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The expression and distribution patterns of the taste signaling molecules, α-gustducin (Gαgust) and α-transducin (Gαtran) G-protein subunits, were studied in the gastrointestinal tract of the chicken (Gallus domesticus) using the immunohistochemical method. Gαgust and Gαtran immunoreactive (-IR) cells were observed in the mucosal layer of all examined segments, except the esophagus, crop, and the saccus cranialis of the gizzard. The highest numbers of Gαgust and Gαtran-IR cells were found in the proventriculus glands and along the villi of the pyloric, duodenum, and rectal mucosa. Gαgust and Gαtran-IR cells located in the villi of the jejunum, ileum, and cloaca were much less numerous, while only a few Gαgust and Gαtran-IR cells were detected in the mucosa of the proventriculus and cecum. In the crypts, IR cells were observed in the small and large intestine as well as in the cloaca. Gαgust and Gαtran-IR cells displayed elongated ("bottle-" or "pear-like") or rounded shape. The demonstration of Gαgust and Gαtran expression provides evidence for taste receptor mediated mucosal chemosensitivity in the chicken gastrointestinal tract., (© 2016 Poultry Science Association Inc.)

Background: The expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (Gαgust) and α-transducin (Gαtran) G protein subunits. This study tested whether Gαtran and Gαgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract., Result: In the stomach, Gαgust and Gαtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, Gαgust and Gαtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of Gαtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P<0.05), while the increase of Gαgust-IR cells in the pyloric mucosa was significant in Hp30 group vs. Ctr and vs. Hp3 (P<0.05); these cells included Gαtran / 5HT-IR and Gαtran / GHR-IR cells (P<0.05 and P<0.001 vs. Ctr, respectively) as well as Gαgust /5-HT-IR or Gαgust / GHR-IR cells (P<0.05 and P<0.01 vs. Ctr, respectively). In the small intestine, we recorded a significant increase in Gαtran-IR cells in the duodenal crypts and a significant increase of Gαgust-IR cells in the jejunal crypts in Hp3 group compared to HP30 (P<0.05). With regard to the number of Gαtran-Gαgust IR cells colocalized with CCK or 5-HT, there was only a significant increase of Gαtran / CCK-IR cells in Hp3 group compared to Ctr (P = 0.01)., Conclusion: This study showed an upregulation of selected subpopulations of Gαgust / Gαtran-IR cells in distinct regions of the pig GI tract by short- and long-term Hp diet lending support to TASR-mediated effects in metabolic homeostasis and satiety mechanisms.

Vertebrates perceive a variety of exogenous substances using two main chemosensory systems, taste and olfaction. The taste perception occurs through the interaction of taste receptors associated with specific G protein subunits such as α-transducin (Gαtran) and α-gustducin (Gαgust). Aquatic vertebrates are also provided with a chemosensory system consisting of solitary chemosensory cells distributed to the oropharynx and skin. In this study, we identified Gαtran and Gαgust-immunoreactive cells intermingled with non-labeled epithelial cells in the gastric mucosa of the common sole. A long-term diet with increasing concentrations of mussel meal in the protein component of a conventional fish meal-based diet induced a dose-dependent increase in the gastric epithelial area and density of Gαtran and Gαgust immunoreactive cells. These findings suggest that taste-related molecules are regulated by changes in diet formulation in common sole aquaculture.

