Vitis vinifera cv. Corvina is the main grape variety used to produce Amarone wine. Before starting the winemaking process, the Corvina grapes are stored in a withering  warehouse until about 30% of the berry weight is lost (WL). This practice is performed to concentrate the metabolites in the berry and enrich the Amarone wine in aroma and antioxidant compounds. In compliance with the guidelines and strict Amarone protocol set by the Consorzio of Amarone Valpolicella, withering must be carried out by setting the grapes in a suitable environment, either under controlled relative air humidity (RH) conditions and wind speed (WS)—no temperature modification is to be applied—or, following the traditional methods, in non-controlled environmental conditions. In general, the two processes have different dehydration kinetics due to the different conditions in terms of temperature, RH, and WS, which affect the accumulation of sugars and organic acids and the biosynthesis of secondary metabolites such as stilbenes and glycoside aroma precursors. For this study, the two grape-withering processes were carried out under controlled (C) and non-controlled (NC) conditions, and the final compositions of the Corvina dried grapes were compared also to evaluate the effects on the organoleptic characteristics of Amarone wine. The findings highlighted differences between the two processes mainly in terms of the secondary metabolites of the dried grapes, which affect the organoleptic characteristics of Amarone wine. Indeed, by the sensory evaluation, wines produced by adopting the NC process were found more harmonious, elegant, and balanced. Finally, we can state how using a traditional system, grapes were characterised by higher levels of VOCs (volatile compounds), whilst wines had a higher and appreciable complexity and finesse.In the Valpolicella area   Wine grapes are generally harvested at the proper technical ripening stage, which is when the major chemical ripening parameters  meet the oenological requirements of each type of wine, according to the oenological goal set by the winemakers and their focus on the market where they are selling . Indeed,Worldwide, Amarone is considered one of the most important Italian wines . Amarone is produced from three native varieties—Corvina, Corvinone, and Rondinella ; however, withered Corvina grapes are still the main variety used to craft this wine [The grape drying process involves two simultaneous transfer phenomena: (i) heat (energy) transferred from the environment to the bunches and (ii) water (mass) transfer from the inside to the outer surface of the berries, followed by evaporation. The drying conditions influence the properties of the resulting grapes and wines; slow drying at a low temperature (T) and aeration rate (W) and higher relative humidity levels (RH) provides more harmonious wines  , whereasThe most marked effect occurs on sugars. Their increase is mainly due to a concentration effect; nevertheless, the composition and nature of the sugars change throughout the drying process, particularly in terms of the glucose/fructose ratio variations . The chatrans-resveratrol, viniferins, cis- and trans-piceid) [Alongside the primary metabolism compounds, the secondary metabolites, mainly present in the hypodermal layer of the berry skin and that affect pigmentation, flavour and aroma of the wine, also need to be considered ,14. The -piceid) .trans-stilbene) has been shown to block LMP2A, a viral protein-tyrosine kinase associated with leukaemia, non-Hodgkin’s lymphoma and other diseases associated with the EBV virus, which also acts on human melanoma cells [13-norisoprenoids, terpenes, benzenoids, furans and aliphatic alcohols [Likewise, piceatannol  , yieldinGiven these circumstances, the choice between a controlled (modern) or non-controlled  system should be pondered by winemakers, since it can hardly influence the berry composition and final wine profile. Yet the withering method and its effects on the above-mentioned berries and wines composition is not well studied and need to be clarified in order to give precise information to winemakers. However, these issues will also be discussed by local authorities. Hence, the primary purpose of this work is to compare natural and artificial Corvina withering processes to assess their effect on dried grape chemical composition and peculiar wine organoleptic evaluations. This aspect is particularly interesting for the few wineries that still adopt the traditional drying synthesis to pursue a true-to-varietal character and vintage-related wine.The experimental data were collected during 2016 and 2017. The vineyard was selected in the Valpolicella classic area , 10.943691 East (E)), Bertani Winery. The location climate is typical of a temperate area located in the Northeast of Italy, with warm summers and cool winters. Annual rainfall is approximately 850 mm, concentrated mainly in spring and autumn. As for temperatures, during the growing season (April–September), the average is about 20–21 °C. Indeed, concerning the vineyard, the vine training system adopted was Guyot with a plant density of 4444 vines per hectare; the variety raised was Corvina, clone ISV-CV48 grafted onto Kober 5 bb, grown predominantly in calcareous soil. Corvina was chosen as it is commonly the main variety for Amarone wine production. Harvest, following local tradition, was scheduled to take place ten days before the standard harvest period for Valpolicella fresh wine, occurring on 17 September in 2016 and on 6 September in 2017. In order to preserve their healthy status, the carefully selected grapes were placed in plastic trays—in the case of the modern warehouse drying process—and in the traditional “arelle” (wooden trays) to wither the grapes under natural conditions. The two withering processes were carried out in two different rooms inside the winery:(A) For the modern warehouses (C process), the appropriate use of an air dehumidifier machine  maintained the air humidity inside the room between 60% and 80% , and fa(B) Instead, a traditional dehydration process (NC) took place without any air humidity control system or mechanical airflow: the warehouse supervisor was in charge of opening  or closing  the windows, to enable the high-moisture air to escape and the dry, fresh air to flow inside the drying chamber . In the Berry chemical composition was analysed at four different stages: (i) T0 fresh berry, (ii) −10% of berry water loss (WL), (iii) −20% WL and (iv) −30% WL, as described in The climatic conditions in both environments, C and NC, were measured. Specifically, temperature and humidity were measured using air sensors , Italy) located in each environment of the study: four sensors per drying chamber, two in contact with the withering grapes and two 50 cm above the grapes. The daily values recorded were the average of the measurements collected every 15 min. The outdoor climatic conditions were measured by the weather station belonging to Avepa (Avepa.it) located in the Negrar area. In 2016, due to contingent technical problems during data download, the initial climatic records (first ten days) collected in the withering chamber were lost.The kinetics of the withering process was evaluated by measuring the post-harvest weight loss; in the NC and C treatments, values are expressed as the percentage (%) of weight loss during withering. Specifically, data were taken by weighing two arella trays of about 250 kg of grapes in the case of the natural environment treatment, and two other pallets of trays (around 200 kg of grapes) with regard to the controlled environment. The arella trays and the pallets were chosen from two representative positions within the drying chambers. As a final consideration, we report that these values do not come from replicates, since they represent the whole weight of a tray (with hundred bunches), only distinguished in C and NC.For both drying processes (C and NC), at harvest time (T0) and during withering , three samples of grapes weighing 1.0 kg each were collected to determine the berry chemical composition. The samples were obtained from several portions of the grape bunches stored on the selected trays and pellets. Samples were taken every 4 days, thus enabling us to understand when the different WL stages were reached.The total soluble sugars in the berries were quantified using an ATAGO PR-32 digital refractometer , Italy) (0–32%) and expressed in Total Soluble Sugars (TSS), Brix degree; in the same samples, the organic acid profile of the berries  was determined by high-pressure liquid chromatography . The samples for HPLC were prepared by taking 250 µL of grape must diluted 1:50 with distilled water. The sample was then filtered through a 0.2 μm filter and analysed. The grape must samples were prepared by pressing three subsamples of 250 g of berries. Following the method described by Di Stefano , in 30-b®-Werke GmbH & Co. KG, Staufen, Germany) and centrifuged. The volume of the supernatant was adjusted to 250 mL by addition of distilled water, and the solution was treated with 2 g of insoluble poly(vinylpyrrolidone) (PVP) to reduce the polyphenolic content. Additionally, the pulp was homogenised and centrifuged, and the volume was adjusted to 250 mL. The solutions were treated with 75 mg of pectolytic enzyme for 4 h at room temperature to release the volatile compounds. A volume of 200 μL 1-heptanol 250 mg/L solution was added to the extract as internal standard, and the aglycones were isolated by solid-phase extraction using a 10 g C18 cartridge  previously activated by successive passages of 30 mL dichloromethane, 30 mL methanol, and 30 mL water. Salts, sugars, and other polar compounds were removed by washing the cartridge with 50 mL of water, and the fraction containing the free compounds was recovered using 50 mL of dichloromethane. The solution was concentrated to 2–3 mL by distillation using a 40 cm length Vigreux column then to 200 μL under a nitrogen flow prior to performing GC/MS analysis.The aglycones liberated from glycoside-bound aroma precursors were analysed by gas chromatography/mass spectrometry  after performing enzymatic hydrolysis as previously described ,35,36. TGas chromatography/mass spectrometry (GC/MS) analysis was performed using a 6850-gas chromatography system by Agilent Technologies , fitted with a fused silica HP-INNOWax polyethylene glycol capillary column  , coupled with HP 5975C mass spectrometer and 7693A automatic liquid sampler injector . Compound identification was performed using the NIST Mass Spectral Libraries Database (rev08) and the in-house CREA-VE database. To perform a comparison among the samples the contents of volatile compounds were expressed as μg of internal standard per kg of dried grape (d.g.).