(J) Bar graph and its representative recording of colonic contractions showing no inhibitory effect on 5-HI-induced response when 1 μM ML 204 together with 1 μM SAR 7334 were added. We expected B. breve to change the pH of the growth media via its production of lactic acid or acetic acid [36], which might have effect on the TnaA activity that is strongly dependent on the pH conditions [37]. To perform the tests with antagonists for the receptors involved in the gut motility, either 1 μM ondansetron (5-HT3 antagonist; O3639, Sigma), 1 μM SB-207266 (5-HT4 antagonist; SML1349, Sigma), 1 μM TTX (sodium ion transport blocker on the enteric neurons; TTX; 1078/1, Tocris Bioscience), 1 μM hexamethonium bromide (a nicotinic cholinergic antagonist; 4111, Tocris Bioscience), 1 μM AQ-RA 741 (muscarinic receptor 2 antagonist (M2R); 2292, Tocris Bioscience), 100 nM DAU 5884 hydrochloride (M3R antagonist; 2096; Tocris Bioscience), 1 μM ML204 (TRPC4 antagonist; 4732, Tocris Bioscience), 1 μM SAR 7334 (TRPC6 antagonist; 5831; Tocris Bioscience), 100 μM ATP (A1335; Duchefa Bioscience), or 1 μM nifedipine (L-type Ca2+ channels antagonist; 1075; Tocris Bioscience) was applied sequentially with either ACh, 5-HT, or 5-HI. Error bars represent SEM. Deciphering the mechanism of 5-HI stimulatory effect on gut motility observed in WTG rats (Fig 4), we first demonstrated that 5-HI significantly increased 5-HT release from a model of enterochromaffin cells, RIN14B (Fig 5A). https://doi.org/10.1371/journal.pbio.3001070.s005, https://doi.org/10.1371/journal.pbio.3001070.s006, https://doi.org/10.1371/journal.pbio.3001070.s007, https://doi.org/10.1371/journal.pbio.3001070.s008, https://doi.org/10.1371/journal.pbio.3001070.s009, https://doi.org/10.1371/journal.pbio.3001070.s010, https://doi.org/10.1371/journal.pbio.3001070.s011, https://doi.org/10.1371/journal.pbio.3001070.s012, https://doi.org/10.1371/journal.pbio.3001070.s013, https://doi.org/10.1371/journal.pbio.3001070.s014. Only the ACh-induced response, but not the 5-HI-induced response, was inhibited (S5I and S5J Fig), respectively. The illustrative contractions (5 min) segments data were extracted from BioPac Student Lab 4.1 and analyzed in GraphPad Prism 7. Data represent 3–5 biological replicates. On the eighth occasion, the 10% sucrose solution was used as a vehicle for the 1.2% carmine red (C1022, Sigma) and 0.5% methylcellulose (M0512, Sigma) mixture. here. Data recording and analysis were performed using Chromeleon software (version 6.8 SR13). We show that a wide variety of gut bacteria convert 5-HTP to 5-hydroxyindole (5-HI) and that the degree of this conversion is highly dependent on the microbial composition and changes in the pH. (C, D) Michaelis–Menten kinetic curves for (C) 5-HTP and (D) tryptophan as substrates for 200 nM or 20 nM TnaABW25113, respectively. TnaA amino acid sequences from E. coli BW25113, B. thetaiotamicron VPI-5482, and F. nucleatum subsp. (2021) Gut bacteria-derived 5-hydroxyindole is a potent stimulant of intestinal motility via its action on L-type calcium channels. Quantitative analysis of the organ bath data is described in Materials and method section. (D) Graph shows OD600 measurements of F. nucleatum subsp. Overnight incubation of E. coli BW25113ΔtnaA and E. coli BW25113WT bacterial cells with 5-HTP showed that the production of 5-HI was completely abolished in the mutant strain as analyzed by HPLC-ED/UV (Fig 2B), indicating that 5-HTP is degraded to 5-HI by TnaA enzyme. GovSpend - A better way for government buyers and sellers to connect. Gode campingmøbler fremstillet specielt til campinglivet. The study protocol was evaluated by the ethical committee of University of Groningen Medical center. (G) Phylogenetic tree created using iTOL online tool showing 15 closest orthologues (indicated in black) to TnaA enzymes from F. nucleatum, E. coli, and B. thetaiotamicron (indicated in blue). To each of the suspensions, 1% of the F. nucleatum subsp. This yielded a GAMMA+LG model with empirical base frequencies. Oral administration of 5-HTP results in detection of 5-HI in fecal samples of healthy volunteers with interindividual variation. ALL RIGHTS RESERVED, Take A Sneak Peak At The Movies Coming Out This Week (8/12), #BanPaparazzi – Hollywood.com will not post paparazzi photos, Everything you need to know before you rent a movie theater, 8 life lessons from the movie Fargo, 25 years later, Hollywood women are up for the challenge. Error bars represent SEM. Error bars represent SEM. Supervision, (A) Bar graph represents remaining percentage of 5-HI after incubation for 90 min and 24 h in rat colonic tissue extracts (n = 3). https://doi.org/10.1371/journal.pbio.3001070.g002. Cell and protein precipitates were removed by centrifugation at 20,000 × g for 10 min at 4 °C. animalis culture was inoculated. 