inoculants (biofertilizers) is a promising technology for future sustainable farming systems in view of rapidly decreasing phosphorus stocks and the need to more efficiently use available nitrogen (N). Various microbial taxa are currently used as biofertilizers, based on their capacity to access nutrients from fertilizers and soil stocks, to fix atmospheric nitrogen, to improve water uptake or to act as biocontrol agents. Despite the existence of a considerable knowledge on effects of specific taxa of biofertilizers, a comprehensive quantitative assessment of the performance of biofertilizers with different traits such as phosphorus solubilization and N fixation applied to various crops at a global scale is missing. We conducted a meta-analysis to quantify benefits of biofertilizers in terms of yield increase, nitrogen and phosphorus use efficiency, based on 171 peer reviewed publications that met eligibility criteria. Major findings are: (i) the superiority of biofertilizer performance in dry climates over other climatic regions ; (ii) meta-regression analyses revealed that yield response due to biofertilizer application was generally small at low soil P levels; efficacy increased along higher soil P levels in the order arbuscular mycorrhizal fungi (AMF), P solubilizers, and N fixers; (iii) meta-regressions showed that the success of inoculation with AMF was greater at low organic matter content and at neutral pH. Our comprehensive analysis provides a basis and guidance for proper choice and application of biofertilizers. The current alarming rate of decline of earth's natural resources, particularly of the reserves of rock phosphate and fossil fuel, is of great concern for the future of agriculture, particularly in developing countries , help to access nutrients such as phosphorus (P) and N from organic fertilizers and soil stocks, improve drought tolerance, improve plant health or increase salt tolerance Vessey, . The effHere, we conducted a quantitative evaluation of the pertinent literature in the form of a meta-analysis. Its objective was to quantify the effect of biofertilizers on the performance indicators crop yield and P and N nutrient use efficiencies.The following hypotheses were addressed: (i) across all studies, biofertilizer show a significant positive effect on crop yield and nutrient use efficiency; (ii) there is a difference in biofertilizer response between categories of crops; (iii) climate is a major factor for the constituency of soil biodiversity, soil fertility and soil carbon content, and thus the performance of biofertilizers; (iv) P availability is a limiting factor in many soils. P levels are expected to influence activity and thus effectivity of biofertilizers. Especially phosphate-solubilizing bacteria and AMF are expected to be affected by P levels.n) to calculate the different use efficiencies and effect sizes. When fertilizer was applied the amount and type of fertilizer was required to calculate nutrient-use efficiencies for phosphorus (P) and nitrogen (N). Field trials were not included when soils were previously fumigated or heat sterilized to obtain a control without soil biota, because nutrients may be released, soil microbial community disturbed and inoculation success put at risk  were searched for between May 2015 and February 2016 in Web of Science by Thomson Reuter, Scopus by Elsevier and Google Scholar with the following keywords “biofertilizer OR biofertiliser OR microbial inoculants.” Only studies using data from field trials to more closely reflect real farming practices and providing separate data for each treatment and written in English language were selected. Studies were only included when they had conducted pairwise comparison between the application of a biofertilizer to a non-treated control under the same pedo-climatic conditions , and if the biofertilizers had been tested under the same input level of inorganic and organic fertilizers as the paired non-inoculated control. Studies had to report the treatment mean of yields, its standard deviation (SD) and number of replications (One hundred and seventy-one studies (see study list in Supplementary Data Sheet http://plotdigitizer.sourceforge.net) was used. The data was structured after biofertilizers, crops and climate. Tables 4 summarize the characteristics of crop and climate categories for the number of included studies, amount of fertilizer applied and climate representation. pH was usually given as measured in water. If pH was measured in CaCl2, conversion was calculated . Selection bias was assessed with funnel plots , which was estimated according to Nelson and Sommer  was 15.2%, for legumes SD 5.5%, for melon and water melon SD 35.9%, for vegetables SD 11.2%. For maize (SD 10.6%), cotton (SD 14.0%), rice (SD 14.18%), mustard and rapeseed (SD 10.2%) values were averaged within each type of crop. Average of all were applied for anise, fennel, dill, sesame, sunflower, coriander, garlic, ryegrass, turmeric, silage maize, potato, sugarcane SD 12.0%. The standard deviation in yield as a percentage was used to estimate the error in PUE and NUE.It cannot be excluded that there was a certain publication bias within the results. In order to find out whether there was a publication bias in the meta-analysis “funnel plots” were used to detect a possible publication bias. The trim and fill method was used to help interpretation as proposed by Duval and Tweedie ,b and DuOur comprehensive meta-analysis with studies from all over the world Figure  revealedAveraged across all biofertilizer categories, yield was increased the most in dry climates (+20.0 ± 1.7%), followed by tropical climates +14.9 ± 1.2%), oceanic climates (+10.0 ± 3.7%), and continental climates (+8.5 ± 2.4%) , hydrogen cyanide (HCN)]. However, in a separate analysis we found no general superiority to mono inoculation (multi inoculation 15.5 ± 1.4% vs. mono inoculation 16.9 ± 1.3% yield increase). P solubilizers and AMF are most successful at the low levels of plant available P prevalent in soils of tropical regions. Biofertilizers were best in both dry and humid tropics. We also found a decrease in yield response for P solubilizers and even more for AMF with increased soil organic matter Figure , which iThere is circumstantial evidence why legumes were most responsive to biofertilizers across all effect sizes. Biofertilizers applied to legumes consisted in 12% of all included studies of rhizobia, which were selected to build compatible symbioses with their host plants, but rhizobial inoculum is already present in many soils anyways. Legumes have evolved specific symbioses with N fixing rhizobia but require also other nutrients; reportedly the phosphorus requirement of nodules is up to three times higher than the needs of the surrounding roots (Vadez et al., We have analyzed three different effect sizes each giving a different perspective on the success of biofertilizers. It was found that dryland agriculture can benefit most from biofertilizers. Due to climate change, in the future there will be even more dryland areas globally. Biofertilizers are thus a promising option for sustainable agriculture. In the future, pretests of the soil community may predict the competitive chance of biofertilizer in a specific soil and help to efficiently produce adapted biofertilizers for each specific application.LS, AG, PM, TB, MM, AM, and NM: Designed research; LS: Performed research and analyzed data; LS, PM, AM, TB, and NM: Wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The objective of this study was to investigate the in vivo effect of bioactive composites with poly(lactic acid) (PLA) or polycaprolactone (PCL) as the matrix, reinforced with bioplastic flax fibers, on the surrounding muscle tissue.Materials of pure PLA and PCL and their composites with flax fibers from genetically modified plants producing poly-3-hydroxybutyrate  and unmodified plants  were placed subcutaneous on the M. latissimus dorsi for four weeks.The analysis of histological samples revealed that every tested material was differently encapsulated and the capsule thickness is much more pronounced when using the PCL composites in comparison with the PLA composites. The encapsulation by connective tissue was significantly reduced around PCL-transgen and significantly increased in the cases of PLA-transgen and PLA-wt. In the collected muscle samples, the measured protein expression of CD45, lymphocyte common antigen, was significantly increased after the use of all tested materials, with the exception of pure PCL. In contrast, the protein expression of caveolin-1 remained unchanged after treatment with the most examined materials. Only after insertion of PLA-wt, a significant increase of caveolin-1 protein expression was detected, due to the improved neovascularization.These data support the presumption that the new bioactive composites are biocompatible and they could be applicable in the medical field to support the regenerative processes. Large bone defects often require