® and kept under stirring for 20 min in the dark at room temperature. The extract was centrifuged, and to the supernatant of 200 μL of trans-4-hydroxystilbene, a 9.6 mg/L solution in methanol was added as internal standard. The solution was evaporated to dryness under vacuum at 40 °C, and the residue was suspended in 10 mL of HCl 10−3 M in water. After the addition of 5 g of NaCl, the solution was extracted to 3 × 5 mL with ethyl acetate. Ethyl acetate extract was evaporated to dryness under vacuum at 40 °C and reconstituted with 4 mL of methanol/aqueous H3PO4 5 × 10−3 M 30:70 (v/v) solution. Finally, the sample was filtered through a 0.22 μm PTFE filter and analysed by HPLC using a 1220 Infinity G4290B pump coupled with a Gilson 170 G1315A DAD-UV detector  equipped with an RP C18 column . Elution was performed using a binary solvent composed of A) methanol and B) H3PO4 5 × 10−3 M in water and the following gradient program: from 25% to 30% of A in 30 min, from 30% to 35% of A in 20 min, from 35% to 75% of A in 10 min, from 75% to 85% of A in 15, from 85% to 25% of A in 5 min, and isocratic for 20 min . trans-Piceid, trans-resveratrol, trans-piceatannol, trans-εε-viniferin and δ-viniferin were quantified by recording the chromatograms at 307 nm, cis-piceid at 285 nm. The UV–Vis spectra in the 200–400 nm range were also recorded. Standard of trans-resveratrol, piceatannol, trans-piceid and trans-4-hydroxystilbene were purchased from Sigma–Aldrich ; δ-viniferin was provided by CT Chrom . cis-Piceid was produced by photoisomerisation of trans isomer; trans-ε-viniferin was extracted from a lignified vine cane of Gamaret following the method described by Pezet et al. [For our study, only visually healthy grapes, including berries affected by little or no stilbene-oxidase activity caused by  botrytis infections were collected. Consequently, differences observed in the two drying processes were mainly related to the different dehydration conditions. Sample preparation for High-Pressure Liquid Chromatography—diode array detector (HPLC/DAD) analysis was performed using the methods already described but modified to our goals ,38. The t et al. .Qualitative profiles of stilbenes were characterised by using an Ultra-High Performance Liquid Chromatography (UHPLC) Agilent 1290 Infinity system coupled to Agilent 1290 Infinity Autosampler (G4226A) and Agilent 6540 accurate-mass Quadrupole-Time of Flight (QTOF) Mass Spectrometer  equipped with Dual Agilent Jet Stream Ionization source . Analyses were performed by using the methods and analytical conditions previously described .®, Oenofrance, Montebello Vicentino, Italy), 5 g/hl of ascorbic acid and 0.5 g/t of tannin. Afterwards, a sample of must was picked up and inoculated with 20 g/hl of selected yeast , with the addition of 2 g/t of thiamine and vitamins (Oenofrance), 2 g/t of yeast extracts rich in amino acids (Oenofrance) and 2 g/t of inactivated yeast and cellulose (Oenofrance) and then re-mixed incorporating it in the fermenter. After 48 h from the inoculation, to favour malolactic fermentation, 1 g/hl of Oenococcus oeni bacteria  was added. Once a determinate alcoholic level had been reached in the must , a high-alcohol-tolerant yeast at a concentration of 30 g/hl was added , also adding 15 g/hl of mineral nutrients . The use of this second strain of yeast is necessary to reinforce the fermentation under high alcoholic level. At the end of the fermentation, a soft pressing of grapes was performed and 5 g/hl of tannin , 5 g/hl of potassium metabisulfite, 15 g/hl of yeast hulls  and 10 g/hl of nutrients  was added. After having decanted the wine for 3 days, the malolactic fermentation was favoured bringing the must at 21 °C. At the end of the malolactic fermentation, another decanting was performed, and 2, 5 g/hl of enzyme  was added. The temperature was reduced to 10 °C, and for four weeks a batonnage was performed twice per week. At the end of the batonnage, another decanting operation was performed, and the same temperature was kept for two weeks. At the end of this period, the wine was filtered with cardboard filters, stabilised with a stabiliser , and brought to −5 °C for 3 weeks. After that, the wine was filtered again with a, firstly, 1 µm, and secondly, 0.45 µm, candle filter and bottled.The winemaking was made via micro-vinification carried out at the vinification centre of the cooperative nurseries of Rauscedo (VCR), where about 150/180 kg of dried grapes were used for each vinification obtaining about 60 L of wine, as previously described . The vinp-values < 0.0001 [The tasting analysis was carried out in Conegliano (TV) at CREA-VE by a test panel made up of 10 specialist judges trained properly plus a panel leader. To test and to confirm the reliability and accordance of the judges, a periodic training among the participants to the panel test was made following precise criteria reported in the literature ,44. For < 0.0001 ). The 20p ≤ 0.05).One-way analysis of variance was performed using “R” freeware. Statistical analysis to determine significant differences between treatment means was carried out using the Student-Newman-Keuls test  were also taken into account to understand the difference between treatments inside and outside the chambers. In 2016 , the C cAs for 2017 , the aveAs previously reported, T and RH are fundamental for the kinetics of WL. Hence, measurements of the kinetics of withering were taken and are indicated as a percentage of weight loss during the period of withering, starting from time 0 (grape set-aside) to 30% WL . 2016 evDuring withering, generally, a concentration of sugars occurs due to the berry water loss. The analysis of the two tested years reported a similar trend . In 2016Because of water evaporation and WL, acids tended to accumulate during withering. The analysis of the two tested years reported a similar accumulation trend in terms of total acidity . In 2016p ≤ 0.05). In 2017, the trend was again similar in C and NC, but the differences became highly significant starting from the 10% WL stage. As for anthocyanins concentration, in 2016, there were no differences during the initial stage (−10% WL), whilst at the end of the drying period, NC grapes had 11% more anthocyanins than C grapes . In the second year (2017), there was a considerably higher and more meaningful content in NC than C at every stage of WL .Different climate conditions inside the withering chambers may affect the berry pigment compounds. At the end of the process, the content in total polyphenols consistently predominated in NC in the two tested years of study . In bothO-monoglucoside is the main compound. No relevant differences between the two processes in the qualitative profile of anthocyanins were observed (data not shown).The anthocyanin profile of Corvina is constituted by malvidin, peonidin, petunidin, delphinidin and cyanidin monoglucosides and their acyl derivatives, where malvidin-3-trans-resveratrol, cis and trans-piceid, piceatannol, E and Z astringin, pallidol, E and Z ε-viniferin, Z-ω-viniferin, δ-viniferin, a resveratrol dimer, caraphenol, pallidol-glucoside, α-viniferin, E and Z miyabenol C and two resveratrol-tetramers . Nineteen compounds were identified; they include etramers . Stilbentrans-resveratrol and total stilbenes were observed throughout both withering processes. In particular, until the −20% WL stage, stilbenes were higher in the NC samples. In the last stage (between 20–30% WL), the level of stilbenes in NC samples remained constant, while in the C samples, it continued to increase, reaching values comparable to those in the other treatment. In the latter year, the trend was different: at −10% WL, both processes induced a low stilbene increase, and the levels remained similar until the −20% WL stage. A sharp increase in the synthesis of stilbenes was observed during the last stage in both processes, as well as higher accumulation in the C samples.In 2016, a constant increase in Glycoside aroma precursors identified in Corvina grape belong to different chemical classes of compounds, and their composition in the grape berry expressed as μg IS/kg d.g., is reported in E and Z, 2-butoxyethanol, and 2-hexenol. In 2016, C samples had higher levels of total aliphatic alcohols, but a significant difference (p ≤ 0.05) between C and NC samples was found only at the highest content at −20% WL stage. In 2017, the total content was higher in NC with a significant difference at −20% and −30% WL, and the highest aliphatic alcohols content was reached at −30% WL in both C and NC , acetophenone, methyl salicylate, guaiacol, benzyl alcohol, β-phenylethanol (rose), eugenol (clove), 4-vinylguaiacol, 4-vinylphenol, syringol, vanillin (vanilla), methyl vanillate, acetovanillone, 4-hydroxybenzeneethanol, vanillic and homovanillic alcohols . In 2016Moreover, regarding the total content of aromatic compounds at every stage, in 2016, at −10% and −20% WL, the content was almost the same in both C and NC, while at −30% WL, the content was higher in NC than in C, with a statistical difference between treatments. In 2017, there were more aromatic substances at every stage in NC than in C. Furthermore, at −20% and −30% WL, statistical differences were found between treatments, with the already-reported higher content in NC.Results of the tastings carried out in 2016 and 2017 are reported in As for the 2017 vintage, the NC wines confirmed the higher pleasantness with a peak in ripe fruit combined with spicy notes. Overall, the NC wine was more harmonious with good scores in terms of taste balance and finesse.The microclimatic analysis of the chambers used for tests confirmed a different air thermal and humid regime in the two situations: lower temperatures and higher humidity in