Recently, deep eutectic solvent (DES) or ionic liquid (IL) analogues have been considered as the newest green solvent, demonstrating the potential to replace harsh volatile organic solvents. DESs are mainly a combination of two compounds: hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD), which have the ability to interact through extensive hydrogen bonds. A thorough understanding of their physicochemical properties is essential, given their successful applications on an industrial scale. The appropriate blend of HBA to HBD can easily fine-tune DES properties for desired applications. In this context, we have reviewed the basic information related to DESs, the two most studied physicochemical properties (density and viscosity), and their performance as a solvent in (i) drug delivery and (ii) extraction of biomolecules. A broader approach of various factors affecting their performance has been considered, giving a detailed picture of the current status of DESs in research and development. Solvents have been utilized for a number of applications, ranging from drug delivery/discovery to extraction of biologically important compounds. Typically volatile organic solvents, have shown tremendous potential for these targeted applications, but they suffer from adverse effects related to low volatility and toxicity, and they may cause harm to the environment. In this regard, the growing interest of chemical industries and the research communities in “green and sustainable solvents” is attributed to incorporating 12 green principles. A significant portion of harsh solvents have been used in multiple applications, i.e., reaction, solubilization, precipitation, and separation. Such solvent systems can contribute considerably to environmental pollution ,4.4), hexafluorophosphate (PF6), chloride (Cl), and bis(trifluoromethane)sulfonimide (NTf2). The structural variance in cation moiety and a wide choice of anions for the preparation of ILs give a broad spectrum of ILs with different physicochemical properties [18 kinds of different combinations for ILs [As in the literature, ionic liquids were studied as the most promising green solvents during the last two decades ,4. ILs aoperties ,6. Theor for ILs . They ha for ILs ,10,11,12 for ILs ,14.Despite their huge success, ILs have been scrutinized for greener credentials, toxicity, higher cost of starting materials, complex synthesis procedure, difficulty in purification, and regeneration cycling . The purDESs can be obtained by mixing two components to form a eutectic mixture. Choline chloride (ChCl), quaternary ammonium salts are amongst the most studied components used for the preparation of DESs. ChCl can readily form a eutectic mixture with urea, carbohydrates, amino acids, and carboxylic acids via hydrogen bonding. DESs have unique characteristics: (i) they are designed in numerous combinations of two readily biodegradable compounds, HBA (hydrogen bond acceptor) and HBD (hydrogen bond donor), in an appropriate molar ratio; (ii) they are cheaper; (iii) they do not need purification; and (iv) they are biocompatible and biodegradable. For these reasons, considerable growth has been observed in the designing and applications of DESs ,19,20,21DESs have been considered superior solvents to several traditional ILs for the following reasons. (i) Ease of preparation: the preparation of most ILs is complex compared to the preparation of DESs, which is prepared quickly by a simple blending of two components at moderate temperature. This involves lower production value with respect to conventional ILs and permits large scale applications. The preparation of DESs can be obtained with nearly 100% atom economy. (ii) Easy availability of components of DESs: ILs are generally expensive and often toxic and non-biodegradable. In the case of DESs, quaternary ammonium/phosphonium/sulfonium salts and various organic compounds used as HBDs are readily available, as they are produced in vast amounts at a lower cost than the conventional reagents. The components of DESs are less toxic to be scaled up for large-scale production ,23,24.Generally, DESs are a highly non-ideal mixture of two biodegradable components (HBA and HBD) associated with strong hydrogen bonding interactions. The resulting liquid will have a melting point lower than the melting points of both HBA and HBD. They are broadly defined as liquid produced due to greater depression in the freezing point. Most DESs are a combination of quaternary ammonium salt, as HBA and HBD have the ability to form hydrogen bonding with HBA . DESs weAs discussed in the introduction, DESs are prepared by 100% atom economy route, as it involves simple mixing of HBA and HBD, and all other steps such as purification and waste disposal were eliminated or not required. (i) The most common preparation method, HBA and HBD of DESs, were heated and continuously stirred together in an inert atmosphere until homogeneous liquids were formed . (ii) In+A−xB, where R+ is ammonium, sulfonium, and phosphonium cation core. A and B are Lewis base with halide anion and Levis acid, respectively [DESs were presented using the general formula: Rectively . The com2, FeCl2, AgCl, CuCl2, CdCl2, LiCl, SnCl2, and SnCl4 [Type I DESs: This type of DES can be prepared from quaternary ammonium salt and metal chloride. Type I DESs formed with imidazolium salts and various metal halides such as ZnClnd SnCl4 ,28. The Type II DESs: This type of DES can be composed of quaternary ammonium salt and hydrate of metal chloride hydrate .Type III DESs: This type of DES can be prepared using quaternary ammonium salt as HBA and HBD. This type of DES is widely studied. These DESs are mainly composed of choline chloride and HBDs  The HBA used in the preparation of this kind of DES are listed in 2 and urea. In non-aqueous solvents, these metal salts usually do not ionize but form a DES with ethylene glycol, acetamide, and 1,6-hexanediol [Type IV DESs: These types of DESs are composed of metal chloride  and HBD. It was reported to form a DES using a mixture of ZnClxanediol ,30.Type V DESs: These types of DESs are a relatively new class mixture of non-ionic molecular HBA and HBD . AlthougLike ILs, DESs are also characterized by a collection of unique physicochemical properties: DESs have been used as potential solvents in the industry, owing to their numerous physicochemical properties such as density, freezing temperature, viscosity, surface tension, miscibility, conductivity, and polarity, etc. Moreover, a huge number of DESs can be obtained using different combinations of HBA and HBD, making DESs more designable, thus named designer solvents. The unique combination of HBA:HBD resulted in DESs with different physicochemical properties. The physicochemical properties of DESs can be manipulated using various combinations of HBA and HBD in different molar ratios. Apart from the variety of HBA to HBD, the physicochemical properties of DESs are also affected by the molar ratio of HBA and HBD, purity of HBA and HBD, temperature, water content, and method of preparation .The determination of physicochemical properties of DESs is in the framework of their applications as a solvent in chemical processes. The design of DESs for specific applications needs knowledge of the physicochemical properties of DESs. Importantly, the accurate knowledge of physicochemical properties such as density, speed of sound, refractive index, and viscosity of DESs are crucial for their industrial exposure as a solvent in various operation unit processes ,33. Henc3 at 298.15 K [2 and different HBDs have densities between 1.3 to 1.6 g cm−3 [−3 [The PVT data of densities of DESs are crucial parameters for designing equipment and processes, liquid–liquid equilibria, mass transfer, the development of equations of state and predicting models, and the calculation of thermodynamic properties such as viscosity, expansion coefficient, and isothermal compressibility . The den298.15 K . The valcm−3 [−3 .2:urea, the average radius of Hole was found to reduce, resulting in a small increase in density value of a DES composed of ChCl:urea [The difference in DES density with respect to various factors can be understood using the Hole theory . The HolhCl:urea . There ahCl:urea ,63,64,655 was the main reason for the lower density. Again, DESs composed of levulinic acid have a lower density than those of glutaric acid due to higher moles of acid (1:2) in the DES. The densities of DESs decrease in the order of oxalic/glycolic acids (C2) > malonic acid (C3) > glutaric/levulinic acids (C5). As the length of alkyl chain increases, the corresponding molar volume increases, and the density of the DES decreases [(1) Nature of HBD: The densities of DESs obviously depend upon the nature of the HBD ,52,55,64ecreases .The hydrogen bonding between HBA and HBD is the main driving factor for DES formation. The increase in the number of −OH functional groups in HBD resulted in a higher number of hydrogen bonds. The higher number of hydrogen bonds reduces the free spaces available and consequently increases the density of DESs. DESs obtained from benzyl trialkylammonium chloride salts as HBAs and ethylene glycol have lower densities than DESs composed using diethylene glycol, triethylene glycol, and glycerol . As the N,N-Diethyl ethanolammonium chloride (DEACl) or methyltriphenylphosphonium bromide (MTPhPBr) as HBA combined with glycerol and ethyl glycol, the densities of DESs were found to decrease [The density of carbohydrate-based DESs containing ChCl, acetylcholine chloride (Ac ChCl), and benzylcholine chloride (BzChCl) as three different HBA have been reported . The cardecrease . For com3) and ethylene glycol (1.605 g cm3), respectively [The densities of DESs composed of tetraethylammonium chloride/bromide (TEACl or TEABr) salts combined with levulinic acid, ethylene glycol, and glycol in a 1:4 molar ratio showed a decrease in density with change in the type of HBD . The samectively .A similar trend was observed for studied carbohydrates when a type of HBA is considered. The densities of DESs decrease as ChCl:carbohydrate > Ac ChCl:carbohydrate > Bz ChCl:carbohydrate . In both−3, respectively. The slight increase in density of DEACl-, MTPBr-, and THABr-based DESs for common HBD glycerol or ethylene glycol at molar ratio 1:2 [(2) Molar ratio of HBA to HBD: The molar ratio or composition of HBA:HBD can be used to manipulate the density of DESs ,45,48,50atio 1:2 .