Review: Connecting circularity to animal welfare calls for a 'novel' conceptual framework based on integrity.

The current food system is not sustainable. Circular agriculture aims to save the environment and produce food sustainably by closing nutrient cycles, possibly without improving animal welfare. This paper proposes a new conceptual framework, called a circular welfare economy (CWE), to facilitate a transition towards a sustainable agriculture based on integrity. The CWE framework explains how welfare relates to circular agriculture, how potential conflicts can be solved and what future livestock farming could look like. CWE applies the notion of circularity to welfare defined as the quality of life as perceived by the individual itself. CWE also identifies human integrity, defined as being open and honest, as a sine qua non for sustainability. Animal-welfare problems arise when animals are merely used as a means, e.g., for profits. Instead, profits and circular agriculture are means to the end of welfare. In a CWE, welfare is promoted sustainably, without causing undue need frustration in other individuals. This requires informed moral decision-making involving human integrity and the closure of welfare-related feedback loops. Conflicts between circular agriculture and animal welfare are solved by weighing all welfare needs impartially. Three future scenarios are presented: Animal-welfare-exclusive circular agriculture, which resembles modern intensive livestock farming, animal rights agriculture without livestock farming, and a CWE-based agriculture which integrates circular agriculture and animal welfare. In the latter case, we will not use animals merely as a means to close nutrient cycles, but take every effort, openly and honestly, to understand and benefit their points of view as we do our own.

Quantitative evaluation of single cell spread on collagen matrices.

Cells change their morphology as a response to environmental cues. The quantitative evaluation of single cell spread on extracellular matrices, such as type I collagen, is a key tool in cancer research. Inherent to the manual scoring of cellular spread is inter-observer but also intra-observer variation. To overcome these problems, we have developed the Morphology Analysis Software (MAS). MAS scores phase-contrast images of cells on native type I collagen gels and identifies whether a cell has a spread or round morphology using a combination of four unique parameters: the presence of a cellular extension, the cell area, the cell eccentricity and cell circularity. The MAS software scores are equivalent to the average score of five independent observers but MAS is faster, more objective and standardized. A functional screening assay using six cytokines identified TGFα as a stimulator of HCT8/E11 and SK-BR-3 single cell spreading on top of type I collagen gels. This change in morphology correlates with increased migration potential as evidenced by xCELLigence migration assays and are counteracted by EGFR signaling pathway inhibitors. This underscores the use of morphology classification on a population of unlabeled cells as read-out of an important cancer cell property and the potential for the MAS software in drug screening strategies.

The serum estradiol concentration is the main determinant of the estradiol concentration in normal breast tissue.

BACKGROUND: The estrogen concentration has been determined in breast tissue, focusing largely on samples obtained from breast cancers. In this study, estradiol concentration was determined in normal breast tissue obtained from women undergoing esthetic, and oncoplastic surgery. METHODS: Normal breast tissue was obtained during 68 operations for esthetic or reconstructive indications in women with and without a history of breast cancer. Our study included six different groups of women. The first three groups were normal cycling women, women taking oral contraceptives, and normal postmenopausal women, all undergoing a bilateral esthetic breast reduction. The second three groups were premenopausal and postmenopausal women, with a history of breast cancer and currently taking tamoxifen treatment, or postmenopausal women currently taking an aromatase inhibitor, needing contra-lateral corrective esthetic surgery. FINDINGS: In the group of women without history of breast cancer, normal cycling women (n=24) presented a strong correlation (r=0.853; P<0.0001) between 17ß-estradiol concentration in serum (median: 29.7 pg/mL; IQR: 10.8-82.3 pg/mL) and in breast tissue (30.6 pg/g; IQR: 18.6-183.8 pg/g). Postmenopausal women had low 17ß-estradiol concentrations both in serum and breast tissue (r=0.813; P<0.0001, n=16). Women taking oral contraceptives (n=12) had low serum and breast tissue levels of estradiol (r=0.376; P=n.s.). Premenopausal women (n=6, mean age: 44.2 years) with a history of breast cancer and currently taking tamoxifen, had very high concentrations of 17ß-estradiol both in serum (277.9 pg/mL; IQR: 96.2-544.7 pg/mL) and in epithelial cells (251.9 pg/g; IQR: 115.0-426.5 pg/g) (r=0.803; P<0.001). Postmenopausal women taking tamoxifen (n=4, mean age: 48.3) had low concentrations of 17ß-estradiol in serum (7.0 pg/mL; IQR: 5.7-16.3 pg/mL) and in epithelial cells (14.6 pg/g IQR: 13.3-16.3 pg/g) (r=0.10; P=n.s.). The estradiol concentration in the breast of premenopausal women taking tamoxifen was 8.2 times higher that observed in the breast of normal cycling women, and 17.3 times higher that observed in postmenopausal women taking tamoxifen. Women taking adjuvant aromatase inhibitors had extremely low concentrations of 17ß-estradiol both in serum and in breast tissue. INTERPRETATION: This study shows that serum estradiol levels largely determine estrogen levels in normal breast tissue.

