All authors read and approved the final manuscript.”
“Background Lead (Pb) is a widely distributed, environmentally persistent, toxic metal. Most bacteria that are tolerant or resistant to lead either precipitate SAR302503 Pb in an insoluble form, or actively export it [1]. Although some metal efflux ATPases, such as ZntA from Escherichia coli, and CadA from Staphylococcus aureus plasmid pI258, can export Pb(II) as well as Zn(II) and Cd(II) [2, 3], the only characterized bacterial Pb(II) specific resistance system is

from Cupriavidus (formerly Wautersia and Ralstonia) metallidurans CH34 [4, 5] – a Gram-negative, multiply metal-resistant, β-proteobacterium originally isolated from a decantation basin at a Belgian zinc production plant (and originally identified as Alcaligenes eutrophus CH34; [6]). Over 150 genes in CH34 are involved in metal resistance, of which at least 70 are carried on the plasmids pMOL28 (171 kb) or pMOL30 (234 kb), and the remainder are carried on the 3.92 Mb chromosome or on a 2.58 Mb second chromosome [7]. Plasmid pMOL30 carries the czc (Cd(II), Zn(II), Co(II)), mer (Hg(II)), STA-9090 in vitro sil (Ag(I)), cop (Cu(II)) and pbr Pb(II) resistance operons [4, 8]. The pbr lead resistance operon from pMOL30 was originally S3I-201 mw predicted to contain

structural genes which encode PbrT, a putative Pb(II) uptake protein belonging to the ILT (Iron Bay 11-7085 Lead Transporter) family [9], a P-type efflux ATPase (PbrA), a predicted inner–membrane protein (PbrB), a predicted prelipoprotein signal peptidase PbrC and a Pb(II) binding protein, PbrD. The regulator of the pbr operon was shown to be PbrR, which is a MerR family regulator [4, 10] Subsequent work has shown that the pbr operon also contains an interrupted orf; pbrU upstream of

pbrT[11, 12] which is predicted to encode a putative inner membrane (Major Facilitator Family MFS1) permease gene, which is probably inactive, but still part of the pbr operon; and that PbrB/PbrC is a fusion protein [11, 12], and encodes an inner membrane bound undecaprenyl pyrophosphate (C55-PP) phosphatase [5]. The pbr operon contains a predicted MerR-like promoter from which pbrRTU are transcribed on one DNA strand, and the pbrABCD genes are transcribed as a polycistronic message on the other [4, 12]. The most recent work on the mechanism of lead resistance encoded by the pMOL30 pbr operon has proposed a model where Pb2+ induces expression of the pMOL30-encoded PbrABCD by PbrR, as well as expression of zinc and cadmium efflux ATPase homologs ZntA and CadA which are carried on the chromosome or second chromosome. Each of these three ATPases is involved in exporting Pb2+ into the periplasm where inorganic phosphates produced by PbrB are involved in precipitating Pb2+ as insoluble lead phosphate.

Current land cover We used the 1 km resolution Global Land Cover 2000 (GLC2000) map [European Commission Joint Research Centre (EU JRC) 2003] to derive the fraction of each cell corresponding to the following three current land cover classes: (1) forested land (GLC2000 classes 1–6); (2) other natural lands (GLC2000 classes 7–15 and 50 % of the mixed classes 17 and 18), such as shrubland, herbaceous land and mangroves; and (3) cultivated or managed areas (GLC2000 classes 16 and 50 % of classes 17 and 18), which include land converted for crop production and

managed pasture (but not Histone Methyltransferase inhibitor unmanaged pasture land, which is included under other natural land cover). GLC2000 land cover data have been produced and validated regionally and are generally Bafilomycin A1 in vitro considered more accurate and identify forest cover more accurately than alternatives (e.g. 81 % accuracy for forest

vs 60 % accuracy for GlobCover 2005; Fritz et al. 2011), and for the purpose of this study were considered the best available data (Mayaux et al. 2006). Biophysical suitability for agriculture We obtained Selleckchem Combretastatin A4 5′ × 5′ resolution data on land suitability for agriculture from the Global Agro-Ecological Zones (GAEZ; van Velthuizen et al. 2007). In their analysis, for each grid cell, suitability was assessed based on biophysical factors (including climate, soil and terrain conditions) for nine major crop groups (cereals, fibre crops, fibres, oil crops, pulses, roots and tubers, sugar crops, tree fruits and vegetables). The

