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Immunology. Jun 2004; 112(2): 183–190.
PMCID: PMC1782474

Tethered ligand-derived peptides of proteinase-activated receptor 3 (PAR3) activate PAR1 and PAR2 in Jurkat T cells

Abstract

Proteinase-activated receptors (PARs) can activate a number of signalling events, including T-cell signal-transduction pathways. Recent data suggest that the activation of PARs 1, 2 and 3 in Jurkat T-leukaemic cells induces tyrosine phosphorylation of the haematopoietic signal transducer protein, VAV1. To activate the PARs, this study used the agonist peptides SFLLRNPNDK, SLIGKVDGTS and TFRGAPPNSF, which are based on the sequences of the tethered ligand sequences of human PARs 1, 2 and 3, respectively. Here, we show that peptides based on either the human or murine PAR3-derived tethered ligand sequences (TFRGAP-NH2 or SFNGGP-NH2) do not activate PAR3, but rather activate PARs 1 and 2, either in Jurkat or in other PAR-expressing cells. Furthermore, whilst thrombin activates only Jurkat PAR1, trypsin activates both PARs 1 and 2 and also disarms Jurkat PAR1 for thrombin activation. We conclude therefore that in Jurkat or related T cells, signalling via PARs that can affect VAV1 phosphorylation is mediated via PAR 1 or 2, or both, and that distinct serine proteinases may potentially differentially affect T-cell function in the settings of inflammation.

Keywords: enzymes: proteases, proteinases; proteins: peptides, tethered ligands, VAV-1; signalling: calcium, PARs, tyrosine phosphorylation; T cells: Jurkat cells

Introduction

Proteinase-activated receptors (PARs) are members of a recently discovered receptor family, belonging to the seven-transmembrane superfamily of cell-surface G-protein coupled receptors.13 To date, four members of this family have been cloned (PAR1, PAR2, PAR3 and PAR4). Their unique mechanism of activation involves enzymatic cleavage of the N-terminal domain. This cleavage allows the newly unmasked N-terminal sequence of the receptor to function as a ‘tethered ligand’, which binds to another site of the receptor to initiate signalling. Remarkably, it has been established that synthetic peptides of five to six amino acids, modelled on the sequences of the PAR-tethered ligands, can also bind to and activate the PARs in lieu of the proteinase. These peptides are called PAR-activating peptides, or PAR-APs. As the proteinases that activate PARs do not necessarily act on a distinct PAR, and because proteinases can cause biological effects via mechanisms other than activating PARs, receptor-selective PAR-APs, acting in the absence of proteinases, have proved of great utility as probes to determine the potential effects of PAR activation in vitro and in vivo. There are, however, problems in using the PAR-APs that are the same in sequence as the tethered ligands, because of a lack of receptor selectivity. For instance, whereas the PAR2-derived peptide, SLIGRL-NH2, can selectively activate PAR2 from a number of species, the human PAR1-derived peptides, SFLLRNPNDKYEPF, SFLLRN and SFLLR-NH2, can all activate both PAR1 and PAR2 with comparable potencies.46 A selective PAR-AP for PAR3 has yet to be described. Furthermore, current data suggest that PAR3 on its own does not signal, but rather serves as a cofactor for the activation of PAR4.79

