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Published in final edited form as: Biochem Biophys Res Commun. 2024 Sep 4;734:150660. doi: 10.1016/j.bbrc.2024.150660

Effects of 17,18-EEQ analog (TZ-1) on brown adipogenesis and browning of human adipose-derived stromal cells

Haoying Wu 1, Adeniyi M Adebesin 2, John R Falck 2, Xinyun Xu 1, Jiangang Chen 3, Thomas Masi 4, Stacy Stephenson 4, Ling Zhao 1,*
PMCID: PMC11490365  NIHMSID: NIHMS2022045  PMID: 39260207

Introduction:

Obesity remains one of the greatest public health challenges in the US and around the world [1]. Obesity increases the risk of developing many chronic diseases, such as type 2 diabetes, cardiovascular disease, dyslipidemia, and some forms of cancer, leading to increases in medical costs and societal burden [2]. Despite recent advances in anti-obesity medications (AOM), such as glucagon-like peptide-1 receptor agonists (GLP-1RA) (e.g., semaglutide [3, 4]), challenges remain in obesity treatment and prevention. Most AOMs have side effects and varying efficacy, and some of the new AOMs are very costly with limited access. While some AOMs target food consumption or appetite, most of the AOMs do not target energy expenditure.

Functional brown adipose tissue (BAT), including classical brown and beige adipose tissue, has emerged as a novel target for obesity treatment and prevention [5–7]. In contrast to the energy-storing of white adipose tissue (WAT), classical BAT is responsible for non-shivering thermogenesis through uncoupling ATP synthesis from respiration in the mitochondria via uncoupling protein 1 (UCP1), leading to energy dissipating as heat [5]. On the other hand, beige fat, brown-like adipocytes formed within WAT have been reported and developed by a “browning” process in response to cold or β-adrenergic stimulations [6, 7], which are not practical for humans due to poor compliance and side effects.

Anti-obesity effects of n-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA), are well recognized [8–10]. Recent studies have shown that EPA, but not docosahexaenoic acid (another n-3 PUFA) or arachidonic acid (an n-6 PUFA), promotes brown/beige adipocyte development and increases thermogenesis in animal models [11–14] and in human WAT-derived stromal cells [15]. However, similar effects of EPA have not been reported in human trials [9], possibly due to higher doses used in animal studies, which are not achievable in humans. It is well known that EPA can be metabolized by cycloxygenase, lipoxygenase, and cytochrome p450 (CYP) pathways. CYP epoxygenases (mainly CYP2C and CYP2J family) convert EPA to epoxyeicosatetraenic acids (EEQs), such as 17,18-EEQ (the most abundant epoxy fatty acids derived from EPA) [16]. EEQs are metabolically labile and can be quickly metabolized by autooxidation, enzymatic oxidation, esterification, or other eicosanoid metabolic pathways, including soluble epoxide hydrolase (sEH, encoded by the Ephx2 gene) [16]. Recently, we have demonstrated potent thermogenic effects of 17,18-EEQ when stabilized with a sEH inhibitor (sEHi) in preventing diet-induced obesity and associated metabolic dysfunction [17]. Through minipump delivery, 17,18-EEQ at 0.05 mg/kg/day combined with a sEHi (t-TUCB) produced thermogenic effects that are comparable to those produced by the fish oil enriched with EPA at 9 g/kg of diet [18]. The EPA at 9 g/kg of diet is roughly equivalent to 0.75 g/kg /day, assuming a 2.5 g daily food intake for a 30 g mouse. To harness the beneficial effects of EPA and 17,18-EEQ, more stable 17,18-EEQ analogs have been developed [19]. In the present study, we investigate the brown adipogenic and browning effects of a metabolically stable 17,18-EEQ analog, TZ-1, in murine brown preadipocytes and human white adipose-derived stromal cells in vitro.