GAEZ methodology provides a suitability index (SI) for each grid cell for each crop under different input levels. We used SI data that assumes “maximised technological 4-Aminobutyrate aminotransferase mix” for rain-fed agriculture (e.g. the higher level of technology and management inputs will be employed only in areas capable of producing high yields under those systems; for details how the SI was derived see van Velthuizen et al. 2007). Although biophysical factors do not ‘drive’ land-cover change directly, they influence land cover allocation decisions (e.g. according to slope or soil quality) (Verburg et al. 2004). Economic Pressure on Land index Our “Economic Pressure on Land” (EPL) index synthesizes distinct, but fundamentally synergistic demographic and economic forces related to land-cover change. Each grid cell is subject to an economic force for conversion that radiates from the nearest market in a direct relation to that market’s demand and in an inverse relation to the travel distance between the grid cell and the market.

Appl Environ Microbiol 1994,60(6):1875–1883.PubMed 59. Pearce BJ, Iannelli F, Pozzi G: Construction of new unencapsulated (rough) strains of Streptococcus pneumoniae . Res Microbiol 2002,153(4):243–247.PubMedCrossRef

60. Bordoni R, Bonnal R, Rizzi E, Carrera P, Benedetti S, Cremonesi L, Stenirri S, Colombo A, Montrasio C, Bonalumi S, et al.: Evaluation of human gene variant detection in amplicon pools by the GS-FLX parallel Pyrosequencer. BMC Genomics 2008, 9:464.PubMedCrossRef 61. Iacono M, Villa L, Fortini D, Bordoni R, Imperi F, Bonnal RJP, Sicheritz-Ponten T, De Bellis G, Visca P, Cassone A, et al.: Whole-genome pyrosequencing of an epidemic multidrug-resistant Acinetobacter baumannii strain belonging to the European clone II group. Antimicrob Agents Chemother 2008,52(7):2616–2625.PubMedCrossRef 62. Yildirim I, Hanage WP, Lipsitch M, eFT-508 cost Shea KM, Stevenson A, A-769662 mouse Finkelstein J, Huang SS, Lee GM, Kleinman K, Pelton SI: Serotype specific invasive

capacity and persistent SAHA HDAC ic50 reduction in invasive pneumococcal disease. Vaccine 2011,29(2):283–288.CrossRef 63. Nakagawa I, Kurokawa K, Yamashita A, Nakata M, Tomiyasu Y, Okahashi N, Kawabata S, Yamazaki K, Shiba T, Yasunaga T, et al.: Genome sequence of an M3 strain of Streptococcus pyogenes reveals a large-scale genomic rearrangement in invasive strains and new insights into phage evolution. Genome Res 2003,13(6A):1042–1055.PubMedCrossRef 64. Maruyama F, Kobata M, Kurokawa K, Nishida K, Sakurai A, Nakano K, Nomura R, Kawabata S, Ooshima T, Nakai K, et al.: Comparative Olopatadine genomic analyses of Streptococcus mutans provide insights into chromosomal shuffling and species-specific content. BMC Genomics 2009, 10:358.PubMedCrossRef 65. Denapaite D, Bruckner R, Nuhn M, Reichmann P, Henrich B, Maurer P, Schahle Y, Selbmann P, Zimmermann W, Wambutt R, et al.: The genome of Streptococcus mitis B6–what is a commensal?

PLoS One 2010,5(2):e9426.PubMedCrossRef 66. Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, et al.: Genome sequencing in microfabricated high-density picolitre reactors. Nature 2005,437(7057):376–380.PubMed 67. Besemer J, Borodovsky M: GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res 2005, (33 Web Server):W451–454. 68. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL: Improved microbial gene identification with GLIMMER. Nucleic Acids Res 1999,27(23):4636–4641.PubMedCrossRef 69. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B: Artemis: sequence visualization and annotation. Bioinformatics 2000,16(10):944–945.PubMedCrossRef 70. Porter RD, Guild WR: Characterization of some pneumococcal bacteriophages. J Virol 1976,19(2):659–667.

