Binary and Ternary Deep Eutectic Solvents for Methylene Green Electropolymerization on Multiwalled Carbon Nanotubes: Optimization, Characterization and Application.

Choline chloride (ChCl) based binary and ternary deep eutectic solvents (DES) were evaluated for methylene green electropolymerization with oxalic acid (OA) and ethylene glycol (EG) as hydrogen bond donors. Binary DES ChCl:OA in molar ratios 1:1 and 2:1 and ChCl:EG 1:2 and ternary DES (tDES) in different molar ratios and percentages of water were evaluated. The highest polymer growth was in ChCl:OA:EG-tDES with added water, that had a lower viscosity and higher ionic conductivity when associated with HCl as dopant. This enhanced the formation of more cation radicals and, consequently, more polymer formation. The PMG/MWCNT/GCE-tDES sensor was successfully applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and acetaminophen (APAP) by differential pulse voltammetry in the concentration range 2 µM - 200 µM, with detection limits of 0.37 µM and 0.49 µM for 5-ASA and APAP, respectively. The sensor demonstrated good repeatability, reproducibility and stability, and was successfully applied in pharmaceutical formulations.

Thermotolerance in S. cerevisiae as a model to study extracellular vesicle biology.

The budding yeast Saccharomyces cerevisiae is a proven model organism for elucidating conserved eukaryotic biology, but to date its extracellular vesicle (EV) biology is understudied. Here, we show yeast transmit information through the extracellular medium that increases survival when confronted with heat stress and demonstrate the EV-enriched samples mediate this thermotolerance transfer. These samples contain vesicle-like particles that are exosome-sized and disrupting exosome biogenesis by targeting endosomal sorting complexes required for transport (ESCRT) machinery inhibits thermotolerance transfer. We find that Bro1, the yeast ortholog of the human exosome biomarker ALIX, is present in EV samples, and use Bro1 tagged with green fluorescent protein (GFP) to track EV release and uptake by endocytosis. Proteomics analysis reveals that heat shock protein 70 (HSP70) family proteins are enriched in EV samples that provide thermotolerance. We confirm the presence of the HSP70 ortholog stress-seventy subunit A2 (Ssa2) in EV samples and find that mutant yeast cells lacking SSA2 produce EVs but they fail to transfer thermotolerance. We conclude that Ssa2 within exosomes shared between yeast cells contributes to thermotolerance. Through this work, we advance Saccharomyces cerevisiae as an emerging model organism for elucidating molecular details of eukaryotic EV biology and establish a role for exosomes in heat stress and proteostasis that seems to be evolutionarily conserved.

Fluorinated Mn(III)/(II)-Porphyrin with Redox-Responsive 1 H and 19 F Relaxation Properties.

A new fluorinated manganese porphyrin, (Mn-TPP-p-CF3 ) is reported capable of providing, based on the Mn(III)/Mn(II) equilibrium, dual 1 H relaxivity and 19 F NMR response to redox changes. The physical-chemical characterization of both redox states in DMSO-d6 /H2 O evidenced that the 1 H relaxometric and 19 F NMR properties are appropriate for differential redox MRI detection. The Mn(III)-F distance (dMn-F =9.7-10 Å), as assessed by DFT calculations, is well tailored to allow for adequate paramagnetic effect of Mn(III) on 19 F T1 and T2 relaxation times. Mn-TPP-p-CF3 has a reversible Mn(II)/Mn(III) redox potential of 0.574 V vs. NHE in deoxygenated aqueous HEPES/ THF solution. The reduction of Mn(III)-TPP-p-CF3 in the presence of ascorbic acid is slowly, but fully reversed in the presence of air oxygen, as monitored by UV-Vis spectrometry and 19 F NMR. The broad 1 H and 19 F NMR signals of Mn(III)-TPP-p-CF3 disappear in the presence of 1 equivalent ascorbate replaced by a shifted and broadened 19 F NMR signal from Mn(II)-TPP-p-CF3 . Phantom 19 F MR images in DMSO show a MRI signal intensity decrease upon reduction of Mn(III)-TPP-p-CF3 , retrieved upon complete reoxidation in air within ~24 h. 1 H NMRD curves of the Mn(III)/(II)-TPP-p-CF3 chelates in mixed DMSO/water solvent have the typical shape of Mn(II)/Mn(III) porphyrins.

