Departments of 1Physiology and Pharmacology and 2Medicine, Lawson Health Research Institute, The University of Western Ontario, London, Ontario, Canada N6A 5C1; and 3Department of Biochemistry, University of Texas Health Center at Tyler, Tyler, Texas 75708-3154
Submitted 7 May 2003 ; accepted in final form 30 July 2003
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ABSTRACT |
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compliance; collagen; fibronectin; surfactant; cecal ligation and perforation
A TIMP-3 murine knockout model has been developed to study potential pathological effects of MMPTIMP imbalance (32). The TIMP-3-deficient mouse model develops a lung phenotype of spontaneous air space enlargement, similar to that of pulmonary emphysema. The TIMP-3 null animals have increased MMP activity with no compensation by other TIMPs. Lung function is impaired in CO uptake tests, and lung collagen content is decreased and disorganized in structure. Although the phenotype associated with TIMP-3 deletion starts to develop as early as 2 wk of age, the mice show normal overall health for the first year of life. However, they do display early mortality with a mean survival of 68 wk as opposed to wild-type (WT) animals, which can live to >135 wk of age. Because this knockout model was only recently developed, it is currently unknown how they may respond to any type of lung stress, many of which are linked to altered MMP and TIMP activity. For example, Carney and others (12) show, in a porcine model of ALI induced by bacterial lipopolysaccharide (LPS), that pretreatment with a synthetic MMP inhibitor reduced the severity of lung injury. Furthermore, intratracheal instillation of human recombinant TIMP-2 is protective in a rat model of LPS-induced lung injury (21) as is administration of two novel inhibitors of ADAM-17/TNF-
-converting enzyme (TACE) and MMPs in rats exposed to LPS (57). Combined, these studies indicate that MMPs are involved in the lung damage caused by LPS induced ALI.
A common clinical lung stress is systemic sepsis, which is a major factor contributing to the etiology of ALI (3). Systemic infections can be induced in the laboratory using the cecal ligation and perforation technique (CLP), which has been previously shown to alter lung function and induce alterations to the pulmonary surfactant and cytokine systems (39, 40, 59). In addition, MMP levels have been found to be increased during septic shock (15) and have been linked to inflammatory cell infiltration (29, 64).
Pulmonary surfactant is a bioactive substance that lines the lung alveoli, where it reduces surface tension and prevents alveolar collapse (47). It is composed of 90% phospholipids and 10% surfactant-associated proteins and is produced by the type II alveolar epithelial cells of the lung (60). Surfactant lipids consist of two subfractions: the large aggregates (LA), which are functionally active, and the small aggregates (SA), which are the nonactive, metabolic byproducts of the LA produced during breathing (9, 34). In murine models, a decrease in the SA subfraction is observed 18 h following CLP (40). There are four surfactant-associated proteins, SP-A, -B, -C, and -D. Similar to the TIMP-3 null animals, recently described SP-C and SP-D knockout models have increased MMP activity and develop spontaneous air space enlargement (22, 62). Thus we explored surfactant biochemistry in the TIMP-3 knockout model.
The objectives of this study were to 1) determine the effects of a CLP lung stress on TIMP-3 null mice and the ways in which the septic lung is influenced by an imbalance in MMP and TIMP activity and 2) to determine the effects of the MMP-TIMP imbalance on the surfactant and cytokine systems, which are known to be altered during sepsis.
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MATERIALS AND METHODS |
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Lung histology. Lungs were removed from the thoracic cavity and immediately fixed at an inflation pressure of 15 cmH2O in 4% PBS-buffered formaldehyde for 24 h. Lungs were rinsed in PBS for 1 h and then stored in 70% ethanol. Lungs were embedded in paraffin and sectioned at 7 µm. For histology, the sections were deparaffinized to water and stained with hematoxylin and eosin. Blinded as to experimental groups, we captured digital images with a Nikon DXM1200 color digital camera and Nikon E1000 microscope (Nikon Canada, Mississauga, ON, Canada). Images of alveolar regions at the lung periphery were taken at x20 magnification. For determination of alveolar diameter, we examined sections without knowledge of genotype at x40 magnification. Twenty random fields within 100 µm of the lung pleura from two different regions of the left lobe were examined by means of a 350-µm straight line scale in the eyepiece of the microscope. The number of alveoli intersected by the line was counted, and the average alveolar size was calculated. The larger alveolar diameter reported here compared with animals of similar age in our previous report (32) can be attributed to the fact the lungs in this study were inflation fixed for 24 h as opposed to inflation fixed for only 1 min previously.