Soon after the activation of PAR1 was shown to mediate the cellular actions of thrombin on platelets and other cells,10,11 studies were published suggesting that the activation of PAR1 induces early events of T-cell activation.1214 Unfortunately, those studies used PAR1-derived peptides that are now known to activate both PAR1 and PAR2.46 More recently, it has been reported that the activation of PARs 1, 2 and 3 in Jurkat T-leukaemic cells induces tyrosine phosphorylation of the haematopoietic signal transducer protein, VAV1.15 VAV1, a guanine nucleotide exchange factor that targets Rho-family GTPases involved in cytoskeletal rearrangement, is recognized as a key regulator of lymphocyte development and function.16,17 Because the study linking PAR activation to VAV1 phosphorylation used the PAR-APs SFLLRNPNDK and TFRGAPPNSF, one of which can simultaneously activate both PAR1 and PAR2 (SFLLRNPNDK) and one of which (TFRGAPPNSF) has yet to be evaluated for its receptor selectivity, there is uncertainty as to which of the PARs might have been responsible for triggering VAV1 phosphorylation.15 We therefore decided to re-evaluate the ability of selective PAR1 activation to affect Jurkat signalling and we sought to assess, critically, the ability of PAR3-derived peptides to activate PARs, not only in Jurkat cells, but also in other cultured cells that either co-express human PARs 1, 2 and 3 (HEK293) or that selectively express only PAR2 (KNRK-PAR2). It was our working hypothesis, based on previous structure–activity data for the PAR-activating peptides,46 that the tethered ligand peptides derived from either human (TFRGAP…) or murine (SFNGGP…) PAR3 would activate either or both of PAR1 and PAR2 because of the N-terminal TF… or SF… motif in these peptides. The sequence motifs SF… and TF… are known to be of importance for activating PAR1 and PAR2. Here, we illustrate, by using cross-desensitization experiments, in the context of a calcium-signalling assay in Jurkat T cells, HEK293 and KNRK-PAR2 cells, that both peptides corresponding to the human and murine PAR3-tethered ligand sequences (TFRGAP-NH2 and SFNGGP-NH2, respectively) activate either or both PAR1 and PAR2, as does the human PAR1-derived peptide, SFLLRN-NH2. Furthermore, we demonstrate, in Jurkat cells, that whilst thrombin activates only PAR1, trypsin not only activates PAR1 and PAR2 but also disarms PAR1 for activation by thrombin.

Materials and methods

Cell culture

Jurkat T cells were kindly provided by Dr Julie Deans, University of Calgary (Faculty of Medicine, Calgary, Alberta, Canada). The Jurkat T cells were propagated at 37° (under an atmosphere of 5% CO2 in room air) in 80-cm2 T-flasks containing RPMI-1640 supplemented with 10% (v/v) fetal calf serum (FCS), sodium pyruvate (1 mm), and the antibiotics penicillin G (100 U/ml) and streptomycin sulphate (100 µg/ml). Human embryonic kidney cells (HEK293) were those used by us for previous work.6 The HEK cells were propagated at 37° (under an atmosphere of 5% CO2 in room air) in 80-cm2 T-flasks containing Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% (v/v) FCS, sodium pyruvate (1 mm) and penicillin G/streptomycin (as described above). Rat and human PAR2-transfected Kirsten virus-transformed rat kidney cells (KNRK; American Type Culture Collection, Bethesda, MD) were those used by us in previous work.1820 The KNRK cells were propagated at 37° (under an atmosphere of 5% CO2 in room air) in 80-cm2 T-flasks using geneticin (0·6 mg/ml)-containing DMEM supplemented with 10% (v/v) FCS, sodium pyruvate (1 mm) and penicillin G/streptomycin (as described above). The KNRK cells were subcultured in the absence of trypsin by resuspension in an isotonic phosphate-buffered cell-dissociation buffer containing EDTA (Invitrogen, Carlsbad, CA).

Peptides and other reagents

All peptides were synthesized by solid-phase methods at the peptide synthesis facility, University of Calgary (director, Dr Denis McMaster). HPLC and mass spectral analysis were used to confirm the concentration and purity of all stock peptide solutions. Stock peptide solutions (3–5 mm) were prepared in 25 mm HEPES, pH 7·4. Porcine trypsin (14 900 U/mg) was obtained from Sigma (St Louis, MO). High-activity human plasma thrombin (3137 NIH U/mg) was obtained from Calbiochem (San Diego, CA). Stock solutions of both proteinases were made in deionized water. To convert the enzyme concentrations to molar values, calculations were based on the following values: 1 U/ml of trypsin ≈ 2 nm; 1 U/ml of thrombin ≈ 10 nm.