NC, higher temperatures, and lower humidity in C . FurtherPlant phenolics have important effects on food quality and human nutrition. Their presence in grapes and red wine may contribute to health benefits because of their antioxidant and anticarcinogenic activities. Hence, the biosynthesis of antioxidant molecules such as anthocyanins and stilbenes represent a defence mechanism for the plant cells, used as strategy for reducing the oxidative cells damage resulting from the drought stress. Moreover, these molecules have a beneficial impact on humans that consume them through drinking the wine, because they have similar antioxidant roles in human cells. For this, the interest in the physiological role of the bioactive compounds present in plants products has increased dramatically over the last decade ,19,49. FRegarding the berry chemical composition , at the Measurements taken included acidity  and its Drying process induces specific chemical changes inside the berry, and most of them involve secondary metabolites . Previously observed drought-induced compositional changes to the grapes were transferred to the wines, with an increase in polyphenols and VOCs. Moreover, in the berry, these changes are known to take place alongside transcriptome-wide reprogramming of metabolism pathways, involving all secondary metabolites ,60,61. FThe accumulation of polyphenols and anthocyanins evidenced an interesting trend showing an initial positive step linked to a new polyphenol and anthocyanin synthesis  followedRegarding stilbenes, several studies have demonstrated that osmotic stress occurring during withering, which is induced by water loss and the higher temperatures of the chamber (30 °C), stimulates stilbenes biosynthesis ,65,66,67In the glycoside aroma precursors profile, aliphatic alcohols that were found to be increased by withering in both years were butanol, 3-methyl-1-butanol, hexanol and 2-hexenol . In geneC13-Norisoprenoids contribute to the aromas of the Amarone with notes of ripe fruit, honey, jam, tea and tobacco. Here, the glycoside derivatives of 3-hydroxy-β-damascenone, 3-oxo-α-ionol and vomifoliol had the highest concentrations in NC.The primary glycoside benzenoids  found are methyl salicylate, guaiacol, benzyl alcohol, β-phenylethanol, syringol, vanillin and vanillic alcohol. In both the dehydration processes, the highest amount of benzenoids in the berry was found at −30% WL, particularly benzyl alcohol, guaiacol, syringol, vanillin, acetovanillone and vanillic alcohol. Specifically, NC treatment evidenced the highest quantity in benzenoids due to the high level in benzyl alcohol, guaiacol, syringol, vanillin, acetovanillone, eugenol and vanillic alcohol  and togep ≤ 0.05 and p ≤ 0.001 in 2016 and 2017, respectively) with higher contents in NC samples (data not shown). The low benzaldehyde level found in the samples compared to benzyl alcohol  indicates that low or no oxidative processes occurred due to Botrytis cinerea infection [The general trend of the content in 4-vinylguaiacol at −30% WL  and a greater structure (given by the whole composition), which is also accompanied by greater finesse, elegance, and olfactory richness. Overall, it was a more harmonious, structured and smooth wine (less tannicity) than C, where the unpleasant herbaceous notes tended to be higher. Similar outcomes confirming our research are reported in the literature ,74,75.In general, the drying process carried out in NC conditions was slower than in C due to higher RH, lower T and no air-forced movement in the NC environment. This slower process led to a major accumulation of sugars, acids, polyphenols and anthocyanins in NC. It is assumed that the slower process in NC was able to maintain vital cell structures for longer, with less water stress effects (lesser synthesis of stilbenes can confirm this observation). In terms of total aroma compounds, we found a higher content in NC, also confirmed by the better wine profile of these wines. Finally, to reach a considerable content of compounds and obtain balanced wines, we can report that it is desirable to reach a −30% WL and adopt NC conditions. Lastly, the sensory evaluation of wines highlighted the positive sensory descriptors present in the wines produced adopting the non-controlled process.The present research confirmed that slowing down the drying process and creating less stressful conditions for the cells could be a strategic option to achieve a more traditional and well-appreciated Amarone wine and that choosing the traditional drying process carried out in natural conditions could also improve overall market appreciation.


Graphene-based nanocomposites possess excellent mechanical, electrical, thermal, optical, and chemical properties. These materials have potential applications in high-performance transistors, biomedical systems, sensors, and solar cells. This paper presents a critical review of the recent developments in graphene-based nanocomposite research, exploring synthesis methods, characterizations, mechanical properties, and thermal properties. Emphasis is placed on characterization techniques and mechanical properties with detailed examples from recent literature. The importance of characterization techniques including Raman spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and high-resolution transmission electron microscopy (HRTEM) for the characterization of graphene flakes and their composites were thoroughly discussed. Finally, the effect of graphene even at very low loadings on the mechanical properties of the composite matrix was extensively reviewed. This paper provides a critical review of the synthesis, properties and characterizations perspectives of recent advances in graphene-based nanocomposites. A critical review of the characterization of graphene and graphene nanocomposites was presented in 2, sp3): zero-dimensional sp2 fullerenes, the two-dimensional sp2 honeycomb lattice of graphene, or three-dimensional sp3 crystals—diamond [2 ~graphitelike to sp3 ~diamondlike bonds [2-bonded carbons with various degrees of graphitic ordering, ranging from microcrystalline graphite to glassy carbon. Accordingly, these materials have been greatly investigated because of the exceptional mechanical and electronic properties.Carbon has several allotropes, which can be classified according to the type of chemical bond related with hybridization  have been successfully used in many applications including in smart phones, ultra-thin flexible displays [Since its discovery in 2004, graphene has become the center of many research activities ,28,29,30displays , hydrogedisplays , transpadisplays , chemicadisplays ,35, biosdisplays ,37,38, adisplays . It has Graphene reveals remarkable optical properties, which makes it very promising for photonic and optoelectronic applications ,40,41. IGraphene has great prospects for industrial applications, such as polymer composites, conductive coatings, fuel cells batteries, and ultracapacitors due to its distinctive properties of high strength and exemplary electrical and thermal conductivity ,3. TheseThere are several approaches available to produce graphene . These tMost of the graphene produced for research over the past decade was fabricated by mechanical exfoliation (scotch tape method) ,52,53. ILiquid-phase exfoliation (LPE) b is one The CVD method is widely used for the synthesis of carbon nanostructures (CNTs) for composite materials with outstanding mechanical properties ,57,58. FResearch on polymer nanocomposites (PNC) has been growing over the past decade due to their remarkable material properties, yield strength, toughness, electrical conductivity, thermal conductivity, and optical properties, and their applications are growing substantially ,78,79,80Graphene possesses many desirable properties such as high strength and elastic modulus, high electrical and thermal conductivity, high aspect ratio, high thermal stability, high gas impermeability, and good dimensional stability ,10,11,12Graphene can be produced in large quantities from graphite precursor by oxidation. Hence, graphene-based polymer nanocomposites have attracted considerable research interest around the globe. Various polymers, such as epoxy ,86,87,88Graphene-based nanocomposites are increasingly being used for the development of new materials for alternative energy sources, for example, (a) in lithium-ion batteries, graphene-based nanocomposites show better performance as they have high power density and energy density and a fast charging speed in hydrogen fuel cells; (b) graphene is used as an electrode material to enhance electrocatalytic activity; (c) in solar cells, graphene-based composites are used in photovoltaic devices because of their unique characteristics of high carrier mobility and low resistivity; and (d) in thermoelectric materials. Characterization of nanocomposites is crucial to understand the basic physical and chemical properties of the nanocomposites. Graphene materials available in the market are made by different companies through different techniques. It is expected that these graphenes are quite different from each other in flake width, thickness, and defect concentration. All the different techniques to modify the filler surface as well as to synthesize the polymer nanocomposites need to be supplemented with robust characterization of these processes as well as resulting composite properties to gain insight into the various factors affecting the nanocomposite microstructure. Several techniques have been used in the characterization of nanocomposites, dispersion, distribution, and orientation within polymer matrix. These techniques include optical microscopy, scanning electron microscopy, high transmission electron microscopy, Raman spectroscopy, atomic force microscopy and X-ray diffraction; these have been shown to be very useful for quantification of nanocomposites. It is also, in many instances, necessary to employ more than one characterization technique in order to accurately characterize the nanocomposite material.Nanocomposites contain matrices of diverse materials such as polymer, metal or ceramic, and also include different nanoparticle fillers . These fillers enhance the mechanical, thermal and electrical properties of the material ,111,112.Depending upon the degree of dispersion of the nano-sized layer structure, polymer composites can be divided into three main categories: microcomposites, intercalated nanocomposites, and exfoliated nanocomposites ,113,114.The properties of graphene nanocomposites are dependent on chemical compatibility between the filler and the matrix, the volume fraction of the filler and the processing conditions such as dispersion and exfoliation of filler. To achieve optimal results, appropriate fabrication methods must be employed ,60. MoreIn situ polymerization is an effective technique for the formulation of highly dispersed graphene in a polymer matrix. This process involves the polymerization of monomer in the presence of the layered materials a. DuringMelt blending is the preferred method for the synthesis of various polymer hybrids with inorganic nanoparticles . It is fThe most common method of synthesizing polymer nanocomposites is melt intercalation or melt blending ,117,119.A more effective way to achieve interfacial adhesion between the filler and matrix is exfoliation adsorption c 114,12,123,124.2/g, high mobility (15000 cm2/V·s) [Graphene is a material with outstanding properties, such as high specific surface area 2600 mcm2/V·s) ,15, supecm2/V·s) , and extcm2/V·s) ,129,130,cm2/V·s) ,133,134.Raman spectroscopy is a standard nondestructive tool for the characterization of crystalline, nanocrystalline, and amorphous carbons ,150,151.