Effect of low concentrations

of dissolved oxygen on zoospore survival As in the dissolved oxygen elevation assays, the greatest colony counts in the control bottles occurred at 10-min exposure for P. megasperma and at 2- or 4-h exposure for the other three species (Table 3). Table 3 Linear regression analyses of colony counts (y) and levels (x) of dissolved oxygen reduction from that in the control Hoagland’s solution by Phytophthora species and exposure time z Species Exposure (h) Intercept ( a ) Slope ( b ) P P. megasperma 0 (10 min) 18.2 -1.0 0.0936   2 11.3 -0.2 0.6267   4 9.9 -0.8 0.0104   8 7.4 -0.3 0.2903   24 8.4 -0.7 0.0292   48 7.6 -0.9 0.0015   72 4.5 -0.3 0.0724 P. nicotianae 0 7.8 0.8 0.1067   2 25.0 -1.2 0.0548   4 28.5 -2.6 0.0008   8 12.3 -0.4 0.4421   24 5.1 -0.2 0.4100   48 3.6 0.0 0.8670

  72 2.2 0.1 0.3973 P. pini #https://www.selleckchem.com/products/crt0066101.html randurls[1|1|,|CHEM1|]# 0 9.1 0.4 0.2462   2 32.6 -0.3 0.6893   4 37.2 -2.1 0.0002   8 20.8 -1.3 < 0.0001 H 89 mouse   24 14.4 -0.8 0.0034   48 7.4 -0.3 0.2382   72 8.3 -0.5 0.0313 P. tropicalis 0 27.8 -1.8 0.0156   2 31.4 -1.3 0.0749   4 29.7 -0.3 0.6712   8 22.5 -0.1 0.8042   24 7.8 -0.3 0.1730   48 0.7 0.4 0.0008   72 0.4 0.2 0.0079 zLinear model: y = a + bx, in which x = 5.3 - meter readings of dissolved oxygen in the Hoagland’s solutions after being bubbled with pure nitrogen, so 0 ≤ x ≤ 5.3 mg L-1. Zoospore survival of the four species assessed in this study also was negatively impacted by low concentrations of dissolved oxygen in two distinct patterns (Table 3). One pattern is represented by P. megasperma and P. pini. The impact on these two species generally occurred at 4-h or longer exposures at which their colony counts decreased with increasing level of dissolved oxygen reduction from the normal concentration of 5.3 mg L-1 in the control Hoagland’s solution. The greatest rate of decrease in colony counts

occurred at 48-h exposure for P. megasperma at 0.9 colony per unit of dissolved oxygen reduction (P = 0.0015) and at 4-h exposure for P. pini at 2.1 (P = 0.0002). Phytophthora Succinyl-CoA nicotianae and P. tropicalis showed an exactly opposite pattern. The colony counts decreased with increasing level of reduction in dissolved oxygen concentration at both 2- and 4-h exposures for P. nicotianae, 10-min and 2-h exposures for P. tropicalis. These results indicate that P. nicotianae and P. tropicalis are more prone than P. megasperma and P. pini to hypoxia stress in aquatic environments. They help understand the more consistent and greater recoveries of P. megasperma and P. pini than other major plant pathogens including P. nicotianae and P.

g., for grain or cellulosic ethanol, for algal or vegetable oils for biodiesel, or biomass gasification and Fischer–Tropsch reforming for

hydrocarbons. The photon energy densities and process productivities, plus the advantage of no arable land or freshwater displacement, create a scenario in which a minimal dedication of marginal land can serve to meet US renewable fuel standards. Comparisons are often made between the energy efficiencies of photosynthesis and those for solar electricity generation. It is important to make these comparisons in the proper context. Solar thermal or photovoltaic systems generate power requiring economical and efficient storage and transmission into the electrical grid, whereas the systems described here generate easily stored energy check details in liquid form. Moreover, values quoted for solar power systems are peak efficiencies that fall off precipitously under even momentary shading (Curtright and Apt 2008). Solar electricity efficiencies are also compounded by battery efficiencies and impedance losses that introduce system-specific variability. Manufacturing fuels to direct them into an existing refining, distribution,