Elastin (desmosine) and collagen (hydroxyproline) assays. The desmosine and hydroxyproline analyses of the mouse lungs were performed by two different approaches. In the first experiment, all lobes of the lungs were trimmed of all major airways and vessels and hydrolyzed in 1 ml of 6 N HCl at 100°C for 24 h. The hydrolysate was dried by evaporation and resuspended in 1 ml of water. Desmosine was determined in 5 µl by RIA (54) and protein in 0.4 µl by a ninhydrin method (53). Hydroxyproline was determined by amino acid analysis on a Beckman 6300 analyzer (Beckman Coulter, Fullerton, CA). In the second approach, desmosine, protein, and hydroxyproline were quantified from paraffin-embedded sections as described previously (55). Briefly, the right caudal lobe was embedded with the lateral surface of the lung facing down. The block was trimmed, and the first 100 µm was discarded. The next section was removed for histology, 200 µm was pooled for biochemical analysis, and a second section removed for histology. Paraffin was removed from the pooled sections with xylene, and the residue was hydrolyzed. After drying, the hydrolysate was redissolved in 200 µl of water and desmosine, and protein and hydroxyproline were determined as described above.
Fibronectin immunohistochemistry. Paraffin-embedded lung sections, cut at 7 µm, were dewaxed in toluene and rehydrated through an alcohol series. After treatment in 3% H2O2 and pepsin digestion, sections were incubated in DAKO blocking solution. (DAKO Diagnostics Canada, Mississauga, ON, Canada) Sections were next incubated with fibronectin antiserum (Sigma-Aldrich, St. Louis, MO; 1:100 dilution with DAKO blocking solution) or blocking solution alone (negative control) for 1 h. After being washed in 0.05% Tris-buffered saline-Tween (TBS-T), sections were incubated in an anti-rabbit secondary antibody conjugated to horseradish peroxidase (Sigma-Aldrich, 1:200 dilution in DAKO blocking solution) for 45 min. After being washed in TBS-T, sections were incubated with 3-amino-9-ethylcarbazole substrate (ICN Biomedicals, Aurora, OH) for 2.5 min, counterstained with Mayer's hematoxylin (Sigma-Aldrich), and mounted under Crystal/Mount (Biomeda, Foster City, CA). Blinded to experimental groups, we captured images under a Nikon E1000 microscope with the camera and software described above at x40 magnification. Images were analyzed using the SigmaScan 5.0 software (SPSS, Chicago, IL), normalizing fibronectin staining to the total tissue per field.
Zymography. Lungs were homogenized in 1 ml of buffer containing 50 mM Tris · Cl (pH 7.5), 150 M NaCl, 1% SDS, and 1 EDTA-free Protease Inhibitor Cocktail Pill (Roche Diagnostics, Laval, QC, Canada) for every 10 ml of buffer. The homogenate was then measured for total protein by a Lowry assay (37). Homogenate was separated on 10% SDS-polyacrylamide gels containing 1 mg/ml gelatin, with 50 µg of homogenate protein combined with nonreducing gel loading buffer loaded into each well. Gels were washed twice in 2.5% Triton X-100 solution and rinsed in distilled, deionized H2O. The gels were incubated for 18 h at 37°C in an incubation buffer of 50 mM Tris · Cl (pH 7.5), 5 mM CaCl2, and 5 µM ZnCl2. Gels were stained in Coomassie brilliant blue dye (Bio-Rad Laboratories, Hercules, CA) and destained for 15 min. Zymography was then analyzed by spot densitometry using an Alpha Innotech Imager 2200 and the AlphaEase image analysis software (Alpha Innotech, San Leandro, CA).
