Although Y14 is known to be a component of the exon junction complex, we previously reported that Y14 regulates IL-6–induced STAT3 activation. In this study, we showed that endogenous Y14 positively regulated TNF-α–induced IL-6 expression in HeLa cells. Small interfering RNA–mediated Y14-knockdown reduced TNF-α–induced and NF-κB–mediated transcriptional activity, phosphorylation/degradation of IκBα, and nuclear localization of NF-κB/p65. As in the case of IL-6 stimuli, Y14 enhanced TNF-α–induced STAT3 phosphorylation, which is important for its nuclear retention. However, our manipulation of Y14 expression indicated that it is involved in TNF-α–induced IL-6 expression via both STAT3-dependent and -independent mechanisms. We screened signaling molecules in the TNF-α–NF-κB pathway and found that Y14 endogenously associated with receptor-interacting protein 1 (RIP1) and TNFR-associated death domain (TRADD). Overexpression of RIP1, but not TRADD, restored TNF-α–induced NF-κB activation in Y14-knockdown cells, and Y14 overexpression restored TNF-α–induced NF-κB activation in TRADD-knockdown cells, but not in RIP1-knockdown cells, indicating that Y14 lies downstream of TRADD and upstream of RIP1. Of importance, Y14 significantly enhanced the binding between RIP1 and TRADD, and this is a possible new mechanism for Y14-mediated modification of TNF-α signals. Although Y14 associates with MAGOH in the exon junction complex, Y14’s actions in the TNF-α–NF-κB pathway are unlikely to require MAGOH. Therefore, Y14 positively regulates signals for TNF-α–induced IL-6 production at multiple steps beyond an exon junction complex protein.

Tumor necrosis factor–α is a pleiotropic cytokine that mediates diverse biological responses (13). During inflammatory responses, TNF-α is a key player in the inflammatory cascade and produces proinflammatory cytokines, chemokines, adhesion molecules, destructive enzymes, and angiogenic factors. Although TNF-α plays a critical role in the immune defense against pathogens, deregulation or hyperactivation of its signaling cascade often results in the development of inflammatory and/or autoimmune diseases. Indeed, blockade of binding of TNF-α to its receptor has been highly successful in treating several autoimmune diseases, including rheumatoid arthritis and Crohn’s disease (46). With regard to intracellular signals, TNFR is composed of TNFR1/p55 and TNFR2/p75, and TNFR1 is primarily involved in the assembly of signaling complexes to activate NF-κB (13). Upon TNF-α stimuli, TNFR1 recruits TNFR-associated death domain (TRADD), receptor-interacting protein 1 (RIP1), and TNFR-associated factor (TRAF)2 into its cytoplasmic regions. These molecular interactions mediate the activation of the inhibitor of κB kinase (IKK) signalosome, leading to serine phosphorylation of IκBα and its subsequent proteasome-dependent degradation. The activated NF-κB is ready to translocate into the nuclei to promote transcription of its target genes, including proinflammatory cytokine IL-6 (1, 2, 7, 8). However, the transcriptional activity of NF-κB is also regulated by its phosphorylation and acetylation states, which modulate its nuclear retention and its interaction with transcriptional coactivators and/or corepressors (9, 10). For this NF-κB–modification mechanism, we (11) and other investigators (12) reported that serine-phosphorylated STAT3 and p300 cooperate to acetylate NF-κB.

Y14 contains an RNA-recognition motif and heterodimerizes with Mago to form the exon junction complex (EJC), an assembly on spliced mRNAs (13, 14). In spliceosomes, the Y14-Mago complex is loaded upstream of splice junctions by a helicase elF4AIII, and this assembly is stabilized by Barentsz. In the nuclei, EJC endows the mature messenger ribonucleoprotein particles with architectural information on the pre-mRNA intron structure after the introns have been excised. EJC is retained during its journey to the cytoplasm, where it functions as a platform for binding of factors involved in nonsense-mediated mRNA decay (15, 16). In Drosophila, the Y14-Mago homolog also participates in the transport and translational control of oskar mRNA during oogenesis (1719). Although roles for Y14 have been actively examined with regard to mRNA control, we previously found that Y14 binds to a signaling molecule STAT3 (20). Notably, small interfering RNA (siRNA)-mediated reduction of endogenous Y14 expression in a hepatoma cell line (Hep3B) greatly decreased IL-6–induced tyrosine phosphorylation, nuclear accumulation, and DNA-binding activity of STAT3, as well as IL-6/STAT3-dependent gene expression. In this case, MAGOH, a human homolog of Mago protein, negatively regulates complex formation between Y14 and STAT3 (21).

