Combined Effect of Light and Nutrients on the Micromorphology of the White rot Fungus Cerrena Unicolor.

Light influences developmental pathways in fungi. Recent transcriptomic and biochemical analyses have demonstrated that light influences the metabolism of a white-rot basidiomycete Cerrena unicolor. However, the expression profile of genes involved in the growth and development, or micromorphological observations of the mycelium in response to variable lighting and culturing media, have not performed. We aim to reveal the effect of light and nutrients on C. unicolor growth and a potential relationship between the culture medium and lighting conditions on fungus micromorphological structures. Confocal laser scanning microscopy and scanning electron microscopy were employed for morphological observations of C. unicolor mycelium cultivated in red, blue, green, and white light and darkness on mineral and sawdust media. A comprehensive analysis of C. unicolor differentially expressed genes (DEGs) was employed to find global changes in the expression profiles of genes putatively involved in light-dependent morphogenesis. Both light and nutrients influenced C. unicolor growth and development. Considerable differences in the micromorphology of the mycelia were found, which were partially reflected in the functional groups of DEGs observed in the fungus transcriptomes. A complex cross-interaction of nutritional and environmental signals on C. unicolor growth and morphology was suggested. The results are a promising starting point for further investigations of fungus photobiology.

Metabolic Imaging Phenotype Using Radiomics of [18F]FDG PET/CT Associated with Genetic Alterations of Colorectal Cancer.

PURPOSE: To understand the association between genetic mutations and radiomics of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET)/x-ray computed tomography (CT) in patients with colorectal cancer (CRC). PROCEDURES: This study included 74 CRC patients who had undergone preoperative [18F]FDG PET/CT. A total of 65 PET/CT-related features including intensity, volume-based, histogram, and textural features were calculated. High-resolution melting methods were used for genetic mutation analysis. RESULTS: Genetic mutants were found in 21 KRAS tumors (28 %), 31 TP53 tumors (42 %), and 17 APC tumors (23 %). Tumors with a mutated KRAS had an increased value at the 25th percentile of maximal standardized uptake value (SUVmax) within their metabolic tumor volume (MTV) (P < .0001; odds ratio [OR] 1.99; 95 % confidence interval [CI] 1.37-2.90) and their contrast from the gray-level cooccurrence matrix (P = .005; OR 1.52; 95 % CI 1.14-2.04). A mutated TP53 was associated with an increased value of short-run low gray-level emphasis derived from the gray-level run length matrix (P = .001; OR 243006.0; 95 % CI 59.2-996,872,313). APC mutants exhibited lower low gray-level zone emphasis derived from the gray-level zone length matrix (P = .006; OR < .0001; 95 % CI 0.000-0.22). CONCLUSION: PET/CT-derived radiomics can provide supplemental information to determine KRAS, TP53, and APC genetic alterations in CRC.

Development of a temperature-responsive yeast cell factory using engineered Gal4 as a protein switch.

Conflict between cell growth and product accumulation is frequently encountered in biosynthesis of secondary metabolites. Herein, a temperature-dependent dynamic control strategy was developed by modifying the GAL regulation system to facilitate two-stage fermentation in yeast. A temperature-sensitive Gal4 mutant Gal4M9 was created by directed evolution, and used as a protein switch in ΔGAL80 yeast. After EGFP-reported validation of its temperature-responsive induction capability, the sensitivity and stringency of this system in multi-gene pathway regulation was tested, using lycopene as an example product. When Gal4M9 was used to control the expression of PGAL -driven pathway genes, growth and production was successfully decoupled upon temperature shift during fermentation, accumulating 44% higher biomass and 177% more lycopene than the control strain with wild-type Gal4. This is the first example of adopting temperature as an input signal for metabolic pathway regulation in yeast cell factories.

Metabolomic study of urinary polyamines in rat exposed to 915 MHz radiofrequency identification signal.