(3) Temperature: The densities of DESs decrease with increases in temperature due to the thermal expansion of DESs. The density at 298.15 K of DESs formed by suitable combination of HBA and HBD is shown in The isobaric thermal expansion coefficient is obtained from experimental densities at different temperature of DESs:Pα values and density with temperature. It can be observed that the values of Pα of DESs are minimal compared to Pα values of common solvents; however, they are quite similar to Pα values of imidazolium-based ILs. The temperature showed little effect on density and P;α therefore, DESs expanded or compressed less in comparison to ILs and other organic solvents.The linear decrease in the density of all DESs was observed with an increase in temperature. The reduction in density of DESs with a rise in temperature results from the availability of more free spaces between the HBA and HBD of DESs. The available space in DESs is related to changes in ρ corresponds to density in g cm−3, a and b are the fitting parameters, and T is the temperature in K.The variation in density with temperature was correlated using a linear equationP for listed DESs are 0.5–0.7, higher than the values for organic solvents.The densities of DESs at different temperatures have been reported extensively by many researchers ,70,71,72Nicolas et al. measured the density of ChCl-based DESs at a pressure of 101.3 kPa and in a temperature range from 293.15 K to 333.15 K. In the study, ChCl was mixed with ethylene glycol, levulinic acid, and phenol as HBD in 1:2 mole ratio HBA:HBD. It is shown that the densities of the above three DESs decrease as the temperature increases in accordance with thermal expansion of the DES. The maximum thermal stability was observed for ChCl:levulinic acid due to the presence of a significant extent of intermolecular interaction between ChCl and acid. This was the main reason for the greater density of this DES among other studied DESs .As discussed above, the densities of all DESs decrease with a rise in studied temperature. The values of density greatly depend upon type of HBD. It is observed that as the number of -OH groups in the DES structure increases, the density of DESs is found to increase. A similar trend was reported when the number of aromatic groups or alkyl chains of organic acids increased. A change in density corresponded to a change in the free volume of the DESs . In termDES = 0.645) at temperature ranges between 288.15 K and 338.15 K for the DES and at a temperature range between 278.15 K to 338.15 K for the aqueous DES. The density of a hydrated DES was found to be less in comparison to pure DES due to a decrease in hydrogen bonding between HBA:HBD with the addition of water [The influence of water content on the density of a ChCl:urea DES was measured by Shah and Mjalli  at two dof water .Water had an effect on a ChCl-based DES composed of oxalic acid, levulinic acid, glutaric acid, malonic acid, and glycolic acid as HBD. The DESs were exposed to an open-air environment for a month, and the amount of water taken by DESs was measured, which was found to be 14 to 20 wt%. The density of DESs saturated with water (5%) was lower than that of the dried DES samples. Therefore, it was reported that trace amounts of water affect the density of DESs. As a result, the presence of water slightly affects the density of these DESs .v/v) water content. Further dilution with water caused the rupture of the DES structure, and HBA and HBD existed as an individual component in the mixture. The study showed a decrease in density of a choline chloride:phenol DES by the addition of water [Dai et al.  studied of water .P for ChCl:glutaric acid than for ChCl:malonic acid suggests more expansion in the former. The densities of an aqueous solution of both the DESs were decreased with an increase in the amount of water and temperature.A temperature variation of densities of three other DESs such as ChCl:ethylene glycol, ChCl:malonic acid, and ChCl:oxalic acid was carried out by Shekaari et al. . The higMjalli and Ahmad  reportedη describes the internal friction or resistance experienced by moving fluid or is defined as the resistance of moving fluid in reference to deformation at a given shear rate. The viscosity of DESs has been extensively reported due to its industrial importance. The applicability of a solvent as a reaction medium can be decided from the viscosity of the solvent. Viscosity values at different temperatures play a vital role in equipment design calculations and understanding the activation energy and mass transport phenomena [The viscosity henomena ,81,82,83henomena . The vishenomena .2 [(1) The nature of DES components: The viscosities of DESs are mainly affected by the chemical nature of HBAs and HBDs. The viscosity of ChCl:ethyleneglycol (1:4) DES exhibits the lowest viscosity (19 cP at 293.15 K), whereas the viscosity of DES increased up to 8500 cP at 298.15 K when HBD was replaced to ZnCl2 . Similar2  and 11242 . This caIt was shown that the DESs with dicarboxylic acid are higher than those containing a monocarboxylic acid. Moreover, the addition of one –OH or –COOH group also contributed to the rise in the viscosity of DESs, probably due to an increase in hydrogen bonding. The viscosities of DESs increase in order of ChCl:oxalic acid > ChCl:gluconic acid > ChCl:malonic acid > ChCl:glycolic acid > ChCl:levulinic acid > ChCl:oxalic acid . For com2) Molar ratio of HBA:HBD: In general, higher molar ratios of HBA to HBD resulted in higher viscosities of DESs. The amount of free volume decreases due to the compact structuring of DESs. The small amount of free volume leads to slow molecular motion and higher viscosity of DESs [ Molar raThe viscosities of DESs decrease with increases in the molar ratio of HBD. The viscosity of a DES increases with an increase in the molar ratio of glycerol for ChCl/glycerol DES. The viscosity values of DESs at 1:2, 1:3, and 1:4 molar ratios were reported around 503, 450, and 376 cP, respectively, at 293.15 K. The addition of ChCl to glycerol leads to a breakdown of hydrogen bonding and decreased viscosity of DES . A simil(3) Water content of DESs: Water content in DES is one of the most important criteria when considering the viscosity of DES. Since most DESs are highly hygroscopic, it is essential to provide the water content of DESs. The viscosity of DES composed of ChCl:urea (1:2) decreased to 200.6 cP from 527.3 cP in the presence of 0.1 mole fraction of water . Dependi(4) Temperature: As per general observation, the viscosity of DESs decreases with a rise in temperature; therefore, DESs with high viscosity at ambient temperature can be used at a higher temperature. The viscosity of ChCl:urea (1:2), ChCl:glycerol (1:2), ChCl:malonic acid, and ChCl:glucose decreases from 750 to 95 mPa s, 259 to 52 mPa s, and 1124 to 161 mPa s and 7992 mPa s to 262 mPa s between temperature ranges between 298 and 328 K ,82.The temperature-dependent viscosity behavior of DES was explained using Hole theory. The application of the Hole theory for the viscosity of DES was studied by Abbott and co-workers ,42,45. Aη∞ is a pre-exponential constant, lnη is logarithm of viscosity, T is temperature, R is gas constant, and The two most commonly used models applied to DESs to study the effect of temperature on viscosity are (1) the Arrhenius model and (2) the Vogel–Fulcher–Tamman (VFT) model ,24,37,74aE) can be obtained using the Arrhenius equation. Less viscous DESs such as ChCl:ethylene glycol show very low aE values, whereas highly viscous DESs such as ChCl:glucose have larger aE [aE were −65.2, −46.7 and −47.6 kJ mol−1, respectively [aE; therefore, HBA:HBD interactions are crucial for viscosity of DES. For molten salts, it was reported that the activation energy for viscosity is linearly related to melting temperature (aE = 3.7 RTm) [The relation of DES viscosity with a strength of intermolecular interaction and temperature can be obtained by fitting viscosity at various temperatures in this equation. The activation energy (arger Ea . In the ectively . The DES3.7 RTm) . A simil3.7 RTm)  for cholη is logarithm of viscosity, and A, B, and T0 are fitting parameters. T0 is the Vogel temperature, where all atoms′ movement is considered frozen.Another approach to correlate the viscosity of DES with temperature is the Vogel–Fulcher–Tammann (VFT) model ,80.(4)lAN, h, and R indicate Avogadro’s number, Planck′s constant, and ideal gas constant, respectively. mV is molar volume, η is dynamic viscosity of liquid, and E0 is activation free energy for viscous flow.The viscosity of ChCl-based DESs composed of p-toluenesulfonic acid, monochloroacetic acid, trichloroacetic acid, and propionic acid as HBDs showed better fitting using the VFT model than the Arrhenius . SeveralThe drug discovery and drug delivery of new drugs often face challenges such as safety, efficacy, cost, and availability ,86,87,88The approved drugs or drugs under development are poorly water-soluble, so one of the characteristics to improve the drug efficiency, permeability, and bioavailability is enhancing drug hydrophilicity ,88. For One such biocompatible, cheap, less toxic solvent being studied is based on DESs ,88,89. DDESs have promising properties required for successful drug formulations. The choice of components for DES preparation provides an opportunity to tailor the solvent system to various applications, including drug formulation, delivery, or dissolution ,90,91. DEnhancing drug solubility and modulating drug release kinetics are two strategies to overcome problems associated with drug molecules′ low bioavailability/low solubility . EnhanceAPIs such as ibuprofen, phenylacetic acid, and acetylsalicylic acid were combined with commonly used HBAs (choline chloride or menthol) to form THEDES. These eutectic solvents can have melting points above or below ambient temperature . The eutstratum corneum) makes penetration of exogenous molecules challenging [stratum corneum using various invasive and non-invasive techniques [stratum corneum [Skin is the largest organ of the body, which acts as a protective barrier against the external environment and absorbs exogenous molecules . This skllenging . Thus, tllenging  or to wechniques ,107,108. corneum . Nonethe corneum ,109. In  corneum ,111,112. corneum ,114.Boscariol et al.  have recstratum corneum was observed for a few concentrations of DES in contact with skin. The DES-based formulation with a DES composition of 2% showed a maximum average permeated curcumin concentration of 375 ngcurcumin/mm2 after 15 min of transdermal application to modelled skin. The results showed that DES promoted transdermal permeation of the bioactive curcumin molecules. Thus, DES can be employed for topical formulations for enhanced delivery by a non-invasive technique to deliver various poorly water-soluble bioactive molecules [A virtually nil genotoxicity effect of DES on 3T3 cell was observed, which indicated a lack of cytotoxicity of DES . As showolecules . For traolecules ,117,118.olecules ,116,117.olecules . The druolecules . The perCnidium monieri (L.) Cusson fruits, have shown various pharmacological functions such as anti-bacterial, anti-inflammatory, anti-oxidant, and anti-cancer properties [For DES formed from choline chloride with API, various interactions can occur, such as alkyl–alky interactions, cation–pi, anion–pi, halogen bonds, and hydrogen bonds ,97. Amonoperties ,121,122.operties .THEDES and microemulsion gel were made using these APIs and evaluated for their transdermal delivery performance . The THEA fatty acid containing THEDES was formulated using the antipsychotic drug risperidone . The ben2 h was achieved for the formulation having the lowest melting point of 17 °C, and the drug permeation studies reveal that high drug accumulation in