−1), G band (~1580 cm−1), and 2D band (~2700 cm−1), were observed for these two graphitic materials. The intensity ratio of D/G (ID/IG) for the prepared graphene was 0.10, lower than that of ultrasonication exfoliated graphene (0.29) [Zhiliang et al.  demonstre (0.29) .60/rGO samples were characterized by Raman spectra. 60, and C60 graphene. The two intense peaks in rGO are designated to the D and G band. The G band and D band are a result of the presence of defects that have been introduced throughout the oxidization and reduction technique. The G band has moved to 1582 cm−1 in the C60/graphene hybrid, indicating the effect of graphene on C60.Zhang et al.  carried −1) emerged as the most important feature of the Raman spectra. In addition, the double 2D band considerably weakened, while the G band around was broadened due to the presence of a defect.Chemical functionalization of graphene (CFG) enables graphene to be processed by solvent assisted techniques, such as layer-by-layer assembly, spin-coating, and filtration. It also prevents the agglomeration of single-layer graphene during reduction and maintains the inherent properties of graphene ,157,158.Atomic force microscopy (AFM) is one of the most powerful microscopy techniques for studying samples at nanoscale. AFM is a type of scanning probe microscopy (SPM), with a resolution on the order of fractions of a nanometer . For exaReduced graphene oxides (rGO) are single-layered sheets derived from the chemical reduction of graphene oxide (GO). The rGO possess electrical and mechanical properties similar to those of graphene; thus, this makes them versatile for a number of applications. Various synthetic routes have been reported for the synthesis of reduced graphene oxide ,167,168.Gurunathan et al., demonstrated the synthesis of water-soluble graphene through reduction of GO using bacterial biomass . The proGraphene fabricated by different techniques contains many defects depends on the process. Defects in graphene have negative impacts on the high mobility and other physical and mechanical properties of graphene. Accordingly, it is essential to characterize these defects using SEM or other advanced microscopy techniques. SEM is an advanced microscopy instrument in characterization of micro- and nanostructured materials. Along with its high power to offer a 2D analysis of substantial aspects of the sample, SEM can also provide information and varied qualitative data on several physical properties such as roughness, morphology, surface consistency, size, and chemical structure of materials. Nano scale features of graphene including wrinkles, grain shapes, and folding lines can be specifically characterized utilizing the SEM ,162.Many studies demonstrated that the state of dispersion of graphene sheets in the matrix has a strong effect on the mechanical properties of the composite ,168,169.2 detection [Graphene is quite a robust material in hydrogen sensors due to a possible improvement of its surface area and susceptibility of its electronic properties to the changes caused by adsorbing atoms and molecules including hydrogen. However, the pristine graphene sensitiveness to hydrogen is limited . Sharma etection . Figure HRTEM is a very powerful structure characterization technique for graphene. It is a unique tool for characterizing the atomic structures and interfaces of graphene ,171. It 2 carbon atoms of the π network into a sp3 configuration, resulting in adjustments of the innate physical and chemical properties of graphene [Due to strong interactions and van der Waals forces, exfoliated graphene sheets have a strong tendency to irreversibly aggregate or even restack, reverting to multilayer structures such as graphite . Therefographene .2O3–graphene structure, namely sheet-on-sheet nanostructure by a solvothermal method. Fe2O3 nanoparticles were also prepared on graphene nanosheets. The Fe2O3–graphene sheet-on-sheet nanostructure was fabricated as an anode for Li-ion batteries. It was confirmed that Fe2O3 nanosheets were uniformly dispersed among graphene nanosheets, forming a unique sheet-on-sheet nanostructure. HRTEM of the morphological analysis of Fe2O3–graphene nanocomposites is shown in 2O3 nanoparticles are shown in this figure. Their particle sizes are 30–50 nm size. The particle-on-sheet and sheet-on-sheet nanostructures can be easily confirmed by the HRTEM images of The lithium-ion battery is considered to be one of the best power sources to maximize the efficiency of energy use. Graphite has been widely used as an anode material for commercial Li-ion batteries because of its good electrochemical properties. Kan et al.  prepared2 carbon materials [XRD is an important analytical tool for the characterization of the intercalated and exfoliated nanocomposites ,175,176.aterials ,150.The application of X-ray diffraction (XRD) for phase determination and confirming the reduction of GO to graphene is a very useful tool. Of course, it is not as powerful as the other approaches  due to the limited information collected. Therefore, its application is always accompanied by other methods such as Raman, XPS, and FTIR to provide more information about the catalyst structure.60) had been effectively integrated into the surface of graphene.Polymer-based thermoelectric materials have been the focus of many studies for the past 10 years ,180,181.Chieng et al.  preparedGraphene-based nanocomposite materials exhibit significant improvement in mechanical properties compared to the standard composites ,186,187.Most of the properties of the nanocomposites were superior compared to the polymer matrix ,210,211.Pinto et al.  investigKing et al.  fabricatLi et al.  prepared1/2 with a graphene loading of 0.5 wt.%, suggesting major improvements due to graphene. The microhardness indentations and radial cracks on the polished surface of 58S-0.5 were characterized by SEM to identify the mechanism responsible for the enhanced mechanical properties  can be produced via the rapid heating of GO under inert gas and high temperature (1000 °C) . TRG is Nanoindentation is a widely used technique for measuring the nano and microscale mechanical properties in nanomaterials and nanocomposites. This technique has been commonly used in determining the mechanical properties of polymers ,207,208.−1. The fracture strength that signifies the intrinsic strength (σint) of the sheet was ~42 N m−1. The corresponding values for the bulk graphite were E = 1.0 TPa and σint = 130 GPa.Shen et al.  used nanAll electronic units produce excessive heat and thus demand thermal management to prevent premature failure ,219,220.−1K−1) compared to copper (400 Wm−1K−1). The thermal properties of graphene have become an important research topic and are attracting tremendous interest in the area of thermoelectric waste heat recovery. Thermal properties of graphene are related to its low mass and the strong bond of carbon atoms. Thermal properties of graphene and its use in electronics and thermal management applications have been the subject of many scientific studies [Graphene has drawn tremendous attention for heat dissipation due to its extraordinarily high in-plane thermal conductivity  was a very effective nanofiller for epoxy composites. They reported a remarkable increase in thermal conductivity (3000%) at 25 vol% loading. They attributed the remarkable increase in thermal properties of this composite to the optimum combination of the high aspect ratio, stiffness, and low thermal interface resistance of the GNPs.Yu et al. prepared graphite nanoplatelets (GNPs) by thermally exfoliating graphite . ResultsZhang et al.  fabricat3O4 nanoparticles allowed them to align the fillers in an external magnetic field during dispersion of the thermal paste to the connecting surfaces [The variation in thermal conductivity with different forms of graphene and graphite nanocomposites is reviewed in surfaces . The filThis article reviewed the recent progress in graphene-based polymer nanocomposites, focusing on two areas: properties and characterization. Emphasis was placed on the unique roles and advantages of advanced graphene characterization techniques, such as Raman spectroscopy, XRD, AFM, and HRTEM. Characterization of 2D graphene involves different types of microscopic and spectroscopic techniques to obtain the structural, morphological, and chemical information of as-synthesized graphene. Raman spectroscopy is a remarkable analytical tool that can detect small changes in the structural morphology of carbon nanomaterials. It is used to determine the number of layers, defects, strain, and chemical modifications. HRTEM is one of the most powerful characterization techniques for graphene’s structural characterizations. Several examples of graphene polymer composite characterizations using Raman, SEN, TEM, AFM, XRD, and