Materials and Methods

Reagents

Insulin, triiodothyronine (T3), 3-isobutyl-L-methylxanthine (IBMX), dexamethasone (Dex), and rosiglitazone (Rosi) were purchased from Millipore Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was from Bio-techne (Minneapolis, MN, USA). Anti-peroxisome proliferator-activated receptor gamma (PPARγ) (Catalog#2443) and anti-ERK1/2 (Catalog# 4695) antibodies and horseradish peroxidase-conjugated goat anti-rabbit were from Cell Signaling Technology (Danvers, MA, USA). Anti-uncoupling protein 1 (UCP1) (Catalog# U6382) was from Sigma Aldrich (St. Louis, MO, USA). The anti-PGC1α antibody (Catalog# AB3242) was from Millipore (Temecula, CA, USA). Other reagents, if not specified, were purchased from Millipore Sigma.

The 17,18-EEQ analog (N1-(15-(1H-tetrazol-5-yl)pentadec-5(Z)-enyl)-N2-methyloxalamide) (TZ-1, Fig. 1A) was synthesized as previously described [19]. The compound was prepared in dimethyl sulfoxide (DMSO) as a 50 mM stock solution and then further diluted to lower stock concentrations for the studies. The final concentrations of DMSO present for each treatment were maintained at 0.1%.

Figure 1: TZ-1 promotes murine brown adipocyte differentiation.

Figure 1:

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Murine brown preadipocytes were induced to differentiate in the presence of increasing concentrations of TZ-1 (1, 5, 10, 20, and 50 μM) or the vehicle control (DMSO) for 6 days. A) Chemical structures of 17,18-EEQ and the analog TZ-1 (top) and schematic diagram of the treatment (bottom). B) Micrographs of oil-red-O-stained brown adipocytes treated with TZ-1 at 10, 20, and 50 μM, compared to the untreated (-) and the vehicle control DMSO group. C) Protein expression of brown marker genes PGC1α and UCP1. D) Quantification of the expression by densitometry presented as folds of the (-) group. Data = Mean ± SEM (n = 3). a, aa, p<0.05 and p<0.01 compared to the DMSO group, respectively; bb, p<0.01 compared to the 10 μM group. Scale bar=100 μm.

Cell culture and treatment

The murine brown pre-adipocyte cell line was cultured in DMEM/high glucose and 20% FBS and kept at 37°C with 5% CO2 and humidified air, as described [20]. Upon reaching confluence, the cells were differentiated in the DMEM/high glucose, 20% FBS, 20 nM insulin, and 1 nM T3 in the presence of TZ-1 at concentrations ranging from 1 to 50 μM. The vehicle control of 0.1% DMSO was also included. This media and the treatment were changed every two days. On the sixth day, the cells were lysed for subsequent analyses.

Murine brown pre-adipocytes with peroxisome proliferator-activated receptor gamma knockdown (PPARγ-KD) and the scrambled non-targeting control cells (SCR) were generated as described [20]. Upon reaching confluence, both PPARγ-KD and SCR cells were differentiated in the DMEM/high glucose, 20% FBS, 20 nM insulin, and 1 nM T3 in the presence of TZ-1 at increasing concentrations or the vehicle control. Fresh treatments were introduced at every media change that occurred every two days. On the sixth day, the cells were lysed for subsequent analyses.

Human adipose tissue-derived stromal cells (hADSCs) from a male patient (41 years old, with a BMI of 26.9, and without diabetes) were isolated at the University of Tennessee Medical Center. Patient consent was obtained prior to surgery as approved by an IRB protocol (#3995). The cells were cultured in DMEM/high glucose and 10% FBS at 37°C with 5% CO2 and humidified air. Upon reaching confluence, hADSCs were differentiated in DMEM/high glucose with 10% FBS, 2 nM T3, 0.5 μM human insulin, 33 μM biotin, 17 μM pantothenate, 0.1 μM dexamethasone, 0.5 mM isobutyl-1-methylxanthine, and 30 μM indomethacin in the presence of increasing concentrations of TZ-1 (10 and 20 μM) or the vehicle control (0.1% DMSO) for fourteen days. Fresh treatments were introduced with every media change that occurred every two days. On the fourteenth day, the cells were lysed for subsequent analyses.

ORO Staining

Differentiated murine brown adipocytes underwent oil red O (ORO) staining as described [21] with modification. Briefly, the cells were first fixed with 10% neutral buffered formalin for a minimum of two hours. Then, they were rinsed with water and stained with a 0.21% ORO solution (dissolved in isopropanol) for 10 min. The cells were again rinsed with water to remove excess dye and visualized using a Micromaster inverted phase contrast microscope equipped with an integrated digital camera.