stratum corneum (hydrophobic part of the skin) may be due to the hydrophobicity of the eutectic mixture containing the drug risperidone [The stable viscous mixture was obtained by gently mixing components without temperature treatment, mechano-chemical agitation, or solvent . The forperidone .Recently, the first report of a clinical translation of choline-based DES for the treatment of rosacea was presented . The res2 technology [2O compositions were optimized for encapsulation in glycerol monostearate particles. The methodology was adopted to administer powdered inhalable particles for fast and localized delivery of the drug [2 phases were used for selecting particles from gas saturated solution (PGSS) for THEDES encapsulation [THEDES based delivery systems have also been studied for tuberculosis therapy by incorporation of L-arginine forming THEDES into a polymer matrix using supercritical COchnology . Citric the drug . Phase esulation .DESs have been employed as vehicles for the solubilization of pharmaceutical ingredients. DESs have also been prepared with one of the components as the active pharmaceutical ingredients API (known as THEDES) . This grAccording to the principle of green chemistry, solvents or auxiliaries can be avoided; if not, they should be non-toxic or harmless . Green sThe development of novel analytical procedures requires miniaturizing the system and improving selectivity and sensitivity . One of DESs have demonstrated excellent ability to be used in the extraction process ,135. As In Improving the extraction process is needed to recover the maximum content of bioactive compounds; in this regard, various advanced technologies have been employed ,136. Ult−1 dw) in large amounts [−1 dw) [−1 dw and oleuropein was 1221.17 mg kg−1 dw) [Recently, the first NADES based on a glucose–fructose–water mixture was used for the extraction of bioactive compounds assisted by ultrasound and was reported in caffeic acid (112.77 mg kg amounts  and oleu [−1 dw)  from olikg−1 dw) . Among tkg−1 dw) .The class of natural compounds with various phenolic structures with anti-cancer, anti-inflammatory, and anti-oxidant properties ,159,160 The carbon atoms are numbered in the rings X and Z as 2–8, and those in ring Y are numbered 2′ to 6′ ,161. ManGenerally, the extraction of bioactive molecules using eutectic systems depends on various factors such as type of DES, moisture, and energy input . The extPolysaccharides have shown various medicinal effects such as antioxidant, anti-diabetic, antiglycation, anti-viral, anti-inflammatory, anti-coagulant, immunostimulatory, and anti-tumor properties ,171. DueLilium lancifolium, edible fungus (Poria cocos), polyculture biofilms, etc. [Polysaccharides can be extracted using hot water; however, such extraction is time consuming and requires a lot of energy . High-tems, etc. ,174,177.ms, etc. ,173.Poria cocos was extracted using DES composed of sorbitol, 1,3-butanediol, and choline chloride [poria cocos powder was irradiated with FIR and provided thermal energy from vortex airflow (hot air) [poria cocos material [Polysaccharide from chloride . The DESchloride . The mixhot air) . The hotmaterial . The polmaterial . The extmaterial .NADES have also been explored to extract biomolecules and were recently used for a biofilm structure breaker . A groupAnother application of DES solvent is for protein extraction ,176,177.Xu et al.  have useCholine-based DES was also used in extracting vegetable protein from natural sources oilseed cakes. Water was used as an anti-solvent for forming protein-rich precipitates, and the methodology can extract 40–50% protein . The proThe DES-based ABS has been used extensively for protein extraction from natural sources ,183,184.DES-based aqueous biphasic system (ABS) with highly concentrated salt solution, as shown in Volatile organic solvents (VOCs) are used extensively due to their widespread industrial and scientific applications. Several advantages and disadvantages are associated with VOCs. Considering the environmental issue, the search for replacement solvents has led to the synthesis, the characterization, and evaluating physicochemical properties and applications of neoteric solvents ,183. ResEutectic mixtures, DES, THEDES, and NADES are recent examples of biocompatible solvent systems , and the


Lippia origanoides (LEO) on the in vitro proliferation of Clostridium perfringens (CP), as well as the performance, intestinal integrity, and necrotic enteritis lesions using a previously established necrotic enteritis challenge model in broiler chickens. The supernatant from digested feed supplemented with LEO and inoculated with CP reduced CP in vitro compared with the positive control. LEO showed a significant reduction of the harmful effects of induced infection/dysbiosis and a significant reduction in NE lesion scores, morbidity and mortality compared with the positive challenge control group These results suggest that the dietary inclusion of L. origanoides could mitigate some of the complex negative impacts caused by necrotic enteritis.In recent years, a considerable expansion in the use of essential oils as feed additives in animal nutrition has occurred, mainly as an alternative to antibiotic growth promoters worldwide. The objective of the present research was to evaluate dietary supplementation of essential oils from Lippia origanoides (LEO) on necrotic enteritis (NE). Chickens were randomly assigned to three groups. Group 1: negative control; Group 2: positive control challenged with Salmonella typhimurium (day 1), Eimeria maxima (day 18), and C. perfringens ; Group 3: dietary supplementation LEO and challenged. On d 25 of age, serum samples were collected to evaluate fluorescein isothiocyanate-dextran (FITC-d), superoxide dismutase (SOD), gamma interferon (IFN-γ), Immunoglobulin A (IgA). Group 3 showed a significant reduction of the harmful effects of induced infection/dysbiosis and a significant reduction in NE lesion scores, morbidity and mortality compared with the positive challenge control group (p < 0.05) compared with Group 2. Digested feed supernatant, supplemented with LEO and inoculated with CP, reduced CP burden (p < 0.05). Group 3 also exhibited a significant reduction in FITC-d, IFN-γ and IgA compared with Group 2. However, a significant increase SOD was observed in Group 3 compared with both control groups. Further investigation to compare the effect of LEO and the standard treatment of clostridial NE is required.The objective of the present research was to evaluate dietary supplementation of essential oils from  Eimeria spp. and Clostridium perfringens, in many cases, EO are combined with other strategic products such as probiotics, prebiotics, organic acids, and enzymes to modulate the intestinal microbiota and immune system of the birds [In recent years, a considerable attention to essential oils (EO) as nutraceuticals in livestock production has occurred, mainly as an alternative to antibiotic growth promoters (AGPs) worldwide. Essential oils are derived from various plants as secondary metabolites with well-documented antibacterial , antivirhe birds ,8,9.C. perfringens (CP). Several virulence factors have been identified in the pathogenesis of NE, including toxins, proteolytic enzymes, bacteriocins, and adherent molecules produced by virulent strains of CP [Eimeria spp. infections, immunosuppression, dysbacteriosis, and removal of AGPs [In poultry, NE is a disease caused by the anaerobe Gram-positive bacterium, ns of CP . In 2000ns of CP ,12. Howens of CP . Based ons of CP ,15. Seve of AGPs ,8,9,10.  of AGPs , organic of AGPs , probiot of AGPs , and pla of AGPs  as an efEimeria maxima (EM) and CP induce a reliable laboratory model for NE. The objective of the present research was to evaluate dietary supplementation of Lippia origanoides essential oils (LEO) on in vitro proliferation of C. perfringens, as well as performance, intestinal integrity, and NE lesions using early Salmonella typhimurium (ST) infection as a predisposing factor for NE in broiler chickens [A recent study has shown that early microbiota disruption (dysbiosis) is a major consequence of necrotic enteritis . In neonchickens ,24,25,26All animal handling procedures complied with the Institutional Animal Care and Use Committee (IACUC) at the University of Arkansas, Fayetteville. Explicitly, the IACUC approved this study under protocol # 21018.®) was provided by Promitec S.A.  and feed inclusion based on the manufacturer’s recommendations. The product contains EO of Lippia origanoides microencapsulated with maltodextrin by spray drying with a diet inclusion rate of 37 ppm [Lippia origanoides essential oils of LEO is summarized in The LEO  sample was submitted to chromatographic analysis 7890A The LEO  to separate the serum. Serum levels of FITC-d (ng/mL) were used as a biomarker to evaluate intestinal permeability as described by Baxter et al. [At the end of the trial, on d 25 of age, two random chickens per cage were selected (n = 20) and orally gavaged with 8.32 mg/kg of body weight of fluorescein isothiocyanate-dextran . One hour after FITC-d administration, chickens were euthanized by COr et al. . Commerc2 inhalation. Lesion score was evaluated as previously described by Hofacre et al. [To evaluate Ileal NE lesion score, four chickens per each cage, separately from those in e et al.  where 0 8 cfu of ST per bird by oral gavage. At day 18, chickens in challenged groups were orally gavage with 40,000 oocyst per mL. The dose was selected based on a previous trial conducted to determine a challenge dose causing sub-clinical coccidiosis and reduction of 35% body weight gain as described previously [C. perfringens culture was administered on days 22 and 23 of age via oral gavage at a concentration of 1 × 109 cfu per bird and was also used in the in vitro proliferation assay (described below).The challenge organisms ST, EM- GS, and CP isolate details and culture conditions used to induce NE are described in previous studies ,25,26,33eviously ,25,26. T5 cfu/mL of CP (positive control); or (Group 3) 3 mL TSB supplemented with THIO + 3 mL supernatant from a digested diet supplemented with LEO inoculated with 105 cfu/mL of CP. Samples were incubated anaerobically at 40 °C, with tubes set at a 30° angle with continuous shaking (200 rpm) for four hours. Following incubation, ten-fold serial dilutions were made from all three experimental groups, plated on TSA supplemented with THIO and incubated for twenty-four hours at 40 °C, anaerobically. Results are reported as log10 cfu of CP per mL.The in vitro digestion model used in the present study was based on previous publications, with minor modifications ,35. The p < 0.05, and the P value was established with an alpha level of p < 0.05. Total mortality were compared by a chi-square test of independence, testing all possible combinations to determine the significance with an alpha level of p < 0.05.For performance variables, each group had ten replicates and ten chickens in each replicate . BW, BWG, FI and FCR were calculated by cage bases (n = 10). The number of samples per variable evaluated was n = 5 