During the transit in the female genital tract, spermatozoa are exposed to an environment that varies in composition from the vagina to the oviduct. Because G proteins, α-gustducin and α-transducin, are accepted as markers of chemosensitive cells, this study was aimed at assessing whether these proteins are expressed in boar germ cells. Ejaculated sperm extracts were analyzed by Western blot, and indirect immunofluorescence was performed on testis sections, smears of epididymal and ejaculated spermatozoa, sperm cells after in vitro induction of capacitation and acrosome reaction (IVAR), and in sperm cells bound to zona pellucida during IVF. Based on immunoblot results, both G proteins are present in boar sperm. In the testicular tissue sections, α-gustducin and α-transducin positivity was recorded in the germinal cells near the tubular lumen, whereas no positive signal was evident in spermatogonia located in the outer region of the seminiferous tubules. α-Gustducin expression in epididymal and ejaculated spermatozoa was mainly detectable in both the acrosome and the principal piece of the tail, whereas α-transducin was confined to the acrosome and the midpiece. No changes after in vitro induction of capacitation and IVAR were observed, except for the disappearance of acrosomal positivity in reacted spermatozoa. In sperm bound to zona pellucida, the G protein signal was congruent with that observed in IVAR cells. To the best of our knowledge, this is the first description of α-transducin in mammalian sperm and the first description of α-gustducin in boar sperm. Further studies are needed to clarify the possible role of these G proteins in sperm physiology., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In vertebrates, chemosensitivity of nutrients occurs through the activation of taste receptors coupled with G-protein subunits, including α-transducin (G(αtran)) and α-gustducin (G(αgust)). This study was aimed at characterising the cells expressing G(αtran) immunoreactivity throughout the mucosa of the sea bass gastrointestinal tract. G(αtran) immunoreactive cells were mainly found in the stomach, and a lower number of immunopositive cells were detected in the intestine. Some G(αtran) immunoreactive cells in the stomach contained G(αgust) immunoreactivity. Gastric G(αtran) immunoreactive cells co-expressed ghrelin, obestatin and 5-hydroxytryptamine immunoreactivity. In contrast, G(αtran) immunopositive cells did not contain somatostatin, gastrin/cholecystokinin, glucagon-like peptide-1, substance P or calcitonin gene-related peptide immunoreactivity in any investigated segments of the sea bass gastrointestinal tract. Specificity of G(αtran) and G(αgust) antisera was determined by Western blot analysis, which identified two bands at the theoretical molecular weight of ~45 and ~40 kDa, respectively, in sea bass gut tissue as well as in positive tissue, and by immunoblocking with the respective peptide, which prevented immunostaining. The results of the present study provide a molecular and morphological basis for a role of taste-related molecules in chemosensing in the sea bass gastrointestinal tract.

A routine use of boar-sexed semen is limited by the long sorting time necessary to obtain an adequate number of sexed spermatozoa for artificial insemination and by the high susceptibility of spermatozoa of this species to damages induced by sorting procedure and subsequent cryopreservation. The aim of this work was to study the impact of encapsulation in barium alginate membrane on sorted boar spermatozoa by evaluating membrane integrity, chlortetracycline staining patterns, protein tyrosine phosphorylation, and Hsp70 immunolocalization during storage over 72 hours in liquid or encapsulated form. The encapsulation procedure significantly (P < 0.05) decreased the overall membrane integrity of control unsorted semen (81.8 vs. 57.4, CTR vs. CPS), but did not negatively affect the overall viability and the chlortetracycline staining patterns of sorted encapsulated cells. Moreover, encapsulation significantly decreased (P < 0.05) the overall phosphotyrosin A pattern cell percentage in unsorted (98.4 vs. 92.6, CTR vs. CPS) but not in sorted semen (64.0 vs. 74.2; SORT CTR vs. SORT CPS). As for Hsp70, the overall percentage of cells displaying the different patterns was significantly influenced (P < 0.05) by treatment but not by storage time. The sorting procedure seems to induce the major changes, whereas encapsulation tends to exert a protective effect on sorted semen by increasing the percentage of spermatozoa displaying the T pattern (2.8 vs. 