Western blot analysis

The cells were rinsed with cold phosphate-buffered saline and were lysed with RIPA buffer (Cell Signaling, Danvers, MA). Protein quantitation was carried out using the bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific, Waltham, MA). The total cell lysates were subjected to 10% SDS-PAGE. The separated proteins were then electro-transferred onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA). The membranes were blocked in 137 mM NaCl, 20 mM Tris HCl, and 0.1% Tween 20 (pH 7.4) solution with 5% non-fat milk, followed by immunoblotting with primary antibodies at 4°C overnight and secondary antibody conjugated with horseradish peroxidase for 1 hr. The signals were developed using the SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific) and detected by the ChemiDoc XRS+ Gel Imaging System (Bio-Rad).

Total RNA isolation and semi-quantitative reverse-transcription PCR analysis

Total RNA was isolated with TRI reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. Total RNA abundance was measured by a NanoDrop One spectrophotometer (ThermoFisher Scientific). Reverse transcription was performed using a High Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific) according to the manufacturer’s instructions. Analysis of mRNA expression of the target genes and the housekeeping gene 36b4 [encodes acidic ribosomal phosphoprotein P0 (RPLP0)] was carried out using the Absolute Blue QPCR SYBR Green ROX mix. PCR reactions were carried out in a QuantStudio 3 Real-Time PCR thermocycler (Thermo Fisher Scientific). The conditions were set at 50°C 2 min and 95°C 15 min, followed by 40 cycles of 95°C 15 s/60°C 1 min. Relative gene expression was calculated using the 2–ΔΔCt method, which normalizes against 36b4. The primer sequences are reported in the supplemental materials.

Reporter gene assays

Murine PPARγ activation reporter assays were performed as described [20]. Briefly, murine brown pre-adipocytes were seeded on a 24-well plate overnight and then transiently transfected with murine PPARγ activation reporters, including mPPARγ-Gal4, which is a murine PPARγ ligand binding domain linked to the Gal4 DNA binding domain, and 4xUAS-TK-Luc, a reporter with an upstream activating sequence linked to luciferase (TK: thymidine kinase) and β-galactosidase (β-gal) plasmid to serve as a transfection control. The transfection was performed using TransIT 2020 (ThermoFisher Scientific). After 24 hr post-transfection, the cells were treated with TZ-1 or 0.1% DMSO for 18 hr before lysis. Luciferase and β-gal activities of the cell lysates were detected using the GloMax Multi Detection System (Promega, Madison, WI).

Cellular bioenergetics measurements

On the fourth day of the differentiation, murine brown cells were seeded into Seahorse XF cell culture plates (Agilent, Santa Clara, CA) and prepared for the mitochondrial stress test, as described [20]. Briefly, the cells were washed three times with XF assay buffer containing DMEM lacking NaHCO3, 10 mM glucose, 2 mM pyruvate, 2 mM GlutaMAX, and 2% bovine serum albumin, adjusted to pH 7.4. The cells were then equilibrated at 37°C in a non-CO2 environment for 1 hr. Oxygen consumption rates (OCR), an indicator of mitochondrial respiration, were measured in an XFe24 Extracellular Flux Analyzer (Agilent, Santa Clara, CA, USA). For the mitochondrial stress test, the cells were treated with oligomycin (1 μM), carbonyl cyanide-ptrifluoromethoxyphenylhydrazone (FCCP; 6.5 μM), rotenone/antimycin A (1 μM each) in sequential order, three measurements of OCR were taken after each treatment. OCR linked to basal and maximal respiration, proton leak, and ATP-linked respiration and coupling efficiency were calculated according to the manufacturer’s instructions.

Statistical analysis

Triplicates were performed in each experiment. Statistical analysis was conducted using Prism 9.3.0 (GraphPad Software, San Diego, CA, USA). Significant differences in group mean among groups were detected using one-way or two-way ANOVA (only for PPAR knockdown studies) with repeated measures followed by Tukey’s multiple comparisons test. The level of significance was set at p<0.05.