for in vitro proliferation of CP; n = 40 for NE lesion score; n = 20 for serum variables. Hence, all these variables implied a normal distribution (Shapiro–Wilk test), and the homoscedasticity was verified (Levene’s test). Accordingly, the parametric test of analysis of variance as a completely randomized design, using the General Linear Models procedure of SAS was used . Signifip < 0.05) in BW at d 7 when compared with negative control chickens. It was interesting to observe that chickens that received the diets with the inclusion of LEO had a similar BW compared with negative control chickens on d 14 and 18, before E. maxima challenge. However, BW of and BWG of positive control chickens that were challenged with EM and CP were severely affected  compared to 1.25% in chickens that received LEO. No mortality due to NE in the negative control group. Overall, chickens in the positive control group had also an increase in mortality rate compared with negative control chickens, or chickens that received LEO .The results of the evaluation of serum levels of FITC-d, SOD, IFN-γ, and IgA in broiler chickens consuming a diet supplemented LEO on a NE challenge model are summarized in Recent studies have demonstrated that the immune modulator properties of synbiotics significantly reduced ST infection, and such reduction was associated with reduction in the severity of NE caused by CP in broilers chickens challenged with both enteropathogens ,38.It is estimated that EO are produced by over 17,500 species of plants; however, only a minor fraction are used commercially . The cheDue to their antimicrobial properties, the use of whole EO is more advantageous than purified components due to synergistic effects, multiple biological properties, and fewer probabilities to select for antimicrobial resistance ,4,39,41.C. perfringens and other pathogenic bacteria compared to antibiotics [C. perfringens in the digested supernatant of an in vitro digestive model compared to the positive control diet by 1.94 logs. While EO have shown enhanced antimicrobial activity, the encapsulation of EO has shown higher antimicrobial activity compared to the use of EO alone by slowing down the degradation and reducing the organoleptic effects of EO [Even at low concentrations, EO have shown substantial and superior antibacterial properties against ibiotics . Similarts of EO ,48. In tts of EO ,28. At ats of EO ,17. Morets of EO ,50. Othets of EO ,52 have ts of EO  describeFurthermore, there was a significant reduction in IFN-γ serum levels in chickens that received LEO compared with positive control chickens, suggesting a reduction in the inflammatory response ,55. MoreL. origanoides have shown to increase the antioxidant response, radical scavenging activity, and apoptosis, suggesting a unique mechanism by these compounds [In addition to their antimicrobial properties, other assets of EO are their antioxidant activity and radical scavenging activities . In factompounds ,66,67,68At an inclusion rate of 37 ppm LEO, this formulation showed a significant reduction of in vitro CP proliferation as well as reduction of the harmful effects induced by the infection/dysbiosis of the NE model compared with the positive challenge control group. These effects were associated with the antimicrobial, anti-inflammatory, and antioxidant properties of LEO. However, further investigation to compare the effect of LEO and the standard antibiotic treatment of clostridial NE is required. It is fair to mention that this was a limitation in the present study. Moreover, it is imperative to establish standardized protocols that consider individual and inter-sample variability and consider the utility of molecular tools and epigenetic adjustments underlying phytobiotics such as EO, where research has not yet been elucidated.


Alpha- and Gammaproteobacteria were the predominant contributors. However, the exoprotein-producing microbial communities varied with depth: SAR11 in the shallow waters, Pseudomonadales and Nitrososphaeria in the mesopelagic layer, and Alteromonadales, Rhizobiales, and Betaproteobacteria in the bathypelagic layer. Besides viral and unknown proteins, diverse transporters contributed substantially to the exoproteomes and varied vertically in their microbial origins, but presented similar patterns in their predicted substrate identities throughout the water column. Other microbial metabolic processes subject to vertical zonation included proteolysis, the oxidation of ammonia, nitrite and carbon monoxide, C1 metabolism, and the degradation of sulfur-containing dissolved organic matter (DOM). Our metaexoproteomic study provides insights into the depth-variable trends in the in situ ecological traits of the marine microbial community hidden in the non-cellular world, including nutrient cycling, niche partitioning and DOM remineralization.The proteins present in the extracellular environment of cells, named the “exoproteome,” are critical for microbial survival, growth, and interaction with their surroundings. However, little is known about microbial exoproteomes in natural marine environments. Here, we used a metaproteomic approach to characterize the exoprotein profiles (10 kDa-0.2 μm) throughout a water column in the South China Sea. Viruses, together with  Exoproteins are present in the extracellular environment in close proximity to specific biological systems after their release by cell secretion, lysis, or leakage . These pin silico analyses have shown that 8–44% of the encoded proteomes of marine bacteria can be exported to the extracellular environment as theoretical exoproteomes  proteins, which act as virulence factors in many pathogens , upper mesopelagic layer (200 m), and bathypelagic layer  at the SEATS station  in the SCS in August 2012. To enrich the exoproteins for analysis, each sample was prepared from 100 L of seawater, and precautions were taken to avoid any possible contaminations during the experiment. In brief, sequential filtration with GF/F glass fiber filters  and Durapore membrane filters  was performed gently at a filtration rate of less than 1 L/min to eliminate any remaining cells once the seawater had arrived onboard. A final concentration of 0.01% (w/v) sodium dodecyl sulfate was added to increase protein recovery and 5 mM of sodium azide was added to inhibit microbial growth. A Pellicon 2 tangential flow filtration system equipped with a 10-kDa-cutoff regenerated cellulose membrane package (Millipore) was used to concentrate the filtrate to a volume of around 450 mL. The concentrates were stored at –80°C until a second concentration procedure was performed in the laboratory to obtain the final metaexoproteomic samples (around 10 mL). Cell counting of the filtrates using flow cytometry showed cell concentrations below the detection limit of flow cytometry (data not shown), indicating little contribution of intact cells to the samples. It should be noted that concentrating large volumes of seawater was a time-consuming, laborious and expensive process, especially with deep seawater. Therefore, we collected duplicate seawater samples from the DCM layer, but only one seawater sample each from the upper mesopelagic layer and the bathypelagic layer due to the limitation of ship time.2PO4 in 25% acetonitrile (ACN), pH 3.0]; 10–40 min, 5–35% buffer B ; 40–41 min, 35–80% buffer B; 41–44 min, 80% buffer B. The peptide fractions were desalted with C18 columns and resuspended in buffer C . The sample was loaded at 8 μL/min for 4 min, and analyzed under a elution gradient  at 300 nL/min on a Shimadzu LC-20AD nano-HPLC in line with a Thermo Q Exactive mass spectrometer using data-dependent fragmentation with a dynamic exclusion duration of 15 s. A normalized collision energy setting of 27.0 in the high-energy collision dissociation operating mode was used to fragment selected peptides. Resolutions of 70,000 and 17,500 for the Orbitrap analyzer were used for the MS and MS/MS scans, respectively, and the MS scans were set to between 350 and 2,000 Da.The experimental procedures for protein precipitation, preparation and final trypsin digestion for the MS analysis were conducted as described previously . BrieflyThe raw MS data for each sample were merged and formatted to MGF files with Proteome Discoverer . Because we did not create sample-specific metagenomes, the proteins were identified and quantified with a two-step iterative strategy similar to the previously described MetaPro-IQ approach . Brieflyi) with the equation:A quantitative analysis was used to estimate the relative abundances of the proteins (Pi) are divided by the protein length (Li). The subscript i or k denotes a protein or a protein group identity, and n is the total number of proteins or protein groups detected in a sample.in which the unique spectral counts (S1 the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (version 81), and the National Center for Biotechnology Information Non-redundant Protein database  with an E-value cutoff of 1e-5 and other default parameters. The taxonomic annotations were inferred from taxonomic information on the hits in the NCBI-nr database. Note that the taxonomic annotation of a bacterial protein was modified to that of a putative viral protein if the protein function was assigned to a viral structural protein.All identified proteins were newly annotated using BLASTP against the Clusters of Orthologous Groups of proteins (COG) database,A high-throughput shotgun metaproteomic approach was used to investigate exoprotein profiles collected from the deep chlorophyll maximum DCM, 75 m), the upper mesopelagic layer (200 m), and the bathypelagic layer  in an oligotrophic basin of the SCS . The tan m, the uProteobacteria (30–58%), especially Alpha- and Gammaproteobacteria. Their descent taxonomic groups, including Pelagibacterales (SAR11), Rhizobiales, Alteromonadales, Pseudomonadales, and the FCB  group, contributed the most proteins to the exoproteomes. Viruses contributed similar amounts of protein (37–41%) as bacteria at the DCM, but the abundance of viral proteins decreased dramatically to 12% at 200 m and to 9% at 3,000 m. Except for the large amount of taxonomically unclassified proteins and putative viral proteins, most of the virus-associated proteins originated from the viral families Siphoviridae (1.1–4.2%), Myoviridae (1.0–3.5%), Podoviridae (0.02–2.1%), and Phycodnaviridae (0.07–2.2%). The abundances of eukaryotic proteins were similar among layers (4.7–8.9%), and the majority of them were assigned to eukaryotic phytoplankton, such as Haptophyceae (0.3–1.2%), Pelagophyceae (0.3–1.2%), and Prasinophyceae (0.2–0.8%). Archaea contributed the least proteins (2.3–7.6%) to the exoproteomes, and Nitrososphaeria [a reclassified class in GTDB database for the NCBI phylum Thaumarchaeota , Nitrososphaeria (6.3%), Candidatus Thioglobus (2.6%), Actinobacteria (2.6%), Nitrospinae (2.3%), Deltaproteobacteria (1.1%), Chloroflexi (1.0%), Nitrospirae (1.0%), and Chromatiales (0.9%) were the most abundant at 200 m, whereas those from Alteromonadales (20.2%), Rhizobiales (8.6%), Betaproteobacteria (7.4%), Oceanospirillales (2.2%), and