Metabolomic analysis of urinary polyamines (PAs) from rat exposed to 915 MHz radiofrequency identification (RFID) signal for 8 h/day for 2 weeks was performed by gas chromatography-mass spectrometry as N-ethoxycarbonyl/N-pentafluoropropionyl derivatives. Large alterations in nine PA levels including four aliphatic and five acetylated PAs were monitored in sham-exposed and RFID-exposed groups. Total PA and urinary levels of N (1)-acetylputrescine, N (1)-acetylcadaverine, putrescine, cadaverine, N (1)-acetylspermidine, N (8)-acetylspermidine, spermidine and spermine were reduced, whereas N (1)-acetylspermine was significantly increased after sham and RFID exposure compared with those before exposure. Their levels were normalized to the corresponding group means before exposure and then plotted into star symbol patterns. N (1)-Acetylspermine after RFID exposure was 54 % higher compared to the level before RFID exposure, while it was elevated by only 17 % in the sham group. The results suggest that 915 MHz RFID exposure may induce metabolic disturbance of PA. It may also elevate spermidine/spermine acetyltransferase (SSAT) activity. Thus, the present metabolic profiling combined with star pattern recognition method might be useful for understanding the complexity of biochemical events after exposure to RFID signal.

Mechanical loading causes site-specific anabolic effects on bone following exposure to ionizing radiation.

During spaceflight, astronauts will be exposed to a complex mixture of ionizing radiation that poses a risk to their health. Exposure of rodents to ionizing radiation on Earth causes bone loss and increases osteoclasts in cancellous tissue, but also may cause persistent damage to stem cells and osteoprogenitors. We hypothesized that ionizing radiation damages skeletal tissue despite a prolonged recovery period, and depletes the ability of cells in the osteoblast lineage to respond at a later time. The goal of the current study was to test if irradiation prevents bone accrual and bone formation induced by an anabolic mechanical stimulus. Tibial axial compression was used as an anabolic stimulus after irradiation with heavy ions. Mice (male, C57BL/6J, 16 weeks) were exposed to high atomic number, high energy (HZE) iron ions ((56)Fe, 2 Gy, 600 MeV/ion) (IR, n=5) or sham-irradiated (Sham, n=5). In vivo axial loading was initiated 5 months post-irradiation; right tibiae in anesthetized mice were subjected to an established protocol known to stimulate bone formation (cyclic 9N compressive pulse, 60 cycles/day, 3 day/wk for 4 weeks). In vivo data showed no difference due to irradiation in the apparent stiffness of the lower limb at the initiation of the axial loading regimen. Axial loading increased cancellous bone volume by microcomputed tomography and bone formation rate by histomorphometry in both sham and irradiated animals, with a main effect of axial loading determined by two-factor ANOVA with repeated measure. There were no effects of radiation in cancellous bone microarchitecture and indices of bone formation. At the tibia diaphysis, results also revealed a main effect of axial loading on structure. Furthermore, irradiation prevented axial loading-induced stimulation of bone formation rate at the periosteal surface of cortical tissue. In summary, axial loading stimulated the net accrual of cancellous and cortical mass and increased cancellous bone formation rate despite prior exposure to ionizing radiation, in this case, HZE particles. Our findings suggest that mechanical stimuli may prove an effective treatment to improve skeletal structure following exposure to ionizing radiation.

Metabolic signature of sun exposed skin suggests catabolic pathway overweighs anabolic pathway.

Skin chronically exposed to sun results in phenotypic changes referred as photoaging. This aspect of aging has been studied extensively through genomic and proteomic tools. Metabolites, the end product are generated as a result of biochemical reactions are often studied as a culmination of complex interplay of gene and protein expression. In this study, we focused exclusively on the metabolome to study effects from sun-exposed and sun-protected skin sites from 25 human subjects. We generated a highly accurate metabolomic signature for the skin that is exposed to sun. Biochemical pathway analysis from this data set showed that sun-exposed skin resides under high oxidative stress and the chains of reactions to produce these metabolites are inclined toward catabolism rather than anabolism. These catabolic activities persuade the skin cells to generate metabolites through the salvage pathway instead of de novo synthesis pathways. Metabolomic profile suggests catabolic pathways and reactive oxygen species operate in a feed forward fashion to alter the biology of sun exposed skin.