bone-graft implantation to promote bone regeneration. Although autograft remains the gold standard for bone repair, the harvesting of autologous bone is limited and for some patients the usage can even be associated with complications . The useBiodegradable synthetic polymers, such as polycaprolactones , poly(lactid acid) (PLA), polyanhydrides (PAHs), poly-3-hydroxybutyrate , poly(propylene fumarate), poly(glycolic acid) (PGA), and associated copolymers are interesting for wide use as matrix or components for scaffolds in tissue engineering and regenerative medicine, due to their various mechanical and physical properties, degradation times, or modes of degradation , 5.Numerous studies have shown the usefulness of P3HB for bone regeneration, and they have also indicated that the degradation products of P3HB are phagocytosed by macrophages and osteoclasts; they show no toxicity and may even stimulate the osteoblasts in a positive manner , 7. P3HBIn order to achieve better mechanical properties, similar to bone tissue, polymer composite materials can be reinforced with natural fibers. It has been shown that the application of such composites as medical devices was beneficial regarding the biocompatibility in the human body compared to single materials such as polymers, ceramics and metals .Natural fibers have the advantage that they are renewable resources and have marketing appeal. It could be proven that some natural fibers prevent stress shielding and increase bone remodeling , 17. FurIt has been shown that P3HB can be successfully synthesized in transgenic plants such as cotton, tobacco, potato or flax , 28. In Recently, flax plants were transformed with three bacterial genes responsible for the synthesis of P3HB . It coulUsing these transgenic bioplastic flax fibers embedded in PLA or PCL matrix, a new generation of biodegradable and bioactive composites was prepared and initially analyzed . A good The question was whether the enrichment of polyesters with flax fibers affected intensified inflammatory reaction in another tissue than bone. In a previous study, we could not detect any enhanced inflammation after insertion these materials on the cranial bone of rat .Thus, thFor the preparation of the composites, two kinds of matrix, PLA and PCL, obtained from Biomer (Germany), were used. The polymers were reinforced with a 20% content of flax fibers (either wt- unmodified flax fibers or transgenic flax fibers which contained P3HB). Pulverized combed flax fibers were mixed with PLA or PCL granules at 170°C. The mixture was mechanically pressed into sheets. These were cut into smaller pieces and heat-pressed between Teflon sheets, 30–60s at 175°C for the PLA composite or 100°C for the PCL composite, to produce final composite sheets of 0.2 mm thickness , 35, 37.A previous study of prepared composites on mechanical analysis showed improved stiffness and a decrease in tensile strength. Furthermore, bacteriostatic and platelet anti-aggregated properties of these new materials could be proven .n = 44). The approvals for all surgical and experimental procedures were issued by the Animal Welfare Committee of the State Government of Mecklenburg-Western Pomerania (LALLF M-V/TSD/7221.3-1.1-094/11). The surgical procedure was performed using a fixed protocol established by Gredes et al., 2010 [Experiments were performed on 2-month old Lewis 1A rats of both sexes  pure PLAgroup 1  biocomposite of PLA and flax fibers containing P3HBgroup 2  biocomposite of PLA and unmodified flax fibersgroup 3  pure PCLgroup 4  biocomposite of PCL and flax fibers containing P3HBgroup 5  biocomposite of PCL and unmodified flax fibersgroup 6  animals without any treatment.group 7 . The sections were stained at first with hematoxylin/eosin (H.E.) for the assessment of the encapsulation of test materials in surrounding soft tissue. The evaluation of the granulo-fibrous connective tissue capsule around the composites and division in grades was done as described by Miura et al. .2O2 were used for 10 minutes. After retrieval of specimens, they were rinsed with distilled water and TBS and thereafter incubated with the primary antibodies in a humid chamber . The visualization of the bound caveolin-1 and CD45 antibodies was done using a New Fuchsine alkaline phosphatase substrate protocol. All sections were then lightly counterstained with hematoxylin and cover-slipped. For negative controls the primary antibody was replaced by PBS. The determination of antigen level was carried out using a blind test, which was conducted at the same time with identical staff, equipment and chemicals. For quantification of the protein expression levels, 5–10 digital pictures with a magnification of x200 were randomly taken in different areas of each section . For all sections, the quantity of pixels that had a positive reaction for cav-1 and CD45 was assessed using cell^F Ink software  as described by Kunert-Keil et al., [For immunohistochemical staining, antibodies against CD45 (lymphocyte common antigen) as well as caveolin-1  were used. For that, the specimens were firstly deparaffinized, rehydrated, rinsed for 10 minutes in tris-buffered saline (TBS) and incubated in citrate buffer in a water bath at 95–99°C for 40 minutes. The slides were cooled down for 20 minutes at room temperature. In order to remove endogenous peroxidase, methanol and 3% Hp < 0.05 was set as the level of statistical significance.All data were analyzed using SigmaPlot software  and statistically analyzed using Student’s unpaired t-test  or Mann-Withney-U rank sum test . Data are shown as mean ± SEM, and Histological specimens showed 4 weeks after insertion of the test materials a well-vascularized connective tissue between the back muscle and the inserted materials. Furthermore, the inserted materials were covered by a compact capsular structure composed of granulo-fibrous connective tissue. This tissue was classified as grade 4, fibrous connective tissue with few cellular components aligned in parallel. Fig. Pure PCL did not induce inflammatory response.When using PCL-flax composites there was a significant increase in the number of CD45 positive cells by 2.5–3fold compared to both, control as well as pure PCL.There is no difference between the composites of transgenic and wild-type flax.P = 0.037; control vs. PLA transgene: 73 ± 21 vs. 433 ± 117, P = 0.023; control vs. PLA wt: 73 ± 21 vs. 421 ± 104, P = 0.017; Fig. In the tissue between pure PLA and the muscle, the evaluation of the expression of CD45 showed a significant increase of CD45 positive lymphocytes to 3.1 times. When using the PLA-flax composites the number of inflammatory cells increased significantly compared to the pure polymer again by about 50% to  or were significantly reduced (PCL-transgen) compared to pure PCL. It is well known, that encapsulation around implants generally occurs as a result of wound healing and foreign body reaction , 45. As Despite the good biocompatibility and the medical usage of PLA , 43, 47,The encapsulation of PLA, accompanied by macrophages and giant cells, could be detected after subcutaneous implantation . SimilarIn addition, a correlation between the degradation of the polymers and the capsule thickness could be observed in many studies. Grayson et al. proved that the fibrous capsules surrounding implants made of polymers, showed greater maturation of the capsules for the more rapidly degrading materials after 21 days (e.g. PLGA), but less mature capsules was observed in case of PLA up to 49 days . Qu et aIn vivo degradation of PCL can be divided into two phases. The first long-term phase is a superficial non-enzymatic hydrolytic cleavage of the ester groups without loss of quantity and deformation of the material. In the subsequent second phase, the molecular weight as well as the volume of polymer significantly decreases and released small particles are further degraded during phagocytosis, and finally excreted , 65. A lA subcutaneous implantation of PCL in the rabbit model was well tolerated and resulted after 6 months in only 1% of mass loss of this polymer. No inflammatory reactions or other pathological effects in the surrounding tissue could be histologically detected neither after 3 nor after 6 months . UltrathDue to their biodegradability and good biocompatibility, both PLA and PCL were also used pure or as copolymer, in order to ameliorate the acidic byproducts, for many new scaffolds in the regenerative medicine which are still of increased interest.This study reports on the suitability and effectiveness of the biomaterials reinforced with flax fibers in the field of regenerative medicine. Although these materials, according to the macroscopic analysis, did not cause any necrotic processes in the surrounding tissue, immunohistological examinations revealed that the expression of inflammatory cells increased significantly compared to the pure polymer. Furthermore, the physical and mechanical properties of the new materials could be improved for their better application and handling.


