Menthol-assisted homogenous liquid-liquid microextraction for HPLC/UV determination of favipiravir as an antiviral for COVID-19 in human plasma.

Favipiravir is a promising antiviral agent that has been recently approved for treatment of COVID-19 infection. In this study, a menthol-assisted homogenous liquid-liquid microextraction method has been developed for favipiravir determination in human plasma using HPLC/UV. The different factors that could affect the extraction efficiency were studied, including extractant type, extractant volume, menthol amount and vortex time. The optimum extraction efficiency was achieved using 300 µL of tetrahydrofuran, 30 mg of menthol and vortexing for 1 min before centrifuging the sample for 5 min at 3467g. Addition of menthol does not only induce phase separation, but also helps to form reverse micelles to facilitate extraction. The highly polar favipiravir molecules would be incorporated into the hydrophilic core of the formed reverse micelle to be extracted by the non-polar organic extractant. The method was validated according to the FDA bioanalytical method guidelines. The developed method was found linear in the concentration range of 0.1 to 100 µg/mL with a coefficient of determination of 0.9992. The method accuracy and precision were studied by calculating the recovery (%) and the relative standard deviation (%), respectively. The recovery (%) was in the range of 97.1-103.9%, while the RSD (%) values ranged between 2.03 and 8.15 %. The developed method was successfully applied in a bioequivalence study of Flupirava® 200 mg versus Avigan® 200 mg, after a single oral dose of favipiravir administered to healthy adult volunteers. The proposed method was simple, cheap, more eco-friendly and sufficiently sensitive for biomedical application.

Lentzeacins A-E, New Bacterial-Derived 2,5- and 2,6-Disubstituted Pyrazines from a BGC-Rich Soil Bacterium Lentzea sp. GA3-008.

Pyrazines (1,4-diazirines) are an important group of natural products that have tremendous monetary value in the food and fragrance industries and can exhibit a wide range of biological effects including antineoplastic, antidiabetic and antibiotic activities. As part of a project investigating the secondary metabolites present in understudied and chemically rich Actinomycetes, we isolated a series of six pyrazines from a soil-derived Lentzea sp. GA3-008, four of which are new. Here we describe the structures of lentzeacins A-E (1, 3, 5 and 6) along with two known analogues (2 and 4) and the porphyrin zincphyrin. The structures were determined by NMR spectroscopy and HR-ESI-MS. The suite of compounds present in Lentzea sp. includes 2,5-disubstituted pyrazines (compounds 2, 4, and 6) together with the new 2,6-disubstituted isomers (compounds 1, 3 and 5), a chemical class that is uncommon. We used long-read Nanopore sequencing to assemble a draft genome sequence of Lentzea sp. which revealed the presence of 40 biosynthetic gene clusters. Analysis of classical di-modular and single module non-ribosomal peptide synthase genes, and cyclic dipeptide synthases narrows down the possibilities for the biosynthesis of the pyrazines present in this strain.

Electron attachment to isolated and microhydrated favipiravir.

Electron attachment and its equivalent in complex environments, single-electron reduction, are important in many biological processes. Here, we experimentally study the electron attachment to favipiravir, a well-known antiviral agent. Electron attachment spectroscopy is used to explore the energetics of associative (AEA) and dissociative (DEA) electron attachment to isolated favipiravir. AEA dominates the interaction and the yields of the fragment anions after DEA are an order of magnitude lower than that of the parent anion. DEA primary proceeds via decomposition of the CONH2 functional group, which is supported by reaction threshold calculations using ab initio methods. Mass spectrometry of small favipiravir-water clusters demonstrates that a lot of energy is transferred to the solvent upon electron attachment. The energy gained upon electron attachment, and the high stability of the parent anion were previously suggested as important properties for the action of several electron-affinic radiosensitizers. If any of these mechanisms cause synergism in chemo-radiation therapy, favipiravir could be repurposed as a radiosensitizer.

Single Origin Coffee Aroma: From Optimized Flavor Protocols and Coffee Customization to Instrumental Volatile Characterization and Chemometrics.