and transportation infrastructure would be more fairly compared to other existing and developing technologies for energy conversion to reasonably storable forms and not to electricity. The aquatic species program report of 1998 (Sheehan et al. 1998) and the recently published National Algal Biofuels Technology Roadmap BIIB057 (2009) each conclude that photosynthesis could support

viable fuel processes given advances in organism and process productivities. Organism Thymidine kinase engineering, direct production, product secretion, and process optimization are areas for improvement to achieve viability. The direct photosynthetic platform is an alternative approach that addresses many of these ideas and offers efficiencies nearest to a thermodynamic maximum with more advantageous process economics. AZD9291 datasheet further application of systems and synthetic biology approaches could extend the range of efficiency for photosynthetic processes. For example, some photosynthetic microorganisms, particularly the nonoxygenic bacteria, have light capture systems allowing them to extend the PAR range into the near infrared (up to ~1,100 nm; Kiang et al. 2007). Incorporating these alternate photon-capturing and reaction center complexes into oxygenic production organisms to supplement endogenous systems and broaden the spectrum of light harvesting could further optimize efficiency relative to PAR. Other innovations that reduce culture reflection, enhance photon capture, and broaden temperature optima can also be envisioned using advanced organism-engineering tools.

The quantitative and spatially explicit results of this study may serve as a base layer within which those more intricate relations will play their role. Our results suggest, however, that this basic model explains a significant proportion of the global land cover, and provides insights about what may be expected over the coming decades. We also demonstrated that interventions

for reducing deforestation without complementary policies addressing the agricultural drivers of forest loss and demand for land, may have limited effectiveness in climate change mitigation. If national REDD + policies are to be effective, they must be accompanied by complementary international measures, such as trade regulation beyond the borders of individual countries to avoid leakage. Scientific Akt activity and policy approaches should therefore encompass both forests and other natural ecosystems, as well as agricultural land, along with the links among them. This perspective incorporates the interdependencies and synergies involved in land-cover LY3039478 price change and adopt the whole-landscape approach (DeFries and Rosenzweig 2010). If the global population stabilizes

at about 9 billion people, the coming 50 years may be the final episode of rapid global agricultural expansion and land-cover change. During this period, Salubrinal research buy fuelled by increasing economic and demographic pressure, agriculture and other human subsistence practices have the potential to have irreversible impacts on the environment. Despite this gloomy prognosis there is evidence from a few countries, such as Costa Rica and Bhutan, that appropriate policies may allow an increase in food production without conversion of all available land (Ewers et al. 2009; Lambin and Tideglusib Meyfroidt 2011; Rudel et al. 2009). Understanding land-cover change trajectories presents a unique opportunity to estimate the size of possible displacement of land-cover, and to test the effects of policies

to limit this problem. In doing so, it may aid in focusing and prioritising conservation efforts, and facilitate environmental management and planning in the context of a continued pursuit of economic development. Acknowledgments This study was supported by the Gordon and Betty Moore Foundation, The Planetary Skin Institute and the UN-REDD Programme. Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. References Baillie JEM, Hilton-Taylor C, Stuart SN (2004) A Global Species Assessment IUCN. Gland, SwitzerlandCrossRef Bouwman AF, Kram T, Klein Goldewijk K (eds) 2006 Integrated modeling of global environmental change. An overview of IMAGE 2.4.

Given the impact of chronic Ion Channel Ligand Library stress on a cancer patient, the confluence of the psychological and physical discomfort places the patient at high risk for the occurrence of stress-induced Tipifarnib solubility dmso behavioral alterations which usually presents depression, anxiety, sadness, fear and hopelessness [4, 11, 31, 32]. We reported previously that 39.5% of cancer patients were unwilling to realize the diagnosis of cancer, 63.0% were burdened with mental stress and 33.0% considered the impact of mental stress above that of somatic symptoms [33]. We hypothesize that the discrepancy of the efficacy of anti-angiogenic drugs between clinical and

preclinical results is caused by chronic stress, which has not been yet identified. So in this research, the goal is to investigate whether NE, one of the most potent stress related hormones, can attenuate the efficacy of sunitinib in a mouse model and whether this effect can be blocked by propranolol. Materials