The cancer stem cell (CSC) and epithelial-to-mesenchymal transition (EMT) models have been closely associated and used to describe both the formation of metastasis and therapy resistance. We established a primary lung cell culture from a patient in a clinically rare and unique situation of primary resistant disease. This culture consisted of two biologically profoundly distinct adenocarcinoma cell subpopulations, which differed phenotypically and genotypically. One subpopulation initiated and sustained in spheroid cell culture (LT22s) whereas the other subpopulation was only capable of growth and proliferation under adherent conditions (LT22a). In contrast to our expectations, LT22s were strongly associated with the epithelial phenotype, and expressed additionally CSC markers ALDH1 and CD133, whereas the LT22a was characterized as mesenchymal with lack of CSC markers. The LT22s cells also demonstrated an invasive behavior and mimicked gland formation. Finally, LT22s were more resistant to Cisplatin than LT22a cells. We demonstrate a primary lung adenocarcinoma cell culture derived from a patient with resistant disease, with epithelial aggressive subpopulation of cells associated with stem cell features and therapy resistance. Our findings challenge the current model associating CSC and disease resistance mainly to mesenchymal cells and may have important clinical implications. With the worldwide leading cause of cancer deaths in men  and second leading cause in women  the necessity for further improvement in the treatment of lung cancer is clearly demonstrated2. These poor survival rates greatly strengthened the efforts to establish biomarkers for earlier diagnosis, prognosis and better treatment prediction. There are two major histological groups of lung cancer: 80–85% of tumors belong to the NSCLC, and 15–20% to the small cell lung cancer (SCLC). In the group of NSCLC the three major types are: adenocarcinomas (AC), squamous cell carcinomas (SCC) and large cell carcinomas. While smoking is strongly associated with an increased risk to develop SCLC or SCC, AC is not only the most common type of NSCLC, but also most commonly diagnosed in patients who have never smoked3.Despite substantial recent progress in the treatment of lung cancer, in particular non-small cell lung cancer (NSCLC), the survival rate remains poor, with approximately 10–20% of patients surviving 5 years after diagnosis4. As their characteristics are similar to embryonic stem cells they are termed cancer stem cells (CSC) or also tumor initiating cells. CSC are hierarchically organized and are capable of symmetric and asymmetric cell division5. The first evidence of CSC was observed in hematological malignancies6, and substantial literature on CSC also exists in various solid tumors10. Typically, CSC have been identified by expression of markers associated with stem cell properties. In NSCLC, CD133 and aldehyde dehydrogenase 1 (ALDH1) have been described as candidate markers for enrichment of CSC. Prognostic impact of cells expressing these markers has already been demonstrated11. Although CD133+ cells found in tumor samples from lung cancer patients constitute a minor population, their tumorigenic potential has been proven in an animal model12. While the CD133 function is still unclear13, a high occurrence of ALDH1 can be related to therapy resistance, as it oxidizes a large number of aldehydes. ALDH1 is also associated with processes of proliferation and survival14. Both markers are associated with aggressive behavior of the tumor, poor prognosis and cancer recurrences16.Within primary tumors a small population of tumor cells has been identified possessing the capability of self-renewal and pluripotency18. Epithelial-to-mesenchymal transition (EMT) has been closely associated with CSC as one of the mechanisms facilitating the formation of metastasis19. EMT is a well-known process occurring during the embryonic development for tissue morphogenesis and in cancer, EMT has been correlated to CSC plasticity20. During the metastasis formation tumor cells are losing their cell-cell contact supposedly accompanied with a downregulation of E-Cadherin (ECad) and epithelial markers such as the epithelial cell adhesion molecule (EpCam), cytokeratin (CK), and an upregulation of Vimentin21. This metamorphosis allows the cells to escape from the epithelial compartment and to proceed to distant sites. Unfortunately, there is only limited clinical data and experimental models providing convincing evidence. Therefore, the effect of EMT for metastasis formation and association with stemness is still a matter of debate22. A recent study in mouse models with different tumor entities showed that metastases are derived from non-EMT tumor cells24. Overall, most data supporting either the EMT theory or the opposite were derived from cell lines and mouse experiments, which are not sufficiently able to capture this transient mechanism. Clinical data from patients proving or disproving that EMT is necessary for metastasis formation is scarce.A further trait of CSC is their capability to metastasize, as we and others have shownHere, we show our established primary cell culture from a patient diagnosed with adenocarcinoma of the lung resistant to conventional treatment, which we believe to challenge the current model associating CSC with EMT and therapy resistance.After isolating a single cell suspension of the primary lung adenocarcinoma tumor, cells were cultured in non-adherent culture conditions, referred to as LT22s, and 10 days were needed for the formation of spheres, as shown in Fig. The presence of putative CSCs was investigated with FACS using CD133 and ALDH1. LT22s showed a continuous increase of the putative cancer stem cell marker CD133 with serial passages, starting from 14% and resulting in more than 80% after passage 19. Additionally, EpCam reached a constant expression of more than 90% (starting from 58%). In contrast, ALDH1 expression was not very prominent in LT22s cells at the beginning (mean 8.4% ± 1.4% SD) and even decreased with serial passages (mean 0.4% ± 0.3% SD) Fig. . Due to For genotypic and phenotypic characterization of the LT22s and LT22a cultures, qRT-PCR and immunostaining was performed, respectively. The expression of selected markers in the cell culture was compared to the respective formalin-fixed paraffin-embedded (FFPE) primary tumor. For the analyses of the primary tumor, only IHC was used.IHC staining of the primary tumor revealed following characteristics summarized in Fig. Corresponding marker analysis was performed in LT22s and LT22a summarized in Fig. Results from qRT-PCR supported the results of the immunostainings. In LT22s cells, CD133 expression increased with serial passages as shown in Fig. In concordance with the immunostaining results, no mRNA expression of the cancer stem cell markers ALDH1 and CD133 could be detected in LT22a cells. While the expression of ECad was not detectable, mesenchymal markers such as Vimentin, NCadherin (NCad) and Fibronectin were increased Fig. . EMT traFinally, stem cell associated markers Oct4, Sox2 and Nanog were analyzed by qRT-PCR Fig. . These mTaken together, these results confirm the epithelial and stem-cell associated phenotype of LT22s cells and the mesenchymal phenotype of LT22a cells at both the mRNA and at the protein level. These results are also consistent with the morphologic characteristics of the two cell populations.25. These multiple aberrations suggest dysregulations of biological functions and confirm the tumorigenic potential. It is of note that aberrant regions were less prominent in the primary tumor than in the cultured cells. Most likely these additional changes result from potential somatic mosaicisms in the primary tumor that were cloned during the establishment of the cell cultures. Interestingly, the SCNA patterns of LT22s compared to LT22a were also dissimilar, indicating that substantial chromosomal alterations contribute to the different phenotypes. The most prevalent differences included 2q, 4p, 6p, 7q, 8q, 9p, 10p, 11, 13q and 20q. However, when we analyzed the two cell populations after cell sorting based on CD133 expression for LT22s and EpCam expression for LT22a, we did not observe a difference between the two cell fractions, indicating that the expression of these markers is driven by other mechanisms than SCNAs, such as point mutations or regulatory mechanisms on the transcriptional level. In order to prove that LT22a and LT22s indeed originate from the same patient, we performed STR analysis and comparisons of mitochondrial haplotypes. Both LT22a and LT22s showed the same STR profiles and mitochondrial SNP patterns than the primary tumor and constitutive DNA of the patient retrieved from leukocytes confirming a common origin , which is pictured in Fig. As mentioned before, LT22a cells did not have the capability to form spheres under non-adherent conditions. This observation is in line with the fact that these cells did not form tight contacts in adherent culture and the lack of cell adhesion marker ECad. Together, this data supports the mesenchymal phenotype of LT22a cells. On the other hand, when LT22s was transferred to adherent cell culture, a large number of cells were needed in order to enable attachment and growth of cells.