{'fraction_non_alphanumeric': 0.03676470588235294, 'fraction_numerical': 0.01904724655819775, 'mean_word_length': 5.112598613435334, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The E3 ligase Itch and deubiquitinase Cyld co-operatively regulate Tak1 and inflammation

Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld form a complex via the interaction through ‘WW-PPXY’ motifs. The Itch-Cyld complex sequentially cleaved K63-linked ubiquitin chains and catalyzed K48-linked ubiquitination on the kinase Tak1 to terminate inflammatory tumor necrosis factor signaling. Reconstitution of wild-type Cyld but not mutant Cyld(Y485A), which cannot associate with Itch, blocked the sustained Tak1 activation and proinflammatory cytokine production by Cyld−/− bone marrow-derived macrophages. Itch or Cyld deficiency resulted in chronic production of tumor-promoting cytokines by the tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have uncovered an Itch-Cyld mediated regulatory mechanism in innate inflammatory cells.


INTRODUCTION
Accumulating evidence indicates that tumor progression is governed not only by genetic changes in cancer cells, but also by epigenetic and environmental factors. Chronic infection and subsequent inflammation are considered critical environmental and epigenetic factors contributing to tumor progression 1,2 . Primary examples are gastric cancer and Helicobacter pylori infections, hepatocellular carcinoma and viral hepatitis, colitis-associated cancer, and pancreatic cancer associated with chronic pancreatitis 1 . Similarly, chronic obstructive pulmonary disease (COPD), cigarette smoking, exposure to asbestos and silica are all associated with lung inflammation and lung carcinoma [3][4][5] . Inflammatory cells found in the tumor microenvironment, particularly tumor-associated macrophages (TAMs), constitute an important interface between tumor cells and the immune system 6 . Production of proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 6 (IL-6) and IL-1β, by TAMs not only trigger pro-survival signals in tumor cells, but also support their growth and metastasis 6,7 . However, the molecular mechanisms that regulate inflammation and tumor progression remain unclear.
Post-translational modifications mediated by ubiquitin conjugation have emerged as a key regulatory mechanism in the immune cells 8,9 . The ubiquitin signal is interpreted based on the number of attached ubiquitin chains and the topology of their linkage. A polyubiquitin chain is formed when one of the seven lysines within ubiquitin is linked to the C-terminal glycine of another ubiquitin. Although ubiquitin contains seven lysine residues, linkage generally occurs via either K48 or K63. K48-linked polyubiquitination predominantly targets proteins for proteasomal degradation, whereas K63-linked polyubiquitination results in non-proteasomal modifications, such as subcellular localization or protein-protein interactions 8 . We and others have demonstrated HECT type E3 ligase Itch as a critical regulator of inflammation 10,11 . On a mouse C57BL/6 background, Itch deficiency results in the development of a late onset lymphoproliferation disorder and chronic pulmonary inflammation 12,13 . Itch possesses four WW domains that recognize the Pro-rich PPXY (PY) consensus sequence in their substrate targets 14 . Although it is clear that Itch regulates inflammation, no systematic study has linked Itch to tumorigenesis. Similar to phosphorylation, protein ubiquitination is reversible, and removal of ubiquitin molecules is mediated by de-ubiquitinating (DUB) enzymes, such as Cyld 15 . Cyld is encoded by a NF-κB-inducible gene 16 , inhibits NF-κB and the kinase JNK by removing K63-linked polyubiquitin chains from signaling pathway molecules such as Bcl-3, TRAF2, TRAF6 and Nemo (also known as IKK-γ) [17][18][19][20][21] . Cyld through its deubiquitinating function has been shown to regulate various types of human cancers, including lung, hepatocellular, colon and cervical cancers 15 . Cyld was also shown to negatively regulate tumor-promoting cytokine secretion by inflammatory cells 22 . Despite these important observations, the molecular mechanisms regulating Cyld function and its role in inflammation remain largely unknown. Here, we demonstrate that Itch interacts with Cyld and cooperatively regulates Tak1 and inflammation.