The possible involvement of Y14 in the regulation of signaling or transcriptional factors is likely to focus on new roles for it, in addition to being an mRNA-shuttling protein. In the current study, we analyzed the effects of Y14 on a proinflammatory cytokine (TNF-α) whose signals are mediated primarily by NF-κB. Of importance, endogenous Y14 enhanced TNF-α–induced IL-6 production and transcriptional activation of NF-κB. We propose a novel role for Y14 in NF-κB activation after TNF-α stimulation via the enhancement of interactions between RIP1 and TRADD.

Recombinant human TNF-α was purchased from Wako Chemicals (Osaka, Japan). Expression vectors for Y14, TNFR, NF-κB/p65, p50, p100, c-Rel, RIP1, TRADD, TRAF2/5, TAK1, NEMO, IKKα/β, IL-6 promoter-luciferase (pIL-6–LUC), and NF-κB–luciferase (NF-κB–LUC) were kindly provided by J. Inoue (Tokyo University, Tokyo, Japan), T. Kobayashi (Keio University, Tokyo, Japan), T. Fujita (Kyoto University, Kyoto, Japan), S. Akira (Osaka University), and H. Sakurai (Toyama University, Toyama, Japan) (2225). Expression vectors for Y14 were described previously (20, 21). Anti-Y14, anti-MAGOH, anti–NF-κB/p65, anti-IκBα, anti-STAT3, anti-p300, anti-TRADD, and anti-Myc Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-FLAG, anti-HA Abs were from Sigma-Aldrich (St. Louis, MO); anti–acetyl–NF-κB/p65 (Lys310), anti-pIκBα (Ser32/36), and anti-pSTAT3 (Tyr705) Abs were from Cell Signaling Technology (Beverly, MA); anti-RIP1 and anti-pSTAT3 (Ser727) Abs were from GE Healthcare Life Sciences (Tokyo, Japan); anti-TRAF2 Ab was from AnaSpec (Fremont, CA); and anti-actin Ab was from Millipore (Billerica, MA).

A human cervix carcinoma cell line (HeLa) and a human embryonic kidney carcinoma cell line (293T) were maintained in DMEM containing 10% FCS. HeLa cells were transfected using jetPEI (Polyplus Transfection, Strasbourg, France), according to the manufacturer’s instructions. 293T cells were transfected using a standard calcium-precipitation protocol (26). The siRNAs targeting human Y14, STAT3, MAGOH, TRADD, RIP1, and TRAF2 used in this study were as follows: Y14 (#1), 5′-CCACCGAAGAAGACAUACATT-3′; Y14 (#2), 5′-GGUAUACUCUAGUUGAAUATT-3′; RIP1, 5′-GGGCGAUAUUUGCAAAUAATT-3′; TRADD, 5′-GGGUCAGCCUGUAGUGAAUTT-3′; TRAF2, 5′-AGAUGUGUCUGCGUAUCUATT-3′; STAT3 (#1), 5′-CCGUCAACAAAUUAAGAAATT-3′; STAT3 (#2), 5′-GCAGCAGCUGAACAACAUGUTT-3′; MAGOH (#1), 5′-GUGUGAUGGAAGAGUUAAATT-3′; and MAGOH (#2), 5′-CCUUAUUGAUGUCAAUCAATT-3′. Control siRNA was obtained from QIAGEN (nonsilencing; cat. no. 1022076). HeLa cells were plated on 24-well plates at 2 × 104 cells/well and incubated with an siRNA–Lipofectamine 2000 (Invitrogen, Carlsbad, CA) mixture at 37°C for 4 h, followed by the addition of fresh medium containing 10% FCS (27). HeLa cells were further transfected or not with NF-κB–LUC or pIL-6–LUC using jetPEI, as described above. At 24 h after transfection, the cells were left untreated or were treated with TNF-α (10 ng/ml) for an additional 12 h and assayed for their luciferase (LUC) activities using a Dual-LUC Reporter Assay System (Promega, Madison, WI), according to the manufacturer’s instructions. Three or more independent experiments were carried out for each assay.