Results

TZ-1 promotes murine brown differentiation

To investigate the effects of TZ-1 on brown adipocyte differentiation, murine brown preadipocytes were differentiated in the presence of increasing concentrations of TZ-1 (1, 5, 10, 20, 50 μM) or the vehicle control (DMSO), as shown in the schematic diagram (Fig. 1A). TZ-1 dose-dependently increased murine brown adipocyte differentiation, as evidenced by ORO-stained lipid accumulation in the brown adipocytes treated at 10, 20, and 50 μM (Fig 1B), and increased protein expression of brown marker genes PGC1α and UCP1 (Fig. 1C), compared to the untreated (−) and the vehicle control DMSO groups. While there was an increasing trend, significant increases in protein expression of PGC1α (p<0.05) and UCP1 (p<0.05) were only detected when TZ-1 was used at the higher concentration of 50 μM (Fig. 1D). There were no significant changes of the PPARγ protein expression by TZ-1 (Data not shown).

TZ-1 increases mitochondrial uncoupling in murine brown adipocytes

Since we did not detect significant changes in lipid accumulation and protein expression by TZ-1 at 1 and 5 μM, we focused on TZ-1 at 10, 20, and 50 μM in the subsequent studies. To investigate whether increased brown adipocyte differentiation by TZ-1 was accompanied by increased thermogenic function, we performed cellular energetic measurements, i.e., oxygen consumption rates (OCR) coupled with mitochondrial stress tests in the treated cells using an XFe 24 extracellular flux analyzer (Fig. 2A). TZ-1 dose-dependently increased basal respiration, maximal respiration, and OCR linked proton leak (i.e., heat production) (Fig. 2B). Statistical significance was observed when TZ-1 was tested at and above 20 μM. No significant changes were observed in OCR linked to ATP synthesis (Fig. 2B). TZ-1 at 50 μM seemed to decrease mitochondrial respiration coupling efficiency (i.e., the ratio of OCR link to ATP synthesis over basal respiration x 100%) (Fig. 2B); however, it did not reach statistical significance.

Figure 2: TZ-1 increases mitochondrial uncoupling in murine brown adipocytes.

Figure 2:

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Murine brown preadipocytes were differentiated and reseeded onto the XF assay plate. The next day, the cells were subjected to real-time measurements of OCR. A) OCR over time during mitochondrial stress tests. B) Basal respiration, maximal respiration, OCR from proton leak and ATP production. Data = Mean ±SEM (n = 3). aa, aaa, p<0.01 and p<0.001 compared to the DMSO group, respectively; b, bbb, p<0.05 and p<0.001 compared to the 10 μM group, respectively; ccc, p<0.001 compared to the 20 μM group.

TZ-1 activates PPARγ, and PPARγ knockdown attenuates the effects of TZ-1

PPARγ activation is critical for brown adipocyte differentiation and browning. To gain insight into the molecular mechanisms by which TZ-1 promotes murine brown differentiation, we investigated TZ-1’s abilities to activate PPARγ using the PPARγ activation reporter assays. Rosiglitazone (Rosi) was included as a positive control. TZ-1 dose-dependently activated PPARγ, reaching statistical significance at and above 20 μM (Fig. 3A). To further evaluate the role of PPARγ, we tested TZ-1 in PPARγ-KD cells compared to the scrambled controls (SCR). We show that the knockdown efficiency at the protein level was ~ 75% (Fig. 3B). The effects of TZ-1 on brown adipocyte differentiation were significantly attenuated, as shown by the attenuated thermogenic PGC1α and UCP1 protein expression (Fig. 3C, D) (p<0.05). As expected, Rosi downregulated PPARγ protein expression, as previously reported [22]. TZ-1 did not induce significant changes in PPARγ protein expression in both cell types (Fig. 3C, D). The results suggest that TZ-1 promotes brown adipocyte differentiation at least in part mediated through PPARγ.

Figure 3: Effects of PPARγ knockdown on TZ-1 treated murine brown adipocytes.