Novel Amperometric Mercury-Selective Sensor Based on Organic Chelator Ionophore.

A novel amperometric sensor for the direct determination of toxic mercury ions, Hg2+, based on the organic chelator ionophore N, N di (2-hydroxy-5-[(4-nitrophenyl)diazenyl]benzaldehyde) benzene-1,2-diamine (NDBD), and multiwalled carbon nanotubes (MWCNT) immobilized on a glassy carbon electrode surface was developed. The parameters influencing sensor performance including the ionophore concentration, the applied potential, and electrolyte pH were optimized. The sensor response to Hg2+ was linear between 1-25 µM with a limit of detection of 60 nM. Interferences from other heavy metal ions were evaluated and the sensor showed excellent selectivity towards Hg2+. The method was successfully applied to the determination of mercury ions in milk and water samples.

A two-tiered system for selective receptor and transporter protein degradation.

Diverse physiology relies on receptor and transporter protein down-regulation and degradation mediated by ESCRTs. Loss-of-function mutations in human ESCRT genes linked to cancers and neurological disorders are thought to block this process. However, when homologous mutations are introduced into model organisms, cells thrive and degradation persists, suggesting other mechanisms compensate. To better understand this secondary process, we studied degradation of transporter (Mup1) or receptor (Ste3) proteins when ESCRT genes (VPS27, VPS36) are deleted in Saccharomyces cerevisiae using live-cell imaging and organelle biochemistry. We find that endocytosis remains intact, but internalized proteins aberrantly accumulate on vacuolar lysosome membranes within cells. Here they are sorted for degradation by the intralumenal fragment (ILF) pathway, constitutively or when triggered by substrates, misfolding or TOR activation in vivo and in vitro. Thus, the ILF pathway functions as fail-safe layer of defense when ESCRTs disregard their clients, representing a two-tiered system that ensures degradation of surface polytopic proteins.

Acetate and hypertonic stress stimulate vacuole membrane fission using distinct mechanisms.

Vacuoles in plants and fungi play critical roles in cell metabolism and osmoregulation. To support these functions, vacuoles change their morphology, e.g. they fragment when these organisms are challenged with draught, high salinity or metabolic stress (e.g. acetate accumulation). In turn, morphology reflects an equilibrium between membrane fusion and fission that determines size, shape and copy number. By studying Saccharomyces cerevisiae and its vacuole as models, conserved molecular mechanisms responsible for fusion have been revealed. However, a detailed understanding of vacuole fission and how these opposing processes respond to metabolism or osmoregulation remain elusive. Herein we describe a new fluorometric assay to measure yeast vacuole fission in vitro. For proof-of-concept, we use this assay to confirm that acetate, a metabolic stressor, triggers vacuole fission and show it blocks homotypic vacuole fusion in vitro. Similarly, hypertonic stress induced by sorbitol or glucose caused robust vacuole fission in vitro whilst inhibiting fusion. Using wortmannin to inhibit phosphatidylinositol (PI) -kinases or rGyp1-46 to inactivate Rab-GTPases, we show that acetate stress likely targets PI signaling, whereas osmotic stress affects Rab signaling on vacuole membranes to stimulate fission. This study sets the stage for further investigation into the mechanisms that change vacuole morphology to support cell metabolism and osmoregulation.

Electrochemical Impedance Spectroscopy in the Characterisation and Application of Modified Electrodes for Electrochemical Sensors and Biosensors.

Electrochemical impedance spectroscopy is finding increasing use in electrochemical sensors and biosensors, both in their characterisation, including during successive phases of sensor construction, and in application as a quantitative determination technique. Much of the published work continues to make little use of all the information that can be furnished by full physical modelling and analysis of the impedance spectra, and thus does not throw more than a superficial light on the processes occurring. Analysis is often restricted to estimating values of charge transfer resistances without interpretation and ignoring other electrical equivalent circuit components. In this article, the important basics of electrochemical impedance for electrochemical sensors and biosensors are presented, focussing on the necessary electrical circuit elements. This is followed by examples of its use in characterisation and in electroanalytical applications, at the same time demonstrating how fuller use can be made of the information obtained from complete modelling and analysis of the data in the spectra, the values of the circuit components and their physical meaning. The future outlook for electrochemical impedance in the sensing field is discussed.