Calcium signalling assay

Measurements of proteinase and peptide-stimulated fluorescence emission (reflecting an increase in intracellular calcium) were performed with Jurkat T cells, permanent rat KNRK cell lines expressing either human or rat PAR2, or HEK293 cells. Jurkat T cells (1–2 confluent 80-cm2 T-flasks), PAR2-transfected KNRK cells, or HEK293 cells (about 80% confluence), were harvested without the use of proteinase, pelleted by centrifugation and resuspended in DMEM (1 ml). The cells were loaded with the intracellular calcium indicator, fluo-3 acetoxymethyl ester (Molecular Probes Inc., Eugene, OR), at a final concentration of 22 µm (25 µg/ml), as described previously.21,22 Indicator uptake was allowed to proceed for 25 min at room temperature in the presence of 0·4 mm sulfinpyrazone, after which the cells were washed twice by centrifugation and resuspended in the following buffer: 150 mm NaCl, 3 mm KCl, 1·5 mm CaCl2, 20 mm HEPES, 10 mm dextrose and 0·25 mm sulfinpyrazone, pH 7·4. Fluorescence measurements, reflecting elevations of intracellular calcium, were conducted at 24° using an AMINCO-Bowman® Series 2 Luminescence Spectrometer (Spectronic Unicam, Rochester, NY), with an excitation wavelength of 480 nm and an emission recorded at 530 nm. The fluorescence signals caused by the addition of test agonists [proteinase or peptides, added to 1 ml of a cell suspension (final concentration of cells: ≈ 1 × 106/ml in the above buffer)] were compared with the fluorescence peak heights yielded by replicate cell suspensions treated with 2 µm of ionophore A23187 (Sigma Chemical Co).

Cross-desensitization assay

The cross-desensitization assay, using an elevation of intracellular calcium as an index of cellular response, was performed exactly as outlined previously for the evaluation of PAR stimulation in HEK293 cells.6 The pharmacological principle of this assay depends on a specific desensitization of one receptor via a selective agonist, without affecting a second distinct receptor that causes the same downstream effect in cells or tissues by presumably the same downstream signal pathways. Thus, a prior desensitization at the ligand-activation site of PAR1, by appropriate concentrations of a receptor-selective agonist (e.g. TFLLR-NH2) that does not desensitize downstream signal pathways, but does silence PAR1 without affecting other PARs, will also result in a desensitization of the cell to all other agonists (e.g. thrombin) that act via PAR1. However, prior desensitization will not desensitize the cell for the same response (calcium signals) triggered by agonists that act via PAR2 (e.g. trypsin) or via other receptors (e.g. lysophosphatidic acid), which also activate calcium signalling in HEK cells.6 In the experiments, TFLLR-NH2 was used for the selective desensitization of PAR1, whereas SLIGRL-NH2 was used for selectively desensitizing PAR2.

Results

PAR3-derived tethered ligand peptides activate Jurkat PAR1 and PAR2

To assess the ability of the human and murine PAR3-derived peptides (TFRGAP-NH2 and SFNGGP-NH2) to activate either PAR1 or PAR2 in Jurkat cells, we made use of a cross-desensitization assay,6 previously developed using human HEK293 cells that, like Jurkat cells, possess all PARs 1–3, but not PAR4. In the Jurkat cells, both the PAR1-selective agonist, TFLLR-NH2, and the PAR2-selective agonist, SLIGRL-NH2, were able to cause a robust calcium signal, with 50% effective concentration (EC50) values of ≈ 10 and 50 µm, respectively (Fig. 1). Based on these concentration–effect curves, cross-desensitization experiments were then carried out for the PAR1- and PAR2-selective agonists, wherein the Jurkat cell was first desensitized either to the PAR1 or PAR2 agonist and then challenged with either of the two PAR3-derived peptides, TFRGAP-NH2 (human) or SFNGGP-NH2 (murine).

Figure 1
Calcium signalling in Jurkat cells by proteinase-activated receptor activating peptides (PAR-APs): concentration–effect curves. The calcium signal, relative to that obtained with ...