In situ zymography. In situ zymography was performed on 7-µm fresh-frozen sections of right lobe lung tissue as previously described (32). To specify that activity was caused by MMPs, negative controls, which contained 5 mM EDTA in the incubation buffer, were processed simultaneously. Samples were incubated for 8 h at 37°C. Results were visualized by florescence microscopy, with exposure time based on minimal fluorescence of negative controls. Images were captured at x10 and x40 magnification under a Nikon E1000 microscope as previously described, with identical light intensity, exposure time, and filter used (FITC, 460-500 nm) for all samples. Green fluorescence was indicative of gelatinolytic activity.
Lung lavage, pulmonary surfactant, and total protein. Total lung lavage was obtained by sequential washing with saline. Three separate volumes of 1 ml of 0.15 M NaCl were instilled and withdrawn in each lung three times. The recovered lavage was combined, and total volume was recorded. The lavage was centrifuged for 10 min at 150 g at 4°C to remove cellular debris. An aliquot of the supernatant from this spin was called the total surfactant and was frozen at -20°C. The remaining volume (1 ml) was removed and centrifuged for 15 min at 40,000 g. The supernatant contained the SA subfraction of total surfactant, and the pellet, which was resuspended in 300 µl of 0.15 M NaCl, contained the LA subfraction. Both the SA and LA were frozen at -20°C. The quantification of the surfactant pool sizes was done by lipid extraction via the Bligh and Dyer method (8) followed by a phosphorus assay using the Duck-Chong protocol (16). Total protein in the lavage was measured in the lavage via a Lowry assay (37).
Total surfactant was used for SP-D protein quantification by Western blot. A 50-µl aliquot was loaded on a 12% polyacrylamide gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Mississauga, ON, Canada). Nitrocellulose membranes were blocked at 4°C overnight in 5% Blotto in TBS (pH 7.6). The nitrocellulose was then incubated in rabbit anti-mouse recombinant SP-D IgG primary antibody in a 1:5,000 dilution with 5% Blotto in 1x TBS. The nitrocellulose was washed for 5 min in TBS, 5 min in TBS with 0.01% Tween 20, 3 x 5 min in TBS, followed by 5 min in 5% Blotto in TBS. After being washed, the nitrocellulose membrane was incubated in goat anti-rabbit horseradish peroxidase conjugated secondary antibody (Amersham Biosciences, Buckinghamshire, UK) diluted 1:1,000 in 5% Blotto in TBS. Subsequently, substrates for enhanced chemiluminescence reaction (Amersham Life Science, Little Chalfont, UK) were applied to all nitrocellulose membranes, which were then exposed to X-ray film (Eastman Kodak, Rochester, NY). Densitometry was performed on the radiographs, expressed in arbitrary units standardized to background.
IL-6 and TNF- cytokine analysis. After compliance measurements, the lungs were lavaged three times with 2x 1 ml of 0.15 M NaCl. The lavage was centrifuged at 2,000 g for 10 min at 4°C, and the supernatant was frozen in liquid nitrogen and stored at -70°C. The concentrations of IL-6 and TNF-
were measured in all samples with separate OPTIEIA ELISA kits specific for each cytokine, following the manufacturer's protocols (PharMingen, San Diego, CA).
Statistical analysis. All values are expressed as means ± SE. Significance was determined by a two-way ANOVA to determine between subject effects, followed by a one-way ANOVA, with a Tukey post hoc test to determined differences among groups. Statistical analysis was performed using the SPSS statistical software package for Windows, version 9.0 (SPSS). Differences were considered statistically significant when the probability value was <0.05. All significant results are displayed where a = (-/-) vs. (+/+), b = CLP vs. Sham, c = (-/-) Sham vs. (+/+) Sham, and d = (-/-) CLP vs. (-/-) Sham.
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RESULTS |
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Histology. Figure 2A displays lung histology showing differences between (-/-) and (+/+) groups upon gross examination. Analysis of mean alveolar diameter (Fig. 2B) demonstrates that both Sham and CLP knockout groups had significantly higher values compared with the WT littermate controls.
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Elastin and collagen levels. Elastin was measured both in whole lung (data not shown) and in sections containing only alveolar tissue (due to the high content of elastin in alveolar bronchioles); however, results were similar from each method. Statistical analysis established there were not any significant differences between any of the experimental groups (Fig. 3).