LXH254 research buy and methods Cell culture The murine melanoma B16F1 cells and human lung adenocarcinoma A549 cells, kind gifts from State Key Laboratory of Biotherapy (Sichuan University, Chengdu), were authenticated by the supplier [29] and cultured in RPMI 1640 complete medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin Nintedanib mouse at 37°C with 5% CO2 in humidified atmosphere. Reagents NE, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), isoproterenol, dobutamine and terbutaline were purchased from Sigma (St. Louis, MO, USA); propranolol and 8-CPT from Enzo (Germany); forskolin from Biovision (USA); H-89 and myristoylated PKI from Calbiochem (USA);

sunitinib from Pfizer (USA); RNAiso plus and One Step SYBR® PrimeScript™ RT-PCR Kit from TaKaRa (Japan). In vitro cell proliferation assays for measuring the IC50 (half maximal inhibitory concentration) of sunitinib in B16F1 cells B16F1 cells were harvested and seeded in 96-well plates (5,000 cells/200 μL complete medium/ well). After 24 hours incubation, the cells were exposed to various concentrations (0–100 μM, each concentration had six replicate wells) of sunitinib for 48 h. Following sunitinib treatment, 20 μL of 5 mg/mL MTT was added to each well and incubated at 37°C for 4 hours. The plates were centrifuged, the supernatants were carefully discarded and formazan crystals were dissolved in 150 μL DMSO. At last, the light absorbance at 490 nm was determined in a luminescence plate reader (PerkinElmer, USA) according to the manufacturer’s instructions. Evaluation of the influence of NE on mRNA and protein expression in vitro B16F1 and A549 cells were dispensed in six-well culture plates (2 × 105/well).

This consideration is in agreement with the observation

that zin T is constitutively expressed in a znu A Temsirolimus mutant strain, but that ZnuA accumulation is not significantly modulated by the absence of zin T (Figure 5). LY2603618 supplier This is likely explained by a decrease of the zinc concentration in the cytoplasm in the absence of ZnuA, but not of ZinT, with the consequent derepression of zin T by Zur. It should be highlighted that the zin T mutant strain exhibits a sharp growth defect either in LB supplemented with 0.5 mM EDTA or in defined medium. This behaviour was not observed in a zin T mutant of S. enterica [18], which showed a clear impairment of growth in LB only in presence of 2 mM EDTA, a concentration at which the E. coli O157:H7 mutant is hardly able to grow. Furthermore, our results indicate that there are differences between E. coli O157:H7 and S. enterica in the regulation of znu A and zin T in response to low zinc availability (Figure 4). In particular, Selleckchem MK-0457 in E. coli O157:H7 ZinT can be easily detected in bacteria growing in a medium supplemented with up to 1 μM zinc, whereas in S. enterica this protein accumulates only in media completely devoid of the metal. This observation, which is in agreement with the different effect of zin T disruption in the

two bacterial species, may suggest that the relative role of ZnuA and ZinT could be slightly different in the two microorganisms. Although several of the bacteria which rely on the ZnuABC transporter to import zinc do not possess DCLK1 ZinT [18], our study suggests that, despite the role of ZinT is clearly dependent on the presence of ZnuA, its contribution to metal recruitment within the periplasmic space is considerable. The exact involvement of ZinT in zinc uptake is yet to be determined, but it is possible to hypothesize that ZinT and ZnuA display a diverse ability to sequester metal ions from different molecules within the periplasm or that the binding of ZinT to ZnuA accelerates the rate of metal transfer to

ZnuB [18]. We have also analyzed the involvement of the zinc uptake system in the interaction between E. coli O157:H7 and epithelial Caco-2 cells. Both ZnuA and ZinT accumulates at high levels in bacteria adhering to the cell monolayer, but not in bacteria cultivated in D-MEM without cells (Figure 9). This finding expands previous observations showing that bacterial pathogens have to face with a problem of zinc paucity within the host [17] and specifically suggests that the host cell surface microenvironment is poor of zinc, possibly due to active metal sequestration mechanism implemented by eukaryotic cells. In line with this observation strains lacking znu A display a reduced ability to adhere to epithelial cells (Table 4).