Epithelial expression of FHL2 is negatively associated with metastasis-free and overall survival in colorectal cancer.

BACKGROUND: Four-and-a-half LIM domains protein 2 (FHL2) is a component of the focal adhesion structures and has been suggested to have a role in cancer progression. It has been shown to be overexpressed in the colorectal cancer (CRC). METHODS: Here, we examined a possible prognostic value of FHL2 in CRC. Immunohistochemistry for FHL2 was performed on 296 CRCs without distant metastases at the time of surgery. Staining in the epithelial compartment was quantitatively evaluated using image analysis, and results were related to clinical variables. Antibody specificity was tested using small-interfering RNA transfection in hTERT-immortalised myofibroblasts. RESULTS: Varying degrees of cytoplasmic FHL2 expression by neoplastic epithelial cells were detectable in all cases. Higher FHL2 expression in the epithelial compartment was an independent adverse prognostic factor. Multivariate Cox analysis shows that expression in the tumour invasion front (P<0.001) as well as in the centre of the tumour (P<0.001) was associated with metachronous metastases independently of the clinicopathological variables; expression in the tumour invasion front was also associated with overall survival independently of the clinicopathological variables (P<0.01). CONCLUSION: Higher FHL2 expression is involved in CRC progression and correlates with the development of metachronous metastases and overall survival, suggesting that FHL2 is an independent adverse prognostic indicator for CRC.

Assessment of the antineoplastic potential of chalcones in animal models.

One part of chemical space that is endowed with interesting biological properties is the area of the chalcones. With this review, we provide a comprehensive overview of the numerous in vivo animal studies on the antineoplastic potential of both natural and synthetic members of this flavonoid subclass (covering: up to mid-2011). The thus far identified modes of action of these compounds are also discussed. We hope that this overview may stimulate deeper investigations into the biochemical mechanisms by which chalcones exert their antineoplastic action. As a result, in the foreseeable future, chalcones may prove suitable lead molecules or early drug candidates for the prevention or treatment of various neoplastic diseases.

Persistent subretinal fluid after surgery for rhegmatogenous retinal detachment: hypothesis and review.

BACKGROUND: Persistent subretinal fluid after rhegmatogenous retinal detachment (RRD) surgery is responsible for delayed recovery, and may affect the final visual outcome. Cause, consequences, and treatment remain elusive. DESIGN: Literature review and case series. METHODS: We reviewed the pathophysiological principles and therapeutic options from the literature, and we report the results from a subretinal fluid cytology study. Nine eyes from nine patients with macula-involving RRD underwent surgical repair. The cellular content of subretinal fluid (SRF) was studied by electron microscopy and anti-rhodopsin immunostaining. All eyes were assessed postoperatively with optical coherence tomography for the detection of persistent submacular fluid (PSF) (Ethics Committee Ghent University Hospital, registration number B6702006169). RESULTS: Certain patient characteristics as well as surgical methods were implicated. PSF appears to occur more frequently in patients with longstanding detachments treated with buckling surgery. Several therapeutic options have been suggested but safety and efficacy remain unclear. We found PSF in three eyes on postoperative OCT scans, which corresponded to the three cell-rich subretinal samples. CONCLUSIONS: PSF after successful RRD repair seems to be related to fluid composition. We hypothesize, in the absence of an effective treatment, that a modified surgical drainage, including a washout of the subretinal space, could evacuate the subretinal fluid more completely, and may prevent this complication.

Soy consumption during menopause.