5-HTP is used as a food supplement or as a drug with or without other medications for the treatment of a wide variety of conditions, including depression, fibromyalgia, binge eating associated with obesity, chronic headaches, and insomnia [22–27]. A plastic collection container, relief container, and gloves were provided to each subject for fecal collection. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, https://doi.org/10.1371/journal.pbio.3001070, https://dataview.ncbi.nlm.nih.gov/object/PRJNA683576?reviewer=vpei68t9t04tnfmlh15f041ggd, https://www.rug.nl/about-ug/work-with-us/rff/, https://cran.r-project.org/web/packages/ggpubr/index.html, http://www.bioinformatics.org/sms/prot_mw.html, Fig 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J and 5K, S5A, S5B, S5C, S5D, S5E, S5F, S5G, S5H, S5I, S5J and S5K Fig. The proteins were considered homologous when the minimal identity percentage was above 30% and query cover was above 90% [31]. An essay is a short piece of writing, and it needs to have the correct level of quality matching your readers’ interests. In contrast, there was a significant increase in the 5-HI-induced response when M3R was inhibited (Fig 5I), and the addition of M2R antagonist did not inhibit the 5-HI-induced response (Fig 5J), suggesting that 5-HI exerts its excitatory effect on the colonic smooth muscles through other receptors. The raw data can be found in S2 Raw images. The study coordinator met with each participant to review the consent and study details. Remarkably, the 5-HI-induced response was completely abolished with the addition of nifedipine (Fig 5K), suggesting that 5-HI elicits its response via L-VDCCs. animalis DSM 19679 and B. breve DSM 20213 were grown anaerobically at 37 °C in an EBB. (G–I) Bar graphs represent an inhibitory effect on ACh-induced response by the addition of either (G) 1 μM AQ-RA 741, (H) 100 nM DAU 5884 hydrochloride, or (I) 1 μM ML 204 and SAR 7334. Participants collected the feces and delivered it immediately to the study coordinator who delivered it to the laboratory for fecal samples extraction of metabolites. Liquid Chromatography–Mass Spectrometry; L-VDCCs, Data were recorded using BioPac Student Lab 4.1 (Build: February 12, 2015). e3001070. The 15 closest (with highest identity, the whole list can be found in Table A in S1 Table) orthologues to each of the initial seed sequences were extracted, and a multiple sequence alignment was performed (S3 Fig). Before lysis, 2 μg/mL DNase and 200 μg/mL lysozyme were added to each solution, samples were transferred to screw-caped tubes containing 0.5 g of 0.1 mm zirconia glass beads and kept on ice for 10 min. Here, we show that a wide variety of gut bacteria can metabolize the dietary supplement and antidepressant 5-hydroxytryptophan (5-HTP) to 5-hydroxyindole (5-HI) via the tryptophanase (TnaA) enzyme. Under these conditions, colonic rings were equilibrated for at least 45 to 60 min with replacement of Krebs–Henseleit solution approximately every 15 min. (C) Cell lysates of B. thetaiotamicron VPI-5482 incubated at 37 °C converted 5-HTP to 5-HI after 48 h. The raw data of B and C can be found in S3 Raw images. Baseline TGTT measurements showed a natural variation among the WTG rats, similar to what exists among humans. tetrodotoxin; WTG, For E. coli BW25113 tnaA mutant experiment and Fusobacterium strains screening experiment, HPLC with electrochemical detection was used (Jasco AS2059 plus autosampler, Jasco Benelux, Utrecht, the Netherlands; Knauer K-1001 pump, Separations, H. I. Ambacht, the Netherlands; Dionex ED40 electrochemical detector, Dionex, Sunnyvale, USA, with a glassy carbon working