+ and EpCam− subpopulations formed colonies to a similar extent . These results indicate that CD133+ cells have a higher tumorigenic potential than CD133- cells.Next, we investigated in LT22a whether the expression of EpCam had an influence on their ability to form colonies in soft agar conditions. However, both EpCament Fig. . The LT2. + Fig.  comparedex ovo chorioallantoic membrane (CAM) assay model, a well-established in vivo tumor system27. One advantage of this model is the independence of the culture conditions, because the cells grow directly in the CAM microenvironment. The LT22s and LT22a cells were applied with 1*106 single cells per on-plant onto the CAM of the chicken embryo to form tumors. The tumor formation differed at both the macroscopic and microscopic level. After 72 hours on CAM the LT22s cells formed a compact round-shaped structure whereas the LT22a showed a centrum with radial arranged branches at macroscopic level  for lung adenocarcinoma were used for viability assays. In both LT22a and LT22s, Pemetrexed did not reduce viability at any concentration tested  mice. Both cell lines started to form tumors, but LT22s cells were significantly more tumorigenic in vivo compared to LT22a cells . PCR revealed the presence of human cells in liver and in lungs in both groups of mice, but to a different degree. In two (A1 and A2) out of the three mice of group A transplanted with LT22s cells we detected micrometastases in liver and lung, while in the mouse A3 only micrometastases in the lung were detected, but to a relatively strong extent Fig. . In bothWith the established primary cell culture, our study challenges the current model associating CSC with EMT and disease resistance mainly to mesenchymal cells. Our cell culture was established from a patient diagnosed with adenocarcinoma of the lung, who was resistant to standard neoadjuvant treatment including Cisplatin, Docetaxel, Vinorelbine, and radiotherapy that revealed two distinct subpopulations of cells with biologically distinct features.Contradicting our expectations, epithelial subpopulation of cells associated with CSC features, higher expression of stem cell marker genes, aggressive phenotype, enhanced invasion, and therapy resistance. Less aggressive cells, only capable of growth in adherent conditions, lacking stem cell and epithelial markers, and more responsive to treatment, were largely of mesenchymal origin.31. The presented cell culture model was generated from a patient in a rare clinical situation since the surgery was eventually performed with palliative intention, while disease progressed on conventional neoadjuvant treatment, including both chemotherapy and radiotherapy. Surprisingly, epithelial cells turned out to be more resistant than the mesenchymal subpopulation, implicating that future research of resistance should also focus on epithelial cells.Our data challenge the CSC model associated with the EMT phenotypeOver serial passages we observed the enrichment of cells with stem cell markers including ALDH1 and CD133 (LT22s cells), respectively. However, the expression of both proteins varied between the used methods, i.e. FACS and immunostaining. Although the anti-CD133 antibody was used from the same clone, it was differently labeled. Moreover, this antibody was specifically designed for FACS analysis, which might be another explanation for the detected discrepancy. ALDH1 staining for FACS analysis results from an enzymatic reaction. Various reasons such as inactivity of ALDH1 or the presence of inhibitors can influence the fluorescent reaction. Here, we observed even greater discrepancy between the immunostaining and FACS. Thus, we confirmed the enrichment of ALDH positive cells at mRNA level which correlated with the results of immunofluorescence, as shown in Fig. In sharp contrast to the expected association between CSC and EMT, LT22s cells enriched for CSC were positive for epithelial markers including EpCam, CK and ECad, but lacked mesenchymal traits. The epithelial CSC phenotype was also consistent during the invasion process of the CAM assay. We could demonstrate that LT22s cells invaded the membrane and formed glandular structures with an inner lumen, which was comparable to the primary tumor cell formation and were also capable to form peripheral metastasis. Thereby, the expression of epithelial and CSC markers persisted. Therefore, it seemed that these cells were able to invade without undergoing EMT.32. The absence of epithelial cell adhesion markers including ECad could be one reason for the inability to form spheres. This is in line with a recent study, showing that sphere formation in breast cancer cell lines depends on expression of ECad33.In addition to the epithelial CSC-enriched spheroid culture (LT22s), we also isolated mesenchymal-like cells of the same tumor, with a high Vimentin expression and a prominent mesenchymal phenotype. These LT22a cells did not express any CSC markers, but revealed a spindle-shaped morphology together with strong expression of mesenchymal markers at protein and mRNA level. We also found that LT22a cells were not able to proliferate and form spheres under spheroid culture conditions. The origin of LT22a cells was rather unclear, because in the primary tumor a Vimentin positive cell subpopulation could not be detected. However, in the primary tumor we found single cells positive for the EMT transcription factor Snail. We therefore hypothesize that these Snail-positive cells may be the origin of the LT22a culture. Under adherent conditions these cells might have gained a considerable growth advantage in the absence of LT22s cells. Furthermore, Snail has been shown to be responsible for downregulation of ECadDue to these remarkable divergences of the two cell subpopulations, we evaluated their genomic relations. STR analysis and mtDNA analysis confirmed that both subpopulations originated from the same patient. Furthermore, genome-wide copy number profiling indicated that both subpopulations were highly degenerated, consistent with a malignant phenotype of these cells. However, SCNAs differed between epithelial and mesenchymal cells, further confirming the presence of two independent subpopulations of cells in the primary tumor.34. Fischer et al. generated a triple transgenic mouse model, where the EMT initiator fibroblast specific protein 1 (FSP-1) served as a switch for a Cre fluorescent alternation, which was not reversible. Cells with epithelial phenotype emitted a red fluorescence, but when EMT occurred, cells emitted a green fluorescence. Surprisingly, cells that traveled to the lung showed a red fluorescence staining and therefore kept their epithelial phenotype during the formation of metastasis23. Similarly, Zheng et al. generated a mouse model of pancreatic cancer to study the influence of EMT. They found that loss of either Snail or Twist suppressed EMT in the primary tumor, but did not reduce invasion and metastasis24. These two seminal papers seriously challenge the role of EMT as a key element involved in cancer metastasis. Together with our observation, these studies question the association of EMT and CSC and formation of metastasis. Nevertheless, it is important to mention that EMT is a dynamic and transient process and therefore, experiments to capture this complicated process are