A low-dose arsenic-induced p53 protein-mediated metabolic mechanism of radiotherapy protection.

Radiotherapy is the current frontline cancer treatment, but the resulting severe side effects often pose a significant threat to cancer patients, raising a pressing need for the development of effective strategies for radiotherapy protection. We exploited the distinct metabolic characteristics between normal and malignant cells for a metabolic mechanism of normal tissue protection. We showed that low doses of arsenic induce HIF-1α, which activates a metabolic shift from oxidative phosphorylation to glycolysis, resulting in increased cellular resistance to radiation. Of importance is that low-dose arsenic-induced HIF-1α requires functional p53, limiting the glycolytic shift to normal cells. Using tumor-bearing mice, we provide proof of principle for selective normal tissue protection against radiation injury.

Effect of culture pH on recombinant antibody production by a new human cell line, F2N78, grown in suspension at 33.0 °C and 37.0 °C.

The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0-7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.

Perfil metabólico y osteogénico de las metástasis óseas por cáncer de próstata / Metabolic and osteogenic pattern of prostate cancer bone metastases

Los protocolos actuales incluyen a la gammagrafia ósea y a la PET/TC con colina para localizar la recidiva en pacientes tratados por un cáncer de próstata que presentan elevación de PSA sérico. Existe una buena concordancia entre ambos métodos en el diagnóstico de las metástasis óseas, pero puedan darse resultados discordantes. Presentamos 3 casos en los hemos realizado una PET/TC con 18F-fluoruro, ante una discordancia entre la gammagrafia ósea y la PET/TC con 11C-colina. En ellos, el uso de 18F-fluoruro ha podido corroborado la existencia de metástasis óseas, por lo que puede ser una alternativa como trazador de segunda modalidad de imagen ósea a la RM, si bien su uso esta limitado por su coste y disponibilidad (AU)

The current protocols include the bone scintigraphy and choline PET/CT to localize recurrence in patients having elevated serum PSA after treatment for prostate cancer. Both methods show good agreement in the diagnosis of bone metastases, however conflicting results can be found. We present three cases in which a PET/CT was performed with 18F-Fluoride due to disagreement between the bone scintigraphy and 11C-Choline PET/CT. The 18F-Fluoride PET/CT was capable of confirming the existence of bone metastasis in all of them, so it may be an alternative to the MRI as a tracer of second bone imaging modality, although its use is limited by cost and availability (AU)

Low-level laser therapy on MCF-7 cells: a micro-Fourier transform infrared spectroscopy study.

Low-level laser therapy (LLLT) is an emerging therapeutic approach for several clinical conditions. The clinical effects induced by LLLT presumably scale from photobiostimulation/photobioinhibition at the cellular level to the molecular level. The detailed mechanism underlying this effect remains unknown. This study quantifies some relevant aspects of LLLT related to molecular and cellular variations. Malignant breast cells (MCF-7) were exposed to spatially filtered light from a He-Ne laser (633 nm) with fluences of 5, 28.8, and 1000 mJ/cm². The cell viability was evaluated by optical microscopy using the Trypan Blue viability test. The micro-Fourier transform infrared technique was employed to obtain the vibrational spectra of each experimental group (control and irradiated) and identify the relevant biochemical alterations that occurred due to the process. It was observed that the red light influenced the RNA, phosphate, and serine/threonine/tyrosine bands. We found that light can influence cell metabolism depending on the laser fluence. For 5 mJ/cm², MCF-7 cells suffer bioinhibition with decreased metabolic rates. In contrast, for the 1 J/cm² laser fluence, cells present biostimulation accompanied by a metabolic rate elevation. Surprisingly, at the intermediate fluence, 28.8 mJ/cm², the metabolic rate is increased despite the absence of proliferative results. The data were interpreted within the retrograde signaling pathway mechanism activated with light irradiation.

Animal studies on growth and development.