Cells were harvested, and total RNAs were prepared using Iso-Gen (Nippon Gene, Tokyo, Japan) and used in RT-PCR. RT-PCR was performed using an RT-PCR high-Plus-Kit (TOYOBO, Tokyo, Japan). Primers used for RT-PCR were IL-6: 5′- ATGAACTCCTTCTCCACAAGCGC-3′ (sense), 5′- GAAGAAGCCCTCAGGCTGGACTG-3′ (antisense); Y14: 5′-AAGATTTCGCCATGGATGAG-3′ (sense), 5′-ATCAAATCCTGGCCATTGAG-3′ (antisense); STAT3: 5′- CCTTTGGAACGAAGGGTACA-3′ (sense), 5′-CGGACTGGATCTGGGTCTTA-3′ (antisense); IκBα: 5′-CCTGTAATGGCCGGACTG-3′ (sense), 5′-AGGAGTGACACCAGGTCAGGA-3′ (antisense); COX-2: 5′-TGAGCATCTACGGTTTGCTG-3′ (sense), 5′-TGCTTGTCTGGAACAACTGC-3′ (antisense); and G3PDH: 5′-GAAATCCCATCACCATCTTCCAGG-3′ (sense), 5′-CAGTAGAGGCAGGGATGATGTTC-3′ (antisense). Quantitative real-time PCR analyses of IL-6, Y14, and STAT3, as well as the control G3PDH mRNA transcripts, were carried out using the assay-on-demand gene-specific fluorescently labeled TaqMan MGB probe in an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) (11). To quantify IL-6 production, cells were stimulated with TNF-α for 1 h, and culture supernatant was measured for IL-6 by ELISA (BD Biosciences; cat. no. KHC0062), according to the manufacturer’s instructions.

The immunoprecipitation and Western blotting assays were performed as described previously (28). The immunoprecipitates from cell lysates were resolved on SDS-PAGE and transferred to polyvinylidene difluoride transfer membrane (PerkinElmer, Boston, MA). The filters were then immunoblotted with each Ab. Immunoreactive proteins were visualized using an ECL-detection system (Millipore). Immunoblotting results were analyzed using Adobe Photoshop (Adobe Systems, San Jose, CA), and densitometric measurements were normalized against the expression level of the respective proteins (11).

Immunofluorescence staining was performed as described (29). The following primary Abs were used: mouse anti-Y14 and rabbit anti–NF-κB/p65. Two secondary Abs were used: FITC-conjugated anti-rabbit IgG or rhodamine-conjugated anti-mouse IgG (Millipore). The intracellular localization of the proteins was determined under a Biozero BZ-8000 fluorescent microscope (Keyence, Osaka, Japan) equipped with a Plan APO VC 60×/1.40 objective lens (Nikon, Tokyo, Japan). Image processing was performed using the BZ-Analyzer 3.5 (Keyence).

The significance of differences between group means was determined by Student t test.

We previously reported that Y14 physically interacts with STAT3 and positively regulates signals induced by IL-6 (20, 21). This led us to investigate the effects of Y14 on another proinflammatory cytokine, TNF-α, whose signals are mediated primarily by NF-κB. We used a human cervix carcinoma cell line (HeLa), which produces IL-6 in response to TNF-α. A specific siRNA for Y14 or a control siRNA was transfected into HeLa cells, and cell lysates and total RNAs were analyzed with Western blotting and RT-PCR, respectively. Y14 mRNA expression was reduced by ∼80% after Y14 siRNA treatment (Fig. 1A). Under these conditions, Y14 siRNA–transfected HeLa cells showed reduced TNF-α–induced IL-6 mRNA levels compared with control siRNA transfectants (Fig. 1A, 1B). Importantly, the expression of other NF-κB–regulated genes, such as IκBα and COX-2, was also reduced in Y14 siRNA–transfected HeLa cells (Fig. 1A). TNF-α–induced synthesis of IL-6 protein was also reduced in Y14 siRNA–transfected HeLa cells (Fig. 1C). Therefore, endogenous Y14 is likely to enhance TNF-α–induced IL-6 production.