Sphingomonadales (1.3%) were particularly abundant at 3,000 m.The distribution patterns of microbial contributors to the exoproteomes varied throughout the water column. The protein abundances of most microbial groups declined with increasing depth. However, proteins from Virus-associated proteins were one of the major components of the exoproteomes throughout the water column, although their abundance decreased from 41% at the DCM to 12% at 200 m and 9% at 3,000 m. Of the virus-associated proteins, 48–70% were viral structural components, such as capsid or tail proteins. The remaining virus-associated proteins were largely functionally uncharacterized (28–45%), with the exception of several virus-encoded auxiliary metabolic proteins (AMPs), including the photosystem II D1 and D2 proteins, chaperonin GroES, and transporters of iron complexes and amino acids .The non-viral proteins included cellular proteins with diverse functional categories (35–71%) and functionally unknown proteins (17–24%) . At all 3+ and iron complexes were abundantly detected throughout the water column and were the dominant transporters at 3,000 m, suggesting that iron was one of the most important nutrients recycled in the deep ocean. Vertical variations in transporters, classified by predicted substrates, were apparent among the different prokaryotic taxonomic groups  and were associated with diverse functions. In the 3,000 m layer, 30, 17, 14, and 8% of eukaryotic exoproteins were associated with cytoskeleton, protein chaperones, translation and ribosomal structure, and energy metabolism, respectively. More than one third of these exoproteins were from eukaryotic phytoplankton, and included actin, tubulin, heat shock proteins, ribosomal proteins, translation factors, and ATPases. Interestingly, most of these exoproteins from eukaryotic phytoplankton, including the rubisco large subunit (rbcL), were also detected in the upper two layers, providing the first exoprotein evidence of biological pump in the ocean.Roseobacter and Synechococcus, have been reported  populations , which was subsequently used for hydroxylamine oxidation, although this was not supported by isotopic trace experiments  from ulations . Moreoveeriments . Insteaderiments . Regardleriments , which ieriments . Similareriments , only amat 200 m . Ammoniaproposed . ConsistNitrospinae and Nitrospirae, consistent with the genetic capacity of their cultured isolates and natural lineages for nitrite oxidation within the nitrification process  were associated with two groups of nitrite-oxidizing bacteria (NOB),  process . All sub process . NXR pro the SCS . The preanalysis . Taken t2, were detected from diverse heterotrophic bacteria, including SAR11, Bacteroidetes, Actinobacteria, and Chloroflexi, indicating that they probably utilize CO. It has been estimated that CoxL genes are as abundant as one per 14 bacterial cells, based on a marine metagenomic library  of aerobic carbon monoxide dehydrogenase (CODH), which oxidizes CO to CO library . Therefo library  demonstr library . Therefo2 occurred in SAR11 because no formate dehydrogenase was detected from SAR11, but only from other bacteria. In addition, methylamine was probably not oxidized by SAR11 but degraded by Alteromonas at 3,000 m, because the MauB and QhpA proteins in another methylamine oxidation pathway were detected. The vertical distribution of these proteins indicated that the roles of SAR11 in the cycling of low-molecular-weight DOM are depth-dependent.It has been experimentally demonstrated that SAR11 are versatile in the utilization of diverse C1 or methylated compounds, mainly for energy production rather than carbon assimilation, in a process called “carboxidovory” . ProteinNotably, a suite of proteins involved in sulfur metabolism were detected, including DmdC, which acts in the pathway of dimethylsulfoniopropionate demethylation; SBP56, which acts in the oxidation of methanethiol; and ssuD, which acts in the degradation of alkanesulfonate . Except psbA gene in the surface ocean (Synechococcus phage in the 3,000 m layer, suggesting that some cyanobacterial cells in the surface ocean were probably phage-infected and were lysed as they sank to the deep ocean. Overall, our results demonstrate the depth-dependence of host-virus interactions and their roles in carbon export in the ocean.The significant contribution of viral structural proteins to the metaexoproteomes throughout the water column demonstrated the presence of abundant viruses in the ocean and indicated the potential interactions between viruses and their hosts. The presence of and variations of host-virus interaction at depths were verified with the detection of viral AMPs in the exoproteomes . AMPs arce ocean  and the ce ocean  have beeAlpha- and Gammaproteobacteria, from the sunlit zone to the deep dark ocean. The origin of exoproteins suggests that these microbial groups make a significant contribution to the oceanic DOM pool. However, the contribution of abundant taxa is depth-dependent, e.g., SAR11 in the shallow waters, Pseudomonadales and Nitrososphaeria in the mesopelagic layer, and Alteromonadales, Rhizobiales and Betaproteobacteria in the bathypelagic layer. Furthermore, we demonstrate the vertical zonation of exoprotein-linked functions, including substrate transport, protein degradation, nitrification, oxidation of C1 and methylated compounds, sulfur metabolism and host-virus interaction. Especially, microbial players are depth-dependent despite of utilizing similar DOM substrates; different extracellular proteases are involved in protein recycling at different layers; nitrification by AOA and NOB is more abundantly present below the euphotic zone, whereas degradation of sulfur-containing DOMs and host-virus interactions are more frequently observed in the shallow layers. The depth-variable trends in these categories of metabolic functions provide new insights into the dynamic interactions between microbes and their surroundings in terms of nutrient uptake, DOM remineralization, and niche partitioning in the ocean.In this metaexoproteomic analysis, we investigate the natural exoprotein profiles throughout an oceanic water column. Our study sheds light on the complex origins of the exoproteins and the predominant contributors to the exoproteomes, such as viruses, in situ metabolic rate measurements. This will comprehensively improve our understanding of microbial functions and roles in the ocean.Cell-free active enzymes have been documented in epipelagic and bathypelagic waters, and their activities are regarded as the “gatekeepers” of biogeochemical cycles . HoweverIt should be pointed out that only one sample was collected in both the meso- and bathypelagic layers owing to the limitation of sampling time. Because the content of protein in particulate organic carbon is very low, large volumes of seawater must be concentrated to obtain sufficient protein for proteomic analysis, which is a time-consuming, laborious and expensive process. This brings forward challenges for repeated sampling, especially in deep waters. In oceanography, oceanographic consistency across the vertical depth structure is a good substitute for biological replicates when validating results, because the fluid feature of the ocean may allow different water masses to be captured, even within a short time, leading to unreal variations in the microbial community and other properties among replicates . The strwww.ebi.ac.uk/pride/archive/, accession number PXD020896.The data presented in the study are deposited in the ProteomeXchange repository available here: D-ZW and Z-XX conceived the study. Z-XX, S-FZ, and M-HW collected the samples. Z-XX and Y-BH analyzed the data. Z-XX and LL performed the proteomic experiment. Z-XX, Y-BH, and D-ZW wrote the manuscript. All authors contributed to the discussion of the results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A protective effect of vegan diets on health outcomes has been observed in previous studies, but its impact on diabetes is still debated. The aim of this review is to assess the relationship between vegan diets and the risk for type 2 diabetes (T2D) along with its effect on glycemic control and diabetes-related complications. In accordance with PRISMA  guidelines, Pubmed and Cochrane library databases were systematically searched for all relevant studies. Seven observational and eight randomized controlled (RCTs) studies were included. The methodological quality of studies was assessed using the National Institutes of Health quality assessment tool for observational cohort and cross-sectional studies and the Cochrane Risk of Bias Tool for RCTs. We found that a vegan diet is associated with lower T2D prevalence or incidence and in T2D patients decreases high glucose values and improves glucose homeostasis, as reported from the majority of included studies. This approach seems to be comparable to other recommended healthful eating models, but as it may have potential adverse effects associated with the long-term exclusion of some nutrients, appropriate nutritional planning and surveillance are recommended, particularly in specific groups of diabetic patients such as frail elderly, adolescents, and pregnant or breastfeeding women. Interest in vegan diets is increasing around the world: it is estimated that the number of vegans in the US grew by 600% from nearly 4 million in 2014 to 19.6 million in 2017 [An association between a vegan diet and a reduced risk of total cancer incidence has been demonstrated in large prospective cohort studies , but itsThe aim of this paper is to conduct a systematic review of all observational and intervention studies that describe the relationship between vegan diets and the risk for type 2 diabetes (T2D), along with its effect on glycemic control and T2D-related complications.Following the PRISMA  statement  we syste1c (HbA1c) levels and risk factors for diabetes related complications—Body Mass Index (BMI), waist circumference, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, systolic and diastolic blood pressure, microalbuminuria or other parameters of renal function, electrochemical skin conductance or other parameters of nerve function in case of randomized controlled studies. Studies were excluded if there was no vegan intervention ; intervention < 4 week; no omnivorous control; were not conducted in humans or in individuals with T2D ; were conducted in children, adolescents, pregnant or breastfeeding women. When multiple articles for a single study were present, we considered the most recent publication, eventually completed with data from the previous reports. Three investigators  independently reviewed and extracted relevant data. All discrepancies were resolved by consensus.We considered all observational  and randomized controlled studies based on the PICO framework  for methodological quality using elements of the National Institutes of Health (NIH) quality assessment tool for observational cohort and cross-sectional studies  and the The initial search identified 898 results  and after the application of inclusion/exclusion criteria, a final sample of 15 