Despite the fact that no plausible biological mechanism has yet been identified how electromagnetic fields below recommended exposure limits could negatively affect health of animals or humans, many experiments have been performed in various animal species, mainly mice and rats, to investigate the possible effects on growth and development. While older studies often suffered from sub-optimal exposure conditions, recent investigations, using sophisticated exposure devices and thus preventing thermal effects, have been performed without these limitations. In principle, two types of studies can be addressed: those which have investigated the carcinogenic or co-carcinogenic effects of exposure in developing animals, and those which have been done in developing animals without the focus on carcinogenic or co-carcinogenic effects. In both areas, the vast majority of publications did not show adverse effects. The largest study so far has been done in normal mice which have been chronically exposed to UMTS signals up to 1.3 W/kg SAR, thus 16 times higher than the whole-body exposure limit for humans. Even after four generations, no systematic or dose-dependent alterations in development or fertility could be found, supporting the view that negative effects on humans are very unlikely. Ongoing experiments in our laboratory investigate the effects of head-only exposure in rats (up to 10 W/kg local SAR) which are exposed from 14 days of age daily for 2 h. A battery of behavioral tests is performed in young, adult, and pre-senile animals. The results will help to clarify possible effects of exposure on brain development.

Comprehensive assessment of host responses to ionizing radiation by nuclear factor-κB bioluminescence imaging-guided transcriptomic analysis.

The aim of this study was to analyze the host responses to ionizing radiation by nuclear factor-κB (NF-κB) bioluminescence imaging-guided transcriptomic tool. Transgenic mice carrying the NF-κB-driven luciferase gene were exposed to a single dose of 8.5 Gy total-body irradiation. In vivo imaging showed that a maximal NF-κB-dependent bioluminescent intensity was observed at 3 h after irradiation and ex vivo imaging showed that liver, intestine, and brain displayed strong NF-κB activations. Microarray analysis of these organs showed that irradiation altered gene expression signatures in an organ-specific manner and several pathways associated with metabolism and immune system were significantly altered. Additionally, the upregulation of fatty acid binding protein 4, serum amyloid A2, and serum amyloid A3 genes, which participate in both inflammation and lipid metabolism, suggested that irradiation might affect the cross pathways of metabolism and inflammation. Moreover, the alteration of chemokine (CC-motif) ligand 5, chemokine (CC-motif) ligand 20, and Jagged 1 genes, which are involved in the inflammation and enterocyte proliferation, suggested that these genes might be involved in the radiation enteropathy. In conclusion, this report describes the comprehensive evaluation of host responses to ionizing radiation. Our findings provide the fundamental information about the in vivo NF-κB activity and transcriptomic pattern after irradiation. Moreover, novel targets involved in radiation injury are also suggested.

18-F-fluorodeoxyglucose-positron emission tomography evaluation of early metabolic response during radiation therapy for cervical cancer.

PURPOSE: To document changes in cervical tumor (18)-F-fluorodeoxyglocose (FDG) uptake during radiation therapy and to correlate those changes with post-treatment tumor response and survival outcome. METHODS AND MATERIALS: A total of 36 patients with Stage Ib1 to IIIb cervical cancer were enrolled in an institutional protocol examining the use of fluorodeoxyglucose-positron emission tomography (FDG-PET) for brachytherapy treatment planning. As part of this study, FDG-PET or PET/computed tomograpy (CT) images were obtained before, during, and after the completion of radiation therapy. Tumor metabolic responses were assessed qualitatively and semi-quantitatively by measurement of the maximal standardized uptake value (SUV(max)). RESULTS: Post-treatment FDG-PET images were obtained for 36 patients in this study. Of the patients, 29 patients had a complete metabolic response on the post-treatment PET, 4 had a partial metabolic response, and 3 had new sites of FDG uptake. Six patients had a complete metabolic response observed during radiation therapy, 26 had a partial metabolic response and 4 had stable or increased tumor metabolic activity. For patients with complete metabolic response during radiation therapy, median time to complete response was 29.5 days (range, 18-43 days). The mean cervical tumor SUV(max) decreased from 11.2 (SD, 6.3; range, 2.1-38.0) pretreatment to 2.4 (SD, 2.7; range, 0-8.8) mid treatment, and 0.5 (SD, 1.7; range, 0-8.3) post-treatment. CONCLUSIONS: During radiation therapy for cervical cancer, FDG-PET can be used to monitor treatment response. Complete metabolic response during radiation therapy was observed for a subset of patients. Recommendations regarding the optimal timing of FDG-PET during treatment for cervical cancer will require further systematic study.