challenging. Interestingly, LT22a cells also formed metastasis to a minor extant, although in vivo tumor growth was limited.Unfortunately, material from metastasis of this patient was not available, thus it remains unclear whether LT22s or LT22a cells were responsible for the formation of metastasis. We could, however, observe in mouse transplantation a higher potential of LT22s cells to form metastasis. Furthermore, a consistent epithelial phenotype of LT22s cells could be observed during invasion into the amnion cavity of the chick embryo, which further questions the necessity of EMT for metastasis. Recently published papers share our observationsBoth previously mentioned papers suggest, on the other hand, that EMT contributed to drug resistance. This suggestion could not be confirmed with our experiments. In fact, LT22a cells showed a significantly better response to Cisplatin than LT22s cells. It is of note that the cell culture was established at a stage of disease progression under primary treatment with potentially curative intention. The surgery was performed as a salvage treatment due to the young age of our patient. Unfortunately, soon after the surgery our patient died. Therefore, this cell culture is representative for disease resistance resulting from a rare and unique clinical situation. Patients who progress under the neoadjuvant or definitive radiochemotherapy represent the minority, and those who undergo surgery are individual cases. Thus, the limitation of our study (that this observation is obtained from only one case) is at the same time its strength, since it is representative of progressive disease, where conventional therapies were ineffective.Overall, we show for the first time the independence of CSC markers and EMT features in primary cells derived from a lung cancer patient resistant to conventional treatment. We believe that our data provide important clinical evidence challenging the current model of EMT in cancer progression, resistance and metastasis. Our data suggest that epithelial tumor cells play a crucial role in progression, therapy resistance, and metastasis, and have to remain in focus of further studies.Detailed methods can be found in the supplementary materials.2) and Docetaxel (75 mg/m2) and during concomitant radiochemotherapy with Cisplatin (75 mg/m2) on day 1 and Navelbine (12.5 mg/m2) on days 1, 7 and 15 of the cycle. At the time of surgery lumbar bone and liver metastasis were suspected and the primary tumor progressed on treatment. The surgery was performed as a palliative treatment. Soon after the surgery, the patient had a further disease progression and did not receive any further systemic tumor specific treatment. The overall survival from the diagnosis to death was 6 months.A 43-year old male patient was diagnosed with a poorly differentiated adenocarcinoma of the lung in September 2014. No genomic aberrations for lung cancer-specific genes EML4-ALK and EGFR were detected. The patient received two cycles of neoadjuvant chemotherapy with Cisplatin . Informed consent for the primary cell culture was obtained from the patient. All experiments were carried out in consideration of the guidelines for good scientific practice as officially required from the Medical University of Graz.35. Isolated cells were cultured in ultra-low attachment flasks containing DMEM/F12 media  supplemented with human recombinant EGF (20 ng/mL), bFGF , B27  and 4 µg/mL Heparin . The media was changed twice a week. At the appearance of brown spots in the center of the spheroids, they were dissociated with TrypLE (Gibco by life technologies). After two spheroid passages, a proportion of the cells was further cultured in adherent conditions, with DMEM/F12 media and 10% fetal bovine serum superior (FBS)  in standard flasks .Tumor cell isolation was performed as previously described with the addition of dissociation steps using a gentleMACS automated dissociator  and some modifications36. For the first staining, single cells were blocked with 0.5% bovine serum albumin/PBS, for 10 min at room temperature (RT). 1*106 cells were incubated with aliquots of antibodies (5 µL anti-EpCam Phycoerythrin (PE) and 10 µL anti-CD133 Allophycocyanin (APC)) for 30 minutes at 4 °C. Another approach was to measure the ALDH1 activity with Aldefluor assay kit , performed according to the manufacturerʼs instructions. All monoclonal antibodies were pretitered to determine their optimal dilutions before use. Flow cytometry sorting was performed on the fluorescence activating cell sorter (FACS) Aria . Cells without staining, single stained cells, and isotype controls (BD Bioscience) were used as controls in the experiments. Side scatter and forward scatter profiles were used to eliminate cell doublets and apoptotic cells were excluded by using 7-aminoactinomycin D . Data analysis was performed using the Diva software 7.0 (BD Bioscience). Sorted cells were seeded into ultra-low attachment flasks for evaluation of sphere formation capacity. Additionally, DNA and RNA were extracted from sorted fractions for sequencing and gene expression analyses.Staining of the cells for flow cytometry analysis was performed following two different protocols as adapted from our previous publicationThe expression of selected cancer related proteins was analyzed with multicolor immunofluorescence staining. Cytospins from single cells or compact spheres, chamber slides with adherently grown cells  and adhesion slides  from spheres were prepared and used. After fixation with 3.7% paraformaldehyde buffer and blocking with normal goat serum for 30 min, samples were incubated with a primary monoclonal antibody cocktail against certain antigens that were obtained from mouse or rabbit hosts for 1 hour at room temperature. After washing with PBS, slides were incubated for 1 hour with secondary fluorescent labeled antibody cocktail consisting of 488 nm goat anti-rabbit and 594 nm goat anti-mouse . The antibody list can be found in Supplementary Table 37 and is further described in Supplementary materials file.Genome-wide somatic copy number aberrations (SCNA) were established using low-coverage whole genome sequencing. Shotgun libraries were prepared using the TruSeq DNA Nano LT Sample preparation Kit  with slight modifications to the manufacturer’s protocol. Detailed description can be found in the Supplementary materials file. Copy number analysis was performed as previously described2O. Colonies were counted with ImageJ software.Sorted cells were seeded at 200 cells per well in 6-well plates with DMEM/F12 media containing FBS and incubated for 8 days. Additional culture media was added every 3 days. Cells were fixed with ice cold methanol for 10 min, following the staining with 0.5% crystal violet  solution for 10 minutes. Staining was rinsed off with ddH38. After 3 weeks of sphere formation, the plate was stained with 0.005% crystal violet (Sigma Aldrich) and the total numbers of colonies were counted under a light microscope Olympus Basic BX51.0.3% soft agar (Sigma Aldrich) in media was mixed with 800 sorted cells and plated onto a 0.6% solid agar layer in a 24-well plateetal.39. Cell culture media was added only once a week. Formed spheres >50 µm were counted under the microscope. Sphere formation assays were performed in 6 technical replicates and three independent experiments  were performed.900 cells were sorted into a 24-well ultra-low attachment plate and cultured in sphere formation media for 3 weeks according to the protocol of Lombardo 6 cells  were grafted in the centre of a 5 mm silicon ring on the surface of the CAM40. Xenografts were allowed to develop for 3 days, photographed using a stereo-microscope  and then excised with the surrounding CAM 4 day after seeding, fixed in 4% paraformaldehyde, paraffin embedded, cut in 4-μm sections. Every 10th section was stained with HE to find the area of interest. For the immunofluorescent analysis of the tumors, the same markers were used as for the primary culture cells. To distinguish the chicken cells, every section was co-stained with Lens Culinaris Agglutinin (LCA) antibody 41.Fertilized white leghorn chicken eggs from local hatchery were incubated at 37.6 °C and 70% humidity . The egg shell was cracked on day 3 of chick development and the embryo decanted to a sterile dish. On day 10, 1*10On day one, 