Augmented tumor growth in Itch −/− and Cyld −/− mice
We have demonstrated earlier that Itch deficiency results in elevated lung inflammation 10,11 . Similarly, Cyld −/− mice exhibit inflammatory infiltrates in numerous tissues including lung, liver, spleen and the salivary glands 22 . Cyld deficiency resulted in increased susceptibility to spontaneous and chemically induced colitis and colon tumorigenesis 22,23 . Cyld was also shown to regulate lung inflammation 24,25 and Cyld expression is frequently downregulated in non-small cell lung carcinoma (NSCLC) cell lines 26 . Over-expression of the full-length but not the catalytically inactive mutant of Cyld in NSCLC cell lines significantly reduces cell growth, suggesting that Cyld acts as tumor suppressor gene for the progression of NSCLC and that its deubiquitin function is important for this mechanism 26 . To determine the contribution of Itch and Cyld in inflammation and metastasis of lung carcinoma, we inoculated 1 × 10 6 Lewis Lung Carcinoma (LLC) cells into Cyld −/− , Itch −/− and control C57BL/6 mice intravenously via tail vein. Both Itch −/− and Cyld −/− mice exhibited higher mortality rate compared to wild-type mice (Fig. 1a). Histological analysis revealed aggressive tumor growth in the lungs of Itch −/− and Cyld −/− mice (Fig. 1b). Similarly, remarkable differences were seen in tumor multiplicity (Fig. 1c) and tumor size (Fig. 1d). To investigate the effect of Itch and Cyld deficiency on metastasis, we inoculated LLC cells subcutaneously (s.c.) and spontaneous lung metastasis was analyzed. A marked increase in the metastasis of LLC tumors was observed in Itch −/− and Cyld −/− mice ( Supplementary Fig. 1). These data suggest that Itch and Cyld deficiency in the hosts result in elevated growth and metastasis of lung carcinoma.
Since proinflammatory cytokines secreted by TAMs have been associated with enhanced tumor growth and metastasis 6 , we analyzed cytokine expression by tumor infiltrated CD11b + cells. Enhanced expression of TNF, IL-6 and IL-1β was observed in both Itch −/− and Cyld −/− cells (Fig. 1e). Similarly, we have observed elevated expression of these cytokines from the whole tumor nodules isolated from the lungs of Itch −/− and Cyld −/− mice (data not shown). No significant difference in cytokine expression was observed in the lung tissues of naïve Itch −/− and Cyld −/− mice that were not inoculated with LLC cells (Fig. 1f), suggesting that Itch and Cyld negatively regulate TAM activity.

Itch interacts with Cyld via the WW-PPXY motifs
We previously reported that Itch regulated TNF-induced NF-κB signaling by modulating cFLIP L turnover 27 . Cyld deubiquitin activity was shown earlier to interfere with the ubiquitination of TRAF molecules and Tak1, which are involved directly in the TNFassociated NF-κB signaling pathway 15 . Since Itch and Cyld can regulate similar pathways, we tested whether these enzymes can cooperate to terminate NF-κB signaling and inflammatory response during tumor growth and metastasis. To determine whether Itch and Cyld can directly interact with each other, we performed sequence analysis and found a highly conserved Cyld 'PPXY' motif ( Fig. 2a) by which it could interact with Itch 'WW' domains. We tested this interaction by transiently transfecting 293T cells with Flag-Itch and Myc-Cyld. Cell lysates were immunoprecipitated using control mouse IgG or antibodies against Flag or Myc. Antibody against Myc immunoprecipitated Flag-Itch and vice versa, suggesting that Itch physically interacts with Cyld (Fig. 2b). Further, we generated a fusion protein of glutathione S-transferase (GST) and Itch (GST-Itch) and performed pull-down assay using lysates of 293T cells transfected with Myc-Cyld, GST-Itch precipitated Myc-Cyld, but not GST alone, confirming the association ( Supplementary Fig. 2). We further tested whether the interaction is direct by using GST-Itch and His-Cyld (expressed in Escherichia coli). GST-Itch precipitated His-Cyld in the pull-down assay confirming a direct interaction between Itch and Cyld (Fig. 2c).