articles  were included for this review .The main characteristics of the included observational studies reporting the effects of vegan or plant-based dietary patterns on risk for T2D  are summarized in 2 vs. 28.8 kg/m2, respectively), but also after adjustment for BMI and others variables , vegans had a lower risk of T2D than nonvegetarians (Odds Ratio (OR) 0.51, 95% Confidence Interval (CI) 0.40–0.66). Different results were observed in India’s third National Family Health Survey [n = 2560) and omnivores  was observed  and the difference between the mean BMI was also low (20.5 kg/m2 in vegans vs. 20.7 kg/m2 in omnivores).Out of the seven observational studies, two cross-sectional studies reported the prevalence of T2D among vegans ,13. In tFrom the seven observational studies, five prospective cohort studies evaluated the incidence rate of T2D among vegans ,11 or pen = 3545), respective to 2.12% of omnivores  after 2 years. The vegan diet was resulted as protective for the development of T2D, respective to non-vegetarians in both non-Black  and Black  participants  after adjustment for age, sex, education, income, television watching, physical activity, sleep, alcohol use, smoking and BMI.In the population of the Adventist Health Study-2  that incSlightly different results were observed in the EPIC–Oxford cohort study of 45,314 UK adults  aged 20–90 years. Over a mean follow-up of 17.6 years, the percentage of incident T2D cases among 1781 vegans was lower than in 22,796 omnivores , and after adjustment for age, education, socio-economic status, physical activity, ethnicity, smoke and alcohol intake, the hazard ratio for developing T2D in vegans was significantly lower than in regular meat eaters . However, when the analysis was further adjusted for BMI, the significance was lost (Hazard Ratio (HR) 0.99, 95% CI 0.66–1.48). It is important to notice that authors have evaluated regular meat eaters (defined as subjects who consumed ≥ 50 g of meat per day) and low meat eaters (consumption of <50 g of meat per day) separately, using regular meat eaters as a reference group but not a whole sample of omnivores.In the ATTICA Cohort Study , the dieThe impact of PBD, and its healthy and unhealthy version, on T2D incidence was studied on the sample of more than 200,000 participants  aged 25–75 years, recruited in three US cohort studies: Nurses’ Health Study, Nurses’ Health Study 2 and Health Professionals Follow-up Study [Additionally, in the Rotterdam Study (RS) , a prospEight randomized controlled studies evaluating the impact of vegan or PBD on glycemic control and diabetes-related complications in T2D patients met all inclusion criteria. The main characteristics of the included studies are summarized in 1c levels after vegan diet, and in five studies, this reduction reached statistical significance [1c levels [All included studies reported a reduction between −0.3%  to −1.4 ificance ,19,20,21c levels ,17,20,21c levels ,20,21) tc levels  to −1.0%c levels . Bunner c levels  describec levels ,20, the Not all studies reported the effect of the vegan diet on fasting plasma glucose (FPG). Out of six studies evaluating this variable ,19,20,212 [2 [2 [Five studies reported the BMI measurement as an outcome ,19,20,212 ,19,20,212 ,21 reach2 [2 [2  reported2 [2 [2 ,19 was tAs regards the waist circumference, three studies evaluated this outcome ,17,20, aAll studies reported the evaluation of systolic (SBP) and diastolic (DBP) blood pressure. In the intervention groups, the changes in SBP varied from −11.5 mmHg  to +1.0 All studies except Lee et al.  reportedAll studies reported the evaluation of HDL cholesterol. In the intervention groups, the changes from baseline to final values varied from −7.72 mg/dL  to + 2.2On the other hand, not all studies evaluated changes in LDL cholesterol ,19,20,21Relatively uneven results were observed assessing triglyceride levels; all studies reported changes from baseline to final values, expressed in mg/dL. Only Wheeler et al.  reportedIt is worth saying that in the study of Ferdowsian et al.  and MishEvaluating renal function, four studies reported diverse outcomes: Nicholson et al.  and BarnChanges in clinical outcomes from baseline to final values by group assignment are summarized in Finally, Bunner et al.  evaluateLower DM prevalence among vegetarians compared to omnivores has been observed for decades, but it was well documented only recently, in particular through population studies of Seventh-Day Adventists. This religious movement originated in the United States and promotes a healthy lifestyle through the adherence to the vegetarianism with the avoidance of alcohol, tobacco, illegal drugs and caffeine-containing beverages. The rate of vegetarianism (including pesco-vegetarians and semi-vegetarians) in the Adventist Health study-2 was high; about fifty per cent and 4.48% of them were following vegan diets : the preNeither in the study of Tonstad et al.  evaluatiThe major limitations of the included observational studies are that the diet assessments were self-reported and that in cross-sectional studies, the dietary habits may not be maintained in time. Nowadays, many individuals define themselves “vegans”, although they may include little animal product in their diet, especially after following the strict diet for a longer time . Another1c levels after the vegan diet, although the duration of the intervention varied from 6 to 74 weeks. Besides the length of follow-up, there are many other factors that make it difficult to compare the results among studies: duration of disease, medication regimens, baseline HbA1c value, changes in medication during the intervention, baseline BMI, etc. It is worth noting that much of the effect of the intervention diets on glycemia appears to be mediated by weight reduction. Even more arduous is the comparison between the vegan and the omnivorous groups, as control diets in the included studies were not always neutral in energy balance and the approaches were different. In the vegan groups, mostly no restrictions in portion sizes, energy or carbohydrate intakes were placed; on the contrary, in some control groups, calorie-restricted diets were prescribed. These limitations are conclusive not only for the glycemic control parameters, but also for other cardiovascular risk factors . We can presume that for some patients with T2D, the vegan diet may be an efficient way to lose weight and maintain glycemic control.All randomized controlled studies reported the reduction in HbABesides the randomized controlled studies included in this systematic review, a recent meta-analysis of prospective cohort studies has also confirmed that the intake of plant-based proteins is significantly associated with a lower risk of cardiovascular disease mortality  .Despite the fact that the vegan diet is high in carbohydrates, all clinical trials included in this review have demonstrated its effect on glucose lowering, which is even stronger than that seen in other hypocaloric conventional diets recommended for people with DM ,20. ThisDietary fibers lower the postprandial glucose response by well-known mechanisms such as the reduction in gastric emptying and consequent slower starch digestion and glucose absorption. Additionally, glucagon-like peptide 1 (GLP-1) plays an important role; besides a slower gastric emptying action, it improves glucose uptake and disposal in peripheral, especially in insulin-dependent tissues. GLP-1 reduces glucose production from the liver through the inhibition of glucagon secretion . Some prAnother important factor by which we may explain the benefits of the vegan diet on different cardiometabolic risk factors is the lower fat content. It has been demonstrated that fat-rich diets lead to an increased intramyocellular lipid (IMCL) concentration through the mitochondrial oxidative phosphorylation genes downregulation in skeletal muscle . ExcessiThe improvement of insulin sensitivity was observed in correlation with lower serum ferritin levels several years ago , and latIt is well known that vegans are at risk of nutritional deficiencies, such as proteins, vitamin B12, calcium, vitamin D, iron, zinc or omega-3, if not well planned .Many clinical studies have demonstrated that plant-based proteins have lower anabolic capacity compared to animal proteins due to their lower digestibility and higher limiting essential amino acid content , which are more likely directed toward oxidation rather than being used for muscle protein synthesis. On the other hand, prospective cohort studies suggest that the difference in the anabolic effects of plant versus animal-based proteins could be reduced with an adequate protein intake (1.0–1.2 g of proteins/kg of body weight) or by blending different plant-based protein sources , which could mitigate sarcopenia, a common condition in elderly, especially in those with diabetes .It is essential that all vegans get enough vitamin B12, as this micronutrient is generally found only in animal-sourced foods and its deficiency prevalence among vegans is high . NeverthCalcium and vitamin D are crucial for maintaining healthy bones and preventing osteoporosis, although the other nutrients listed above may also play a role. Vegans may be at higher risk of lower bone mineral density (BMD) and fractures due to an inadequate intake of these nutrients. Iguacel et al.  has confWe can conclude that many large observational studies have demonstrated that the vegan diet is associated with lower T2D prevalence or incidence, although in some cohorts, it is not possible to distinguish if the beneficial effects derive from the vegan diet alone or from the overall healthy lifestyle. Furthermore, the results of randomized controlled studies performed in T2D patients have indicated its antihyperglycemic effect, even in the long-term.Moreover, clinical trials with vegan diet in pre-diabetes and T2D people, in which the quantity of simple and complex carbohydrates and the quality of the nutrients taken will be evaluated, should be conducted. In this way, some unevenness of results obtained in the different reported clinical trials could be clarified and an appropriate vegan diet for pre-diabetic and T2D individuals could be created to be used as a nutraceutical intervention.Finally, the vegan diet may be considered an acceptable and safe alternative to Western diets and seems to be comparable to other recommended healthful eating models , etc.), but it must be stated that it has been considered to be a therapeutic diet with adverse effects associated with the long-term exclusion of some nutrients. Appropriate nutritional planning and surveillance conducted by dietitians and nutritionists trained in a vegan diet are recommended, as vegans are more likely to require vitamin B12 , vitamin D, calcium and iron supplementation, as well as a sufficient amount of protein. This is important, particularly in specific groups of diabetic patients such as frail elderly, adolescents, and pregnant and breastfeeding women.Larger randomized controlled studies are necessary to confirm the effectiveness and safety of the vegan diet for diabetic patients.