1000 cells were seeded into 96-well plates. On day two cells were treated with chemotherapeutics including Cisplatin , Pemetrexed  and Salinomycin (Sigma Aldrich) at varying concentrations. Untreated cells and cells treated with solvent of chemotherapeutics (vehicle control VC) served as controls. After 72 h, Prestoblue  was added according to manufacturer’s instructions and the fluorescence emission was measured on the FLUOstar omega plate reader . Half-maximal inhibitory concentrations (IC50) were calculated using GraphPad Prism6 non-linear regression analysis . Experiments were done in five technical and three biological replicates.7 cells were re-suspended in 100 µL PBS/Matrigel (1:1) and transplanted subcutaneously to the left flank of each mouse. LT22s cells were transplanted to mice of group A, while LT22a cells were transplanted to mice of group B. Tumor diameters were measured two times a week with a caliper. Tumor volumes were calculated according to V = (length × (width)2)/2. For calculation of the relative tumor volume (RTV), the tumor volumes at each measurement day were related to the day of first treatment. Mice were sacrificed by cervical dislocation and liver and lung tissue was removed for inspection of metastatic spread. After isolation of genomic DNA from two samples of each tissue, a human specific DNA was performed amplifying a fragment of the alpha satellite region of human chromosome 17. Indicated are the ΔCt values, which were calculated by subtracting the experimental mean Ct-value from that of the negative control (mouse tissue). Genomic DNA of the human mammary carcinoma xenograft 3366 was used as positive control, and water as negative control.These experiments were performed by an external facility , which was completely blinded to the results of our study. All animal experiments were done in accordance with the United Kingdom Coordinating Committee on Cancer Research regulations for the Welfare of Animals and the German Animal Protection Law and with approval by the local responsible authorities . Female NOD/SCID mice , kept under conditions summarized in Supplementary Table Statistical significance was evaluated by either Student’s t-test or analysis of variance (ANOVA). To control for multiple testing, Sidak adjustment method was used. All p values were two-sided and considered statistically significant when <0.05. All statistical analysis was performed using GraphPad Prism 6 .Supplementary information


Combining the synoptic mass balance approach with principal components analysis (PCA) can be an effective method for discretising the chemistry of inflows and source areas in watersheds where contamination is diffuse in nature and/or complicated by groundwater interactions. This paper presents a field-scale study in which synoptic sampling and PCA are employed in a mineralized watershed  under low flow conditions to (i) quantify the impacts of mining activity on stream water quality; (ii) quantify the spatial pattern of constituent loading; and (iii) identify inflow sources most responsible for observed changes in stream chemistry and constituent loading. Several of the constituents investigated  fail to meet chronic aquatic life standards along most of the study reach. The spatial pattern of constituent loading suggests four primary sources of contamination under low flow conditions. Three of these sources are associated with acidic (pH <3.1) seeps that enter along the left bank of Lion Creek. Investigation of inflow water  chemistry using PCA suggests a hydraulic connection between many of the left bank inflows and mine water in the Minnesota Mine shaft located to the north-east of the river channel. In addition, water chemistry data during a rainfall-runoff event suggests the spatial pattern of constituent loading may be modified during rainfall due to dissolution of efflorescent salts or erosion of streamside tailings. These data point to the complexity of contaminant mobilisation processes and constituent loading in mining-affected watersheds but the combined synoptic sampling and PCA approach enables a conceptual model of contaminant dynamics to be developed to inform remediation.The online version of this article (doi:10.1007/s11356-017-9038-x) contains supplementary material, which is available to authorized users. Contaminated drainage from historical and contemporary hard rock mining activities is recognised as one of the most pressing global water quality issues  is a powerful statistical method for determining chemical similarity or distinction between spatially dense synoptic samples. PCA effectively rotates chemical data so as to visualise the greatest distinctions among groups of samples  to quantify the impacts of mining activity on stream water quality; (ii) to quantify the spatial pattern of constituent loading; and (iii) to identify inflow sources most responsible for observed changes in stream chemistry and constituent loading.Lion Creek is located within the Empire Mining District in the Clear Creek Watershed, Colorado, USA Fig. . The strLion Creek now forms part of the Clear Creek/Central City Superfund Site (EPA ID: COD980717557) due to extensive historical mining operations. Water quality in the Clear Creek watershed is a significant issue as it is a major drinking water source, fishery, and recreation area. The study reach originates just upstream from Minnesota Mine  and synoptic sampling (to quantify stream and inflow chemistry)  sites, and 2 mine water sites  cation concentrations were determined from unfiltered and filtered samples, respectively, by inductively coupled plasma–mass spectroscopy (ICP-MS). Trace element concentrations are reported for Al, Ag, As, Ca, Cu, Ba, Cd, Co, Cr, Fe, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Si, Sr, U, V, and Zn , measurement of pH and specific conductance, and preservation of samples for iron speciation. Anion concentrations were determined from filtered, unacidified samples by ion chromatography. Anion concentrations are reported for Cl, Br, F, NOZn Table  for all Zn Table .Table 2DQ) at each synoptic site relates the injected tracer to the observed dilution at the site  was therefore calculated based on the observed Li dilution:Sites MN-0370 to MN-1469. pH is <3.5 in this subreach. Although Br appears to be conservative , there may be small losses of Br at low pH. Li, in contrast, should be conservative at these pH values. Streamflow at the downstream site in a site pair  may be developed using simple mass balance calculations on individual stream segments, yielding . Effective inflow concentrations . All constituents (dissolved concentrations) except tracer variables  were included in the analysis. Data were log10 (x + 1) transformed before analysis to reduce the clustering of common and abundant measurements at the centre of the ordination plot and also the effect of outliers. All constituent concentrations (including pH) were changed to units of millimoles per litre.Patterns in the chemistry of the stream inflow, mine water, and pore water samples were investigated further using PCA, a multivariate statistical analysis. PCA is an ordination method that aims to reduce a large number of measured variables down to a smaller number of principal components that summarise the information provided by each contributing variable. This method allows the important variables and patterns in multivariate data to be summarised and visualised more easily. Analyses were performed in the program SPSS (Study data are available in the 4) that are common to mine drainage and identified in subsequent analyses as a potential cause for concern in Lion Creek.The following sections focus on stream pH and nine constituents  and right bank (n = 7). Only two stream segments contribute more than 5% to overall flow. Segment 300 to 344 m contributes 5.5% to overall streamflow and contains three distinct inflows that includes the seepage face at 324 m. Segment 545 to 579 m contributes 5.1% to streamflow due to two small left bank inflows at 550 m. Only 12 out of the 20 stream segments had identifiable inflows and these segments accounted for 84% of the total streamflow, suggesting some unidentified or unknown dispersed surface and/or subsurface water inputs.Streamflow estimates increase linearly with distance from ∼3 L/s just downstream