To determine if this interaction occurs endogenously in primary cells, we stimulated bone marrow derived macrophages (BMDMs) with TNF for 30 min followed by immunoprecipitation experiments using antibodies against Itch and Cyld. Itch coimmunoprecipitated with Cyld (Fig. 2d). Itch and Cyld interaction in BMDMs seems to be stimulation dependent as we did not detect the association in unstimulated cells (data not shown). To test whether Itch-Cyld interaction occurs via the 'PPXY' motif, we generated Y to A mutant Cyld (Cyld(Y485A)) by site-directed mutagenesis. The Cyld(Y485A) mutation disrupted Itch-Cyld interaction (Fig. 2e). Taken together these data collectively indicate that Itch forms a complex with Cyld via its interaction through 'PPXY' motif.

Itch targets Tak1 for K48-linked ubiquitination
Next, we sought to understand the role of Itch-Cyld complex in the regulation of inflammatory response. Inflammatory stimuli driven signaling converge at the IκB kinase (IKK) complex, a trimeric holoenzyme comprised of catalytic subunits IKKα, IKKβ, and the regulatory subunit Nemo 28 . Tak1 is a key intermediate that transmits signals from receptor complexes to IKK. Tak1 associates with TNF-receptor associated factors TRAF2 or TRAF6 following stimulation with TNF or Toll-like receptor ligands and IL-1, respectively, to initiate a signaling cascade. Both TRAF2 and TRAF6 are RING domain ubiquitin ligases that catalyze K63-linked polyubiquitin chains, which results in Tak1 autophosporylation 29 . Activated Tak1 phosphorylates IKKβ within the activation loop, leading to the activation of IKK 30 . IKK in turn phosphorylates IκBα, resulting in NF-κB nuclear translocation and transcription of inflammatory target genes 28 . Tak1 activation is transient and is rapidly inactivated, which is important for preventing prolonged inflammatory responses [31][32][33] . Cyld was previously shown to cleave K63-linked polyubiquitin chain on Tak1 23 . Because Itch deficiency resulted in persistent IKK and Jnk kinase activation 34 , similar to Cyld deficiency [17][18][19][20][21] , we reasoned that Itch may participate in terminating Tak1 activation by K48-linked polyubiquitination. To test this notion, we co-expressed Flag-Tak1 with Myc-Itch and either Hemaglutinin (HA) tagged Ub(WT), Ub(K48) [in which all the lysine residues, except K48, are mutated], or Ub(K63) [in which all the lysine residues, except K63, are mutated]. Slow-migrating high molecular weight polyubiquitinated forms of Tak1 was observed when Itch and Tak1 were co-expressed with Ub(WT) (Fig. 3a, lane1). Interestingly, Itch catalyzed Tak1 polyubiquitination when co-expressed with Ub(K48) (lane3), but not with Ub(K63) (lane5) suggesting that Itch targets Tak1 for K48-linked but not K63-linked ubiquitination. To confirm the specificity of Itch as the E3 ligase, we generated an Itch(C830A) mutant in which the conserved cysteine is mutated to alanine 35 . Expression of the Itch(C830A) mutant with either Ub(WT) or Ub(K48) did not result in Tak1 ubiquitination (Fig. 3a, lanes 2 and 4). To further confirm that Itch targets Tak1 for K48-linked polyubiquitination, we utilized Ub(K48R) in which only K48 was mutated to arginine. Co-expression of Ub(K48R) and Itch failed to polyubiquitinate Tak1 (Fig. 3b). Next, we tested whether another functionally related E3 ligase, Cbl-b 8 ubiquitinates Tak1. Co-expression of Cbl-b with Tak1 did not result in Tak1 ubiquitination ( Supplementary Fig.  3), suggesting that Itch is the specific ligase for Tak1.