In this study, the aroma profile of 10 single origin Arabica coffees originating from eight different growing locations, from Central America to Indonesia, was analyzed using Headspace SPME-GC-MS as the analytical method. Their roasting was performed under temperature-time conditions, customized for each sample to reach specific sensory brew characteristics in an attempt to underline the customization of roast profiles and implementation of separate roastings followed by subsequent blending as a means to tailor cup quality. A total of 138 volatile compounds were identified in all coffee samples, mainly furan (~24-41%) and pyrazine (~25-39%) derivatives, many of which are recognized as coffee key odorants, while the main formation mechanism was the Maillard reaction. Volatile compounds' composition data were also chemometrically processed using the HCA Heatmap, PCA and HCA aiming to explore if they meet the expected aroma quality attributes and if they can be an indicator of coffee origin. The desired brew characteristics of the samples were satisfactorily captured from the volatile compounds formed, contributing to the aroma potential of each sample. Furthermore, the volatile compounds presented a strong variation with the applied roasting conditions, meaning lighter roasted samples were efficiently differentiated from darker roasted samples, while roasting degree exceeded the geographical origin of the coffee. The coffee samples were distinguished into two groups, with the first two PCs accounting for 73.66% of the total variation, attributed mainly to the presence of higher quantities of furans and pyrazines, as well as to other chemical classes (e.g., dihydrofuranone and phenol derivatives), while HCA confirmed the above results rendering roasting conditions as the underlying criterion for differentiation.

The antibacterial activity of Libanstin from Ilex paraguariensis (Yerba Mate).

Infectious diseases are reported to be one of the major causes of death in the world. The World Health Organization (WHO) warns of an increase in the deaths number because of antibacterial resistance. Lately, a trend towards searching for new active antibacterial compounds in plants has been observed. Ilex paraguariensis, known as Yerba Mate, is a plant known to be rich in numerous bioactive compounds that have an important role in human health. In this study, Yerba Mate was extracted with acetone: water (1:1) and further fractionated with hexane, chloroform and ethyl acetate. The obtained fractions were tested for antibacterial activity against Staphylococcus aureus and Salmonella species. The minimum inhibitory concentration (MIC) values on S. aureus ranged from 1.56 to 3.12 mg/mL for both the chloroform and ethyl acetate fractions. Whereas for the water fraction, the MIC values ranged from 0.78 to 3.12 mg/mL on S. aureus and ranged from 1.56 mg/mL to 3.12 mg/mL on Salmonella species. The aqueous fraction was further treated with different enzymes to mimic in vivo digestion and the fractions obtained were then tested for antibacterial activity. Furthermore, the Yerba Mate aqueous fraction was run on High Performance Liquid Chromatography (HPLC) and collected fractions were tested for antibacterial activity, to identify the active metabolite. Fraction 3 was tested on different strains of S. aureus and the MIC values ranged from 0.19 to 1.56 µg/mL. A novel pyrazinone, Libanstin, from Ilex paraguariensis was identified using NMR spectroscopy.

Research Progress of the Biosynthesis of Natural Bio-Antibacterial Agent Pulcherriminic Acid in Bacillus.

Pulcherriminic acid is a cyclic dipeptide found mainly in Bacillus and yeast. Due to the ability of pulcherriminic acid to chelate Fe3+ to produce reddish brown pulcherrimin, microorganisms capable of synthesizing pulcherriminic acid compete with other microorganisms for environmental iron ions to achieve bacteriostatic effects. Therefore, studying the biosynthetic pathway and their enzymatic catalysis, gene regulation in the process of synthesis of pulcherriminic acid in Bacillus can facilitate the industrial production, and promote the wide application in food, agriculture and medicine industries. After initially discussing, this review summarizes current research on the synthesis of pulcherriminic acid by Bacillus, which includes the crystallization of key enzymes, molecular catalytic mechanisms, regulation of synthetic pathways, and methods to improve efficiency in synthesizing pulcherriminic acid and its precursors. Finally, possible applications of pulcherriminic acid in the fermented food, such as Chinese Baijiu, applying combinatorial biosynthesis will be summarized.

Determination of phase-partitioning tracer candidates in production waters from oilfields based on solid-phase microextraction followed by gas chromatography-tandem mass spectrometry.