electrode (DC amperometry at 1.0 V, with Ag/AgCl as reference electrode), for every other experiment HPLC with UV detection (280 nm) was used (Waters 2695 Alliance Separations module, Milford, Massachusetts, USA; UV6000LP Detector, Thermo Fisher Scientific, Waltham, Massachusetts, USA). These data clearly show that the reduction in the pH results in a complete inhibition of the production of 5-HI via its effect on the bacterial growth. Constipation is a common, debilitating motility disorder affecting up to 27% of the population [14]. RDA constrained to 5-HI levels as a single explanatory variable was performed in the same manner, while also testing for significance via an anova-like permutation for the significance of the constraint. Chimeric pseudoreads were removed using USEARCH 9.240, and the remaining reads were aligned to the RDP 16S rRNA gene database (RDP Release version 11). Exploded slices represent the difference in tnaA-encoding bacterial phyla among High, Intermediate, and Non-Converters. To test whether the augmented amplitude enhancement of the 5-HT-induced response upon addition of 5-HI was due to the action of 5-HI on the 5-HT3 or 5-HT4 receptors, the main 5-HT receptors involved in the gut motility [41], the 5-HT3 antagonist ondansetron (1 μM) or the 5-HT4 antagonist SB-207266 (1 μM) were added to the colonic tissue to abolish the 5-HT-induced response [42,43]. (B) Increased defecation frequency per 24 h per rat in rats treated with 30 mg/kg of 5-HI compared to the vehicle group (10% sucrose). A must-read for English-speaking expatriates and internationals across Europe, Expatica provides a tailored local news service and essential information on living, working, and moving to your country of choice. Validation, 5-HI, 5-hydroxyindole; 5-HTP, 5-hydroxytryptophan; PCA, principal component analysis; RDA, redundancy analysis; TnaA, tryptophanase. (A) Formation of 5-HI was identified and confirmed by LC-MS. Chromatograms show mass [M+H]+ m/z = 134,0602, which corresponds to exact molecular weight of 5-HI in positive mode. Apply to the latest job opportunities for freshers and experienced professionals in the fields of IT, HR, Admin, Pharma, Teaching, top MNC jobs and more across India. Similarly, RDA showed significant influence of 5-HI levels, being the explanatory variable (p-value = 0.017), which was positively related to the Bifidobacterium in the Intermediate and Non-Converters group. Overnight culture was diluted 1:100 and grown to OD600 = approximately 0.8. The observed 5-HI effect occurred via a competitive or noncompetitive interaction at low (10 mM) or high (10 to 50 mM) concentrations, respectively. The (n) refers to the number of individuals, rats, or rats’ tissues used for each experiment. Based on the alignment scores of the pseudoreads, the taxonomic depth of the lineage is based on the identity threshold of the rank; Species 99%, Genus 97%, Family 95%, Order 90%, Class 85%, Phylum 80%. (A) Graph shows effect of 5-HI on TGTT in WTG rats. Funding: S.E.A. Gut bacteria convert 5-hydroxytryptophan to 5-hydroxyindole. At the beginning of the experiment, tension of the intestine of 0.5 to 1 g was obtained by adjusting the stretcher. F. nucleatum subsp. (B–D) Overnight cultures of F. nucleatum subsp. Yes Samples were collected after 48 h and were analyzed using HPLC-ED/UV. 5-HI, 5-hydroxyindole; ACh, acetylcholine; n.s., not significant; TTX, tetrodotoxin. Copyright: © 2021 Waclawiková et al. Kooyman and colleagues have shown that 5-HI, when used as a chemical to mimic the aromatic moiety of 5-HT, could modulate 5-HT-induced desensitization of the 5-HT3 receptor-mediated inward current in murine neuroblastoma cells [68]. Data are presented as mean ± SEM, and p < 0.05 was considered statistically significant. As expected, Michaelis–Menten kinetics indicated TnaA had a lower affinity (KM) and catalytic efficiency (kcat/KM) for 5-HTP than its natural substrate tryptophan as illustrated by the KM and kcat/KM values for 5-HTP (3.7 ± 0.2 mM and 21.9 min−1 mM−1, respectively) compared to those for tryptophan (0.19 ± 0.009 mM and 2823.7 min−1 mM−1, respectively) (Fig 2C and 2D and Table 1). Microbiome analysis was done with the statistics software R (R version 