Tak1 activation in Itch −/− and Cyld −/− BMDMs
Subsequently, we investigated the effect of Itch and Cyld deficiency on Tak1 activation in BMDMs following treatment with TNF. The cell lysate was immunoprecipitated using antibody against TRAF2 and immunoblotted with antibody against phospho-Tak1 to test TRAF2-associated Tak1 activation. Tak1 phosphorylation peaked within five minutes following stimulation in wild-type macrophages and the phosphorylation was reduced to minimal within thirty minutes, however, we have observed sustained Tak1 phosphorylation in Itch −/− and Cyld −/− BMDMs (Fig. 5a). We reprobed the membranes with Tak1-specific antibody to analyze total TRAF2-associated Tak1. Similar to Tak1 phosphorylation, association of Tak1 with TRAF2 was transient in wild-type macrophages; however, Itch and Cyld deficiency prolonged the association. To analyze the effect of Itch and Cyld deficiency on total cellular Tak1 abundance, we immunoblotted the cell lysate with antibody against Tak1. We observed only a marginal decrease in the total Tak1 protein in wild-type BMDMs stimulated with TNF. This finding suggests that Itch-Cyld complex degrades the Tak1 that is associated with TRAF complex but not the entire pool of Tak1 within the cell during an inflammatory response.
Because Tak1 activation is directly linked to pro-inflammatory cytokine secretion, we analyzed the expression of IL-6, TNF and IL-1β in BMDMs after stimulation with TNF. Expression of proinflammatory cytokine mRNA was sustained until 12 hours in Itch −/− and Cyld −/− BMDMs (Fig. 5b). We also analyzed the cytokine concentrations in the culture supernatant by ELISA. Itch and Cyld deficiency resulted in elevated secretion of proinflammatory cytokines (Fig. 5c). This result provides evidence that cleavage of K63linked polyubiquitination is not sufficient to deactivate Tak1 and downstream effector function, rather sequential Itch-mediated K48 linked ubiquitination is essential. Similarly, sustained Tak1 phosphorylation and elevated cytokine mRNA expression were observed in Itch −/− and Cyld −/− BMDMs stimulated with lipopolysaccharide (LPS) (Supplementary Fig.  7).
To further confirm the role of Itch and Cyld interaction for terminating Tak1 activation in macrophages, we reconstituted Cyld −/− BMDMs with Cyld(WT) or Cyld(Y485A) mutant that fails to interact with Itch. Expression of Cyld(WT) but not Cyld(Y485A) mutant restored transient Tak1 activation (Fig. 8a). Similarly, wild-type Cyld almost completely reverted elevated cytokine production by Cyld −/− BMDMs; however, expression of Cyld(Y485A) mutant had only minimum effect (Fig. 8b-c). These data clearly suggest that Itch and Cyld cooperatively regulate cytokine production by the inflammatory cells.

DISCUSSION
In this study, we demonstrate that Itch forms a ubiquitin-editing complex with Cyld via the interaction through WW-PPXY motifs. Itch-Cyld complex promotes the transition of K63linked to K48-linked ubiquitination by sequentially cleaving K63-linked ubiquitin chain and by catalyzing K48-linked ubiquitination on Tak1. During the early phase of TNF-or TLRinduced inflammatory response RING type E3 ligases TRAF2 and TRAF6 promote K63linked ubiquitination within the kinase domain 29 , promoting phosphorylation of Tak1 that leads to IKK-NF-κB activation and transcription of proinflammatory cytokine gene expression 42 . We propose that the combined activity of Itch and Cyld shifts Tak1 ubiquitination from K63-linked toward K48-linked chains that triggers the proteasomal degradation of Tak1, thus terminating the inflammatory response. Itch-Cyld association was detected only following stimulation of BMDMs with TNF suggesting the requirement of extracellular signals. At present the molecular mechanisms that regulate Itch-Cyld complex formation remain unclear. One possibility is that Cyld expression is upregulated by TNF and IL-1β, via IKK-NF-κB pathway 16,24,25 triggers its association with Itch. Alternatively, Itch-Cyld association could be regulated by NF-κB-dependent phosphorylation mediated mechanisms. Such a phenomenon has recently been demonstrated for the recruitment of Itch to A20 complex by TAXIBP1 34,43 .
The molecular basis for the chronic multi-organ inflammatory disorder observed in Itch −/− mice has been attributed to biased T H 2 differentiation due to the defect in the degradation of JunB 13 . Our previous studies have shown that Itch also regulates T cell tolerance, as Itch −/− T cells are resistant to anergy induction 44 and TGF-β mediated suppression 11 . Our present results indicate a critical role for Itch in the regulation innate immune cells via targeting Tak1 for proteasomal degradation. These findings along with recent reports that Itch is a component of A20, TAXIBP1 and RNF11 complex 34, 43 raise the possibility that Itch has more prominent role as a negative regulator of inflammatory signaling in the innate immune cells.