Highly sensitive and selective nanostructured microbiosensors for glucose and lactate simultaneous measurements in blood serum and in vivo in brain tissue.

Highly sensitive and selective nanostructured lactate and glucose microbiosensors for their in vivo simultaneous determination in rat brain were developed based on carbon fiber microelectrodes (CFM) modified with nanoporous gold (NPG) using the Dynamic Hydrogen Bubble Template (DHBT) method. Electrodeposition of platinum nanoparticles (PtNP) onto the NPG film enhances the sensitivity and the electrocatalytic properties towards H2O2 detection. The nanostructured microelectrode platform was modified by glucose oxidase (GOx) and lactate oxidase (LOx) enzyme immobilization. High selective measurements were achieved by covering with a perm-selective layer of electropolymerized m-phenylenediamine, deposition of a Nafion® film and by using a null sensor. The morphological characteristics and electroanalytical performance of the microbiosensors were assessed, by scanning electron microscopy and electrochemical techniques, respectively. The PtNP/NPG/CFM shows a high sensitivity to H2O2 (5.96 A M-1 cm-2) at 0.36 V vs. Ag/AgCl, with a linear range from 0.2 to 200 µM, and an LOD of 10 nM. The microbiosensors were applied to the simultaneous determination of lactate and glucose in blood serum samples. Moreover, the basal extracellular concentrations of lactate and glucose were measured in vivo in four different rat brain structures. These results support the potential of the microbiosensor to be used as a valuable tool to investigate brain neurochemicals in vivo.

Magnetic-field-assisted deposition of self-assembling crystallite layers of Co2+-containing layered double hydroxides.

Precipitation of nanocrystallites of cobalt-aluminium layered double hydroxides in a magnetic field has been studied. In a magnetic field perpendicular to the substrate, dense and homogeneous films have been obtained. Magnetic anisotropy of the crystallites is explained by deviation from the statistical cation distribution in favour of honeycomb-like coordination of cobalt.

Polyphenazine and polytriphenylmethane redox polymer/nanomaterial-based electrochemical sensors and biosensors: a review.

In recent years, an increasing number of studies has demonstrated that redox polymers can be used in simple and effective electrochemical sensing platforms due to their fast electron transfer and electrocatalytic ability. To develop more sensitive and selective electrochemical (bio)sensors, the electrocatalytic properties of redox polymers and the electrical, mechanical, and catalytic properties of various nanomaterials are combined. This review aims to summarize and contribute to the development of (bio)sensors based on polyphenazine or polytriphenylmethane redox polymers combined with nanomaterials, including carbon-based nanomaterials, metal/metal oxide, and semiconductor nanoparticles. The synthesis, preparation, and modification of these nanocomposites is presented and the contribution of each material to the performance of (bio)sensor has been be examined. It is explained how the combined use of these redox polymers and nanomaterials as a sensing platform leads to improved analytical performance of the (bio)sensors. Finally, the analytical performance characteristics and practical applications of polyphenazine and polytriphenylmethane redox polymer/nanomaterial-based electrochemical (bio)sensors are compared and discussed.

Hybrid Nanocomposite Platform, Based on Carbon Nanotubes and Poly(Methylene Blue) Redox Polymer Synthesized in Ethaline Deep Eutectic Solvent for Electrochemical Determination of 5-Aminosalicylic Acid.

A novel hybrid composite of conductive poly(methylene blue) (PMB) and carbon nanotubes (CNT) was prepared for the detection of 5-aminosalicylic acid (5-ASA). Electrosynthesis of PMB with glassy carbon electrode (GCE) or with carbon nanotube modified GCE was done in ethaline deep eutectic solvent of choline chloride mixed with ethylene glycol and a 10% v/v aqueous solution. Different sensor architectures were evaluated in a broad range of pH values in a Britton-Robinson (BR) buffer using electrochemical techniques, chronoamperometry (CA), and differential pulse voltammetry (DPV), to determine the optimum sensor configuration for 5-ASA sensing. Under optimal conditions, the best analytical performance was obtained with CNT/PMBDES/GCE in 0.04 M BR buffer pH 7.0 in the range 5-100 µM 5-ASA using the DPV method, with an excellent sensitivity of 9.84 µA cm-2 µM-1 (4.9 % RSD, n = 5) and a detection limit (LOD) (3σ/slope) of 7.7 nM, outclassing most similar sensors found in the literature. The sensitivity of the same sensor obtained in CA (1.33 µA cm-2 µM-1) under optimal conditions (pH 7.0, Eapp = +0.40 V) was lower than that obtained by DPV. Simultaneous detection of 5-ASA and its analogue, acetaminophen (APAP), was successfully realized, showing a catalytic effect towards the electro-oxidation of both analytes, lowering their oxidation overpotential, and enhancing the oxidation peak currents and peak-to-peak separation as compared with the unmodified electrode. The proposed method is simple, sensitive, easy to apply, and economical for routine analysis.