HRTEM were discussed.Most common graphene synthesis techniques have been discussed. The mechanical exfoliation of graphene is a simple technique for graphene fabrication. The process has the ability to fabricate a single crystal of graphene with various numbers of layers. However, control over the wafer scale synthesis and reproducibility is very difficult. Liquid-phase exfoliation is one of the most practical methods for commercial manufacturing of graphene because of its low cost and potential scalability. Graphene prepared by this technique exhibited a low percentage of defects and oxygen functional groups.Chemical synthesis techniques are suitable for low volume production of graphene at low temperature. Furthermore, the process can yield graphene film or graphene coatings on several substrate materials. Graphene synthesized using chemical processes exhibits a high surface area and easy functionalization. CVD is widely used for the synthesis of carbon nanostructures. This method has been effectively used for producing carbon filaments which are the basis for composite materials with outstanding mechanical properties.Graphene–polymer nanocomposites display remarkable mechanical properties compared to the pure polymer. −1K−1). Graphene and MLG, produced by the liquid-phase exfoliation technique, were successfully used to fabricate novel nanocomposites to be used as TIM in electronic applications. Remarkable enhancement of thermal conductivity of the epoxy matrix (increase by 23×) was achieved at the 10% volume of graphene loading. Graphene and xGnP are potential key materials for the next generations of ICs and 3D electronics. Graphene chemical functionalization caused outstanding improvement in the thermal conductivity of the polymer composites. Thermal conductivity is affected by several factors, such as the graphene loading, dispersion, and the thermal resistance of the interface.Graphene and xGnP have attracted tremendous attention for heat removal due to their extraordinarily high in-plane thermal conductivity (2000~4000 Wm


Despite an increase in the prevalence of sleep problems, few studies have investigated changes in the prescribing of drugs that are often used to manage insomnia.To explore changes in the pattern of benzodiazepine (BZD), Z-drug , and non-BZD prescriptions.Open-cohort study comprising 1 773 525 patients (55 903 294 consultations) who attended one of 404 Australian general practices at least three times in two consecutive years between 2011 and 2018.Data were extracted from MedicineInsight, a database of 662 general practices in Australia. Prescription rates per 1000 consultations, the proportion of repeat prescriptions above recommendations, and the proportion of prescriptions for patients with a recent (within 2 years) recorded diagnosis of insomnia were analysed using adjusted regression models.Rates of BZD, Z-drug, and non-BZD prescriptions were 56.6, 4.4, and 15.5 per 1000 consultations in 2011 and 41.8, 3.5, and 21.5 per 1000 consultations in 2018, respectively. Over the whole study period, temazepam represented 25.3% of the prescriptions and diazepam 21.9%. All BZD and zolpidem prescriptions declined over the whole study period , but non-BZD and zopiclone prescriptions increased in the same period . Repeat prescriptions that exceeded recommended levels remained at <10% for all medications, except melatonin (64.5%), zolpidem (63.3%), zopiclone (31.4%), and alprazolam (13.3%). In 2018, >50% of Z-drug and melatonin prescriptions were for patients with insomnia. There was an annual increase of 0.8–5.9% in the proportion of prescriptions associated with a recently recorded diagnosis of insomnia.Overall, BZD prescriptions in Australia declined between 2011 and 2018. However, the prescription of some of these drugs increased for patients with a recently recorded diagnosis of insomnia. This is concerning because of the potential adverse effects of these medications and the risk of dependence. For example, studies using prescribing data demonstrated that BZD prescribing prevalence among adults ranged from 2.2% of all prescriptions written by GP registrars in Australia (2011–2013)13 and Sweden (2006–2013),21 but has declined in Canada ,19 Spain ,20 South Korea ,22 Ireland ,23 and England .24 Moreover, an upsurge in the prescription of Z-drugs and other non-BZDs has been reported in the last decade in Canada,19,25 Ireland,26 and other European countries.24,27 In Norway, Z-drugs have become the most prescribed medications for insomnia management.28 Although BZD use has been linked with adverse health outcomes, Z-drugs were thought to have a good safety profile due to a shorter half-life and lower residual drowsiness;29 however, long-term use of Z-drugs is also associated with negative consequences such as risk of dependence, falls, and fractures.10–11,30–31The prescribing and dispensing of BZDs and other related drugs has increased in high-income countries, such as the US (1996–2013)17,32–33 In the US, data from the National Health and Nutrition Examination Survey (1999–2010) revealed that 3% of adults used prescribed medications for insomnia management.34 In Australia, a cross-sectional analysis of data from 645 Australian GP trainees (2010–2013) showed that BZDs constituted 2.2% of all prescriptions, and 28.2% of BZD prescriptions were associated with a diagnosis of insomnia.17Evidence about drug prescribing for insomnia usually comes from small surveys or studies using data on drug dispensing.Using data from MedicineInsight, a national general practice database in Australia, the study presented here aimed to explore current trends in the prescription of BZDs, Z-drugs, and other non-BZD medications usually prescribed for insomnia management. In addition, it investigated whether repeat prescriptions for these drugs exceeded Australian recommendations, as well as the proportion of these prescriptions that were for patients with a recently (within 2 years) recorded diagnosis of insomnia.35 Australia has a universal healthcare system called Medicare, which is funded by the Australian Government through taxation revenue; it covers GP visits , hospitalisations, and most medication costs  for Australian nationals, permanent residents, and people from countries with reciprocal healthcare agreements. Most general practices are privately owned and operated, but the cost of GP consultations in all practices are fully or partially covered by Medicare rebates for clinical services.36 The characteristics of practices recruited by MedicineInsight are reflective of all Australian practices.35This open-cohort study used longitudinal de-identified data from MedicineInsight, a large-scale primary care Australian database containing electronic health records (EHRs) from approximately 2700 GPs and 662 general practices across Australia .MedicineInsight extracts de-identified EHRs from all patients attending these participating practices every month, including the exact date when associated diagnoses, reasons for encounter, reasons for prescription, prescribed medications, laboratory results, or clinical assessments were performed or recorded, as well as current data on sociodemographic characteristics. During data extraction, every patient registered with a participating practice receives a unique identification number that allows them to be tracked over time.35 as recorded on MedicineInsight between 1 January 2011 and 31 December 2018. Data were only kept for analysis if they were from practices with a consistent number of consultations over time — that is, a ratio <5 between the maximum and minimum number of consultations in the same practice between 2011 and 2018, with no gaps of more than 6 weeks in the previous 2 years in practice data. This strategy was used to reduce the risk of selection bias, as changes in patterns of prescribed medications could reflect abrupt changes in the number of patients in certain practices, rather than changes in prescribing.This study includes all patients of any age or sex who attended the practices on at least three visits in two consecutive years,Administrative contacts  and duplicated records were excluded — that is, only one consultation per day per patient was retained for analysis. The final sample consisted of 55 903 294 consultations of 1 773 525 patients regularly attending 404 general practices.In routine clinical practice, Australian GPs can record clinical data — that is, diagnosis, reason for encounter, reason for prescription — in a patient’s EHR using either precoded fields or free text that may include standard clinical terminology, spelling variations, and misspellings. Prescriptions are recorded using standardised terms. Data on patients’ demographics, clinical information, and prescribed medications were extracted from MedicineInsight using Stata MP (version 15.1).9 were extracted from the ‘script item’ dataset, which used either the active ingredient or commercial brand name.37Prescriptions and the number of repeats of 11 different BZDs approved for use in Australia , two Z-drugs (zolpidem and zopiclone), and four related non-BZDs  traditionally used for insomnia managementRecords of insomnia or sleep issues were extracted from the ‘diagnosis’, ‘reason for encounter’, and ‘reason for prescriptions’ datasets. Algorithms for data extraction included pre-coded terms, synonyms, and misspellings related to these diagnoses, but excluded obstructive sleep apnoea.more than one prescription for BZDs and Z-drugs;more than one prescription for melatonin;more than three prescriptions for amitriptyline; and9,37more than six prescriptions for mirtazapine or quetiapine.BZD, Z-drug, and related non-BZD prescription rates (per 1000 consultations) were estimated for each year from 2011 until 2018 inclusive. The repeated annual period prevalence was computed by taking the number of prescriptions  in a year as the numerator and the total number of consultations in the corresponding year as the denominator. The researchers then calculated the annual proportion of prescriptions that exceeded the number of repeats  recommended by the Pharmaceutical Benefits Scheme (PBS) guidelines, namely:More than one prescription was used for Z-drugs and melatonin, even though the PBS provides recommendations