24.3; SORT CTR vs. SORT CPS). In conclusion, our data confirm that the damaging impact of the encapsulation in barium alginate capsules seems to be limited when compared with that of the sorting procedure and, moreover, the association of the two procedures does not result in an algebraic sum of the negative effects. These results suggest the possibility of a future utilization of the encapsulation technology in order to store sorted spermatozoa and permit their controlled release in the female genital tract., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Single layer centrifugation (SLC) is a useful technique to select porcine spermatozoa for further artificial insemination practices. The aim of this study was to determine possible side-effects related to capacitation due to the process. Semen viability, acrosome integrity and capacitation status were determined through fluorescent probes (SYBR14-PI, FITC-PSA, CTC staining) and Hsp70 immunolocalization and protein tyrosine phosphorylation (by western blotting and immunolocalization) in different groups: control, after SLC with Androcoll (AND), after SLC and washing (AND-Wash) and after SLC, washing and storage for 2h at 17°C with 2.5% of seminal plasma (AND-Wash-SP). Neither viability nor acrosome integrity were impaired by the different treatments; as far as CTC staining, we observed a significant increase (p<0.05) in the capacitation related pattern in AND and AND-Wash, while after exposure for 2h to seminal plasma (AND-Wash-SP group), the increase became less evident; the same trend was observed in Hsp70 immunolocalization for the EL pattern. Neither immunolocalization nor western blotting for tyrosine phosphorylated proteins had an increase in capacitated pattern or in phosphorylation status, except for a 25kDa band that increased in AND and AND-Wash groups and decreased in AND-Wash-SP group. SLC using Androcoll-P induces some capacitation-related changes in boar sperm membrane, as demonstrated by CTC staining and Hsp70 immunolocalization. For protein tyrosine phosphorylation, only a 25kDa protein showed some changes that should be investigated further., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Taste signalling molecules are found in the gastrointestinal (GI) tract suggesting that they participate to chemosensing. We tested whether fasting and refeeding affect the expression of the taste signalling molecule, α-transducin (Gαtran ), throughout the pig GI tract and the peptide content of Gαtran cells. The highest density of Gαtran -immunoreactive (IR) cells was in the pylorus, followed by the cardiac mucosa, duodenum, rectum, descending colon, jejunum, caecum, ascending colon and ileum. Most Gαtran -IR cells contained chromogranin A. In the stomach, many Gαtran -IR cells contained ghrelin, whereas in the upper small intestine many were gastrin/cholecystokinin-IR and a few somatostatin-IR. Gαtran -IR and Gαgust -IR colocalized in some cells. Fasting (24 h) resulted in a significant decrease in Gαtran -IR cells in the cardiac mucosa (29.3 ± 0.8 versus 64.8 ± 1.3, P < 0.05), pylorus (98.8 ± 1.7 versus 190.8 ± 1.9, P < 0.0 l), caecum (8 ± 0.01 versus 15.5 ± 0.5, P < 0.01), descending colon (17.8 ± 0.3 versus 23 ± 0.6, P < 0.05) and rectum (15.3 ± 0.3 versus 27.5 ± 0.7, P < 0.05). Refeeding restored the control level of Gαtran -IR cells in the cardiac mucosa. In contrast, in the duodenum and jejunum, Gαtran -IR cells were significantly reduced after refeeding, whereas Gαtran -IR cells density in the ileum was not changed by fasting/refeeding. These findings provide further support to the concept that taste receptors contribute to luminal chemosensing in the GI tract and suggest they are involved in modulation of food intake and GI function induced by feeding and fasting., (© 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Although sorted semen is experimentally used for artificial, intrauterine, and intratubal insemination and in vitro fertilization, its commercial application in swine species is still far from a reality. This is because of the low sort rate and the large number of sperm required for routine artificial insemination in the pig, compared with other production animals, and the greater susceptibility of porcine spermatozoa to stress induced by the different sex sorting steps and the postsorting handling protocols. The encapsulation technology could overcome this limitation in vivo, protecting and allowing the slow release of low-dose sorted semen. The aim of this work was to evaluate the impact of the encapsulation process on viability, acrosome integrity, and on the