As shown in Fig. 2 (right-hand tracings in panels a, b, d and e), both PAR3-derived peptides, at a concentration of 500 µm, caused a calcium signal that was 15–55% of the signal maximally caused by the PAR1- and PAR2-selective agonists, TFLLR-NH2 (TFL-NH2, 100 µm) and SLIGRL-NH2 (SL-NH2, 200 µm). However, prior desensitization of PAR1 in the Jurkat cells with TFLLR-NH2 caused a reduction of 74 ± 5% (n = 3) in the Jurkat calcium signal caused by the human PAR3-derived peptide, TFRGAP-NH2 (TFR-NH2; Fig. 2, tracing a), whereas prior desensitization of Jurkat cell PAR2 with SLIGRL-NH2 diminished the Jurkat calcium signal, caused by TFRGAP-NH2, by 29 ± 3% (n = 3) (Fig. 2, tracing b). In contrast, prior desensitization of the Jurkat cells with the PAR2-targeted agonist, SLIGRL-NH2, had a greater effect (61 ± 1%, n = 3, reduction; Fig. 2, tracing e) on the signal caused by the murine-derived PAR3 peptide, SFNGGP-NH2 (SFN-NH2), than did prior desensitization of PAR1 using TFLLR-NH2 (17 ± 6%, n = 3, reduction in the calcium signal caused by SFNGGP-NH2; Fig. 2, tracing d). Furthermore, predesensitization of the Jurkat cells with the non-selective PAR-agonist, SFLLR-NH2, at a concentration that can affect both PAR1 and PAR2, greatly reduced the Jurkat cell signal caused by either the human or murine-derived PAR3 peptides (100% reduction in the calcium signal caused by TFRGAP-NH2: data not shown; 61 ± 3%, n=3, reduction in the calcium signal caused by SFNGGP-NH2; Fig. 2, tracing f). Finally, there was no calcium signal generated by thrombin treatment of cells preactivated with SFLLR-NH2 in order to co-desensitize both PAR1 and PAR2 (Fig. 2, tracing c), indicating that PAR3, although present, was unable to cause a signal in response to thrombin.

Figure 2
Desensitization of the TFRGAP-NH2 (TFR-NH2)-, SFNGGP-NH2 (SFN-NH2)-, and thrombin-mediated calcium signal by desensitization of proteinase-activated receptor 1 (PAR1), proteinase-activated receptor 2 (PAR2), or both. Jurkat cell suspensions were first ...

The effects of the peptides TFRGAP-NH2 and SFNGGP-NH2 are not unique to Jurkat cells. Similar experiments to those described above were performed in the HEK cell assay system.6 As in the Jurkat cell assay, in the HEK cell assay the human-derived peptide, TFRGAP-NH2, acted preferentially via PAR1, and the murine-derived peptide, SFNGGP-NH2, acted preferentially through PAR2 (data not shown).

To confirm that the murine-derived PAR3 peptides can activate PAR2, we evaluated their ability to generate a calcium signal in rat and human PAR2-expressing cell lines (KNRK-PAR2) with which we have had considerable experience.23,24 As shown in Fig. 3, the peptide SFNGGP-NH2 elicited a concentration-dependent calcium response in the cell lines that express PAR2 but do not express other PARs. The human PAR3-derived peptide, TFRGAP-NH2, was less potent in this regard, although it too was able to cause a calcium signal in both KNRK-PAR2 cell lines. Clearly, the PAR3-derived peptides were much less potent than the PAR2-selective agonist, SLIGRL-NH2, in activating either human or rat PAR2. Additionally, the PAR3-derived peptides were even less potent than the PAR1-selective agonist, TFLLR-NH2, which, at concentrations of > 100 µm can activate PAR2 (Fig. 3). In terms of their potencies for activating PAR2, relative to the potency of the PAR-AP TFLLR-NH2, the murine PAR3-derived peptide, SFNGGP-NH2, was more potent for activating the rat receptor than human PAR2 (compare upper and lower curves for human and rat KNRK-PAR2, respectively, in Fig. 3), whereas, relative to SFNGGP-NH2, the human PAR3-derived peptide, TFRGAP-NH2, displayed a low potency for both the human and rat receptors. KNRK cells transfected with the vector alone, but not the receptor, failed to respond to the PAR3-derived peptides (data not shown).

Figure 3
Calcium signalling in Kirsten virus-transformed rat kidney (KNRK) cells transfected with human proteinase-activated receptor 2 (PAR2) (a) or rat PAR2 (b): concentration–effect curves. The calcium signal, relative to that obtained with ...