The effects of Y14 on transcriptional activation of the IL-6 promoter were examined with a transient reporter assay. As shown in Fig. 2A and 2B, overexpression of Y14 resulted in a significant enhancement of TNF-α–induced pIL-6–LUC activity in 293T and HeLa cells. In addition, Y14 knockdown resulted in a significant reduction in TNF-α–induced pIL-6–LUC activity in HeLa cells (Fig. 2C). Because the induction of IL-6 after TNF-α stimulation is largely dependent on NF-κB, the effects of Y14 on the transcriptional activation of NF-κB were also examined. Similarly, NF-κB–LUC activity after TNF-α stimulation was significantly upregulated by Y14 overexpression in a concentration-dependent manner (Fig. 2D, 2E). Y14 knockdown then resulted in a significant reduction in TNF-α–induced NF-κB–LUC activity in HeLa cells (Fig. 2F). Therefore, Y14 positively regulates TNF-α–induced transcriptional activation of the IL-6 promoter through NF-κB.

To delineate the molecular mechanisms mediating the upregulation of TNF-α–induced NF-κB activation, the effects of Y14 knockdown on TNF-α–induced IκBα degradation were analyzed. As shown in Fig. 3A and 3B, the reduction in Y14 proteins decreased IκBα degradation 15 min after TNF-α stimulation. Phosphorylation of IκBα, which occurs immediately after TNF-α stimulation and triggers its degradation, was also decreased by Y14 knockdown (Fig. 3C, 3D). With regard to signals after TNF-α stimulation, activation of IKK-α/β and TAK1, but not ERK, p38, or JNK, was reduced in Y14-knockdown HeLa cells when assessed using site- and phospho-specific Abs (Supplemental Fig. 1A, 1B). Ubiquitination of IκBα was also reduced by Y14 knockdown in HeLa cells (Supplemental Fig. 1C). Therefore, Y14 directly affects molecular events upstream of IκBα phosphorylation and degradation, which occur at an early phase after TNF-α stimulation.

The influence of Y14 knockdown on NF-κB/p65 nuclear translocation after TNF-α stimulation was analyzed using confocal microscopy. As shown in Fig. 4, NF-κB/p65 translocated from the cytoplasm into nuclei within 15 min after TNF-α stimulation. In Y14 siRNA–transfected HeLa cells, the nuclear translocation and/or retention of NF-κB/p65 was markedly reduced. Therefore, Y14 positively regulates TNF-α–induced nuclear localization of NF-κB/p65.

Our previous studies showed that phosphorylated STAT3 is involved in the activation of NF-κB/p65 and that Y14 binds to STAT3 and modifies its phosphorylation status (11, 20, 21). Thus, we examined how STAT3 modification is involved in Y14-mediated regulation of TNF-α–induced NF-κB activation. Several reports demonstrated that STAT3 was phosphorylated and activated in various cells by TNF-α stimulation (3032). Indeed, TNF-α induced STAT3 activation in human prostate cancer DU145 cells, as well as HeLa cells (Fig. 5A, Supplemental Fig. 2A). STAT3 phosphorylation after TNF-α stimulation was observed at early (within 15 min) and at late (>120 min) phases. Treatment with anti–IL-6R Ab suppressed STAT3 phosphorylation at late, but not early, phases (Supplemental Fig. 2B). In addition, TNF-α–induced IL-6 mRNA expression was detected after 30 min (Fig. 1A). Thus, STAT3 activation is directly mediated by TNF-α-stimulation at early phases, and TNF-α–induced IL-6 affects STAT3 activation at late phases. As expected from our previous studies, both Ser727 and Tyr705 phosphorylation after TNF-α stimulation was decreased in Y14-knockdown cells (Fig. 5A–C). STAT3 knockdown resulted in a great reduction in TNF-α–induced NF-κB acetylation (Fig. 5D) but not IκBα degradation (Fig. 5E). We then transfected STAT3 siRNA, with or without Y14 siRNA, into HeLa cells and analyzed TNF-α–induced IL-6 expression. As shown in Fig. 5F, STAT3 knockdown itself decreased TNF-α–induced IL-6 mRNA expression. Of importance, TNF-α–induced IL-6 mRNA expression in STAT3 siRNA–transfected HeLa cells was further reduced by the additional knockdown of Y14, indicating that these inhibitory effects were additive. Therefore, Y14 is involved in TNF-α–induced IL-6 expression via both STAT3-dependent and -independent mechanisms.