The global COVID-19 pandemic has led to extensive development in many fields, including the diagnosis of COVID-19 infection by mass spectrometry. The aim of this systematic review and meta-analysis was to assess the accuracy of mass spectrometry diagnostic tests developed so far, across a wide range of biological matrices, and additionally to assess risks of bias and applicability in studies published to date.23 retrospective observational cohort studies were included in the systematic review using the PRISMA-DTA framework, with a total of 2858 COVID-19 positive participants and 2544 controls. Risks of bias and applicability were assessed via a QUADAS-2 questionnaire. A meta-analysis was also performed focusing on sensitivity, specificity, diagnostic accuracy and Youden's Index, in addition to assessing heterogeneity.Sensitivity averaged 0.87 in the studies reviewed herein (interquartile range 0.81–0.96) and specificity 0.88 (interquartile range 0.82–0.98), with an area under the receiver operating characteristic summary curve of 0.93. By subgroup, the best diagnostic results were achieved by viral proteomic analyses of nasopharyngeal swabs and metabolomic analyses of plasma and serum. The performance of other sampling matrices  was less good, indicating that these protocols are currently insufficiently mature for clinical application.This systematic review and meta-analysis demonstrates the potential for mass spectrometry and ‘omics in achieving accurate test results for COVID-19 diagnosis, but also highlights the need for further work to optimize and harmonize practice across laboratories before these methods can be translated to clinical applications. •Mass spectrometry complements RT-PCR in COVID-19 diagnostics.•Meta-analysis of mass spectrometry diagnostics for COVID-19 shows accuracy of 87%.•Diagnosis in admissions settings is a promising use-case for mass spectrometry.•Future work should focus on validating features and protocols across laboratories. These included accuracy, sensitivity, specificity, and Youden's Index, as well as an assessment of heterogeneity. Any diagnostic test must have a relevant use-case, and in this review we focused on applicability to hospital admissions , given t1.2The objective of this review was to benchmark a series of MS based diagnostic index tests against each other using RT-PCR as a reference test. We also aimed to identify how well new tests might meet a clinical role of accurate identification of COVID-19 infection, with a focus on admission settings. The review also sought to identify areas in existing research where bias or applicability issues may occur, and how future research may mitigate against these issues.22.1This study was conducted based on the principles of the Preferred Reporting Items for a Systematic Review and Meta-analysis of Diagnostic Test Accuracy (PRISMA-DTA) statement . Searche2.2For all articles identified under the search strategy, titles and abstracts were screened for eligibility. The relevant articles were then read in full, including data extraction for meta-analysis. In this work, the eligibility criteria for inclusion in the systematic review and meta analysis were set as follows: (a) evaluation of a diagnostic method for COVID-19 using mass spectrometry, based on ‘omics approaches, (b) using human biological matrices and (c) including diagnostic analyses, at a minimum reporting sensitivity and specificity by confusion matrix, or receiver operating characteristic (ROC) curves provided that the sensitivity/specificity trade-off was unambiguous. Articles in non-Roman characters were not included.The above search and eligibility steps were carried out by two researchers, with differences in identified articles reviewed by a third author for inclusion/exclusion.2.3The following items were collected by two researchers from articles identified above: key metadata for each article ; methods employed  and diagnostic outcomes . Diagnostic outcomes were taken directly from research where possible, or were calculated using confusion matrices based on reported sensitivity and specificity outcomes as applied to cohort data, or in one case by use of a reported ROC chart.2.4Two researchers independently evaluated risks relating to both bias and applicability using the Diagnostic Precision Study Quality Assessment Tool (QUADAS-2) , with th2.5Meta-analysis was performed for the aggregate of mass spectrometry ‘omics based approaches. Given the small sample sizes, not all subgroups offered meaningful results, but subgroups comprising viral proteomics, blood-based metabolomics, and novel ‘omics approaches  were reviewed independently from the aggregate. The following ratios were calculated: sensitivity, specificity, diagnostic accuracy, Youden's Index, positive likelihood ratio (PLR) and negative likelihood ratio (NLR).Sensitivity was defined as the true positive rate, i.e. the probability that a positive test result will be obtained when the disease is present, and calculated as TP/(TP + FN). Specificity was defined as the true negative rate, i.e. the probability that a negative test result will be obtained when the disease is not present, and calculated as TN/(TN + FP). Youden's Index was defined as sensitivity - (1 - specificity), or alternatively, one minus the sum of the error rates. The PLR was defined as the true positive rate/false positive rate. The NLR was defined as false negative rate/true negative rate.2. In this work, a p-value below 0.10 or I2 value greater than 50% was taken as evidence of substantial heterogeneity of diagnostic power; it should be noted however, that lower values do not necessarily confirm homogeneity, only an absence of evidence for heterogeneity [Heterogeneity of diagnostic power across the different biofluids investigated in this work was investigated by measuring Cochran's Q and Higgins Iogeneity ,12. A su2.6All statistical analysis was performed in the R Studio environment ,14, with33.1In total, 253 articles were identified in the initial search strategy by the terms described, after removing 308 duplicate results. From this initial list, 51 were identified as meeting the eligibility criteria and 202 were excluded. The articles on this shortlist were then read in full. 23 of the 51 identified articles contained the complete set of diagnostic accuracy data to allow for meta-analysis, albeit for one article  the dataThe studies analysed in this review were all conducted in the years 2020–2021, and in total recruited 5402 participants. Reflecting the difficulties of conducting research in a pandemic, all 23 studies were based on retrospective observational cohorts. 11 of the 23 studies separated participants into training and testing groups. Only 1 study included training, testing and blind validation cohorts. The 23 studies were conducted in 12 different countries, mainly in Europe and the Americas, coincident with high prevalence of COVID-19 infection in 2020 and the first half of 2021 [3.2In the analysis that follows, Unclear does not denote ‘medium’ risk of concern; rather it denotes that insufficient information was provided, and there is no basis to consider the study to be at ‘low’ risk of bias or inapplicability.bias around patient selection, 30% of the studies provided no cohort analysis, making it impossible to ascertain whether the work was free from bias in this regard. Furthermore, only 9% studies specified whether participants were recruited consecutively or at random. Only 23% of studies explicitly stated that asymptomatic patients were included, 39% stated that they were excluded, and 39% provided no information, potentially biasing results. Overall, 57% of the studies were assessed as high risk of bias in the patient selection domain, and 22% unclear, with only 22% studies assessed as at low risk of bias.In terms of risks of applicability of patient selection, 61% of the studies reviewed in this report stated explicitly that participant recruitment took place in a hospital setting with 17% obtaining samples from a biobank, and 22% not stating recruitment location. Hospital-based participant recruitment is relevant if MS based testing is considered to have a use-case as a clinical admissions tool. Although hospital recruitment can introduce selection bias by providing the index test with participants with high viral load and low incidences of asymptomatic disease, from an applicability perspective a hospital setting mirrors application in a clinical setting for use on admission for triage or allocation of patients to COVID-19 specific wards. 57% were considered low risk for applicability of patient selection  and the rest were assessed as of unclear risk.In terms of risks of bias in the index test, 52% of studies operated with separate training and test/validation tests. 