Low concentrations of trypsin disarm PAR1 and activate PAR2 in Jurkat cells

As the data presented above indicated that the Jurkat calcium signal caused by the PAR3-derived peptides could be desensitized by both selective PAR1 and PAR2 agonists, we also wanted to determine the effect of the proteinases thrombin and trypsin themselves on Jurkat T cells in the calcium-signalling assay. As shown in Fig. 4, both thrombin (5 U/ml; 50 nm) and trypsin (25 U/ml; 50 nm) were able to generate a calcium signal in Jurkat cells (Fig. 4, right-hand tracings), in keeping with previous reports.14 Prior desensitization of the Jurkat cell with the PAR1-selective agonist, TFLLR-NH2, completely eliminated the response to 50 nm thrombin (Fig. 4, tracing a). However, desensitization of PAR1 reduced the signal generated by 50 nm trypsin by only 35 ± 3% (n=3) (Fig. 4, tracing b). Conversely, prior desensitization of PAR2 with SLIGRL-NH2 had less of an effect on the response to thrombin (17 ± 5%, n = 3, reduction in the calcium signal; Fig. 4, tracing c), but markedly reduced the Jurkat cell response to trypsin (97 ± 3%, n = 3, reduction in the calcium signal; Fig. 4, tracing d). Thus, trypsin was able to stimulate Jurkat cells by activating both PAR1 and PAR2, with the main signal generated via PAR2.

Figure 4
Desensitization of the thrombin (Thr)-, and trypsin (Trp)-mediated calcium signal by desensitization of proteinase-activated receptor 1 (PAR1) or proteinase-activated receptor 2 (PAR2). Jurkat cell suspensions were first exposed to receptor-desensitizing ...

Finally, although the data presented the paragraph above indicated that the calcium signal caused by trypsin was attenuated by prior desensitization of both PAR1 and PAR2, we wished to determine whether prior treatment of the cells with trypsin might also affect their response to thrombin. We have previously documented the ability of trypsin to disarm PAR1, for both thrombin and TFLLR-NH2 activation, using HEK cells that possess both PAR1 and PAR2.6 As shown by the concentration–response curves in Fig. 5, trypsin was also able to disarm PAR1 for both thrombin and TFLLR-NH2 activation in Jurkat cells. At concentrations lower than those required to generate a substantial calcium signal in Jurkat cells (white squares, Fig. 5), trypsin treatment (< 10 U/ml or < 20 nm) was able to attenuate the subsequent cellular response to thrombin (5 U/ml or ≈ 50 nm) by up to 80% (white circles, Fig. 5). Interestingly, trypsin was not able to desensitize the Jurkat cells to activation by the PAR1-activating peptide, TFLLR-NH2, by more than 30% (top tracing, black circles, Fig. 5), even at concentrations of trypsin that caused a maximal calcium signal in the Jurkat cells (white squares, Fig. 5).

Figure 5
Concentration–desensitization (•, ○) and concentration–stimulation (□) curves for trypsin-mediated activation of Jurkat cells. Jurkat cell suspensions were first stimulated by the addition of trypsin (2–200 ...

Discussion

The main finding of our study was that the human and murine PAR3-derived tethered ligand sequences, TFRGAP-NH2 and SFNGGP-NH2, can activate both PAR1 and PAR2 in either Jurkat or HEK cells. Furthermore, in a calcium signalling assay wherein both PARs 1 and 2 were first co-desensitized by treatment of the Jurkat cells with the peptide SFLLR-NH2, the calcium signal generated by either the PAR3-derived peptides or thrombin, was greatly reduced. However, both PAR3-derived peptides and thrombin did cause a calcium signal prior to desensitization. Thus, the PAR3-derived peptides significantly activate PARs 1 and 2, despite the expression of PAR3 in the cells, as shown by us (by reverse transcription–polymerase chain reaction) and others (by Western blot).15 The use of PAR3-derived peptides to determine the effects of PAR3 is confounding in cases where PARs 1 and 2 are also expressed. Even if PAR3 were the only PAR expressed, it has been shown by others that COS 7 cells expressing murine PAR3 do not respond to the PAR3-derived peptide, SFNGGP.8