3.6.1, https://www.R-project.org/). Search through our exhaustive list of best job vacancies for women graduates. Participants collected the feces and delivered it immediately to the study coordinator who delivered it to the laboratory for processing. (A) β-elimination reaction for tryptophan and 5-HTP. Investigation, Whether you are looking for essay, coursework, research, or term paper help, or with any other assignments, it is no problem for us. Methodology, Subsequently, samples containing 5-HTP were further incubated anaerobically at 37 °C for 6 and 24 h, prior to HPLC-UV analysis. Chromatograms on the left show variation in conversion of 5-HTP to 5-HI after 24 h of anaerobic incubation with 5-HTP. Derniers chiffres du Coronavirus issus du CSSE 08/03/2021 (lundi 8 mars 2021). Samples were incubated anaerobically (10% H2, 10% CO2, 80% N2) in a Don Whitley Scientific DG250 Workstation (LA Biosystems, Waalwijk, the Netherlands) at 37 °C overnight prior to the addition of 100 μM 5-hydroxy-L-tryptophan (5-HTP; H9772, Sigma). Create an account or log into Facebook. 5-HTP was supplemented during the lag or stationary phase depending on the experiment. Interestingly, when applied after 10 min of the 5-HT exposure (this timing is used in all the following organ bath experiments), the addition of 5-HI elicited significantly higher amplitude in the tissue contractility compared to the 5-HT-induced response (Fig 5D). Quantitative analysis of the organ bath data is described in Materials and method section. The level of conservation in the multiple active sites of TnaA is highlighted in red. Finally, to recover the spontaneous contractions of the colonic tissue after the nifedipine treatment (nifedipine was washed away from the tissue in the organ bath), only 5-HI but not ACh was able to restore the tissue to the baseline contractions before nifedipine treatment (Fig 5L) showing 5-HI to be a potent stimulant of the colonic contractility. Therefore, gut microbiota-derived molecules appear to functionally link the microbiota activity to the host gut motility. Interestingly, there was a similar variation among the tested participants in their ability to convert 5-HTP into 5-HI as observed in the ex vivo screening (Fig 1A). RIN14B (ATCC; Cat# CRL-2059) cells were seeded in 12-well plates at the rate of 2 × 105 cells/1 mL in RPMI1640 containing 10% FBS/well and cultured for 72 h. The medium was removed before washing the cells with HBSS (+Ca2+, +Mg2+) containing 0.1% BSA and 2 μM fluoxetine (F132, Sigma) (HBSS-S). https://doi.org/10.1371/journal.pbio.3001070.s003. Specific test, significance, and (n) number are indicated in the Fig legends. Therefore, to validate an independent contractile agent in the presence of nifedipine, 100 μM of ATP, which causes contraction mainly by the release of Ca2+ from intracellular Ca2+ stores, was added to the colonic tissue. Roles Levels of the conserved amino acids are highlighted in the shades of blue (dark blue = higher level of conservation, light blue = lower level of conservation). transient receptor potential channel 6; TTX, After fecal specimen collection, part of the sample was weighed, and MilliQ-filtered water was added (1:5 w/v). Methodology, A pressure transducer and data-acquisition software were employed to display the measurement of tension generated by the smooth muscle of intestinal walls. Error bars represent SEM. Moreover, altered expression or function of L-VDCCs in the gut was shown to cause GI motility disorders, including constipation [53,74,75]. This claim is further strengthened by our results where the oral administration of 5-HI reduced TGTT in WTG rats (Fig 4A). The supernatants were collected and stored at −20 °C until 5-HT measurement using an enzyme immunoassay (EIA) kit (Abnova). Illumina 16S rRNA gene amplicon libraries were generated and sequenced at BaseClear (Leiden, the Netherlands). One part of the fecal specimen was homogenized in Liquid Amies medium containing 20% glycerol (Table E in S1 Table), snap-frozen, and stored in −80 °C. To determine the possible cause of variation in the bacterial conversion of 5-HTP into 5-HI in human fecal samples (Fig 1), 16S rRNA sequencing data of the fecal samples were analyzed (Table B in S1 