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Collagen abundance, assessed by hydroxyproline levels, is designated by solid bars in Fig. 3. Collagen abundance was found to be significantly decreased in the (+/+) CLP to 52.8 ± 1.7 nanomolar hydroxyproline per milligram protein (nM HP/mg P) from 59.1 ± 2.3 nM HP/mg P in the (+/+) Sham animals. There was also a significantly lower collagen level in the (-/-) Sham group to 52.2 ± 1.5 nM HP/mg P compared with the (+/+) Sham controls. The lungs of the (-/-) CLP animals displayed a further significant decrease in collagen to 45.0 ± 1.9 nM HP/mg P compared with (-/-) Sham controls.
Fibronectin immunohistochemistry. Fibronectin abundance, measured by immunohistochemical staining and normalized to total tissue area, is shown in Fig. 4; it was significantly decreased in the (+/+) CLP vs. (+/+) Sham controls. In the (-/-) Sham animals, there was also a significant decrease in fibronectin observed compared with the (+/+) Sham controls. The (-/-) CLP lungs did not show any decrease in fibronectin in relation to the (-/-) Sham. Negative controls lacking primary antibody demonstrated no signal.
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Gel zymography. Figure 5 shows gelatinolytic activity as measured through densitometric analysis of gelatin polyacrylamide gel zymography. Figure 5A shows a representative electrophoretic profile. Figure 5B shows MMP-2 levels increased in the CLP groups compared with Sham controls. Of particular interest is the finding that although no difference was found between (-/-) Sham and (+/+) Sham or between (+/+) CLP and (+/+) Sham, the latent MMP-2 levels were increased in the (-/-) CLP compared with the (-/-) Sham. Comparing the activation of MMP-2 via cleavage of the propeptide, although not statistically significant, the (-/-) animals showed a slight increase in MMP-2 activation compared with their respective (+/+) control animals. Moreover, this activation was not strongly affected by the CLP procedure. Figure 5C displays both propeptide and activated forms of MMP-9, which were both increased in the (+/+) CLP compared with (+/+) Sham. There was not a significant difference in the MMP-9 levels in the (-/-) Sham vs. (+/+) Sham group. However, the pattern of increased MMP-9 (both pro- and activated forms) following CLP seen in the WT animals was also observed in the (-/-) CLP vs. (-/-) Sham groups.
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In situ zymography. In situ zymography (Fig. 6) shows the localization of MMP activity in the presence of their natural inhibitors, the TIMPs. The results show that there are slight increases in alveolar gelatinase activity in the (+/+) CLP lungs vs. (+/+) Sham controls. Similarly, there was a slight increase in activity in the (-/-) Sham groups compared with the (+/+) Sham control. Additionally, there was a larger increase in activity in the alveolar tissue of the (-/-) CLP group compared with (-/-) Sham. After examination of pulmonary airways, very low gelatinase activity was observed surrounding the airways in both the (+/+) Sham and (+/+) CLP groups. However, there was increased gelatinase activity around airways in the (-/-) Sham lungs compared with (+/+) Sham controls. Airway-associated gelatinase activity was highly elevated in (-/-) CLP animals compared with all other groups. For all samples analyzed, the negative controls showed undetectable levels of activity, demonstrating the observed activity came from a metalloproteinase.
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Pulmonary surfactant and total protein. The total volume of lavage recovered was not significantly different between groups. Table 1 displays the levels of pulmonary surfactant pools and total protein in total lung lavage. Quantitative analysis of pulmonary surfactant pools shows that there was no change between (+/+) CLP and (+/+) Sham controls. The pool size of total surfactant was significantly increased in the (-/-) Sham group compared with the (+/+) Sham control. This increase in total surfactant was found to be due to a significant increase in the LA subfraction. The (-/-) CLP group had surfactant pools that were not significantly different from that of the WT controls. Total protein levels were not significantly different between any of the experimental groups.
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Although the knockout groups showed a trend for decreased SP-D in lavage, no significant differences were found between groups (Table 1).
IL-6 and TNF- cytokine analysis. Table 2 displays cytokine levels in the lavage, with IL-6 levels significantly increased in response to the (+/+) CLP treatment vs. (+/+) Sham controls. IL-6 was not significantly different in the (-/-) Sham group compared with the (+/+) Sham group. The (-/-) CLP animals responded in the same fashion to the CLP procedure as WT controls, showing an increase in IL-6 levels.