In the present document, we report the development of an analytical method consisting of a sequential direct-immersion/headspace solid-phase microextraction (DI-HS-SPME) followed by gas-phase chromatography and tandem mass spectrometry (GC-MS/MS) for simultaneous analysis of 4-chlorobenzyl alcohol, 2,6-dichlorobenzyl alcohol, 4-methoxybenzyl alcohol, 3,4-dimethoxybenzyl alcohol, pyridine, and 2,3-dimethylpyrazine in oilfield production waters. These compounds are under evaluation for use as phase-partitioning tracers in oil reservoirs. To the best of our knowledge, this is the first time SPME has been applied to the analysis of these compounds in production waters, or any other type of matrix where the compounds targeted are the base for a technical application. Relevant extraction parameters, such as the adsorbent phase of the fiber, direct immersion or headspace, addition of salt, temperature and time of extraction were investigated. The final optimal operation conditions consist on extracting 5 mL of sample at pH 9.0 with 1.8 g of NaCl with constant stirring during 5 minutes of DI-SPME followed by 15 minutes of HS-SPME at 70 °C using a DVB/CAR/PDMS (50/30 µm) fiber. The limits of quantification (LOQ), linearity, precision and accuracy of the method were evaluated. Analyses of the tracer compounds and recovery studies were also performed on production waters from 8 different oilfields of the Norwegian continental shelf. LOQs between 0.080 and 0.35 µg L-1 were obtained. The recovery yields of the method were consistently higher than 85% and RSDs less than 13%. None of the tracer compounds was found in the real samples processed, which is consistent with one of the requirements for an artificial tracer in an oilfield: absence or constant and low background in the traced fluid. The performance of the method developed, combined with its easiness to automate, introduce a new, accurate and cost-efficient technique to process the hundreds of samples required by an inter-well tracer test.

Comparison of volatile trapping techniques for the comprehensive analysis of food flavourings by Gas Chromatography-Mass Spectrometry.

Trapping volatiles is a convenient way to study aroma compounds but it is important to determine which volatile trapping method is most comprehensive in extracting the most relevant aroma components when investigating complex food products. Awareness of their limitations is also crucial. (Un)targeted metabolomic approaches were used to determine the volatile profiles of two commercial flavourings. Four trapping techniques were tested as was the addition of salt to the mixture. Comprehensiveness and repeatability were compared and SBSE proved particularly suitable for extracting components such as polysulfides, pyrazines and terpene alcohols, and provided an overall broader chemical spectrum. SPME proved to be more suitable in extracting sesquiterpenes and DHS in extracting monoterpenes. Adding salt to the sample had only quantitative effects on volatiles as detected by SPME. These results help clarify the advantages and limitations of different trapping techniques and hence deliver a valuable decision tool for food matrix analysis.

Optimal extraction bioactive components of tetramethylpyrazine in Chinese herbal medicine jointly using back propagation neural network and genetic algorithm in R language.

A combinational approach of back propagation neural network (BPNN) and genetic algorithm (GA) was proposed in the present study to optimize the extraction technology of tetramethylpyrazine (TMP) in Ligusticum wallichii Franchat. Based on the single factor test, the orthogonal experiment design method of four factors and three levels was adopted, and the concentration of TMP was measured by high performance liquid chromatography (HPLC). Subsequently, BPNN model was trained for a predictive computational model of the performance indices via experimental data, and GA was exploited to find the optimization con ditions for extraction technology of TMP. Meanwhile, both the model and algorithm were implemented in R language. Ethanol concentration of 80%, extraction time of 1.5h, extraction temperature of 55℃ and liquid-solid ratio of 8:1 were derived as optimal conditions with a maximum content of TMP of 2.04 mg/g, which was confirmed with the relative error 2.63% through the validation of the experiments. This mathematical model could be used to analyze and predict the extraction technology of TMP in Ligusticum wallichii Franchat and provide a new reference for screening optimization of Chinese medicine effective parts and components.

Terezine E, bioactive prenylated tryptophan analogue from an endophyte of Centaurea stoebe.

Fungal endophytes are considered promising sources of new bioactive natural products. In this study, a Mucor sp. has been isolated as an endophyte from the medicinal plant Centaurea stoebe. Through bioactivity-guided fractionation, the isolation of the new bioactive terezine E in addition to the previously reported 14-hydroxyterezine D was carried out. The isolated compounds were fully characterised by HRESIMS and 1D and 2D NMR analyses. Both compounds exhibited potent antiproliferative activity against K-562 and HUVEC cell lines and antifungal efficacy against the tested fungal strains.