In developed countries, the life expectancy of women is currently extending more than 30 years beyond the age of menopause. The menopausal transition is often associated with complaints. The conflicting results on the effectivity of phytoestrogens to alleviate menopausal symptoms. This discrepancy in treatment effect may be due to the large interindividual variation in isoflavone bioavailability in general and equol production in particular. Equol, a microbial metabolite of daidzein, has been hypothesized as a clue to the effectiveness of soy and its isoflavones, but only about 30-50% of the population harbor an intestinal microbial ecosystem supporting the conversion of daidzein into equol. There is much concern on breast cancer, since this incidence of this disease increases with age. There is indication that soy phytoestrogens may decrease this breast cancer incidence. In order to evaluate the estrogenic potential of these exposure levels, we studied the isoflavone-derived E2α- and E2ß-equivalents (i.e. 17ß-estradiol (E2)-equivalents towards ERα and ERß, respectively) in human breast tissue. Total isoflavones showed a breast adipose/glandular tissue distribution of 40/60 and their derived E2ß-equivalents exceeded on average 21 ±â€ˆ4 and 40 ±â€ˆ10 times the endogenous E2 concentrations in corresponding adipose and glandular biopsies, respectively, whereas the E2α/E2 ratios were 0.4 ±â€ˆ0.1 and 0.8 ±â€ˆ0.2 in adipose and glandular breast tissue, respectively. These calculations suggest that, at least in this case, soy consumption could elicit partial ERß agonistic effects in human breast tissue. We are currently characterizing the differential activation of estrogen-responsive genes between dietary isoflavones, the chemopreventive selective ER modulators tamoxifen and raloxifene and exogenous estrogens in a controlled dietary intervention trial that integrates data on the exposure to estrogenically active compounds, expression of isoflavone and estrogen target genes, and epigenetic events. During the menopause, there is a close relation between the drop in serum estrogen and negative metabolic changes such as the increase in bone resorption and negative change in the serum lipid profile. Randomized controlled trials measuring bone turnover markers in menopausal women revealed that soy isoflavone supplements significantly but moderately decrease the bone resorption marker urinary deoxypyridinoline without significant effects on the bone formation markers serum bone alkaline phosphatase and osteocalcin.

Individual piglets' contribution to the development of tail biting.

Conflicting hypotheses exist about the contribution of individual pigs to the development of a tail-biting outbreak, but there is limited quantitative information to support or dismiss them. This study aims to quantify the development of tail-biting behaviour at pen and individual piglet level, before and after the first visible tail damage. Video recordings of 14 pens with tail-biting outbreaks and individually marked weaned piglets were used to observe tail-biting incidents (TBIs; piglet biting a penmate's tail). When visible tail damage was first observed in a pen (i.e. day of tail biting outbreak; D0), the video recordings of the previous 6 (till D-6) and the following 6 days (till D6) were analysed every other day for TBIs and the identities of the biter and bitten piglet were recorded. The average TBIs per individual piglet (within each pen) per observation day were analysed to quantify the development of tail-biting behaviour and to identify pronounced biters and/or bitten piglets. The (absence of) coherence for TBIs in a pen was used to test whether biters preferred a specific penmate. There was an exponential increase in the intensity (linear on log scale) of the TBIs from an average of 0.7 bites/h at D-6 to 2.3 bites/h at D6. An additional negative quadratic component suggests that a plateau for tail-biting behaviour was reached by the end of the observation period. Before any visible tail damage was observed (i.e. before D0), 82% of the piglets performed and 96% of them received tail bites. After D0, the figures were 99% and 100%, respectively. One or a few pronounced biters could be identified in almost all pens. These biters already showed more tail biting at D-6 than their penmates. Furthermore, these biters showed a greater increase in tail-biting behaviour during the observation period than the average scores of their penmates. In contrast, there was no apparent increase in the receipt of bites among the piglets that had already been bitten more than their penmates at D-6. Finally, there was no significant coherence between biters and bitten piglets, indicating that biters showed no preference for biting particular penmates, even when some of them had a damaged tail. These results show that, by using observations of TBIs, possible biters or bitten piglets can already be identified 6 days before tail damage is first apparent in a pen.

Characteristics of biter and victim piglets apparent before a tail-biting outbreak.