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TNF- levels were also analyzed in lavage showing no significant differences among any of the groups. However, there was a trend toward increased TNF-
in the (-/-) CLP vs. (-/-) Sham, which was not detected in (+/+) controls.
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DISCUSSION |
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Previous characterization of the TIMP-3 knockout mouse had demonstrated phenotypic alterations over a prolonged period of time; however, the immediate response of these animals to a lung stress was not known (32). Sepsis induced by CLP is a clinically relevant model in which lung stress is induced via an inflammatory pathway (40, 48). Others have reported increased MMP activity in sepsis, although the model in those studies involved infusion of endotoxin (12, 15). In our experiments, animals were studied at 9-12 wk of age, a time point at which some phenotypic differences between knockout and WT mice are present (32), but these differences do not have an impact on the general health status of the animal.
The most intriguing finding of the current study was that lung compliance rapidly and significantly increased in TIMP-3 knockout animals in response to CLP-induced sepsis over the sham-operated controls. Compliance is a measure of the distensibility of the lung, resulting from contributions from 1) the elastic and structure components of the lung, as well as 2) surface tension and the pulmonary surfactant system (36). Increased compliance and air space enlargement are phenomena that generally involve prolonged periods of time (19, 56), whereas in our study this was observed after a 6-h period. We are not aware of other studies demonstrating such a rapid increase in compliance in response to a lung stress. The typical response to an acute lung insult is a decrease in lung compliance (17); however, due to the predisposed increase in MMP activity in the TIMP-3 null, the opposite phenomenon occurs. Here, we have observed that CLP-induced sepsis in young adult TIMP-3 animals resulted in an enhancement of the preexisting abnormal phenotype as measured by lung compliance.
To explain the rapid changes in lung compliance and lung structure, we examined the abundance of several of the major components of the lung ECM, namely elastin, collagen, and fibronectin, as well as MMP abundance and activation. No changes in steady-state elastin levels were detected between any of the experimental groups; therefore, it is unlikely that degradation of elastin contributed to the observed changes in compliance. In contrast, collagen abundance was significantly reduced in knockout vs. WT animals in both Sham and CLP groups and in CLP compared with Sham. Furthermore, these changes in collagen were additive, such that TIMP-3 knockout mice that underwent the CLP procedure had significantly lower collagen values than all other groups. This result, in combination with the relative changes in fibronectin, a linking protein between collagen molecules and cells (27, 41, 42), would suggest that decreases in collagen may have contributed to the significant increase in compliance of the knockout CLP animals. This is consistent with our previous report, which identified collagen as the primary ECM molecule to be targeted by MMP activation in the lungs of TIMP-3 null mice (32). TIMP-3 knockout animals also have disorganized collagen in the lung, as determined by electron microscopy (32). Thus in addition to the absolute amount of collagen, the molecular organization of this molecule may also have contributed to the observed compliance changes. Although not investigated in the current study, disorganization of collagen fibers could explain increased compliance in the (-/-) Sham compared with (+/+) CLP, despite the similar absolute levels of collagen in these two groups.
Our analysis of MMP activities focused on the gelatinases MMP-2 (72 kDa) and MMP-9 (105 kDa), which are known to degrade collagen (1, 25, 43, 45, 58). Gelatin SDS-PAGE zymography demonstrated increased activities of these enzymes in response to CLP-induced sepsis in both the knockout and WT animals. However, gelatinase activities in (-/-) CLP lung extracts were not significantly higher than that of the (+/+) CLP lung. The limitation of the gelatin zymography is that it fails to examine MMP activity in the presence of endogenous inhibitors; therefore, to determine the activity of the gelatinase within the tissue, that is, in the presence (+/+) or absence (-/-) of endogenous TIMP-3, we performed in situ zymography. In general, the results complement the gel zymography, in that there was increased activity in situ in the groups that had increased activation detected by the gel zymography. Although not quantitative, in situ zymography also showed a relatively high activity in the (-/-) CLP group, which may reflect an alteration to endogenous inhibitor levels. Therefore, it can be concluded that MMP activity was increased due to CLP, and this was further enhanced in the knockout animals. These results, combined with the known roles of MMP in tissue remodeling, lead to the conclusion that increased MMP activity was a likely contributor to the altered compliance observed in our study. Although we found increased MMP activity associated with abnormal lung structure and function, others have shown that deficiency for MMP-9 is protective in an LPS model of ALI (15).