Enhypyrazinones A and B, Pyrazinone Natural Products from a Marine-Derived Myxobacterium Enhygromyxa sp.

To date, studies describing myxobacterial secondary metabolites have been relatively scarce in comparison to those addressing actinobacterial secondary metabolites. This realization suggests the immense potential of myxobacteria as an intriguing source of secondary metabolites with unusual structural features and a wide array of biological activities. Marine-derived myxobacteria are especially attractive due to their unique biosynthetic gene clusters, although they are more difficult to handle than terrestrial myxobacteria. Here, we report the discovery of two new pyrazinone-type molecules, enhypyrazinones A and B, from a marine-derived myxobacterium Enhygromyxa sp. Their structures were elucidated by HRESIMS and comprehensive NMR data analyses. Compounds 1 and 2, which contain a rare trisubstituted-pyrazinone core, represent a unique class of molecules from Enhygromyxa sp.

Isolation and Purification of Pyrazines Produced by Reaction of Cellulosic-Derived Sugars with NH4OH and Selected Amino Acids.

Various pyrazines have been synthesized via reaction of selected cellulosic-derived sugars, ammonium hydroxide and amino acids at 110°C for 2 hours. Different methods of sample cleanup such as liquid-liquid extraction (LLE), liquid-solid extraction, column chromatography and distillation were employed to isolate pyrazines from the reaction mixture. Effective LLE of pyrazines from aqueous solution using either hexane, methyl-t-butyl ether (MTBE) or ethyl acetate required multiple extraction steps with fresh solvent each time. When hexane was used as the extraction solvent, no imidazole derivatives were extracted with the pyrazines. However, when MTBE or ethyl acetate was employed, 4-methyl imidazole was co-extracted and further cleanup was required. Passing the organic solvent extracts through a column of silica revealed that the silica retained the undesirable imidazoles, such as 4-methyl imidazole. A mixture of 90/10 hexane/ethyl acetate as eluting solvent provided the desirable pyrazines, but it also provided a desirable separation of pyrazines as a function of total alkyl substituent content. Distillation of the aqueous reaction mixture was also used to isolate the pyrazines, leaving the undesirable imidazoles in the undistilled portion of the reaction. Additional chromatographic methods were used to isolate pyrazines from the aqueous distillate including a column packed with C18-bonded silica.

Optimization of Headspace Solid-Phase Microextraction (HS-SPME) Parameters for the Analysis of Pyrazines in Yeast Extract via Gas Chromatography Mass Spectrometry (GC-MS).

Yeast extract was analyzed through headspace solid-phase microextraction (HS-SPME) in combination with (GC-MS) for its pyrazine compounds. Four different types of SPME fibers with various polarities were selected for preoptimization. The three coated fiber 50/30 µm DVB/CAR/PDMS showed the maximum volatile extraction efficiency and was selected for further analysis. Twenty-eight volatile compounds were tentatively identified through GC-MS including eight pyrazines and were categorically characterized as major volatile compounds responsible for the flavor enhancing notes in YE. Response surface methodology encoded with face centered central composite design was employed to optimize the experimental design. Average peak area of selected pyrazines; methylpyrazine, 2,3-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-5-methylpyrazine, trimethylpyrazine, 3-ethyl-2,5-dimethylpyrazine, tetramethylpyrazine, 3,5-diethyl-2-methylpyrazine, and 2,3,5-trimethyl-6-ethylpyrazine were optimized through RSM-CCD to get the best conditions for HS-SPME. The HS-SPME variables X1 (equilibrium time), X2 (extraction time), and X3 (extraction temperature) were programed into the run sheets to opt an optimistic statistical approach. Among these, the variable X2 and X3 showed the most significant results with the response variable R and could be concluded as the most tantalize variables while practicing pyrazines extraction through HS-SPME method. Resultantly, the optimization methodology was successfully applied for the extraction of pyrazines from yeast extract. PRACTICAL APPLICATION: The selection of optimal conditions to conduct a HS-SPME experiment can dramatically affect the sensitivity and accuracy of aroma extraction process. Optimizing the SPME conditions is the best way to identify the role of all the possible factors that can fluctuate the volatile profile of any sample. This type of statistical approach to optimize the HS-SPME conditions for pyrazines in yeast extract was practiced for the very first time and could be considered as a prerequisite strategy to proliferate future projects related to some novel studies in terms of pyrazines flavor perception.