from the tracer injection site to ∼17 L/s just downstream from the confluence with North Empire Creek Fig. . Aside f4-type and the right bank samples are mostly Ca-HCO3-type  indicating there was enough relationship between variables to perform the PCA. Two principal components were extracted from the analysis. A primary principal component (PC1—x-axis) explains 76% of the data variance and is characterised by the major contaminants including Al, Cd, Cu, Co, Cr, Fe, Mn, Ni, Pb, Zn, SO4, and pH. A second minor principal component (PC2—y-axis) explains a further 5% of the data variance and is characterised by V and As. Considering PC1, the contamination gradient, three right bank inflows  representing background water chemistry with low contaminant concentrations plot to the left of the biplot  value was 0.86 indicating very good sampling adequacy and the Bartletts test of sphericity was highly significant . Source area 2, North Empire Creek, is the largest source of Fe, Mn, Ni, and SO4  and the second largest source of Cd and Zn . All of these constituents (except SO4) fail to meet chronic aquatic life standards along most of the study reach. Source areas 3  and 4  are responsible for less than 20 and 10% of constituent loading, respectively. These results suggest that remediation activities that are focused on source area 1 (MN-550 and vicinity) and North Empire Creek may have the greatest overall benefit under low flow conditions. Whilst the data presented in this study can be used to help prioritise remediation in Lion Creek, a separate synoptic sampling study and loading analysis would need to be undertaken in North Empire Creek upstream from the Lion Creek confluence in order to identify the primary sources of constituent loading in that watershed.Examination of the spatial pattern of constituent loads in Lion Creek suggests four important source areas of contamination under low flow conditions Fig. , Table 44 loading (six out of the seven constituents that exceed aquatic standards). Effective inflow concentrations suggest the primary source of contamination within this zone is the left bank inflow at MN-0550. On the day of synoptic sampling, this inflow consisted of two small surface inflows from a denuded area. A range of small-scale passive remediation options could be considered here to neutralise pH and remove dissolved metals  would be necessary that account for pre-mining water quality and how remediation will change mass loading and instream geochemistry  accounts for >30% of Al, Cd, Cu, and Zn loading and ∼20% of Mn, Ni, and SO4, and Zn) has been reported at PRB installations worldwide  pathways that link the stream bank to the streambed sediments , North Empire Creek (1403 m), the left bank seepage face (300–344 m), and the left bank eroding area (232–300 m). Multi-variate analysis (PCA) of stream inflow chemistry suggests a hydraulic link between left bank inflows and mine water in the Minnesota Mine shaft. Together, synoptic sampling and PCA suggest constituent loading from North Empire Creek, streamside tailings in Lion Creek, and underground leakage of contaminated mine water from Minnesota Mine are the primary factors limiting water quality in the study reach.Previous remediation of the Minnesota Mine site focussed on stabilisation of the large tailings deposits near the mine and an eroding gully that focussed storm water during high rainfall. However, many tailings deposits remain in the watershed and there is strong evidence for contaminated water from Minnesota Mine entering Lion Creek via subsurface pathways. As a result, remediation to improve water quality to acceptable standards may require a multi-method approach. The greatest benefit for low flow concentrations and loads may be achieved by preventing contaminated water from Minnesota Mine from entering Lion Creek. Stabilisation or removal of remaining streamside tailings may be an effective measure to reduce the potential for contamination episodes during rainfall-runoff events. Additional investigations focussed on establishing the location of potential leakage of the Minnesota Mine workings and also constituent loading in North Empire Creek are needed prior to the implementation of remedial actions.ESM 1(PDF 598 kb).


Mentha piperita L. is a flowering plant belonging to the Lamiaceae family. Mentha plants constitute one of the main valuable sources of essential oil used in foods and for medicinal purposes.Mentha piperita leaf essential oil (MpEO). A single dose of CCl4 was used to induce oxidative stress in rats, which was demonstrated by a significant rise of serum enzyme markers. MpEO was administrated for 7 consecutive days  to Wistar rats prior to CCl4 treatment and the effects on serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ -glutamyl transpeptidase (γ-GT) levels, as well as the liver and kidney superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity and thiobarbituric acid reactive substances (TBARS) levels were evaluated. In addition, histopathological examinations of livers and kidneys was performed.The present study aimed to investigate the composition and in vitro antioxidant activity of 4-induced stress. Whereas pretreatment with MpEO at doses of 15 and 40 mg/kg prior to CCl4, significantly reduced stress parameters  compared to the CCl4-only group. Moreover, a significant reduction in hepatic and kidney lipid peroxidation (TBARS) and an increase in antioxidant enzymes SOD, CAT and GPx was also observed after treatment with MpEO (40 mg/kg) compared to CCl4-treated rats. Furthermore, pretreatment with MpEO at 40 mg/kg can also markedly ameliorate the histopathological hepatic and kidney lesions induced by administration of CCl4.The in vitro antioxidant activity of MpEO was lower than that of silymarin. Pretreatment of animals with MpEO at a dose of 5 mg/kg did not have a significant effect on ALT, AST, ALP, LDH, γGT, urea or creatinine levels in CCl4-induced oxidative stress and thus substantiate the beneficial effects attributed traditionally to this plant.We could demonstrate with this study that MpEO protects liver and kidney from CCl The results were analyzed by One-Way Analysis of Variance (ANOVA) followed by Tukey test for multiple comparisons using SPSS for Windows (version. 12) or ANOVA-on-ranks with Dunn’s correction. Differences were considered significant at Chemical composition of MpEO was determined by GC/MS analysis. The compounds, their percentages as well as their retention indices are listed in Table The antioxidant activity of MpEO was compared to that of silymarin, a well-known antioxidant, using two different assays, namely DPPH and superoxide oxygen radicals inhibition, the results are reported in Table 4 caused severe hepato and reno-toxicity in the treated rats, as evidenced by the significant elevations of serum ALT, AST, ALP, LDH, γGT, total cholesterol, triglycerides, LDL urea and creatinine levels, while HDL level was decreased compared to control animals.The results of biochemical indicators of liver and kidney function are summarized in Tables 4 group. It is worth noting that the treatment with 5 mg/kg MpEO did not induce any significant changes in the biochemical parameters  when compared to the CCl4 group. Treatment of rats with only MpEO (40 mg/kg BW) did not result in significant alterations in biochemical parameters compared to control rats.Pretreatment with the MpEO at doses of 15 or 40 mg/kg significantly reduced levels of ALT, AST, ALP, LDH, γGT, total cholesterol, triglycerides, LDL urea and creatinine and increased the level of HDL compared to the CCl4 group. Its effect was comparable in reducing of liver and kidney damage induced by CCl4 with that observed for the highest dose of MpEO (40 mg/kg).Pretreatment with silymarin (50 mg/kg), used as positive control, significantly decreased the elevated levels of ALT, AST, ALP, LDH, γGT, total cholesterol, triglycerides, LDL urea and creatinine and increased of HDL level as compared to CCl4-treated animals when compared to control untreated rats.TBARS level is widely used as a marker for free radical mediated lipid peroxidation injury. We determined TBARS levels in the liver and kidney tissues of the investigated animals and our results are shown in Fig. 4 group. There was a dose effect; treatment with MpEO at 5 mg/kg BW did not