for zopiclone only (government subsidised). There are PBS guidelines for the drugs that are subsidised by the PBS scheme. Zopiclone is subsidised by the government; zoplidem and melatonin are not subsidised by the government. The authors followed the PBS recommendation regarding repeat prescription of zopiclone, and for zolpidem and melatonin as well.Finally, estimates were calculated for the annual proportion of BZD, Z-drug, and non-BZD prescriptions for patients with a recently recorded insomnia diagnosis — that is, prescriptions for patients for whom a diagnosis of insomnia was identified in the same year or the year preceding that prescription.Age- and sex-adjusted annual prescribing prevalence of BZDs, Z-drugs, and non-BZDs were estimated using logistic regression. Marginal adjusted rates per 1000 consultations were then computed and presented graphically. A similar approach was used to estimate changes in the proportion of repeat prescriptions that exceeded recommended limits. Logistic regression was also used to analyse the proportions of these medications that were prescribed to patients with a recently recorded diagnosis of insomnia (age and sex adjusted). The annual change in prescription rates, repeat prescriptions, and the prescriptions associated with insomnia diagnosis were estimated using Poisson regression.Sensitivity analyses were performed to investigate whether any increase in the proportion of prescriptions linked to a recently recorded diagnosis of insomnia was related to surveillance bias . For that purpose, analyses were repeated considering incident/first-time recorded prescriptions of BZDs and related drugs, incident/first-time recorded insomnia diagnosis, and only including EHRs from patients with recorded data for the whole period .All analyses were conducted using Stata/MP (version 15.1), considering the practice as a cluster and using robust standard errors.The sample included 1.7 million patients contributing to a total of 55 903 294 consultations between 2011 and 2018. Females attended 60.0% of all consultations; 40.6% of the consultations occurred among individuals aged ≥65 years, 50.3% were among patients aged 18–64 years, and 9.1% were for those aged <18 years. Missing data on any covariate represented <0.5% of the sample. Of all the consultations over the study period, 3 740 458 (6.7%) were linked to a BZD, Z-drug, or non-BZD prescription; 896 138 (24.0%) of the prescriptions were given to patients with a recent record of insomnia diagnosis.The most commonly prescribed drugs from those investigated over the whole period were temazepam (25.3%) and diazepam (21.9%), followed by amitriptyline (12.9%), oxazepam (10.9%), mirtazapine (6.8%), quetiapine (4.6%), nitrazepam (3.6%), melatonin (3.3%), and zolpidem .In this large, open-cohort study, the researchers explored trends in the prescribing of BZDs, Z-drugs, and non-BZDs using a nationwide sample of Australian general practices. Four main findings can be highlighted.19,22–24 BZD prescriptions have reduced over time, especially for nitrazepam and alprazolam; there has been a rise in the prescription of non-BZDs (melatonin in particular) and zopiclone, but not zolpidem. Nonetheless, temazepam and diazepam are still two or three times more likely to be prescribed than any of the other investigated medications, accounting for half of all BZD, Z-drug, and non-BZD prescriptions.Consistent with international trends,9,37More than half of medications not funded by the PBS (melatonin and zolpidem are not funded) were provided with one or more repeats, as well as a third of prescriptions for the PBS-funded zopiclone. The prescription of multiple repeats for alprazolam reduced dramatically in 2017; for all other drugs, <10% of prescriptions had repeats that exceeded the recommended levels.More than half of Z-drug or melatonin prescriptions in 2018 were for patients with a recently recorded diagnosis of insomnia (34 551 of 67 443 prescriptions for the three drugs combined). However, in absolute terms for each individual drug, temazepam (53 469 out of 141 994 prescriptions) had the highest number of prescriptions written for patients with a recently recorded insomnia diagnosis, followed by diazepam (30 062 out of 154 562 prescriptions).Finally, overall BZD prescribing reduced, but the prescription of some BZDs for insomnia increased over the study period.There are strengths to this study, namely its use of a large, longitudinal national general practice database of 8 years, and data being recorded by GPs, not self reported.32Some limitations should also be recognised. The indications for prescribing are not commonly recorded and, as such, conditions may not be directly linked with BZD prescribing. In addition, the completeness and accuracy of recorded information may vary between GPs. This study was also based on the number of written prescriptions, but it is not known whether the patient filled the prescription and used the medication; therefore, the estimations may represent an overestimation of actual medication use. Similarly, it has not been possible to account for patients visiting other practices and obtaining and using additional prescriptions for BZDs. However, the researchers believe that these factors would only affect a small proportion of patients because they used data of regular patients with three visits to the same practice in consecutive 2 years. Lastly, data can be recorded as free text and algorithms for data extraction may lead to under/overestimation (measurement bias); however, the findings presented here regarding trends are consistent with previous reports/papers.26 but higher than that for Canada .25 Although the burden and impact of mental health disorders are similar across these countries,38 study methodologies, different anti-BZD campaigns across countries, adherence to guidelines, and different healthcare-seeking behaviour may explain these prescribing discrepancies.27,39–40Prescribing rates were estimated using the total number of consultations as the denominator and are not directly comparable with other studies that used total population, person years, or other denominators. However, sensitivity analyses using patients as the denominator showed that the prevalence of BZD prescribing in Australian general practice in 2018 (89.3 per 1000 population) was lower than that for Ireland ,32,41 and other countries.22–23,25–27 In Europe, a decline in BZD prescription ranging from 4% to 26.5% was observed when analysing EHRs (2001–2009)39 or drug-dispensing data (2005–2015).26Overall, an annual decline of 4.2% in the prescription of BZDs was identified for the period 2011–2018. Similar declining trends were previously reported in Australia 27 It was reported that, in Australia, the introduction of the government-funded medication reviews programme in 2001 may have been responsible for an initial decline in BZD prescribing;42 however, according to the findings presented here, the release of new Australian guidelines9 in 2015 to address the concerns regarding BZD harms and misuse was not followed by any substantial change in trends.Campaigns to reduce long-term BZD prescriptions and use of Z-drugs in nine European countries showed limited efficacy, except when subsidised alternatives were made available, such as the availability of prolonged-released melatonin.37 The large reduction in multiple repeats for alprazolam could be attributed to its rescheduling in 2014 (addiction category), with further restrictions introduced in 2017 .43 Other Australian studies also reported decline in alprazolam use after the resheduling.44–45In general, there was a small increase in the proportion of BZDs prescribed, with repeat prescriptions exceeding recommended levels. Nonetheless, the proportion of prescriptions with multiple repeats remained at <10% for most BZDs and non-BZDs; this was expected, given that multiple repeats of these drugs are only allowed for people in certain situations or with specific conditions .25–27,46–47 However, in the study presented here, 45% of Z-drug prescriptions did not have any indication of recently recorded insomnia diagnosis. Similarly, a high proportion of prescriptions for BZDs or Z-drugs without any recorded indication were reported in a study using seven European EHR databases.39 Not all fields that may contain the diagnosis are extracted by MedicineInsight because of confidentiality issues; one example of this is progress notes. It is also possible that these medications were prescribed for other conditions associated with insomnia , or insomnia itself may have been recorded before the allowable window used in the study presented here.39 Further analysis showed that, in 2018, 76.6% of Z-drug prescriptions from the cohort in this study were for people with any record of insomnia, and 11.1% were for people who had any record of ill mental health between 2011 and 2018.Z-drugs are primarily indicated for insomnia, and their use has increased in high-income countries, including Australia.48 The proportion of prescriptions with multiple repeats for the government-subsidised zopiclone remained steady at ∼30%, whereas multiple prescriptions for the non-subsidised zolpidem increased from 50% to 63% between 2011 and 2018. The finding that more than half of non-PBS-funded medications  were provided with repeat prescriptions suggests long-term treatment. Although Australian guidelines suggest a similar level of caution when prescribing zopiclone and zolpidem,9 explicit recommendations seem necessary regarding repeated prescriptions for zolpidem. Z-drugs are not innocuous and are also associated with increased risk of dependency, drug tolerance, falls/fractures, and poor quality of life.10,11,30 Although melatonin is considered safer than BZDs or Z-drugs, its long-term effects have not been fully explored.49Overall, BZD prescriptions in this study cohort in Australia declined between 2011 and 2018, but the prescription of some BZDs for insomnia management has increased. Despite the observed decline, current BDZ prescribing rates are higher than expected, based on current recommendations. This perspective should be viewed in the context of a 70% increase in the number of deaths involving BZDs between 2009 and 2018 in Australia.1,2 they highlight the need to improve access to recommended treatments for insomnia management  and reduce sedative-hypnotic prescribing.50In spite of declining trends in overall BZD and Z-drug prescriptions, the increasing proportion of them provided to patients with an insomnia diagnosis is concerning, as it could increase the likelihood of dependence. The findings reported here are consistent with the reported rise in the prevalence of sleep issues in Australia during the past decade;9,37 Although there are clear regulations for the prescription of drugs included in the government-subsidised PBS scheme, there is a paucity of explicit guidance for non-PBS drugs;37 therefore, explicit recommendations regarding repeated prescriptions for non-subsidised drugs seem necessary.The findings presented here also suggest that, if restrictions on the number of repeats for some medications on private prescriptions were introduced, this might discourage long-term prescribing.51 and introducing real-time prescription monitoring to identify patients who visit multiple doctors for prescriptions.52Other interventions may also help reduce the number of BZD prescriptions sought by patients and prescribed by GPs, such as reducing pack sizes where possible, increasing the availability of non-pharmaceutical interventions for insomnia management,