Novel Antimicrobial Indolepyrazines A and B from the Marine-Associated Acinetobacter sp. ZZ1275.

Two new alkaloids indolepyrazines A (1) and B (2) were isolated from the marine-derived Acinetobacter sp. ZZ1275. Their structures were elucidated through extensive nuclear magnetic resonance (NMR) spectroscopic analyses, high resolution electrospray ionization mass spectroscopy (HRESIMS) data, and electronic circular dichroism (ECD) calculation. Indolepyrazine A represents the first example of alkaloids with an indole-pyrazine-oxindole skeleton. Both 1 and 2 showed antimicrobial activities against methicillin-resistant Staphylococcus aureus, Escherichia coli, and Candida albicans with minimum inhibitory concentration (MIC) values of 12 µg/mL, 8⁻10 µg/mL, and 12⁻14 µg/mL, respectively.

N-containing compounds from Periplaneta americana and their activities against wound healing.

Three new compounds, periplanamides A (1) and B (2), periplanpyrazine A (3), a new naturally occurring compound salicyluric acid methyl ester (6), and seventeen known compounds were isolated from the medicinal insect Periplaneta americana. The structures of the new compounds were elucidated on the basis of spectroscopic methods. The absolute configurations of 2 were assigned by computational methods. Biological activities of these isolates except 1, 9, 11, and 13 toward nitric oxide (NO) production, cell proliferation in HDFs, cell migration and angiogenesis in HUVECs were evaluated.

PYRROLO isolated from marine sponge associated bacterium Halobacillus kuroshimensis SNSAB01 - Antifouling study based on molecular docking, diatom adhesion and mussel byssal thread inhibition.

In the present study, an attempt has been made to explore the antifouling potential of bioactive compound isolated from sponge associated bacterium Halobacillus kuroshimensis SNSAB01. The crude extract of SNSAB01 strongly inhibited the growth of fouling bacterial strains with least minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The bioactive compound was characterized through FT-IR, HPLC, GCMS and NMR predicted as 'pyrrolo". From the mass spectral library, structure was elucidated as pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro. The in silico studies provided encouraging docking scores with two interactions by GLN 200 and GLU 304. The extract inhibited 89% diatom adhesion at 350 µg/ml concentration against Amphora sp. An EC50 value of 150 µg/ml for 50% inhibition of byssal thread of Perna viridis and LC50 was found to be 500 µg/ml. The LC50/EC50 ratio of 3.0 indicated nontoxic to nature. The result suggested that pyrrolo[1,2-a]pyrazine-1,4-dione can be used for antifouling coating.

Chemical and Sensory Evaluation of Magnetic Polymers as a Remedial Treatment for Elevated Concentrations of 3-Isobutyl-2-methoxypyrazine in Cabernet Sauvignon Grape Must and Wine.

3-Isobutyl-2-methoxypyrazine (IBMP) is a potent odorant present in grapes and wines that is reminiscent of green capsicum. Suprathreshold concentrations can lead to obvious vegetative characters and suppress desirable fruity aroma nuances in wines, but options to manage IBMP concentrations are limited. This work investigated pre- and postfermentation addition of a putative imprinted magnetic polymer (PIMP) as a remedial treatment for elevated concentrations of IBMP in Cabernet Sauvignon grape must in comparison to nonimprinted magnetic polymer (NIMP) and to a commercially available polylactic acid (PLA) based film added postfermentation. Chemical and sensory analyses of wines showed that PIMP treatments were more effective than PLA film for decreasing "fresh green" aroma nuances without negatively impacting overall aroma profiles and that postfermentation addition of a magnetic polymer removed up to 74% of the initial IBMP concentration compared to 18% for PLA. Prefermentation addition of magnetic polymers removed 20-30% less IBMP compared to that of postfermentation addition but also had less of an effect on other wine volatiles and color parameters.

Pyrazinone derivatives from the coral-derived Aspergillus ochraceus LCJ11-102 under high iodide salt.