induce any significant decrease in the levels of TBARS in liver and kidney as compared to CCl4 group. When rats were treated with only MpEO (40 mg/kg BW), no significant differences in the TBARS values was observed compared to control rat. Pretreatment with silymarin (50 mg/kg) significantly decreased the elevated levels of TBARS in both liver and kidney compared to CCl4 control. Moreover, the effect of silymarin (50 mg/kg) in attenuation of TBARS levels in liver and kidney was comparable with highest dose of MpEO (40 mg/kg).Pre-treatment with the MpEO at doses of 15 and 40 mg/kg BW significantly reduced levels of TBARS in liver and kidney tissues as compared to CCl4-treated group when compared to control group. The decrease in hepatic and kidney CAT, SOD and GPx levels induced by CCl4 injection were significantly restored (elevated) in the MpEO and silymarin groups, and this effect was more pronounced with the increase of essential oil concentration. Pretreatment with MpEO at doses of 15 and 40 mg/kg significantly increased levels of hepatic and kidney CAT, SOD and GPx as compared to CCl4 group. It is worth noting that the treatment with MpEO at a dose 5 mg/kg did not induce any significant increase in the levels of hepatic and kidney CAT, SOD and GPx as compared to CCl4 group. No significant differences in the values were observed in rats treated with MpEO only (40 mg/kg) compared to control rat values. Moreover, the effect of silymarin (50 mg/kg) was comparable in attenuation of levels of hepatic and kidney CAT, SOD and GPx with highest dose of MpEO (40 mg/kg).Results presented in Tables 4 group , Norway and Poland that also had menthone and menthol as their most important components [Chemical composition of the essential oil obtained from MpEO was determined by GC-MS analysis. The compounds, their percentages as well as the retention indices are listed in Table mponents –32. On tmponents , 34.M. piperita L. extracts with various major oil components. Differences in chemical composition observed for essential oils is likely related to abiotic factors such as soil type and climate specific regions of provenance samples and geographical factors [4. To be noted: working with natural extracts, the antioxidant activity is considered to be primary related to the major active compounds in the essential oil such as menthol and its derivatives [These studies showed variable chemotypes of  factors . Further factors , making ivatives . Howeverivatives , 37, thi4 exposure is characterized by the elevated levels of serum hepatic marker enzymes indicating the cellular leakage and loss of functional integrity of hepatic membrane architecture. High levels of ALT, AST, ALP, LDH and γGT activities are sensitive indicators of liver cell injury and are most helpful in recognizing hepatic diseases [4-treated rats show increased activities of these enzymes, reflecting damage to the liver cells or changes in the cell membrane permeability leading to leakage of enzymes from cells to the circulation [4 due to increased lipid peroxidation which had the ability to cause membrane damage. It is now generally accepted that CCl4 hepatotoxicity is the result of reductive dehalogenation, which is catalyzed by its specific isoenzyme of cytochrome P450 2E1, and which forms the highly reactive free radical. Hence, the suppression of P450 2E1 could result in reduced levels of reactive metabolites, and thus decreased tissue damage [Liver injury after CCldiseases . CCl4-trculation . In the e damage .4 caused a significant increase in the triglyceride, total cholesterol, and LDL levels and decrease in HDL level. Increase in the cholesterol levels might be due to the increased esterification of fatty acids, inhibition of fatty acid β-oxidation, and decreased excretion of cellular lipids [4 stimulates the transfer of acetate into liver cells (probably by increasing access to acetate) and leads to an increase in cholesterol synthesis. It also increases the synthesis of fatty acids and triglyceride from acetate and enhances lipid esterification [The liver plays a fundamental role in the metabolism of lipids. Injection of CClr lipids . CCl4 stfication . The accfication .4 induced also renal toxicity evidenced by an elevation of serum creatinine and urea [The administration of CCland urea , 45. Theand urea , which iand urea .4 exposure resulted in a dramatically protective effect against acute hepato and renotoxicity and oxidative stress, which was further also confirmed by the hepatic histopathological examinations. The stimulation of hepatic regeneration makes the liver more resistant to damage by the toxin [4 group. Pharmacological studies have shown that essential oil derived from various plant materials possesses anti-inflammatory activities [M. piperita L. leaf essential oil could be partly explained by the presence of sesquiterpenes, such as spathulenol, cadinene, caryophyllene and caryophyllene oxide. The ethanolic extract of parsley leaves also showed significant anti-inflammatory [Treatment of rats with MpEO prior to CClhe toxin . Treatmetivities , 50 Knowtivities , the antammatory  and antiammatory , 54 whicammatory .4-intoxicated rats. Silymarin significantly reversed CCl4-induced TBARS levels elevation but values obtained with MpEO at the highest dose was comparable in attenuation of TBARS levels in liver and kidney. Silymarin reversed CCl4-induced SOD, CAT and GPx activities decrease but values obtained with MpEO at the highest dose (40 mg/kg) was comparable in attenuation of SOD, CAT and GPx activities in liver and kidney.In the present study, the two fold increase in the TBARS levels and reduce activity of SOD, CAT and GPx observed in liver and kidney homogenate of CCl4 metabolism as well as ROS and peroxidation products. This hypothesis is supported by the recent findings on the in vitro antiradical and antioxidative activities of MpEO [These results suggested that MpEO could exert its antioxidant and/or radical scavenging activities thus preventing the formation of the carbon free radicals originated from CCl of MpEO . Previou of MpEO .4-hepato and renotoxicity control and rats of groups 3, 4, 5 and 6 were injected with 1 ml/kg BW of CCl4 and olive oil mixture on day seven . It has already been shown that a single dose of CCl4 initiates lipid peroxidation [4 is further well known to induce fibrosis of the hepatic tissue that may further progress to cirrhosis if the stimuli persists [4 injection in mice can be used as an attractive and highly reproducible model of liver regeneration after toxic injury. The first appearance of histological fibrosis and scarring fibers is usually observed after repeated CCl4 treatment for 2 to 3 weeks, depending on the dosage and mouse strains used [4-treated rats resulted large numbers of inflammatory cells such as lymphocytes along with hepatic sinusoidal inflammation, hepatocyte necrosis and devastating liver architecture The highest dose of MpEO (40 mg/kg) or silymarin (50 mg/kg) significantly attenuated the damaged liver depicting marked focal regenerative changes which are illustrated by presence of actively dividing cells with a prominent nucleolus. The administration of MpEO reducing the histological alterations in liver provoked by CCl4 was quite noticeable. In fact, the histological changes seen in the kidney of rats treated with CCl4 were characterized by some nephrotoxic lesions, as evidenced by the glomerular and tubular necrosis. Our results confirmed previous findings of Ozturk et al. [4. The results suggest that MpEO treatment prior to CCl4 intoxication could prevent the CCl4-induced alterations in kidney tissues of treated animals.In the present study, the rats of group 2 served as CClxidation –58 that xidation , 60. CClpersists –65. Thusins used . In the k et al.  who had 4 induced oxidative stress in rats. Further investigations are essential to elucidate the precise mechanism of active agents of MpEO protection against CCl4-induced hepatotoxicity and nephrotoxicity and it has to be tested against other biological parameters.The contents of MpEO not only protect the integrity of plasma membrane but, at the same time, increased the regenerative and reparative capacity of the liver and kidney. These results suggest that the compound present in MpEO has hepatorenal protective effects against CCl