Little is known about the characteristics of biters and victims before the appearance of a tail-biting outbreak in groups of pigs. This study aimed to characterise biters and victims (according to gender and performance) and to quantify their behavioural development during the 6 days preceding the tail-biting outbreak. The hypotheses tested were: (a) biters are more often female, are the lighter pigs in the group, are more restless and perform more aggressive behaviour; and (b) victims are more often male, heavier and less active. Using video recordings we carried out a detailed study of 14 pens with a tail-biting outbreak among the weaned piglets. All piglets were individually marked and we observed the behaviour of biters, victims and control piglets (piglet types). In every pen, each piglet type was observed every other day from 6 days before (D-6) to the day of the first visible tail damage (i.e. day of tail biting outbreak; D0). While the number of male biters (6 of the 14 biters) and male victims (11 of the 14 victims) was not significantly different (P = 0.13), this numerical contrast was considerable. The start weight of victims was significantly (P = 0.03) higher (8.6 kg) than those of biters (7.5 kg) and control piglets (8.0 kg). Biters tended (P = 0.08) to spend longer sitting/kneeling (3.1 min/h) than controls (1.7 min/h), but no differences were seen in the time spent lying or standing. Victims tended (P = 0.07) to change posture more often (restlessness) than controls and chased penmates more (P = 0.04) than biters. Victims also performed more (P = 0.04) aggressive behaviour than biters and controls. In contrast, biters tended (P = 0.08) to be chased by penmates more often and tended (P = 0.06) to receive more aggressive behaviour than controls. Furthermore, biters spent longer manipulating the enrichment device (P = 0.01) and the posterior/tail (P = 0.02) of their penmates than controls and tended (P = 0.06) to perform more tail bites than victims. Victims received more posterior/tail manipulation (P = 0.02) and tail bites (P = 0.04) than controls. It was also noticed that, independent of piglet type, restlessness (P = 0.03) increased and the frequency of performed tail bites tended (P = 0.08) to increase in the 6 days preceding a tail-biting outbreak. These findings may contribute to the early identification of biters or victims and support the development of strategies to minimise the occurrence of tail biting.

Extracellular cleavage and shedding of P-cadherin: a mechanism underlying the invasive behaviour of breast cancer cells.

Cell-cell adhesion is an elementary process in normal epithelial cellular architecture. Several studies have shown the role mediated by cadherins in this process, besides their role in the maintenance of cell polarity, differentiation and cell growth. However, during tumour progression, these molecules are frequently altered. In breast cancer, tumours that overexpress P-cadherin usually present a high histological grade, show decreased cell polarity and are associated with worse patient survival. However, little is known about how this protein dictates the very aggressive behaviour of these tumours. To achieve this goal, we set up two breast cancer cell models, where P-cadherin expression was differently modulated and analysed in terms of cell invasion, motility and migration. We show that P-cadherin overexpression, in breast cancer cells with wild-type E-cadherin, promotes cell invasion, motility and migration. Moreover, we found that the overexpression of P-cadherin induces the secretion of matrix metalloproteases, specifically MMP-1 and MMP-2, which then lead to P-cadherin ectodomain cleavage. Further, we showed that soluble P-cadherin fragment is able to induce in vitro invasion of breast cancer cells. Overall, our results contribute to elucidate the mechanism underlying the invasive behaviour of P-cadherin expressing breast tumours.

Development of in vitro models for investigating spatially fractionated irradiation: physics and biological results.

We present different in vitro experimental models which allow us to evaluate the effect of spatially fractionated dose distributions on metabolic activity. We irradiated a monolayer of MCF-7/6 human breast cancer cells with a steep and a smooth 6 MV x-ray dose gradient. In the steep gradient model, we irradiated the cells with three separate small fields. We also developed two smooth gradient models. In the first model, the cells are cultured in a T25 flask and irradiated with a smooth dose gradient over the length of the flask, while in the second one, the cells are cultured in a 96-well plate and also irradiated over the length of the plate. In an attempt to correlate the spatially fractionated dose distributions with metabolic activity, the effect of irradiation was evaluated by means of the MTT assay. This assay is used to determine the metabolic activity by measuring the amount of formazan formed after the conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by cellular dehydrogenases. The results obtained with our different models suggest a dose-specific effect on metabolic activity, characterized by an increased formazan optical density occurring in the dose range 1.0-4.0 Gy in the steep dose gradient model and in the dose ranges 4.2-6.5 Gy and 2.3-5.1 Gy in the two smooth dose gradient models. The corresponding times for maximal formazan accumulation were 5-7 days in the steep dose gradient model and day 9-13 and day 9-11 in the smooth dose gradient models. Altogether, our results suggest that the MTT assay may be used as a biological dose-response meter to monitor the radiotherapeutic effectiveness.