Five new pyrazin-2(1H)-one derivatives, ochramides A-D (1-4) and ochralate A (5), as well as three known analogues (6-8) were isolated from the fermentation broth of the marine coral-derived halotolerant Aspergillus ochraceus LCJ11-102 in a nutrient-limited medium containing 10% NaI. Their chemical structures were determined by analyzing NMR and X-ray diffraction data. Compounds 2, 5 and 6 showed antimicrobial activities against Enterobacter aerogenes with the MIC values of 40.0, 18.9, and 20.1 µM, respectively.

Isolation and identification of new chemical constituents from Chinese chive (Allium tuberosum) and toxicological evaluation of raw and cooked Chinese chive.

Chinese chive (jiu cai) is a popular vegetable in China and has a unique flavour and aroma. The molecular basis of the characteristic fragrance and nutritional properties of Chinese chive has not been previously identified. Sequential extractions in a series of solvents and high-performance liquid chromatography were used to isolate 40 compounds from Chinese chive. The compounds were identified based on high-resolution electrospray ionization mass spectra, 1D and 2D nuclear magnetic resonance techniques, and circular dichroism spectra. Eight novel compounds were identified-four new pyrazines, which have distinctive flavour; one new lignan; and three new flavonoids-together with 32 known compounds. Several of these compounds have potential applications as health-promoting dietary supplements, food additives, or seasonings. Additionally, the volatile organic compounds in fresh and steamed Chinese chive were compared, and the toxicological activity of extracts from fresh and steamed Chinese chive was tested in normal rat liver (IAR20) and kidney (NRK) cells. The results showed that Chinese chive is toxic to liver and kidney cells when fresh, but is safe after heating. This could explain why it is traditional to eat cooked Chinese chive. A possible metabolic rule regarding pyrazines is postulated based on this data, and a human metabolic pathway is suggested for two of the novel compounds which have the highest amount of Chinese chive extracts.

Ligustrazine suppresses neuron apoptosis via the Bax/Bcl-2 and caspase-3 pathway in PC12 cells and in rats with vascular dementia.

The aim of this study was to examine the comprehensive neuroprotective mechanism of ligustrazine, which is extracted from Ligusticum Chuanxiong Hort., against vascular dementia (VD) in rats and apoptosis in oxygen and glucose deprivation (OGD) PC12 cells. Rats were subjected to bilateral common carotid artery occlusion (BCCAO) surgery and administered ligustrazine intragastrically for 6 weeks. At the end of the experiments, the hippocampal biomarkers brain-derived neurotrophic factor (BDNF), monocyte chemotactic protein 1 (MCP-1), and homocysteine (Hcy) were examined. In experiments in vitro, OGD PC12 cells were treated with ligustrazine for 0.5, 1, 3, 6, 12, or 24 h. The cell-released biomarkers BDNF, MCP-1, and Hcy were examined. Microscopy, acridine orange-ethidium bromide (AO/EB) staining, and flow cytometry assays were performed to investigate apoptosis. Cleaved caspase-3, Bcl-2 associated X protein (Bax), and B cell lymphoma 2 (Bcl-2) expression was examined using Western blot assays. The results showed that biomarkers, including MCP-1 and Hcy, were significantly increased in both the in vivo and in vitro models, while the BDNF level was significantly decreased compared with the sham or vehicle models. Microscopy, AO/EB staining, and flow cytometry analysis showed that severe cell damage occurred in OGD PC12 cells, and apoptosis played a major role in this environment. Further Western blot studies showed that the apoptosis-related Bax/Bcl-2 protein ratio and cleaved caspase-3 were significantly increased in the experiment. However, ligustrazine profoundly suppressed the imbalance of these biomarkers, reduced cell damage, decreased the Bax/Bcl-2, and downregulated cleaved caspase-3. Pro- and anti-apoptotic biomarkers of multiple pathways including BDNF, MCP-1, and Hcy played a joint role in triggering the activation of the mitochondria-related Bax/Bcl-2 and caspase-3 apoptosis pathway in VD. Ligustrazine attenuated VD by comprehensively regulating BDNF, MCP-1, and Hcy and inactivating the Bax/Bcl-2 and caspase-3 apoptosis pathway. Our data provide novel insight into ligustrazine, which is a promising neuroprotective agent for VD disease treatment strategies. © IUBMB Life, 70(1):60-70, 2018.