Comparison of cellular metabolic responses of (18)F-FDG according to the effect of beta-irradiation in p53 wild and deleted cell lines.

OBJECTIVE: The tumor suppressor gene, p53, plays a pivotal role for cell survival and apoptosis, which could cause different responses to therapeutic agents. Rhenium-188 ((188)Re) decays with the emission of a beta-particle with high energy and is expected to be an important candidate of radiotherapy. We investigated the cellular response of (18)F-fluorodeoxyglucose ((18)F-FDG) uptake and the effect of p53 according to beta-irradiation by (188)Re. METHODS: The HCT116 human colon adenocarcinoma cell lines, containing a wild-type p53 (p53(+/+)) and a p53-deleted derivative (p53(-/-)), were gifts from Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). Cells were plated in 24-well plates at 1.0 x 10(5) cells, then (188)Re perrhenate was added and incubated for 24 hours. After irradiation, we performed a cellular uptake assay of (18)F-FDG (370 kBq, 60 minutes). We assayed the hexokinase, cell viability, and cell cycle. RESULTS: p53-deleted HCT116 cells showed a higher (18)F-FDG uptake and increased hexokinase activity after (188)Re treatment. p53-deleted cells showed a higher G2/M (Gap2/Mitosis) arrest in a relatively low dose of beta-irradiation. However, cell viability was not different, according to the p53 status, after (188)Re treatment. CONCLUSIONS: Therefore, p53 seemed to have a significant role in cellular glucose metabolism and G2/M checkpoint, according to beta-irradiation, and could cause a different therapeutic response of (18)F-FDG uptake in cancer cells.

Distinct effects of ionizing radiation on in vivo murine kidney and brain normal tissue gene expression.

PURPOSE: There is a growing awareness that radiation-induced normal tissue injury in late-responding organs, such as the brain, kidney, and lung, involves complex and dynamic responses between multiple cell types that not only lead to targeted cell death but also acute and chronic alterations in cell function. The specific genes involved in the acute and chronic responses of these late-responding normal tissues remain ill defined; understanding these changes is critical to understanding the mechanism of organ damage. As such, the aim of the present study was to identify candidate genes involved in the development of radiation injury in the murine kidney and brain using microarray analysis. EXPERIMENTAL DESIGN: A multimodality experimental approach combined with a comprehensive expression analysis was done to determine changes in normal murine tissue gene expression at 8 and 24 hours after irradiation. RESULTS: A comparison of the gene expression patterns in normal mouse kidney and brain was strikingly different. This observation was surprising because it has been long assumed that the changes in irradiation-induced gene expression in normal tissues are preprogrammed genetic changes that are not affected by tissue-specific origin. CONCLUSIONS: This study shows the potential of microarray analysis to identify gene expression changes in irradiated normal tissue cells and suggests how normal cells respond to the damaging effects of ionizing radiation is complex and markedly different in cells of differing origin.

[Polysystemic assessment of the state of sanogenesis in workers employed in nuclear fuel plants. The analysis of metabolism regulation processes]. / Polisistemnaia otsenka sostoianiia sanogeneza rabotnikov predpriiatiia iaderno-toplivnogo tsikla.Analiz reguliatsii obmennykh protsessov.

Using the method of laser correlation spectroscopy of biological fluids (blood serum, urine, oropharyngeal washout fluid) we studied the types of metabolic shifts in workers employed in nuclear fuel complex plant. In was found that the incidence of catabolic shifts considerably increased in workers with higher level of occupational exposure. In individuals contacting with open radiation sources we found the contribution of anabolic immunomodifying shifts with predominance of autoimmune sensibilization. A risk group for blood diseases was identified.

[Nonmonotonous metabolic response of mammalian cells and tissues to ionizing radiation]. / Nemonotonnost' metabolicheskogo otveta kletok i tkanei mlekopitaiushchikh na vozdeistvie ioniziruiushchei radiatsii.