Data

analysis Conditional logistic regression analysis was used to estimate the risk of hip/femur fracture associated with the use of Captisol manufacturer dopaminergic drugs and were expressed as odds ratios (OR) with corresponding 95% confidence intervals (CI). Adjusted odds ratios (ORadj) for hip/femur fracture were estimated after adjustment for the various confounding variables. Final regression models RXDX-101 cell line included all potential confounding factors that changed the natural logarithm of the risk estimate with more Selleckchem RG7420 than 5%. Stratified analyses within current dopaminergic drug users were performed regarding gender, age category,

type of current dopaminergic drug (dopamine agonist, levodopa-containing drug, or combined use) and concomitant use of anticholinergics, antidepressants, antipsychotics or benzodiazepines. In order to differentiate between onset and offset of the effect of dopaminergic drugs on hip/femur fractures, two separate analyses were performed: (1) the onset was investigated by calculating the risk of hip/femur fractures in relation to continuous duration of dopaminergic drug use within current users; (2) the offset was investigated by calculating the risk of hip/femur fractures in relation to the recency of use of dopaminergic drug treatment within ever users. In both analyses, the dopaminergic drug users were subdivided into 10 subgroups based on deciles of the continuous duration of use (or recency of use). An OR was calculated for each of the subgroups.

Spline regression was then used to smooth these estimates and to visualise Tau-protein kinase any trends. This method has been advocated as an alternative to categorical analysis [31]. Analyses were performed with SPSS 16.0. Spline regression was performed with SAS 9.1.3. Results We identified 6,763 cases with a fracture of the hip or femur and 26,341 matched controls (Table 1). Almost three-quarters (73%) of the study population was female. The mean duration of follow-up before the index date was 5.8 years for cases and 5.7 years for controls. The median age was 79 years for cases and controls.

Therapeutic effects of the MLs were inconsistent and not very impressive in the reviewed experiments. However, in other tumour types, MLs have also shown substantial growth-inhibiting effects (e.g. [154–157]). Interestingly, in two experiments, the application of VAE-activated macrophages in mice not directly treated with VAE also showed tumour-growth inhibiting effects, while the application of non-activated macrophages had no effects [121]. Similarly in melanoma, the application of VAE-activated splenocytes inhibited

metastasis [158, 159]. In general, the predictive reliability of the preclinical studies for clinical application is #OSI-906 supplier randurls[1|1|,|CHEM1|]# fairly limited in most instances. Clinical cancer disease is insufficiently mimicked by animal models, with major differences regarding age, general condition, co-morbidity, invasiveness, metastases, antigenicity, immune system etc. The results of preclinical screening, especially for treatment of solid tumours, have therefore been largely disappointing. The models currently regarded as best for cytotoxic substances use patient-derived tumours that grow subcutaneously or orthotopically in nude mice, as in several cases reviewed here. Immuno-active substances FK228 chemical structure may however still be insufficiently assessed in immune-deficient animals, as the main components of the immune system

are missing (nude mice, for instance, cannot generate mature T-lymphocytes). Nevertheless, these preclinical experiments can provide important additional information for detecting the possible anti-cancer effects of medicinal

plants, their active compounds, their mode of action and potential risks [20, 160–162]. Safety aspects Mistletoe therapy was well tolerated in the reviewed studies. Mild flu-like symptoms and local reactions at the injections sites are frequent, dose-dependent and self-limited. Allergic reactions can occur, and a few case reports of anaphylactic reactions exist [163–166]. A phase I study, conducted at the NCCAM/NCI, investigated safety, toxicity and drug interactions between VAE and gemcitabine see more [73] and reported good tolerability, with neither dose-limiting toxicity of the VAE nor any effects on the plasma concentration of gemcitabine [44]. Combination of VAE with chemotherapy or radiotherapy did not negatively influence remission rate in clinical and in animal studies [56, 63, 118]. A higher prevalence of depression in VAE-treated patients in one study was observed in raw data of a self-selected population, without adjustment of baseline imbalances. This difference can be ascribed to variations in the patient population; for instance, they differed markedly in the prevalence of hormone treatment. No toxicity was observed in animal experiments.