Our data demonstrating, first, an ability of the PAR3-derived peptides to activate either or both of PAR1 or PAR2, depending on peptide concentration and, second, the ability of the human PAR1-derived peptide, SFLLR-NH2, to activate both PAR1 and PAR2 simultaneously, require a reinterpretation of experiments carried out previously with Jurkat and other T-lymphoblastoid cells using these receptor-derived tethered ligand peptides and thrombin as agonists.1215 What is clear from the previous studies is that activation of PAR2 by the PAR2-selective agonists, SLIGRL or SLIGKVDGTS-NH2, can elevate Jurkat cell intracellular calcium and can induce tyrosine phosphorylation of VAV1 and other VAV1-associated signalling intermediates, such as ZAP-70 and SLP-76.14,15 As Jurkat cells do not express PAR4,15 and because our new data show that the thrombin signal in Jurkat cells is completely eliminated by prior desensitization of PAR1 alone, signalling by PAR3 appears not to occur and the ability of thrombin to trigger early events of T-cell activation can be attributed solely to PAR1.13 That said, the ability of PAR2 to synergize with PAR1 cannot be ruled out, as the non-selective PAR agonist, SFLLRNP, which simultaneously activates both PAR1 and PAR2, caused marked effects on VAV1 phosphorylation in Jurkat cells.15 It will be important to establish whether the activation profile triggered by PAR1, in terms of the downstream signal pathways in T cells, will be identical to that activated by PAR2, as, even in the same assay system, activation of these two distinct PARs can have either common (e.g. vasodilatory, via endothelial cell activation) or opposing (vasodilator versus vasoconstrictor in a perfused renal assay) effects.1 Notwithstanding, the results we describe here establish unequivocally that the selective activation of either PAR1 or PAR2 causes an elevation of intracellular calcium in Jurkat cells and, presumably, tyrosine phosphorylation of VAV1.15

In work that used the human PAR3-derived tethered ligand sequence as an agonist,15 the elevated tyrosine phosphorylation observed for VAV1, ZAP-70 and SLP-76 was probably caused by the combined activation of PARs 1 and 2, with a possible predominance of PAR1, because of the ability of the human PAR3-derived peptide to activate PAR1 somewhat more than PAR2 (Fig. 2). Thus, the potential role that PAR3 may play in T cells remains an open question (e.g. as an adsorbent for thrombin), especially because, as already mentioned, the Jurkat cell does not express PAR4, for which PAR3 might act as a cofactor.8,25 Finally, it is an open question as to how tyrosine phosphorylation of VAV1 stimulated by PAR1/PAR2 activation will interact with T-cell receptor-activated signal pathways that also involve VAV1.16,17

Added to the data obtained with the PAR-activating peptides, our work sheds further light on the actions of thrombin and trypsin on Jurkat cells. In keeping with previous observations,14 our results confirm the ability of thrombin and trypsin to induce calcium mobilization via distinct Jurkat cell receptors. However, the cross-desensitization data reveal that whereas thrombin triggers PAR1 (but not PAR3) to cause an increase in intracellular calcium, trypsin can affect both PARs 1 and 2. In addition, our data show that the PAR-activating peptide used by Mari et al.13 (SFLLRN) would have activated both PAR1 and PAR2, thereby necessitating a reinterpretation of the data obtained in those studies. Although PAR2 represents the main receptor responsible for a trypsin-triggered calcium signal in Jurkat cells, trypsin is also capable of activating PAR1 to a small degree. However, more significantly, trypsin is able to disarm PAR1, rendering it insensitive to thrombin activation (Fig. 5). Importantly, the concentration range over which trypsin was able to disarm the receptor for thrombin activation (presumably via cleavage downstream from the PAR1 tethered ligand sequence), was below the concentrations at which trypsin, via PAR2, maximally stimulated a calcium signal. What our data highlight is the potential importance of proteinases as differential agonists/antagonists for T-cell signalling, in addition to antigens and cytokines. Thus, in an inflammatory milieu containing serine proteinases akin to trypsin, the PAR1 signal derived from thrombin might be abrogated and all signalling might then be caused solely by the activation of PAR2. Alternatively, in some situations, thrombin alone might signal. Clearly, the impact that serine proteinases may have on lymphocyte function, in the setting of inflammation, merits further study. In this regard, the pro-inflammatory role of PAR226 can be seen to take on added significance. Without doubt, further work is warranted to determine more precisely the signalling pathways in T cells, other than an elevation of intracellular calcium, which may be selectively regulated by PARs 1 and 2. In this context, the receptor-selective activating peptides will be of particular utility.

Acknowledgments

This study was supported, in large part, by a grant from the Canadian Institutes of Health Research and by ancillary funds from grants provided by the Heart and Stroke Foundation of Canada, the Kidney Foundation of Canada and by a Focused Giving grant from Johnson & Johnson. We are grateful to Dr Julie Deans for providing the Jurkat cells and for her thoughtful review of the manuscript.

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