Figure 3:

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A) Effects of TZ-1 on PPARγ activation. Murine PPARγ activation reporters were transiently transfected in murine brown preadipocytes. Relative luciferase activities are calculated as the detected luciferase activities normalized by β-gal activities and shown as folds of the (-) group, which was set to 1. B, C, D) PPARγ knockdown attenuated the effects of TZ-1. Murine brown preadipocytes with PPARγ knockdown (PPARγ-KD) and the scrambled control (SCR) were differentiated in the presence of TZ-1 (50 μM) or the vehicle control. PPARγ knockdown efficiency is shown in B). The effects of PPARγ knockdown on protein expression of PPARγ, PGC1α, and UCP1 are shown in C). Quantification of the expression by densitometry presented as the folds of the (-) group are shown in D). ERK1/2 is the loading control. Rosi, rosiglitazone. Data = Mean ± SEM (n = 3). a, aa, p<0.05 and p<0.01 compared to the DMSO group, respectively. *, p< 0.05.

TZ-1 promotes the browning of human adipose-derived stromal cells (hADSCs)

To gain translational insights on the efficacy of TZ-1 on browning, hADSCs were induced to undergo beige adipocyte differentiation in the presence of increasing concentrations of TZ-1 (10 and 20 μM) or the vehicle control (Fig. 4A). Differentiated beige adipocyte morphology were shown (Fig. 4B). TZ-1 dose-dependently increased mRNA expression of brown markers PGC1α, PPARγ, PRMD16, and TFAM, reaching statistical significance when tested at 20 μM for PPARγ and TFAM (Fig. 4C). Interestingly, TZ-1 dose-dependently increased mRNA of UCP2 and UCP3 with statistical significance detected at 20 μM, but not UCP1, in the treated cells (Fig. 4C). Moreover, TZ-1 seemed to increase UCP1, but not PGC1α, protein expression in the treated hADSCs (Supplemental Fig. 1).

Figure 4: TZ-1 induces browning of hADSCs.

Figure 4:

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hADSCs were induced to undergo beige adipocyte differentiation in the presence of TZ-1. A) Schematic diagram of the treatment. B) Micrographs of differentiated hADSCs. C) TZ-1 induced upregulation of mRNA expression of brown marker genes in hADSCs. Relative gene expression is presented relatively to the loading control 36b4. Data = Mean ± SEM (n = 3). a, aa p<0.05 and p<0.01 compared to the DMSO group, respectively. b, p<0.05 compared to the 10 μM group. Scale bar=100 μm.

Discussion

In the present study, the effects of TZ-1, a 17,18-EEQ stable analog, on brown adipocyte differentiation and browning were investigated. The results reveal that TZ-1 dose-dependently promotes brown adipocyte differentiation accompanied by increased thermogenic function in murine brown adipocytes and browning of hADSCs, suggesting the therapeutic utility of TZ-1 to increase thermogenesis and energy expenditure for obesity treatment and prevention. To our knowledge, this is the first report demonstrating the thermogenic potential of a 17,18-EEQ analog.

Several studies have reported the effects of EPA on brown adipogenesis and browning. EPA at 100 μM promoted brown adipogenesis of the precursor cells from interscapular BAT [12] and increased mRNA expression of Pgc1α and Ucp2, mitochondrial content, maximal respiration, and spare respiration in differentiated murine HIB1B brown adipocytes [13]. Similarly, EPA, but not DHA, at 200 μM increased mRNA expression of Ucp1-3 and other brown markers, accompanied by increased mitochondrial biogenesis, in the differentiation of inguinal adipocytes derived from C57BL/6 mice [11]. Moreover, EPA promoted stronger brown-like differentiation of the subcutaneous white fat-derived stromal cells from lean women compared to DHA [15]. However, the effects of epoxy fatty acids from EPA on brown adipogenesis or browning have not been reported. Our unpublished results indicate that 17,18-EEQ combined with t-TUCB, but not alone, promoted brown adipogenesis and thermogenic function. The brown adipogenic and browning effects reported for TZ-1 align with these previous findings.