A novel nanostructured poly(thionine)-deep eutectic solvent/CuO nanoparticle film-modified disposable pencil graphite electrode for determination of acetaminophen in the presence of ascorbic acid.

A new electrochemical sensor based on thionine (TH), an electroactive polymer, and CuO nanoparticle (CuONP)-modified pencil graphite electrode (PGE) has been developed. Poly(thionine) (PTH) was formed on the CuO/PGE surface by electropolymerisation in ethaline deep eutectic solvent (DES) containing acetic acid dopant to form PTHEthaline/CuO/PGE. Cyclic voltammetry, electrochemical impedance spectroscopy, and differential pulse voltammetry were utilized to evaluate the fabrication process, electrochemical properties, and performance parameters of the modified electrodes. The analytical performance of the PTHEthaline/CuO/PGE was evaluated with respect to linear range, limit of detection, repeatability, and reproducibility for the detection of acetaminophen (APAP) by electrooxidation in the presence of ascorbic acid (AA). Analytical parameters such as pH were optimized. The combined use of PTH and CuONP led to enhanced performance towards APAP due to the large electroactive surface area and synergistic catalytic effect, with a wide linear working range and low detection limit. The reliability of the proposed sensor for the detection of APAP was successfully tested in pharmaceutical samples containing APAP and AA, with very good recoveries. Graphical abstract.

Multiple Epithelial Origin Complications Following Subepithelial Connective Tissue Graft for Root Coverage.

INTRODUCTION: The subepithelial connective tissue graft (SCTG) and flap combination is a highly predictable root coverage procedure, with low complication rates. To our knowledge, this article reports the first case of two late SCTG complications, epithelial cell discharge, and subsequent epidermal inclusion cyst (EIC) formation. CASE PRESENTATION: A 35-year-old male presented with a 3-mm deep Miller Class II recession defect on the mandibular right canine and mesial root of mandibular right first molar. A mild discomfort was reported at 8 weeks after envelope flap+SCTG in #27. At 4 months after the procedure, the patient presented with persistent discomfort and minimally compressible recipient site diffuse swelling with discharge, which was cytologically diagnosed as normal epithelial cells. One year postoperatively, enlargement of the lesion was seen, and excisional biopsy was performed simultaneously with SCTG in #30. The lesion was diagnosed as EIC. At 8 months follow-up, the site healed uneventfully, the patient remained asymptomatic, and the site exhibited scar formation and no recurrence of the lesion. CONCLUSION: This report highlights epithelial cell discharge and EIC formation as a rare yet possible SCTG complication and emphasizes the importance of an excisional biopsy as the means to obtain a definitive diagnosis and manage this complication.

Novel Workflow for Conversion of Catheter-Based Electroanatomic Mapping to DICOM Imaging for Noninvasive Radioablation of Ventricular Tachycardia.