48%, however, did not, introducing bias due to test parameters being defined to maximise sensitivity, specificity and AUROC. Due to overfitting, this can lead to sensitivity and specificity being overstated versus the results that could be obtained in a blind validation population. In addition, one of the studies split the sample population into too small a subgroup for appropriate analysis. Consequently, 52% of studies were assessed as high risk in the index test domain and 48% as low risk. Because all tests reviewed here had diagnosis of COVID-19 as their primary objective, no issues relating to applicability of the index test were identified.Looking at risks of bias to the reference test, the studies reviewed in this work used RT-PCR as the gold standard. Mass spectrometry approaches may not be as clinically sensitive as RT-PCR, but offer an alternative approach potentially capable of a faster route to identify more at risk individuals  and support clinical decisions. As many diagnostic laboratories have the technology to perform such analyses. Their use would also capitalise on this alternative established capacity. All 23 studies were considered to be of uniformly low risk of bias with regard to appropriateness of the reference test. Similarly, in terms of applicability, the reference test has as its primary purpose the diagnosis of COVID-19 positive status in participants, and so the risk of inapplicability of the reference test was deemed to be low in the reference test domain for all but two studies, which did not uniformly use RT-PCR as the reference standard for all participants and substituted alternatives in a small number of cases.Regarding the flow and timing, whilst all participants in all but one of the studies reviewed received the same reference tests, only 9% of studies specified the time difference between index test and reference test, but with one of these the interval was too large to be considered a low risk. 43% of studies were classified as low risk due to nasopharyngeal swabs being used for both the index MS-based test and the RT-PCR test, making the timing of both tests simultaneous. The remaining 52% of studies were classified as unclear risk in the flow and timing domain.Finally, in terms of 3.3The key extracted diagnostic indicators are summarised in t-test .Across the studies reviewed, sensitivity ranged from 0.62 to 1.00 (aggregate sensitivity of 0.87), and specificity ranged from 0.72 to 1.00 (aggregate specificity of 0.88). Specificity was greater than sensitivity on average, albeit the difference was not statistically significant based on a two-tailed In terms of biofluids analysed, sebum was analysed in 2 papers, and delivered the lowest aggregated sensitivity (0.76) and specificity (0.82), calculated by summing confusion matrices. Saliva was investigated in 2 studies, with sensitivity and specificity of 0.74/0.75 for metabolomic analysis of saliva, and 1.00/0.93 for proteomic analysis. Breath was analysed in 4 studies, with comparable sensitivity (0.78) and specificity (0.81) to sebum. Nine (9) studies sampled nasopharyngeal swabs, with high sensitivity (0.89) and specificity (0.88). The remaining 5 studies sampled blood (either plasma or serum), with aggregated sensitivity of 0.89 and specificity of 0.96. Proteomic approaches that targeted the virus reported higher sensitivity and specificity than approaches that targeted the impact on the host, albeit within the latter category there was considerable variation.m/z only, but 2 studies targeted peptides originating from spike proteins, and 2 identified peptides originating from nucleocapsid proteins. For the 4 studies analysing breath, a wide variety of alcohols, aldehydes and ketones were found to differentiate the populations, but there was limited overlap, with heptanal and octanal featuring in 2 of the 4 studies. In terms of sebomics, the studies described in this review found no differentiating features in common. Within plasma and serum, 2 papers identified ratios of amino acids (kynurenine in particular) as key differentiating features, and 2 papers focused on lipid dysregulation.As a single measure of performance, estimates of Youden's Index including confidence intervals are shown in 3.4p-value of 0.24, and Higgins' I2 was calculated as 16%. The latter value should be treated with caution given the small samples sizes assessed in this meta-analysis as Higgins I2 tends to be underpowered in the meta-analysis of studies with small n and therefore lower precision [2 does not represent evidence of homogeneity per se, but may indicate that the variability in results could be due to wide confidence intervals rather than unexplained heterogeneity, as is this case in this work .The studies show variation in their diagnostic performance measured by either sensitivity, specificity or Youden's Index  and - parecision . A low I2 was 0%. For blood-based analyses, Cochrane's Q was calculated as 4.1, and Higgins' I2 was calculated as 3%. For saliva, sebum and breath , Cochrane's Q was calculated as 3.2 and Higgins’ I2 was calculated as 0%.Heterogeneity was also investigated by broad method employed, specifically proteomics versus metabolomics, and also by subgroup. Heterogeneity was notably low for proteomics including viral proteins, with Cochrane's Q calculated as 7.6, and Higgins' IVisual inspection also illustrates the differences between, but similarity within, these methods A and B. 4For the studies considered in this work, there were variations in diagnostic metrics , but allIt should be noted, however, that the features identified in the studies reviewed here showed limited commonality, unsurprisingly given the varied sampling matrices and methods employed. Within subgroups of study, viral proteomics showed the greatest agreement in differentiating features, via both spike and nucleocapsid proteins . SeveralRT-PCR as a reference standard achieves very high analytical sensitivity and specificity and is generally seen as the clinical gold standard for release of patients from isolation , but theCompared with these benchmarks, MS-based approaches show promise based on achieved sensitivity and specificity and - given that mass spectrometry facilities are often available in hospital settings - may find a use-case by offering faster turnaround than RT-PCR and so supplementing clinical diagnosis. In addition, MS-based approaches offer alternatives in the initial stages of a pandemic, when supplies for PCR or other tests may be in short supply. Because of the ability of MS based techniques to identify dysregulation involving many pathways, such tests could provide information on the wider host metabolome and proteome. This potentially allows for prognosis as well as diagnosis, and promising results have already been obtained for mass spectrometry-based prognostic analyses of serum, plasma and saliva ,52,53.In this work, the best results were found to be delivered by metabolomic study of homeostatically regulated biofluids (serum and plasma) and by proteomic study of nasopharyngeal swabs, with areas under their respective sROC curves of xxx and xxx respectively. These results were mainly obtained by UHPLC-MS (for blood metabolomics) and MALDI-TOF-MS (for proteomics). These sampling methods are, of course, more invasive than skin swabs or exhaled breath, but based on the studies reviewed here, the invasive methods deliver the greatest diagnostic accuracy and are most concordant with the WHO's Target Product Profile, which targets 90% sensitivity and 99% specificity as desirable for point of care tests . At thisWhilst other ‘omics approaches may offer alternative solutions that are less invasive, results from alternative matrices show lower sensitivity and specificity and a lack of commonality of features identified. This heterogeneity suggests that considerable further work is needed in validation and optimisation. A further caveat is that host-based approaches have not been tested in an environment with widespread respiratory viruses, which could challenge specificity ,56. Of cIn addition, other limitations in the works reviewed are evident relating to bias and applicability, and the studies reviewed here were hampered by the natural difficulties in conducting research in a pandemic setting. Biases relating to patient selection and to the creation of separate testing and training sets for machine learning were common in the work described herein, reflecting the exigencies of the pandemic and acknowledged by all the articles reviewed. An overarching issue is the small sample sizes. As well as limiting the ability to create separate testing and training sets, the numbers recruited typically fall short of best practise for developing diagnostic tests, such as the Target Product Profile set by the UK Department for Health and Social Care which mandates testing in the validation set of 150 COVID-19 positive and 250 COVID-19 negative participants . Whilst 5The detection and diagnosis of COVID-19 by mass spectrometry has made substantial progress over the course of the SARS-CoV-2 pandemic. Achieved sensitivity and specificity of the diagnostic tests discussed in this review are encouraging, but with clear limits in the biases and applicability of the research undertaken so far. Whilst results based on proteomics and blood metabolomics delivered the most compelling performance, and these methods are most promising in terms of clinical application in the near term, more validation studies are still needed to reduce risks of bias and applicability. In the case of less invasive matrices, whilst the potential advantages are attractive, as yet there is little agreement between studies on suitably robust and reproducible targets.Whilst mass spectrometry techniques may show promise, and advances in this field could be applicable to disease diagnosis beyond COVID-19, future research should focus on reducing bias by recruiting larger numbers of participants without inappropriate exclusions, especially to meet thresholds for determining suitability for point of care or other use-cases. In addition, greater use of blind test sets for validation would reduce bias from over-fitted machine learning models in MS based diagnostic testing. Furthermore, and especially for the less invasive sampling matrices, considerable work is required to harmonize and optimize methodologies so that features can be validated between labs.10.13039/501100000266EPSRC Fellowship Funding EP/R031118/1 and EP/P001440/1 in addition to funding by the 10.13039/501100003513University of Surrey and 10.13039/501100000268BBSRC BB/T00212/1.The authors would like to acknowledge Matt Spick: Conceptualization, Methodology, Software, Formal analysis, Writing – original draft, Visualization. Holly M. Lewis: Data curation, Validation. Michael J. Wilde: Data curation, Validation. Christopher Hopley: Conceptualization, Writing – review & editing. Jim Huggett: Conceptualization, Writing – review & editing. Melanie J. Bailey: Supervision, Project administration, Funding acquisition.The authors declare no competing interests.