Compared to 17,18-EEQ, TZ-1 has the following modifications (i) only one double bond at Δ11,12, rendering it stable to auto-oxidation and COX/LOX metabolism; (ii) an oxamide epoxide bioisostere, rendering it sEH stable; and (iii) a tetrazole carboxylate bioisostere, abrogating esterification. Our results suggest structural modifications can make 17,18-EEQ more metabolically stable and enhance its potency. Compared to sEHi, TZ-1 is not associated with the accumulation of endogenous epoxy-PUFAs (e.g., leukotoxins, a.k.a. EpOMEs and epoxyeicosatrienoic acids, which may lead to unpredictable consequences [19]. Together, the results suggest that TZ-1 has the potential for further development as an effective and potent thermogenic agent to combat obesity; moreover, stable 17,18-EEQ analogs are valuable pharmacological tools to further elucidate the anti-obesity mechanisms of EPA and 17,18-EEQ.

A few studies have explored the molecular mechanisms underlying EPA’s regulation of brown adipogenesis and browning. EPA is thought to activate free fatty acid receptor 4, leading to cAMP activation and up-regulation of miR-30b and miR-378 [11]. Others have attributed EPA’s effects to its upregulation of PGC1α and SIRT2 [13], which play important roles in mitochondrial biogenesis and energy metabolism. We found that TZ-1 upregulated PGC1α protein expression in murine brown adipocytes, which could lead to the observed upregulated UCP expression and enhanced mitochondrial respiration. As PPARγ activation is critical for both white and brown adipocyte differentiation [23, 24] and browning [25, 26], we explored its role in our TZ-1 studies. The findings using PPARγ activation reporter and PPARγ-KD cells suggest that brown adipogenic effects of TZ-1 are at least in part through PPARγ activation.

One interesting finding is that the TZ-1 upregulated the mRNA of UCP2 and UCP3, not UCP1, in the treated hADSCs (Fig. 4). The fact that TZ-1 may increase UCP1 protein expression (Supplemental Fig. 1), but not UCP1 mRNA, suggests that it may regulate the UCP1 gene at post-transcriptional levels in hADSCs, similar to that of EPA reported [13]. All UCP1-3 mRNA were detectable in the differentiated hADSCs, and their mRNA abundance was UCP2>UCP1>UCP3 (data not shown). The roles of UCP2 and UCP3 in adipocytes are not clearly understood. In contrast to UCP1’s mediating adaptive thermogenesis through transporting protons in the absence of activators, UCP2 and UCP3 may mediate inducible proton conductance in the presence of activators, such as fatty acids and free radical-derived alkenes [27]. Moreover, UCP2 and UCP3 may be involved in reducing mitochondrial free radicals and protecting from oxidative damage [27]. Due to limited quantities of the analog, we did not measure the effects of the analog on the thermogenic activities or oxidative damage in these hADSCs, nor did we test the responses of hADSCs from subjects with different sex, BMI, and other disease statuses. Future studies of the impact of TZ-1 on the thermogenic functions of the hADSCs with different characteristics are warranted.

In conclusion, TZ-1, a 17,18-EEQ analog with improved metabolic stabilities, has demonstrated efficacies in promoting brown adipogenesis and browning, possibly through activating PPARγ. 17,18-EEQ analogs, such as TZ-1, may be better alternatives to EPA and the use of the sEHi needed to stabilize 17,18-EEQ as a thermogenic agent to promote energy expenditure for obesity treatment and prevention. The thermogenic potential of TZ-1 as a therapeutic warrants further investigation.

Supplementary Material

1

Highlights.

  1. EPA produces 17,18-EEQ through cytochrome P450 pathway.

  2. 17,18-EEQ analog TZ-1 is resistant to auto-oxidation, esterification, sEH and COX/LOX.

  3. TZ-1 promotes brown adipogenesis and browning.

  4. TZ-1’s thermogenic potential as a therapeutic for obesity warrants further investigation.

Acknowledgment

The work is supported by the National Institute of Health grants 1R15DK132728-01 and R01DK126452 and the Robert A. Welch Foundation (I-0011). The funding sources were not involved in the study design, data analysis and interpretation, writing, or decision to submit the paper for publication.

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Ling Zhao and John R. Falck report financial support was provided by the National Institutes of Health. Dr. John R. Falck has a patent, “Preparation of Metabolically Robust Analogs of Cypeicosanoids for the Treatment of Cardiac Disease” licensed to the University of Texas Southwestern (UTSW) and Max Delbruck Center for Molecular Medicine. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

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