Because NF-κB seemed to be a suitable candidate Y14 target following TNF-α stimulation, physical interactions of Y14 with some signaling molecules in the TNFR1–NF-κB pathway were screened. When 293T cells were transfected with TNFR1, NF-κB/p65, p50, NEMO, IKK-α/β, TRAF2/5, RIP1, TAK1, or TRADD together with Y14, Western blot analyses of immunoprecipitates with Y14 revealed that Y14 interacted with RIP1 and TRADD but not with the other proteins (Fig. 6A–H). These results suggested that, among the components of the TNFR1–NF-κB pathway, Y14 specifically interacts with RIP1 and TRADD. With regard to endogenous interactions of Y14 with RIP1 and TRADD, the immunoprecipitates for endogenous Y14 contained significant levels of the two proteins in HeLa cells (Fig. 6I). The role of Y14 in the complex formation between RIP1 and TRADD were then examined. When 293T cells were transfected with TRADD and/or RIP1, with or without Y14, Western blot analyses of the immunoprecipitates for TRADD showed that TRADD interacts with RIP1. Importantly, their interactions increased in the presence of Y14, suggesting that Y14 may induce TRADD–RIP1 complex formation (Fig. 6J, 6K). Significance of the complex formations among Y14, RIP1, and TRADD on NF-κB activation were also evaluated. As shown in Fig. 6L, combined transfection of RIP1, TRADD, and Y14 resulted in remarkably enhanced NF-κB activation compared with the transfection of each one separately. Therefore, enhancement of complex formation between RIP1 and TRADD by Y14 is a possible mechanism to enhance NF-κB signaling after TNF-α stimulation.

Involvement of Y14 in RIP1- or TRADD-mediated transcriptional activation of NF-κB was evaluated with a transient reporter assay. As shown in Fig. 7A and 7B, overexpression of Y14 resulted in a significant enhancement of RIP1-induced NF-κB–LUC activity in 293T and HeLa cells. Overexpression of Y14 induced a significant enhancement of TRADD-induced NF-κB–LUC activity (Fig. 7C, 7D). In addition, siRNA-mediated Y14 reduction resulted in a significant reduction in RIP1- or TRADD-induced NF-κB–LUC activity in HeLa cells (Fig. 7E, 7F).

We next attempted to determine where Y14 interacts with RIP1 and TRADD downstream of TNFR1. As shown in Fig. 8A–C, the Y14 siRNA–mediated decrease in NF-κB activation was restored by overexpression of RIP1 and TRAF2 but not TRADD. Furthermore, the TRADD siRNA–mediated decrease in NF-κB activation was restored by the overexpression of Y14, whereas the RIP1 siRNA– or TRAF2 siRNA–mediated decrease in NF-κB activation was not (Fig. 8D–F). Taken together, Y14 acts downstream of TRADD and upstream of RIP1 and TRAF2 in the TNFR1–NF-κB pathway.

Y14 associates with MAGOH to act in the EJC (1319). We examined the involvement of MAGOH in the TNF-α–NF-κB pathway. MAGOH knockdown resulted in impaired IL-6 production after TNF-α stimulation (Fig. 9A). MAGOH siRNA–transfected HeLa cells showed phosphorylation of Ser727 and Tyr705 of STAT3 after TNF-α stimulation that was as strong as that caused by control siRNA transfectants (Fig. 9B). Similarly, IκBα degradation after TNF-α stimulation was not influenced by the reduction in MAGOH. Furthermore, MAGOH knockdown showed no effect on TNF-α–induced NF-κB–LUC activity in HeLa cells (Fig. 9C). Therefore, MAGOH is not involved in TNF-α–induced STAT3 phosphorylation or IκBα degradation.