Interestingly, the in situ zymography also demonstrated that a very significant proportion of the gelatinase activity in the CLP knockout animals was observed along the airways; however, the implications of this unexpected finding on pulmonary compliance are currently unknown and require further investigation. Some studies have previously linked airway inflammation with increased compliance (19, 20, 30); therefore, this may be a contributing mechanism to the rapid compliance changes in this model. Our result is consistent with that of Corry and coworkers (13), who demonstrated elevated abundance of MMP-2 mRNA in mesenchyme surrounding the airways of mice in response to inflammatory challenge.
In addition to the outcome parameters discussed above, we also analyzed the pulmonary surfactant system and two inflammatory cytokines in our study. The reason for analyzing surfactant in our study was threefold. First, targeted deletion of SP-C or -D leads to air space enlargement and increased MMP activities, as well as an accumulation of lipids in the air space (22, 62), indicating a potential relationship between the surfactant system and the MMP-TIMP balance within the lung. Second, the pulmonary surfactant system is altered in sepsis (14, 40). The specific change appears to be a shift in the surfactant subtypes as observed in sepsis models 18-22 h after the induction via CLP (40, 48). Third, the status of the surfactant system may influence lung compliance, although in general, impairment of surfactant would lead to a decrease in compliance (4, 34) rather than the increase we observed in our experimental group.
Our results show that the (-/-) CLP animals had a normal surfactant system, including surfactant subtypes and SP-D levels, that was not significantly different than all other groups. This leads to the conclusion that pulmonary surfactant did not play a major role in the phenotypic alterations we observed in the knockout animals in response to CLP-induced sepsis. The only significant difference observed in our surfactant analysis was in the (-/-) Sham animals. The higher amounts of surfactant in the (-/-) Sham are interesting, considering that, due to the presence of enlarged air spaces, the actual surface area of the lung may be smaller than those in the WT controls. However, previous studies have shown that higher surfactant levels did not increase compliance over control levels (48). Additionally, since the (-/-) CLP displayed a higher compliance than the (-/-) Sham animals, while having lower levels of total surfactant, it does not appear that the pulmonary surfactant system is involved in the rapid compliance changes observed in the TIMP-3 knockout animals following CLP.
We also examined the cytokines TNF- and IL-6 since these are representative markers of the inflammatory status of the lung (63). Furthermore, TIMP-3 has the ability to inhibit TACE (2, 33). Thus the absence of TIMP-3 could possibly influence the levels of this specific proinflammatory cytokine. The results showed that TNF-
was not significantly different among groups and that IL-6 concentrations were increased following CLP-induced sepsis, similar to past studies. Both knockout groups had similar IL-6 and TNF-
concentrations compared with their WT controls. Thus the absence of TIMP-3 and the CLP procedure did not affect the concentrations of inflammatory cytokines in the lavage. Despite the results of the statistical analysis, it should also be noted that the TNF-
values in the (-/-) CLP tended to be more variable and higher than all other groups. In a different lung injury model of intraperitoneal injection of bacterial LPS, concentrations of TNF-
in both serum and peritoneal lavage peaked at 1 h postinjection (26). It is tempting to speculate that during different stages of the inflammatory cascade, the presence or absence of TIMP-3 may still influence TNF-
concentrations due to its TACE inhibitory activity. This possibility, as well as the other inflammatory aspects of sepsis in this experimental model, is obviously an important area that requires further study.
In conclusion, this investigation shows that TIMP-3 knockout animals had increased lung compliance compared with WT controls and that compliance was further increased only 6 h following a CLP procedure. These changes in compliance are likely due to increased MMP activity degrading ECM components of the lung such as collagen and fibronectin.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* K. J. Leco and R. A. W. Veldhuizen are cosenior authors and contributed equally to this study.
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REFERENCES |
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