in vitro fertilizing potential of sorted boar semen. Our results indicate that the encapsulation technique does not damage boar sorted semen; in fact, during a 72-hour storage, no differences were observed between liquid-stored sorted semen and encapsulated sorted semen in terms of plasma membrane (39.98 ± 14.38% vs. 44.32 ± 11.72%, respectively) and acrosome integrity (74.32 ± 12.17% vs. 66.07 ± 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, P < 0.0001), and a significant reduction of polyspermic fertilization (60.76% vs. 36.43%, respectively, polyspermic ova/total ova; P < 0.0001). However, no difference (P > 0.05) was observed in terms of total efficiency of fertilization expressed as normospermic oocytes/total oocytes (18.45% vs. 15.43% for sorted diluted and sorted encapsulated semen, respectively). The encapsulation could be an alternative method of storing of pig sex sorted spermatozoa and is potentially a promising technique in order to optimize the use of low dose of sexed spermatozoa in vivo., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Oocyte and embryo cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig oocytes, because of their greater susceptibility to cryoinjuries, have shown a reduced ability to be fertilized in vitro and a lower developmental competence. The aim of this study was to evaluate the apoptotic status of porcine oocytes vitrified by Cryotop method. We assessed three parameters linked to the activation of the apoptotic cascade: the exteriorization of phosphatidylserine using Annexin V, the caspase activation using FITC-VAD-FMK and the alteration of plasma membrane permeability employing YO-PRO-1. These assays were performed on control oocytes, oocytes exposed to vitrification solutions (toxicity control) and vitrified oocytes either immediately after warming or after incubation for 2h into maturation medium. The exposition to vitrification solutions triggered an increase of the percentage of oocytes both faintly (VAD+ PI-) and strongly (VAD++ PI-) labeled by FITC-VAD-FMK but not a significant modification of the number of oocytes Annexin V (A+ PI-, early apoptotic) and YO-PRO-1(YP+ PI-, apoptotic) positive in comparison with control oocytes. Oocytes submitted to the whole vitrification procedure showed a rise of the percentage of early apoptotic oocytes (A+ PI-) and FITC-VAD-FMK positive oocytes (VAD+/VAD++ PI-) and a contemporaneous increase of the number of dead oocytes (PI+). On the contrary, vitrified oocytes analyzed immediately after warming did not show a significant increase in the percentage of apoptotic oocytes (YO-PRO-1+/PI-) as compared with the control. Post warming incubation for 2h into maturation medium, in comparison with oocytes analyzed immediately after warming, did not induce any increase in the percentage of early apoptotic (A+ P-) oocytes while a decrease of the percentage of VAD+/PI- oocytes and a contemporaneous increase of VAD-/PI- oocytes were observed. Moreover, the post-warming incubation induced a rise of the percentage of apoptotic oocytes (YO-PRO-1+/PI-). All these data confirm the involvement of apoptotic mechanisms on the injuries induced by vitrification procedure in pig oocytes; explanation of this phenomenon could be useful to improve oocytes' cryopreservation protocols., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In the present study, acetylation status of histone H4 and methylation status of the lysine 9 residue of histone H3 (H3K9) were assessed by immunofluorescence in order to determine the effect of vitrification on epigenetic status of pig MII oocytes. Hyperacetylation of H4 and dimethylation of H3K9 were assessed in control oocytes, after cryoprotectant treatment and after vitrification at two time points, immediately after warming and after a post-warming incubation for 2 h. While no changes in the immunopositivity for both the epitopes were recorded after cryoprotectants, the percentage of negative oocytes for dimethyl H3K9 was observed to increase immediately after devitrification. The influence of vitrification was more evident after 2 h post-thaw incubation when acetylation status of H4 significantly increased and a rise in the percentages of both oocytes exhibiting strong positivity and negative oocytes for dimethyl H3K9 was observed. In conclusion, acetylation of H4 and methylation of H3K9 are altered by vitrification procedure that may lead to an aberrant epigenetic presentation of female chromatin to the fertilizing event and may be, at least in part, responsible for the reduction of developmental competence of vitrified pig oocytes.