Corticosterone is the major stress hormone in birds and research has shown that an increase in corticosterone can have adverse effects on bird health  production performance metrics. However, it is not currently possible to monitor commercial flocks for stress before performance is affected. A popular model of chicken stress involves administering corticosterone to chickens though their drinking water. However, corticosterone is non-polar so it must first be dissolved in ethanol, which means that the chickens are also drinking ethanol. In this study, an untargeted nuclear magnetic resonance-based metabolomics approach was used to investigate the effects of this model of stress in chickens, as well as the effects corticosterone on the chicken kidney, liver, and breast muscle metabolomes. We hypothesized that physiological stress modulates the metabolome of liver, kidney, and breast muscle due to increases in catabolism and gluconeogenesis. The administration of corticosterone altered the chicken liver, kidney, and breast muscle metabolomes. However, the ethanol carrier affected the metabolome of all three tissues, which indicated that corticosterone should be administered in an alternate fashion in future metabolomics studies to remove the confounding effects of ethanol. Furthermore, future research should focus on relating metabolite changes in tissues to non-destructive markers like blood, feces, or feathers to develop new diagnostic tools to better monitor on-farm stress during production.−1), and they were sampled 1, 5, and 12 days after the start of the CORT administration. To solubilize CORT, it was first dissolved in ethanol and then added to water. The administration of ethanol alone significantly altered branched chain amino acid metabolism in both the liver and the kidney, and amino acid and nitrogen metabolism in breast muscle. CORT significantly altered sugar and amino acid metabolism in all three tissues, but to a much greater degree than ethanol alone. In this regard, CORT administration significantly altered 11, 46, and 14 unique metabolites in liver, kidney, and breast muscle, respectively. Many of the metabolites that were affected by CORT administration, such as mannose and glucose, were previously linked to increases in glycosylation and gluconeogenesis in chickens under conditions of production stress. Moreover, several of these metabolites, such as dimethylglycine, galactose, and carnosine were also previously linked to reduced quality meat. In summary, the administration of CORT in chickens significantly modulated host metabolism. Moreover, results indicated that energy potentials are diverted from muscle anabolism to muscle catabolism and gluconeogenesis during periods of stress.The impact of physiological stress on the metabolomes of liver, kidney, and breast muscle was investigated in chickens. To incite a stress response, birds were continuously administered corticosterone (CORT) in their drinking water at three doses (0, 10, and 30 mg L Chickens are exposed to many stressors during production. These include transportation from the hatchery to the barn, handling by people, and exposure to temperature fluctuations. When chickens experience a stressor, it stimulates the hypothalamic-pituitary-adrenal (HPA) axis and causes behavioral and physiological changes that help the chickens to cope with the stressor . The gluOne way to investigate the effects of stressors on metabolic processes is through metabolomics. Metabolomics is the study of the small molecular weight compounds, or metabolites, present in a tissue or biofluid, and allows for the analysis of the metabolic responses of living systems to external stimuli, such as stressors. An understanding of this response can provide valuable insight into the biochemical pathways that are potentially dysregulated due to physiological stress. Metabolomics can be used to study both pathological and physiological states, as well as to identify biomarkers in biological fluids and tissues. The metabolomics research that has been conducted in chickens to date is often focused on the detection of drugs and drug metabolites such as anti-virals , antibioNuclear magnetic resonance spectroscopy (NMR) is an experimental technique used to characterize the metabolome of various biological tissues and fluids that is high throughput, non-destructive, and highly reproducible ,20,21. RUnderstanding the effects of physiological stress on metabolism in a comprehensive and untargeted manner is an important step towards identifying biomarkers of stress. This may facilitate the development of tools to help producers identify when stress may be adversely affecting their flocks so that mitigations can be employed early to prevent production losses. In the current study, we applied an untargeted NMR-based metabolomics approach to investigate the effects of both the ethanol-based delivery method of CORT administration and physiological stress, through dosage effects of CORT, on the metabolome of chicken kidney, liver, and breast muscle. We hypothesized that physiological stress modulates the metabolome of liver, kidney, and breast muscle due to increases in catabolism and gluconeogenesis.The study was carried out in strict accordance with the recommendations specified in the Canadian Council on Animal Care Guidelines. The project was reviewed and approved by the Lethbridge Research and Development Centre (LeRDC) Animal Care Committee  before commencement of the research.n = 9 chicks); (2) ethanol control birds provided with 0.2% ethanol in drinking water (n = 9); (3) 10 mg L−1 CORT in drinking water (n = 9); and (4) 30 mg L−1 CORT in drinking water (n = 9). The dose and method of CORT administration are detailed for a previous study [The study was designed as a factorial experiment with four levels of stress treatment and three levels of time arranged as a completely randomized design. The four stress treatments were: (1) control birds provided untreated drinking water  was dissolved in 2 mL of anhydrous ethanol and added to 1 L of drinking water. Water containing CORT was prepared fresh each day and added to animal cages twice daily.2) for 10 days and had access to a brooder . Birds had ad libitum access to a non-medicated starter diet  and water. Birds were maintained on a 12 h light: 12 h dark cycle. At 11 days-of-age, birds were randomly assigned to the four stress treatments and housed within individually ventilated cages (IVCs) . Each animal cage contained a companion bird to ensure no birds were socially isolated. Corticosterone treatment began when chicks reached 14-days-of-age and continued until the end of the experiment. The experiment was repeated twice with individual birds treated as replicates .Thirty-six specific-pathogen-free white leghorn chickens were used in this study. Eggs were purchased from the Canadian Food Inspection Agency , and upon arrival at Lethbridge (shipped by air) were incubated in a Brinsea Octagon 40 Advanced Digital Egg Incubator according to the manufacturer’s recommendations . In this regard, eggs were maintained at 37.5 °C and 45% humidity with hourly turning for the first 18 days of incubation. Thereafter, eggs were set flat for hatching and humidity was increased to 60%. All hatched chicks were acclimatized in a group within one large animal pen (1.1 m2 min−1) and euthanized by cervical dislocation under general anesthesia. The abdomen was opened with a ventral midline incision and the liver, a kidney, and a sample of breast muscle were aseptically removed and stored at −80 °C until processing. Liver, kidney, and breast muscle were examined because the metabolome of these tissues was previously shown to be significantly altered in response to stress in chickens [One bird per IVC was randomly sampled at each of the three sample times. Birds were anesthetized with isoflurane . A 120 μL aliquot of deuterium oxide containing 0.05% v/v trimethylsilylpropanoic acid (TSP) was added to each sample ; TSP was used as a chemical shift reference for 1H-NMR spectroscopy. The solution was vortexed and then centrifuged at 12,000× g for 5 min at 4 °C to pellet any particulate matter. Following centrifugation, a 550 μL aliquot was loaded into a 5 mm NMR tube and run on a 700 MHz Bruker Avance III HD spectrometer  for spectral collection.Tissues from liver, kidney, and breast muscle were homogenized in 4 mL gSpectra were collected on a 700 MHz Bruker Avance III HD spectrometer . The Bruker 1-D NOESY gradient water suppression pulse sequence ‘noesygppr1d’ was used with 10 ms mixing time. Each sample was run for 512 scans to a total acquisition size of 128 k, a spectral window of 20.5 ppm, a transmitter offset of 4.7 ppm, and a recycle delay of 4 s. All measurements were recorded using a Bruker triple resonance TBO-Z probe. The Bruker automation program “pulsal” was used on each sample before data acquisition to guarantee that the 90-degree pulse