Itch seems to be a specific ligase that targets Tak1 for K48-linked ubiquitination as Cbl-b a functionally related E3 ligase 8 which also regulates NF-κB pathway 45 but failed to ubiquitinate Tak1. Similarly, several other DUBs including A20, Cezanne, USP4, and USP21 are shown to negatively regulate NF-κB pathway 39 . In our assay system we did not detect DUB activity of A20 or Cezanne towards Tak1, suggesting the specificity of Cyld. However, we do not completely rule out the involvement of additional DUBs or E3 ligases in the regulation of Tak1 and downstream signaling.
An additional observation from our studies is that Itch −/− and Cyld −/− TAMs produced elevated tumor promoting inflammatory cytokines. Consistently, Itch −/− and Cyld −/− mice exhibited aggressive growth and metastasis of transplanted LLC tumors. However, it is not clear whether the enhanced tumor growth observed is exclusively due to the chronic production of tumor promoting cytokines by TAMs. Aberrant T cell response in Itch −/− and Cyld −/− mice 8,10,11,13 may also contribute to tumor growth. Further detailed analysis is essential for a clear and better understanding of these mechanisms that could lead to innovative therapeutic targeting.
Consistent with our findings, cytokines produced by activated innate immune cells, particularly TAMs, have been associated with inflammation and cancer 6 . Clinical studies have shown that increased numbers of TAMs are correlated with angiogenesis, metastasis and poor prognosis 6 . Tumor-promoting role of TAMs is linked to NF-κB-mediated production of pro-inflammatory cytokines, including TNF and IL-6 7 supporting the notion that excessive, chronically produced pro-inflammatory cytokines contribute to tumor progression. Our findings have identified an unanticipated co-ordination between Itch and Cyld that restricts prolonged inflammatory signaling and secretion of tumor promoting inflammatory cytokines by the macrophages.
Our studies also show that Tak1 selective inhibitor, (5Z)-7-Oxozeaenol inhibits tumor promoting cytokine production by BMDMs. Since NF-κB has emerged as a key internal factor that promotes inflammation and cancer 1, 2 , several approaches have been designed to block NF-κB, including inhibitors of IKK and proteasome inhibitors to inhibit IκB degradation 46 . Although proteasome inhibitors like bortezomib block NF-κB in a variety of cells and are being used in clinical trials against multiple myeloma, the results of the phase I/II clinical trials indicate the drug has limited activity against advanced NSCLC 47,48 . In addition, complications have been reported after global inhibition of NF-κB using genetic and pharmacological approaches 49 . This highlights the requirement for more specific inhibitors to dampen inflammation and tumor progression. Our results provide a mechanistic insight into regulation of Tak1 for termination of inflammatory cytokine production which can be exploited therapeutically to target inflammatory cells in the tumor microenvironment. By targeting inflammatory cells, which are genetically normal and stable compared to genetically unstable carcinoma cells, development of drug resistance could be minimized. In addition, a combination of conventional cytotoxic cancer therapy along with inhibitors that block inflammatory responses could potentially enhance current therapeutic strategies.

Animals
Cyld −/− and Itch −/− mice were described before 12,18 . Control C57BL/6 mice were purchased from Charles River Laboratory. All the mice were housed in microisolator cages in the barrier facility of Karmanos Cancer Institute. Animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Wayne State University/Karmanos Cancer Institute.