Sperm sorting is a useful technology that permits sex preselection. It presents some troubles because of low fertility after the process. The main aim of this work was to analyze the putative existence of capacitation-like changes in both boar and bull sperm subjected to sex sorting that could lead to a detriment of semen quality. The parameters used were CTC staining patterns, actin cytoskeleton organization and tyrosine phosphorylation patterns; the last two were determined by both western blotting and immunofluorescence. Sex sorted spermatozoa were compared with fresh, in vitro capacitated and in vitro acrosome reacted sperm. In both species, sex sorted sperm showed a CTC staining pattern similar to that observed after in vitro capacitation. The actin pattern distribution after sperm sorting also tended to be similar to that observed after in vitro capacitation, but this effect was more pronounced in bull than in boar spermatozoa. However, actin expression analysis through western blot did not show any change in either species. The tyrosine phosphorylation pattern in boar sperm was practically unaltered after the sex sorting process, but in bulls about 40% of spermatozoa had a staining pattern indicative of capacitation. Additionally, western blotting analysis evidenced some differences in the expression of protein tyrosine phosphorylation among fresh, capacitated, acrosome reacted and sex sorted sperm cells in both species. Our results indicate that not all the sex-sorted-related modifications of the studied parameters were similar to those occurring after "in vitro" capacitation, thus suggesting that sex sorting-induced alterations of sperm function and structure do not necessarily indicate the achievement of the capacitated status of sorted sperm., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Three experiments were designed to evaluate the effects of vitrification using Cryotop method on MII porcine oocyte viability, chromosomes configuration, meiotic spindle morphology and in vitro fertilization; to do this, in vitro matured oocytes were subjected to the cryoprotectant treatment excluding the plunging into liquid nitrogen, the whole vitrification/warming/rehydration procedure or no treatment (control). In experiment 1 viable oocytes were not reduced by either cryoprotectants or vitrification when they were evaluated immediately after warming and cryoprotectant dilution. However, after a 2 h incubation, the survival rate significantly decreased (P<0.05). In experiment 2 cryoprotectant exposure significantly (P<0.05) influenced spindle morphology even if chromosome organization did not vary, while vitrification significantly (P<0.05) increased oocytes with damaged spindles and chromosomes displaced from the metaphase plate. No significant improvements in these parameters were observed after 2 h of incubation but, on the contrary, the rate of oocytes with normal chromosome configuration was reduced. In experiment 3 significant differences among the three groups in the fertilization rate but not in the percentages of monospermy fertilization were recorded; in addition, exposure to cryoprotectants and vitrification significantly (P<0.05) increased degenerated oocyte rate. Overall, these findings confirm that porcine oocytes at MII stage are very sensitive to vitrification, which reduces the rate of viable oocytes and alters microtubule organization, thus impairing fertilization; in addition, incubation of oocytes for 2 h after devitrification seems to be detrimental rather than ameliorative. Further improvements of the current protocol will be necessary in order to optimize the Cryotop method for vitrifying pig matured oocytes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mammalian cells use glucides as a substrate that can be catabolized through glycolitic pathways or oxidative phosphorylation, used as a source of reducing potential, or used for anabolic aims. An important role in supplying cells with energy is played by different membrane proteins that can actively (sodium-dependent glucose transporters) or passively (glucose transporters; GLUT) transport hexoses through the lipidic bilayer. In particular, GLUTs are a family of 13 proteins that facilitate the transport of sugars and have a peculiar distribution in different tissues as well as a particular affinity for substrates. These proteins are also present in mature sperm cells, which, in fact, need carriers for uptake energetic sources that are important for maintaining cell basic activity as well as specific functions, such as motility and fertilization ability. Likewise, several GLUTs have been studied in various mammalian species (man, bull, rat, mouse, boar, dog, stallion, and donkey) to point out both their actual presence or absence and their localization on plasma membrane. The aim of this work is to give an overall picture of the studies available on GLUTs in mammalian spermatozoa at this moment, pointing out the species peculiarity, the possible role of these proteins, and the potential future research on this item.