PURPOSE: A recent clinical trial has demonstrated that noninvasive radioablation (NIRA) has the potential to reduce recurrent ventricular tachycardia (VT) that is refractory to drugs and standard catheter ablation. Electroanatomic mapping (EAM) that would be useful for planning is obtained during catheter ablation, but incompatibility between EAM and DICOM formats required for radiation planning has impeded the use of existing catheter-based mapping to guide NIRA and is an important hurdle for its wider adoption. In this paper we define a process to facilitate the fusion of catheter-based EAM with DICOM imaging for radiation planning. METHOD AND MATERIALS: The raw data export of the CARTO3 EAM system (version 6.0.45.171, ".mesh" file) was processed with a MATLAB script to generate 3-dimensional (3D) visual took kit files containing X, Y, Z coordinates obtained during mapping and corresponding impedance, voltage, and other point-based information. The image could then be visualized with standard image processing software (3D Slicer) and the target outlined on the image surface. This structure was in turn converted to a DICOM image and fused with patient thoracic imaging using anatomic landmarks. Robustness of the workflow was assessed through implementation with a second magnetic resonance imaging based VT ablation planning system, ADAS-VT. RESULTS: This process facilitated the fusion of EAM and DICOM imaging to inform selection of NIRA targets. The workflow was found to be robust and compatible with a second VT ablation planning system. CONCLUSIONS: The conversion of catheter-based EAM to a DICOM compatible format permits the fusion of images for radiation planning and provides an avenue for the wider application of NIRA. Further improvements in the compatibility of these imaging formats would be expected to improve quality and reproducibility of image fusion.

Electrochemical synthesis and characterization of poly(thionine)-deep eutectic solvent/carbon nanotube-modified electrodes and application to electrochemical sensing.

Electropolymerization of thionine (TH) on multiwalled carbon nanotube (MWCNT)-modified glassy carbon electrodes (GCE) in ethaline deep eutectic solvent (DES) was carried out for the first time, to prepare poly(thionine) (PTH) films with different nanostructured morphologies. PTH films were formed on MWCNT/GCE by potential cycling electropolymerization in ethaline with the addition of different acid dopants CH3COOH, HClO4, HNO3, H2SO4 and HCl, acetic acid being the best. The electropolymerization process was monitored with an electrochemical quartz crystal microbalance. The polymerization scan rate was a key factor affecting the electrochemical and morphological properties of the PTHEthaline-CH3COOH/MWCNT/GCE; electrodeposition at 200 mV s-1 showing the best performance. The PTH/MWCNT/GCE platform was characterized using cyclic and differential pulse voltammetry, electrochemical impedance spectroscopy and scanning electron microscopy. The analytical characteristics of the PTH films were evaluated for sensing of ascorbic acid and biosensing of uric acid. The developed sensor exhibited a low detection limit (1.1 µM), wide linear range (2.8-3010 µM) and high sensitivity (1134 µA cm-2 mM-1) for ascorbic acid. After immobilization of uricase, UOx, on PTH/MWCNT/GCE, the biosensor was successfully applied to the determination of uric acid, with fast response (˂ 7 s), good sensitivity (450 µA cm-2 mM-1, wide linear range (0.48-279 µM) and low detection limit (58.9 nM), better than in the literature and than with PTH prepared in aqueous solution. The determination of uric acid in synthetic urine samples was successfully tested and the mean analytical recovery was 100.8 ± 1.4%. This is a promising approach for the determination of uric acid in real samples. Graphical abstract.

Immortal Time Bias in National Cancer Database Studies.

PURPOSE: In studies evaluating the benefit of adjuvant therapies, immortal time bias (ITB) can affect the results by incorrectly reporting a survival advantage. It does so by including all deceased patients who may have been planned to receive adjuvant therapy within the observation cohort. Given the increase in National Cancer Database (NCDB) analyses evaluating postoperative radiation therapy (PORT) as an adjuvant therapy, we sought to examine how often such studies accounted and adjusted for ITB. METHODS AND MATERIALS: A systematic review was undertaken to search MEDLINE and EMBASE from January 2014 until May 2019 for NCDB studies evaluating PORT. After appropriate exclusion criteria were applied, 60 peer-reviewed manuscripts in which PORT was compared with postoperative observation or maintenance therapy were reviewed. The manuscripts were reviewed to evaluate whether ITB was accounted for, the method with which it was adjusted for, impact factor, year of publication, and whether PORT was beneficial. RESULTS: Of the 60 publications reviewed, 23 studies (38.3%) did not include an adjustment for ITB. Most studies that did adjust for ITB employed a single landmark (LM) time (n = 31), 4 used a sequential landmark analyses, and 2 used a time-dependent Cox model. In 23 of 31 studies (74.2%) that did adjust for ITB via a single LM time, the rationale behind why the specified LM time was chosen was not clearly explained. There was no relationship between adjusting for ITB and year of publication (P = .074) or whether the study was published in a high-impact journal (P = .55). CONCLUSIONS: Studies assessing adjuvant radiation therapy by analyzing the NCDB are susceptible to ITB, which overestimates the effect size of adjuvant therapies and can provide misleading results. Adjusting for this bias is essential for accurate data representation and to better quantify the impact of adjuvant therapies such as PORT.