Lung metastasis assays
Sub-confluent LLC cells were trypsinized, washed three times with PBS, and filtered through a cell strainer to obtain single-cell suspension. Cells of >90% viability (Trypan Blue staining) were used. Seven weeks old mice were injected with 1×10 6 cells via tail vein. Mice were sacrificed at indicated days and lungs were removed for histological examination while some mice were kept until death and survival data were obtained. Lung tumor multiplicity count, the number of incidences of tumors in lungs per mouse, was scored under the microscope after the lungs were paraformaldehyde fixed, paraffin embedded, sectioned and stained by hematoxylin and eosin staining. For spontaneous lung metastasis, 2×10 6 cells were subcutaneously injected into the left flank of mice and tumor growth was monitored till the primary tumors reached a size of 2000 mm 3 and after which mice were sacrificed to determine tumor multiplicity count.  Itch and Cyld form a complex via interaction through PPXY motif. (a) Sequence alignment of PPXY motif of Cyld which is conserved across species. Y485 critical for interaction with Itch is indicated (*). (b) 293T cells transiently co-transfected with expression vectors for Flag-Itch and Myc-Cyld. The cell lysate was immunoprecipitated using mouse IgG, antibodies against Flag and Myc. The immunoprecipitates were blotted using antibodies against Flag and Myc. (c) Immunoblot of His-Cyld precipitated using GST or GST-Itch purified from E. coli. The input controls for His-Cyld, GST and GST-Itch are shown below (d) BMDMs were stimulated with TNF for thirty minutes. The cell lysates were immunoprecipitated using mouse IgG, rabbit IgG, antibodies against Itch and Cyld. The immunoprecipitates were analyzed by immunoblot with antibodies against Itch and Cyld. (e) 293T cells were co-transfected with expression vectors for HX-Cyld(Y485A) and Flag-Itch. The cells lysate was immunoprecipitated using antibody against Flag and immunoblotted using antibody against HX. Data are representative of three (b, c and e) and two (d) experiments.  Itch-mediated polyubiquitination results in Tak1 degradation. (a) 293T cells were cotransfected with HA-Tak1 (1μg) and increasing concentrations of Flag-Itch or Itch(C830A). The cellular Tak1 level was analyzed by immunoblotting the lysate using antibody against HA. The level of expression of Itch and Itch-C830A was analyzed by reprobing the membranes using antibodies against Flag and HX. (b) 293T cells were transfected with HA-Tak1 (1μg) and increasing concentrations of Flag-Itch, the cells were cultured in the presence or absence of MG132. The cellular Tak1 level was analyzed by immunoblotting the lysate using antibody against HA. Data are representative of two experiments.  Inhibiting Tak1 activation rescues elevated inflammatory cytokine production by Itch −/− and Cyld −/− BMDMs (a) BMDMs were pre-incubated with indicated amounts of (5Z)-7-Oxozeaenol(Ox) and stimulated with TNF for 6 hours, expression of IL-6, TNF, and IL-1β was analyzed by real-time PCR. (b) BMDMs were pretreated with 100 nM of Ox for 30 minutes and then stimulated with TNF for the indicated time points. The cell lysates were immunoprecipitated using antibody against TRAF2 and immunoblotted using antibody against p-Tak1. The total lysate was immunoblotted using antibodies against TRAF2 and actin. (c) WT, Itch −/− and Cyld −/− BMDMs were transduced with a lentiviral vector encoding dominant negative Tak1 (dnTak1). The efficiency of ectopic expression was analyzed by immunoblotting the total cell lysate using antibody against V5. (d) WT, Itch −/− and Cyld −/− BMDMs transduced with dnTak1 were stimulated with TNF and expression of IL-6, TNF and IL-1β was analyzed by real-time PCR. Data are representative of three experiments. (a, d mean and s.d. of triplicate wells). Reconstitution of Cyld −/− BMDMs with WT but not Cyld(Y485A) mutant rescues defects in termination of Tak1 activation and chronic production of inflammatory cytokines. (a) V5-Cyld(WT) or V5-Cyld(Y485A) mutant was ectopically expressed in Cyld −/− BMDMs using a lentiviral transduction system. The cells lysed at indicated time points post TNF stimulation. The lysate was immunoprecipitated using antibody against TRAF2 and immunoblotted with antibody against p-Tak1. The same membrane was reprobed with antibody against Tak1. The cell lysate from WT BMDMs stimulated with TNF was used as control in these experiments. (b) WT and Cyld −/− transduced with V5-Cyld(WT) or V5-Cyld(Y485A) mutant were stimulated with TNF and expression of IL-6, TNF and IL-1β was analyzed by real-time PCR. (c) IL-6 and IL-1β concentration were assayed in the culture supernatant by ELISA. Data are representative of at least three independent experiments (bc mean and s.d. of triplicate wells).