was calibrated correctly, ensuring quantitative and comparable data across samples . The spet-test and a multivariate variable importance analysis based on random variable combination (VIAVC) [t-test each comparison was paired within each replicate, and samples from all three time points,  were pooled to provide an adequate sample size. The univariate measures were calculated using a decision tree algorithm as described by Goodpaster et al. [p-values obtained from this analysis were Bonferroni-Holm corrected for multiple comparisons. The VIAVC algorithm uses both Partial Least Squares Discriminant Analysis (PLS-DA) and the area under the Receiver Operating Characteristics (ROC) curve to determine the best subset of bins for group classification while also accounting for the significance of the bins; thus the algorithm considers the synergistic effects of bins [Spectral bins were subjected to both a univariate paired  (VIAVC)  analysisr et al. . All p-v of bins . MATLAB  of bins  was used of bins ; the KEG of bins  for chict-test and VIAVC tests revealed the bins that were significantly altered (p ≤ 0.05) between the control and ethanol control treatments in kidney , liver , and breast . Examination of bins that were observed to be significantly altered via the paired t-test and/or the VIAVC analysis was done using a PCA scores plot which showed little to no unsupervised group separation in any of the tissues (2) in liver  and breast , but not kidney  tissues (Paired  tissues . Subsequ tissues .p < 0.001), but not kidney (p = 0.076) tissues. Quantities of specific metabolites differed (p < 0.050) between the control and ethanol control treatments as determined by paired t-test and/or VIAVC analysis. In the kidney, 5/10 (50%), in liver, 11/27 (40%), and in breast muscle, 1/5 (20%) unique metabolites were significantly up-regulated (p < 0.001), and alanine, aspartate, and glutamate metabolism (p < 0.050) in kidney and liver, as well as D-glutamine and D-glutamate metabolism and nitrogen metabolism (p < 0.050) in kidney and breast muscle (p < 0.050). In the liver, other amino acid metabolism pathways that were altered included the phenylalanine metabolism pathway (p < 0.050) and the arginine and proline metabolism pathway (p < 0.050).Double cross-validation and permutation tests validated the observed supervised separation results as a function of ethanol in the drinking water in liver and breast  and breast muscle , but not in kidney  , breast muscle , and kidney   of the unique metabolites in the kidney, 20/115 (17%) in the liver, and 20/25 (80%) in breast muscle. At 30 mg L−1, CORT significantly up-regulated 13/36 (36%) of the unique metabolites in the kidney, 26/93 (28%) in the liver, and 17/96 (18%) in breast muscle (data not shown). There were 11 metabolites that were significantly altered at both the 10 and 30 mg L−1 CORT doses in kidney, 46 in liver, and 14 in breast muscle  A,C,E. Li< 0.050) B,D,F. Att muscle . Glucose−1 CORT in the drinking water affected galactose (p < 0.001); starch and sucrose (p < 0.050); and inositol phosphate (p < 0.050) metabolism. In liver and kidney alanine, aspartate, and glutamate (p < 0.050); and histidine (p < 0.050) metabolism were altered (data not shown). For the 30 mg L−1 CORT, treatment pathway topology analysis indicated that two pathways were altered in all three tissues; these were the aminoacyl-tRNA biosynthesis (p < 0.001) and galactose metabolism (p < 0.050) pathways (p < 0.050), and starch and sucrose (p < 0.050) metabolism were also altered. In liver and breast muscle, pantothenate and CoA biosynthesis (p < 0.050), histidine (p < 0.050), and purine (p < 0.050) metabolism were also all significantly altered. In kidney and breast muscle, alanine, aspartate, and glutamate metabolism (p < 0.050), pyruvate metabolism (p < 0.050), and glycine, serine, and threonine metabolism (p < 0.050) were also altered.Pathway topology analysis indicates that the inclusion of 10 mg Lpathways . In liveMetabolomics provides a comprehensive understanding of all the responses of whole living systems to pathophysiological stimuli, genetic modification, diet, and the environment. Furthermore, metabolomics is particularly adapted to identify biomarkers as it provides a snapshot of all the metabolic activity occurring in a system ,36. For −1) on white leghorn chickens using 1H-NMR metabolomics, and we observed distinct changes in the metabolomes of liver, kidney, and breast muscle regardless of the dose administered. Moreover, CORT administration was observed to alter several metabolite concentrations that can be linked to alterations in metabolic pathways related to both bird health and meat quality.When chickens are exposed to a stressor, the HPA axis is activated to help the bird cope with the stressor ,39.This Previous studies reported that rats administered ethanol in drinking water exhibit reduced renal function due to kidney injury ,47. BothThe liver is the primary site of ethanol catabolism, and chronic ethanol ingestion and metabolism causes mild damage to the liver though oxidative stress  and can In breast muscle of birds administered ethanol, the metabolites of the glutamine and glutamate metabolism pathways, along with the metabolites of the nitrogen metabolism pathway, were significantly altered. Glutamine is normally the most abundant amino acid in healthy skeletal muscle; however, when muscle is stressed, the concentration of glutamine drops significantly. Therefore, glutamine is designated as a conditionally essential amino acid during periods of stress ,58. Stud1H-NMR spectra. The lower concentration of glutamate that we observed in the kidney of birds administered CORT could be caused by greater amounts of the metabolite being bound to its receptor. The activation of these receptors affects renal function, and in some cases may induce renal dysfunction; for example, a previous study showed that prolonged exposure to NMDA caused excessive NMDA receptor activation that leads to an accumulation of reactive oxygen species [The concentration of glutamate in the kidney was decreased in CORT stressed birds regardless of the concentration administered. Glutamate is a neurotransmitter, but is also present in peripheral tissues, like kidney, where it plays an important role in energy production, nitrogen metabolism, and the body’s response to oxidative stress ,60. Glut species ,51,52,53−1 dose relative to the 10 mg L−1 dose. For example, the concentration of 4-hydroxyproline was substantially increased for birds administered CORT at the higher dose. In most animals, including chickens, hydroxyproline is required for the synthesis of glycine. Glycine is known to scavenge oxidants and help regulate the redox state of cells [All metabolites altered in the kidney following CORT administration were affected to a greater extent in birds administered CORT at the 30 mg Lof cells . Moreoveof cells ,63. Elevof cells , and theA key function of the liver is to provide glucose to tissues. Under conditions of stress the liver increases rates of both glycogenolysis and gluconeogenesis to meet the body’s increased energy demands . Amino a−1 as compared to the ethanol control treatments. Previous studies observed up-regulation of mannose-6-phosphase, which is indistinguishable from mannose in 1D 1H-NMR spectra; mannose-6-phosphate is an integral metabolite in the formation of GDP-mannose which is important for N-linked glycosylation [In contrast to essential amino acids, mannose concentrations were significantly increased in the livers of birds administered CORT at 10 and 30 mg Lsylation . Glycosysylation , and mansylation . The funsylation .Previous studies observed an increase in glucose in chicken livers when the birds were stressed by high temperatures . The up-We observed that BCAAs in breast muscle were significantly down-regulated in CORT-treated birds as compared to control treatment birds. BCAAs are mostly metabolized in skeletal muscle and other peripheral tissues , and theWe observed an increased concentration of glucose in breast muscle of birds administered CORT as compared to control treatment birds. As indicated previously, stress increases gluconeogenesis in the liver ,67. ThisGalactose metabolism is a pathway that has previously been determined to be significantly up-regulated in lower quality breast meat samples . MetabolIn the current study we showed that ethanol altered the metabolomes of the liver, kidney, and breast muscle of chickens; however, the effects of ethanol alone were substantially less than the metabolomic effects of CORT orally administered in ethanol. It is possible that CORT and ethanol together exerted a larger effect than either alone would have. Study results indicate that CORT should be administered in an alternate fashion  in future studies to remove the confounding effects of ethanol on the metabolome. Despite the observed effects of ethanol, the data from this study showed that the stress hormone, CORT, was linked to changes in metabolite concentrations across several important tissues in chickens that could be linked to overall health and production. This includes kidney glutamate concentrations, liver amino acid concentrations, and increases in glucose concentrations in the kidney, liver, and breast muscle of white leghorn chickens. Significantly, several stress induced changes in metabolite concentrations observed in breast muscle may be associated with meat quality which warrants further investigation. Moreover, alterations to the metabolome of chickens incited by CORT were consistent with increases in metabolism due to stress induction, which were consistent with a previously published reduction in the mass of CORT administered birds relative to control treatments in the same chicken cohort. Future research should focus on relating metabolite changes in tissues to non-destructive markers like blood, feces, or feathers relative to metrics of birds’ health and meat quality with the goal of developing new diagnostic tools to better monitor on-farm stress. Ultimately, the identification of quantitative biomarkers of stress will allow producers to utilize the markers to develop and objectively evaluate mitigation strategies to proactively enhance bird health and flock performance.