Biotoxic trace metal ion detection by enzymatic inhibition of a glucose biosensor based on a poly(brilliant green)-deep eutectic solvent/carbon nanotube modified electrode.

A highly sensitive glucose oxidase (GOx) electrochemical biosensor for the determination of the biotoxic trace metal ions Hg2+, Cd2+, Pb2+ and CrVI by enzyme inhibition has been developed. GOx was immobilized on a novel sensing platform consisting of poly(brilliant green) films formed by potential cycling electropolymerization in sulfuric acid doped ethaline deep eutectic solvent on multiwalled carbon nanotube modified glassy carbon electrodes. Polymer films produced in this medium presented more uniform morphology and better electrochemical sensing properties than those prepared in aqueous solution. The inhibitor concentration necessary to give 50% inhibition, I50, was used for the determination of the type of reversible inhibition, and the relationship between I50 and the inhibition constant Ki is discussed. The new biosensor was successfully used for the determination of biotoxic trace metal ions with a nanomolar limit of detection, lower than in the literature, very good repeatability, stability and selectivity, and was applied successfully to detection of the toxic trace metal species in milk samples.

A biocompatible redox MRI probe based on a Mn(ii)/Mn(iii) porphyrin.

For the development of redox responsive MRI probes based on the MnIII/MnII couple, stable complexation of both reduced and oxidized forms of the metal ion and appropriate tuning of the redox potential in the biologically relevant range are key elements. The water soluble fluorinated Mn-porphyrin derivative Mn-3 satisfies both requirements. In aqueous solutions, it can reversibly switch between MnIII/MnII oxidation states. In the presence of ascorbic acid or ß-mercaptoethanol, the MnIII form undergoes reduction, which is slowly but fully reversed in the presence of air oxygen. A UV-Vis kinetic study of MnIII/MnII reduction under oxygen-free conditions yielded second-order rate constants, k2, of 46.1 M-1 s-1 and 13.8 M-1 s-1 for the reaction with ascorbic acid and ß-mercaptoethanol, respectively. This could correspond, in the absence of oxygen, to a half-life of a few minutes in blood plasma and a few seconds in circulating immune cells where ascorbic acid reaches 20-40 µM and a few mM concentrations, respectively. In contrast to expectations based on the redox potential, reduction with glutathione or cysteine does not occur. It is prevented by the coordination of the glutathione carboxylate group(s) to MnIII in the axial position, as was evidenced by NMR data. Therefore, MnIII-3 acts as an ascorbate specific turn-on MRI probe, which in turn can be re-oxidized by oxygen. The relaxivity increase from the oxidized to the reduced form is considerably improved at medium frequencies (up to 80 MHz) with respect to the previously studied Mn-TPPS4 analogues; at 20 MHz, it amounts to 150%. No in vitro cytotoxicity is detectable for Mn-3 in the typical MRI concentration range. Finally, 19F NMR resonances of MnIII-3 are relatively sharp which could open further opportunities to exploit such complexes as paramagnetic 19F NMR probes.

Tyrosinase based amperometric biosensor for determination of tyramine in fermented food and beverages with gold nanoparticle doped poly(8-anilino-1-naphthalene sulphonic acid) modified electrode.

The aim of the present work was to develop an amperometric biosensor for tyramine (Tyr) measurement in food and beverages. The biosensor architecture is based on tyrosinase (Tyrase) immobilization on glassy carbon electrode modified by a nanocomposite consisting of gold nanoparticles (AuNP) synthesized by a green method and poly(8-anilino-1-naphthalene sulphonic acid) modified glassy carbon electrode. Under optimized experimental conditions for fixed potential amperometric detection, the biosensor exhibited a linear response to tyramine in the range 10-120 µM and the limit of detection was estimated to be 0.71 µM. The novel platform showed good selectivity, long-term stability, and reproducibility. The strong interaction between tyrosinase and the nanocomposite was revealed by the high value of the Michaelis-Menten constant (79.3 µM). The fabricated biosensor was successfully applied to the determination of Tyr in dairy products and fermented drinks with good recoveries, which makes it a promising biosensor for quantification of tyramine.