In vitro cytotoxicity of paclitaxel/beta-cyclodextrin complexes for HIPEC.

Hyperthermic intraperitoneal chemotherapy (HIPEC) is a promising strategy in the treatment of peritoneal carcinomatosis. To perform HIPEC, a tensioactive- and solvent-free paclitaxel formulation consisting of water-soluble paclitaxel/randomly methylated-beta-cyclodextrin (Pac/RAMEB) complexes was developed previously. Using MTT and SRB assays the cytotoxic activity of this formulation versus Taxol, was evaluated as well as the cytotoxicity of the different formulation excipients (RAMEB and Cremophor EL. The possible synergistic effect of heat and paclitaxel-based chemotherapy during HIPEC was also evaluated in vitro. The cytotoxicity assays revealed differences in viability between Cremophor EL and RAMEB treated cells of 40 and 50% for the CaCo-2 human and the CC531s rat colon cancer line, respectively, in favour of RAMEB. Despite the higher cytotoxicity of Cremophor EL, Pac/RAMEB complexes and Taxol were equipotent. Using the MTT and SRB assays the average difference in viability between both cell lines was below 10% and IC50 values showed no significant difference. Hyperthermia after drug administration (41 degrees C during 1h) had no effect on cell viability. These results indicated that it was possible to reformulate paclitaxel with a less cytotoxic vehicle while maintaining the cytotoxic activity of the formulation and that there is no synergism between paclitaxel and heat for in vitro cytotoxicity.

Autocrine induction of invasion and metastasis by tumor-associated trypsin inhibitor in human colon cancer cells.

From the conditioned medium of the human colon carcinoma cells, HT-29 5M21 (CM-5M21), expressing a spontaneous invasive phenotype, tumor-associated trypsin inhibitor (TATI) was identified and characterized by proteomics, cDNA microarray approaches and functional analyses. Both CM-5M21 and recombinant TATI, but not the K18Y-TATI mutant at the protease inhibitor site, trigger collagen type I invasion by several human adenoma and carcinoma cells of the colon and breast, through phosphoinositide-3-kinase, protein kinase C and Rho-GTPases/Rho kinase-dependent pathways. Conversely, the proinvasive action of TATI in parental HT29 cells was alleviated by the TATI antibody PSKAN2 and the K18Y-TATI mutant. Stable expression of K18Y-TATI in HT-29 5M21 cells downregulated tumor growth, angiogenesis and the expression of several metastasis-related genes, including CSPG4 (13.8-fold), BMP-7 (9.7-fold), the BMP antagonist CHORDIN (5.2-fold), IGFBP-2 and IGF2 (9.6- and 4.6-fold). Accordingly, ectopic expression of KY-TATI inhibited the development of lung metastases from HT-29 5M21 tumor xenografts in immunodeficient mice. These findings identify TATI as an autocrine transforming factor potentially involved in early and late events of colon cancer progression, including local invasion of the primary tumor and its metastatic spread. Targeting TATI, its molecular partners and effectors may bring novel therapeutic applications for high-grade human solid tumors in the digestive and urogenital systems.

Plant polyphenolics as anti-invasive cancer agents.