Table). L-tryptophan and 5-HTP degradation by purified TnaA from E. coli BW25113 was tested by measuring indole or 5-HI formation, respectively, as previously described [34]. https://doi.org/10.1371/journal.pbio.3001070.g005. The raw data can be found in S2 Raw images. Data Availability: All relevant data are within the paper and its Supporting Information files. Methodology, A total of 100 μL samples were collected and quenched in 400 μL ice-cold 100% methanol at 0 and 48 h after initiation. Campingudstyr.dk har alt, du skal bruge. https://doi.org/10.1371/journal.pbio.3001070.g004. Next, the neurotransmitter ACh (50 μM) was applied to the tissue to induce a maximum increase in the intestinal smooth muscle tone [47], which has been long recognized as a key component in gut motility [48]. animalis and Fusobacterium simiae displayed a similar ability to fully metabolize 5-HTP into 5-HI after 24 h of anaerobic incubation (Fig 2E and 2F). Here, we describe how 5-HTP is metabolized by gut bacteria. Gut motility is a tightly controlled system involving the main excitatory neurotransmitter, acetylcholine (ACh), whose excitatory effect on the intestinal smooth muscle is mediated through the muscarinic ACh receptors [6]. For ex vivo organ-bath measurements, the Wilcoxon matched-pairs (before/after) signed rank test was used. Moreover, production of 5-HI was detected in Bacteroides thetaiotamicron cell lysates (S2C Fig), suggesting that TnaA from high abundant Bacteroides genus is also able to convert 5-HTP to 5-HI. The rest of the samples were stored in −80 °C. Amplified tnaA gene was cloned in pET15b, resulting in pBW002 (Table I in S1 Table). Department of Behavioral Neurosciences, Cluster Neurobiology, Groningen Institute of for Evolutionary Life Sciences (GELIFES), University of Groningen, Groningen, The Netherlands, Roles Reactions were initiated by addition of prewarmed cell lysate to prewarmed reaction mixture (100 μM 5-HTP, 100 μM PLP, and either phosphate of citrate buffer). All participants signed the confidentiality and consent form. Total protein concentration was determined by Bradford assay. décès, hospitalisations, réanimations, guérisons par département animalis (S4E Fig) and from 28 ± 0.3 and 0.2 ± 0.2 μM at pH = 7 and 6, respectively, to no production of 5-HI at pH = 5, 4, and 3 in B. thetaiotamicron (S4F Fig), confirming that TnaA activity is inhibited at low pH [37]. As expected, the ACh-induced response was inhibited by the addition of either the M2R or M3R antagonist (S5G and S5H Fig). (K) Bar graph represents contractile agent (ATP) acting independently of L-type Ca2+ channels. Samples were stored at −20 °C until analysis by HPLC-UV. Fecal pellets were monitored every 30 min for the presence of carmine red in the fecal pellet. (A) Microbial profiling of human fecal samples from High, Intermediate, and Non-Converters shows lower relative abundance of tnaA-encoding gut bacterial genera and higher relative abundance of Bifidobacteria genera, respectively. A total of 100 μL samples were collected and quenched in 400 μL ice-cold 100% methanol at 0 and 90 min and 24 h after initiation. Metabolites with similar chemical structures, such as indole and indole derivatives, have large effects on host [58–61], thus it was expected that 5-HI will also influence host physiology [62–64]. Evolutions des sociétés ces dernières années Ci-dessous, l'évolution par an (depuis 2012) des créations et suppressions d'entreprises en France, par mois avec des courbes en moyenne mobile de 12 mois afin de voir l'évolution et les tendances, idem par semaine avec des moyennes mobiles sur 4 semaines. redundancy analysis; TGTT, Only E. coli BW25113ΔtnaA mutant was tested since previous reports showed the same function of TnaA enzyme among different bacterial species [34,35]. To investigate what the effect of pH change in the extracellular environment on the F. nucleatum subsp. animalis and B. breve were first restreaked on Fastidious Anaerobe Agar supplemented with 5% sheep blood or on MRS agar, respectively, and grown overnight before inoculation to liquid EBB culture. Targeted metabolomic analysis revealed that the levels of 5-HI in the fecal samples gradually increased during the period of 5-HTP intake (Fig 1B). First, list of reviewed TnaAs was downloaded from Uniprot (https://www.uniprot.org/), and a BLAST [79] search was locally carried out against the translated HMP Reference Genome (HMRGD) sequence database, which was subsetted to contain only GI tract as body site. Our data showed that 5-HI is able to increase the stimulatory effect of 5-HT and that this effect is not via stimulation of 5-HT3 and 5-HT4 receptors or its action via neuronal action potentials (Figs 5E–5G and S5F). No, Is the Subject Area "Escherichia coli" applicable to this article? Click to see our best Video content. The reaction mixture was prewarmed for 5 min at 37 °C and was initiated by adding an enzyme solution and was terminated afterwards by the addition of 100 μL of 1 M HCl, following the addition of 100 μL of Kovac’s reagent for indoles (60983, Sigma, the Netherlands). (H–J) Bar graphs and illustrative recordings in the rat colon represent enhanced response of the ACh-induced contractility by (H) 100 μM 5-HI, which was significantly increased after adding (I) 100 nM DAU 5884 hydrochloride and was not inhibited after the addition of (J) 1 μM AQ-RA 741. enteric nervous system; GI, 5-hydroxyindole; 5-HT, Following bacterial conversion of 5-HTP to 5-HI, serotonin (5-HT) is released from EC cells to possibly modulate the intestinal motility via activation of 5-HT3 and 5-HT4 receptors on afferent nerve terminals from the ENS. Data were analyzed using the 2-tailed unpaired t test (****p < 0.0001). By combining a suite of in vitro biochemical, culture-based, and organ bath assays, microbiota sequencing, in vivo animal experiments, and human intervention study, we have identified the potency of 5-HI, a product of gut microbial conversion of the dietary supplement and antidepressant 5-HTP, to accelerate GI motility directly via activation of L-type calcium channels located on the colonic smooth muscle cells and possibly through its induction of 5-HT production from enterochromaffin cells. Rats from the vehicle group were treated for the same period of 11 days, but 5-HI was not added to the sucrose solution. Stimulation of the muscarinic receptors by ACh induces contractions that depend on voltage-dependent and voltage-independent Ca2+ entry and intracellular Ca2+ release [7] (cholinergic neurotransmission and smooth muscle biology is extensively discussed in [6,8]). The raw data can be found in S2 Raw images. Citation: Waclawiková B, Bullock A, Schwalbe M, Aranzamendi C, Nelemans SA, van Dijk G, et al. Taken together, the results show that gut bacteria can metabolize 5-HTP with interindividual variation in their ability to produce 5-HI. All animal procedures were approved by the Groningen University Committee of Animal experiments (approval number: AVD1050020197786) and were performed in adherence to the NIH Guide for the Care and Use of Laboratory Animals. Our newfound understanding of the formation of 5-HI by a wide range of TnaA-harboring bacteria will guide how therapeutic communities of microorganisms can be designed in which the production of 5-HI can be genetically specified. The ACh response is mainly exerted through muscarinic ACh receptor subtypes 2 (M2R) and 3 (M3R) located on the smooth muscle cells in the GI tract [6]. (A) From left to right: chromatograms showing bacterial conversion of 100 μM 5-HTP to 5-HI after 6 and 24 h of incubation of human fecal samples (upper panel). Chopped meat medium (CMM; 10 g/L beef extract, 30 g/L casitone, 5 g/L yeast extract, 5 g/L K2HPO4, 0.1% Tween 80, after autoclavation CMM was supplemented with 1 μg/mL vitamin K3, 0.5 g/L cysteine and 0.005 mg/mL hemin). Error bars represent SEM. Notably, no basal levels of 5-HTP were detected by HPLC-ED/UV in the control samples (S1B Fig; 0 h time point). Moreover, 5-HI stimulation of a cell line model of intestinal enterochromaffin cells results in significant increase in serotonin production. Quantitative analysis of the organ bath recordings was performed in BioPac Student Lab 4.1, where each 10-min recording segment was selected and FFT analysis was done with following settings: Data were padded with zeros, mean was removed, magnitude was displayed with linear transform, and signal was processed using Hamming window.