Changes in the activity of ornithindecarboxylase in various tissues and in the amount of catecholamine in rat hypothalamus by the action of acute and chronic ionizing radiation were studied. A nonmonotonous relationship between the metabolic parameters of animal tissues and cells and the radiation dose was revealed. It was assumed that the nonmonotonous character of the dose-response dependence results from the nonmonotonous time course of the metabolic response to irradiation. It was also assumed that living systems have the property of responding to stress agents by nonmonotonous changes in metabolism. In the case of acute irradiation, this response manifests itself as oscillations of metabolic parameters about the control. The oscillations occur with a particular amplitude and periods, which vary with radiation dose, and damp out with time. As a result, in a fixed time interval, the dose-response curve may be nonmonotonous. Reverse dose-response relationships are also possible. In the case of chronic irradiation, the metabolic and functional parameters oscillate throughout irradiation time, and a modification of the response occurs. A prolong exposure to ionizing radiation causes strong changes in the metabolism of lipids of cell membranes, organelles and chromatin, as well as in the functional properties of some mammalian cells and tissues. The necessity of constructing quantitative models for explaining the nonmonotonous dose-response dependence is discussed.

Effect of pre-exposure to beta rays of tritium on some biochemical parameters measured in organs of rats subsequently irradiated with fast neutrons.

The experiment examined biological responses produced by combined sequential exposure to low-level tritium contamination, followed by challenging irradiation with fast neutrons. Modifications of endogenous antioxidant potential of different organs in rats were discussed in relation to tissue radiosensitivity. Rats pre-contaminated to 7 cGy and 35 cGy have been additionally irradiated to 1 Gy with fast neutrons. Lipid peroxide level was determined in liver, kidney, small intestine, spleen, bone marrow, and plasma. Reduced glutathione (GSH) level and glucose-6-phosphate dehydrogenase (G6PDH) activity were determined in erythrocytes. An in vitro thymidine uptake assay was performed in isolated bone marrow cells. The lipid peroxide level decreased significantly only in liver and kidney from rats pre-exposed to 35 cGy. For small intestine and spleen, tissues of comparatively higher radiosensitivity, no induced radioprotection was observed, as reflected in the homeostasis of the lipid peroxides. The same behavior was observed in bone marrow, the most radiosensitive tissue studied. However, the bone marrow thymidine-incorporation assay revealed a possible adaptive-type reaction in rats pre-exposed to 35 cGy. We conclude that for radiosensitive tissues pre-exposure to chronic low doses of low linear energy transfer (LET) irradiation has no protective effect on their antioxidant status, whereas a protective effect is observed in radioresistent tissues.

Do metabolic responses to solar radiation scale directly with intensity of irradiance?

Endotherms exposed to air temperatures below thermal neutrality reduce their metabolic heat production when exposed to sunlight. The physiological effects of this additional source of heat gain from the environment usually are assumed to be proportional to the intensity of irradiance if other factors are held constant. We test this assumption by measuring changes in metabolic heat production produced by exposing a small mammal, the Siberian hamster (Phodopus sungorus) to four intensities of simulated solar radiation (0 W m-2, 317 W m-2, 634 W m-2 and 950 W m-2). In the absence of solar radiation, metabolic heat production is inversely correlated with air temperature over the measured range of 3-27 degrees C. The respiratory quotient varies significantly with ambient temperature, indicating that the catabolic substrate and the thermal equivalent of oxygen consumed or carbon dioxide produced also vary with temperature. The depression of metabolic heat production resulting from exposure to simulated solar radiation is not simply a multiple of the intensity of irradiance. Rather, metabolic responses to higher levels of irradiance are blunted by 14-29% compared with those expected on the basis of the response to less intense irradiance. Because changes in irradiance levels do not have simple linear effects upon the animal's metabolic heat production, even in a simplified situation, significant errors may accumulate in biophysical analyses in which an animal's responses to a restricted set of radiative conditions are measured and the results are extrapolated to a wider range observed in nature.