Because invasion is, either directly or via metastasis formation, the main cause of death in cancer patients, development of efficient anti-invasive agents is an important research challenge. We have established a screening program for potentially anti-invasive compounds. The assay is based on organotypic confronting cultures between human invasive cancer cells and a fragment of normal tissue in three dimensions. Anti-invasive agents appeared to be heterogeneous with regard to their chemical nature, but plant alkaloids, polyphenolics and some of their synthetic congeners were well represented. Even within this group, active compounds were quite diverse: (+)-catechin, tangeretin, xanthohumol and other prenylated chalcones, 3,7-dimethoxyflavone, a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin. The data gathered in this system are now applied in two projects. Firstly, structure-activity relationships are explored with computer models using an artificial neural network approach, based on quantitative structural descriptors. The aim of this study is the prediction and design of optimally efficient anti-invasive compounds. Secondly, the metabolism of orally ingested plant polyphenolics by colonic bacteria is studied in a simulator of the human intestinal microbial ecosystem (SHIME) and in human intervention trials. This method should provide information on the final bioavailability of the active compounds in the human body, with regard to microbial metabolism, and the feasibility of designing pre- or probiotics that increase the generation of active principles for absorption in the gastro-intestinal tract. The final and global aim of all these studies is to predict, synthesize and apply in vivo molecules with an optimal anti-invasive, and hence an anti-metastatic activity against cancer.

Synthesis of semantic modelling and risk analysis methodology applied to animal welfare.

Decision-making on animal welfare issues requires a synthesis of information. For the assessment of farm animal welfare based on scientific information collected in a database, a methodology called 'semantic modelling' has been developed. To date, however, this methodology has not been generally applied. Recently, a qualitative Risk Assessment approach has been published by the European Food Safety Authority (EFSA) for the first time, concerning the welfare of intensively reared calves. This paper reports on a critical analysis of this Risk Assessment (RA) approach from a semantic-modelling (SM) perspective, emphasizing the importance of several seemingly self-evident principles, including the definition of concepts, application of explicit methodological procedures and specification of how underlying values and scientific information lead to the RA output. In addition, the need to include positive aspects of welfare and overall welfare assessments are emphasized. The analysis shows that the RA approach for animal welfare could benefit from SM methodology to support transparent and science-based decision-making.

Use of inhaled and oral glucocorticoids, severity of inflammatory disease and risk of hip/femur fracture: a population-based case-control study.

BACKGROUND: Patients using higher dosages of inhaled or oral glucocorticoids (GCs) have an increased risk of hip/femur fractures. The role of the underlying disease in the aetiology of this increased risk has not been widely studied. OBJECTIVE: To evaluate the contribution of the underlying disease to the risk of hip/femur fracture in patients using inhaled or oral GCs. DESIGN AND SUBJECTS: A case-control study within the Dutch PHARMO-RLS database was conducted. Cases (n = 6763) were adult patients with a first hip/femur fracture during enrolment. Each case was matched to four controls by age, gender and region. RESULTS: The risk of hip/femur fracture increased with current use of inhaled GCs (crude OR 1.30, 95% CI:1.16-1.47) and with current use of oral GCs (crude OR 1.66, 95% CI: 1.46-1.90). After adjustment for disease severity, the risk of hip/femur fracture was no longer statistically significantly increased in inhaled GC users (adjusted OR 1.08, 95% CI: 0.91-1.27), whilst it remained elevated in oral GC users (adjusted OR 1.43, 95% CI: 1.22-1.67). Patients using inhaled GCs without any exposure to oral GCs had no increased risk of fracture (adjusted OR 0.98, 95% CI: 0.79-1.22). CONCLUSION: Inhaled GC users had no increased risk of femur/hip fracture after adjustment for underlying disease severity. Our data suggest that, even at higher dosages, inhaled GC use is not an independent risk factor for fracture. In contrast, oral GC use was associated with an increased risk of fracture, which was not fully explained by the underlying disease severity.

Aggregation of measures to produce an overall assessment of animal welfare. Part 1: a review of existing methods.

Several systems have been proposed for the overall assessment of animal welfare at the farm level for the purpose of advising farmers or assisting public decision-making. They are generally based on several measures compounded into a single evaluation, using different rules to assemble the information. Here we discuss the different methods used to aggregate welfare measures and their applicability to certification schemes involving welfare. Data obtained on a farm can be (i) analysed by an expert who draws an overall conclusion; (ii) compared with minimal requirements set for each measure; (iii) converted into ranks, which are then summed; or (iv) converted into values or scores compounded in a weighted sum (e.g. TGI35L) or using ad hoc rules. Existing methods used at present (at least when used exclusively) may be insufficiently sensitive or not routinely applicable, or may not reflect the multidimensional nature of welfare and the relative importance of various welfare measures. It is concluded that different methods may be used at different stages of the construction of an overall assessment of animal welfare, depending on the constraints imposed on the aggregation process.