Sorting procedures submit sperm cells to a set of stressful steps that can trigger an increase of ROS production and consequently reduce sorted semen quality. This study evaluated the effect of supplementation with different antioxidants (EGCG, Na pyruvate+catalase, SOD) on acrosome and plasma membrane integrity, activation of caspases (as assayed by FITC-VAD/PI staining) and immunolocalization of Hsp70 of boar spermatozoa after sperm preparation (Hoechst 33342 staining) and sorting procedure. The effect of antioxidants, with or without seminal plasma, on sorted spermatozoa stored for 24h at 15°C was also evaluated. Antioxidants did not exert any preventive action on sperm modification induced by staining and sorting. After 24h of storage at 15°C, sorted samples supplemented with either EGCG or SOD plus seminal plasma showed a significant (p<0.05) increase of the percentage of viable spermatozoa, while no positive effect on the other tested parameters was observed; EGCG seems to exert an inhibition on caspase activation in that a decrease of the number of dead cells FITC-VAD+/PI+ was recorded. In conclusion, our results indicate that EGCG and SOD in association with seminal plasma are effective in exerting some compensatory protection against the detrimental effects of storage of sorted semen while their action is not evident during steps of the sorting procedure., (Copyright © 2010 Elsevier B.V. All rights reserved.)

GLUTs are a family of proteins that facilitate the transport of glucose and other hexoses through the plasma membrane of the cells. GLUTs are present in mammalian spermatozoon's membrane in different isoforms and they supply metabolic substrates for all the cell's activities such as motility, homoeostasis and fertilization. As studies about donkey spermatozoa and their metabolism are lacking, this study was aimed at detecting GLUTs 1, 2, 3 and 5 presence by western blotting technique and at determining their localization on the plasma membrane by indirect immunofluorescence. Each protein showed a typical localization on the sperm cells' plasma membrane, differencing the one to the other on the basis of the hexose they transport. We also highlighted some differences between GLUTs distribution and molecular weight in donkey spermatozoa and its nearest relative, the horse., (© 2009 Blackwell Verlag GmbH.)

A routine use of boar sexed semen is far from being a reality due to many limiting factors among which is the long sorting time necessary to obtain the adequate number of sexed spermatozoa for artificial insemination and the high susceptibility to damages induced by cryopreservation. The aim of this study was to evaluate the modification induced by 24-26 h storage on sorted boar spermatozoa on the basis of their viability, acrosome status, Hsp70 presence, and in vitro fertilizing ability. The percentage of viable cells, according to SYBR green/PI staining, was negatively affected (P < 0.05) by sorting procedure. Moreover, liquid storage significantly (P < 0.05) reduced membrane integrity of sorted spermatozoa as compared to all the other groups. Neither sorting nor storage influenced the percentage of live cells with reacted acrosome, according to FITC-PNA/PI staining. Sorted samples, after 24-26 h storage, were characterized by an increase (P < 0.05) of sperm cells negative for Hsp70, as observed by immunofluorescence, and by a decrease (P < 0.05) in Hsp70 content, as evidenced by western blot. While sorting procedure did not adversely affect both penetration rate and total efficiency of fertilization, these parameters were negatively (P < 0.05) influenced by storage after sorting. In order to minimize damages that compromise fertility and function of sex-sorted boar spermatozoa, the mechanisms by which sorting and liquid storage cause these injures require further study., (Copyright 2010 Elsevier Inc. All rights reserved.)

Daidzein, an isoflavone abundant in soybeans and other legumes, displays estrogen like properties. This study was aimed at evaluating the effect of daidzein (1 and 10 microM) on nuclear and cytoplasmic maturation of pig oocytes and on steroidogenic activity of cumulus cells. Daidzein supplementation during IVM had no effect on nuclear maturation and on fertilization traits. By contrast, both concentrations significantly (P < 0.05) inhibited progesterone production by cumulus cells after 24 and 48 h of culture while they did not induce any effect on estradiol production. Furthermore, daidzein did not exert any effect on the percentage of embryos that developed to blastocyst stage, on the number of blastomeres per blastocyst, or on the level of Hsp-70 and -90 gene transcript. Overall, our data demonstrate that daidzein added during oocyte maturation does not affect pig embryo development even if it markedly inhibits progesterone production by cumulus cells. Further studies are needed to evaluate the possible effect of daidzein during embryonic development., (Copyright 2010 Elsevier Inc. All rights reserved.)