Development of innate and adaptive immunity in response to proinflammatory cytokines, such as TNF-α and various pathogen-associated molecular patterns, requires the activation of NF-κB, which involves nuclear translocation, followed by the expression of a variety of inflammatory genes (10, 33, 34). Structurally, NF-κB consists of five Rel-related proteins, and the prototypical NF-κB complex is a p65/p50 heterodimer, which efficiently mediates gene expression (35). In this study, we identified Y14 as a key regulatory component of TNF-α–NF-κB signal transduction, leading to the enhancement of TNF-α–induced IL-6 production. Our results also indicated that Y14 positively regulates NF-κB activation by influencing complex formation between RIP1 and TRADD.

Binding of TNF-α to TNFR1 ordinarily activates related signaling molecules (13, 7, 8), among them, Y14 which selectively associates with RIP1 and TRADD. Our LUC experiments showed that Y14 lies downstream of TRADD and upstream of RIP1, and immunoprecipitation experiments revealed that association of TRADD with RIP1 is greatly enhanced in the presence of Y14. This is likely to be a mechanism to enhance IκBα degradation; however, Y14 could act in the absence of TRADD. Thus, Y14 may have more direct roles in the TNF-α–NF-κB pathway. Furthermore, Y14 augments TNF-α–induced STAT3 phosphorylation, which leads to NF-κB acetylation, as in the case of IL-6 stimulation. Recently, cross-talk between STAT3 and NF-κB was demonstrated (1012), although both stimuli are highly overlapping repertoires of prosurvival, proliferative, and proangiogenic genes. In normal immune cells, STAT3 regulates IKK activity, which induces serine phosphorylation and subsequent proteasome-mediated degradation of IκBα. There are some reports of a relationship between TRADD–RIP1 and STAT3 signals. In non-small cell lung cancer cells, RIP1-Src-STAT3–dependent signals are involved in TRAIL-induced migration and invasion (36). During TNF-induced necroptosis, STAT3 undergoes Ser727 phosphorylation, which is dependent on RIP1 expression and activation (37). Our previous reports (20, 21) and the present study suggest that Y14 is involved in TNF-α–mediated signals through at least two possible mechanisms: enhancement of the association between TRADD and RIP1 and augmentation of NF-κB modification via STAT3. Manipulation of Y14 expression in the present and previous studies clearly demonstrated involvement of Y14 in the TNF-α–NF-κB pathway and suggested meanings of Y14 in the wide fields because TRADD and RIP1, as well as STAT3, are common signaling molecules.

In EJC, Y14 is involved in mRNA quality control via the nonsense-mediated mRNA decay pathway together with MAGOH (1319). However, Y14 does not seem to require MAGOH to interact with TRADD, RIP1, and STAT3. Knockdown of MAGOH did not influence STAT3 phosphorylation, IκBα degradation, or NF-κB–LUC activity at the early phase after TNF-α stimulation. It is possible that the independence of MAGOH suggests that Y14 acts outside EJC. Further experiments are needed to analyze whether Y14 can directly associate with and influence other intracellular proteins. MAGOH knockdown suppressed TNF-α–induced IL-6 protein production, but MAGOH did not influence NF-κB activity. This seems to suggest that MAGOH acts at a late stage after IL-6 mRNA synthesis. Although direct proof is lacking, the inhibitory effects of MAGOH on IL-6 production may be related to mRNA control in EJC.

The TNF-α–NF-κB pathway creates central signals to mediate inflammatory responses. The success of anti–TNF-α Abs in the treatment of patients with rheumatoid arthritis, Crohn’s disease, and other related chronic inflammatory conditions has validated TNF-α as a key mediator of inflammation (46). Given the importance of TNF-α blockage, understanding how it manifests its activities and what signaling pathways are involved could provide insight into the molecular pathways driving inflammation and suggest more specific therapies for inflammation, infectious diseases, and cancers. In addition, detailed information about the molecular mechanisms of Y14-mediated modification of NF-κB and STAT3 activities could be useful in the development of a novel therapeutic strategy for NF-κB–STAT3–associated malignancies and autoimmune diseases.

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Abbreviations used in this article:

EJC

exon junction complex

IKK

inhibitor of κB kinase

LUC

luciferase

NF-κB–LUC

NF-κB–luciferase

pIL-6–LUC

IL-6 promoter–LUC

RIP1

receptor-interacting protein 1

siRNA

small interfering RNA

TRADD

TNFR-associated death domain

TRAF

TNFR-associated factor.

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The authors have no financial conflicts of interest.