C*) and color (ΔE) were calculated to quantify differences between T0₋T1 and T1₋T2 on the fruit surface. Enzymatic activity  and phenolic composition were also quantified for each cultivar. ‘Granny Smith’ and ‘Cripps Pink’ browned minimally. In contrast, ‘Fiesta’ and ‘Mondial Gala’ browned severely, reporting high enzymatic activity and quantified phenolic concentration (QPC). Phenolic compound polymerization appears to play a significant role in enzymatic inhibition. ‘Topaz’ does not fit the high QPC, PPO, and browning formula, suggesting alternative pathways that contribute to apple browning.Phenolic compounds in fruit provide human health benefits, and they contribute to color, taste, and the preservation of post-harvest fruit quality. Phenolic compounds also serve as modifiers of enzymatic activity, whether inhibition or stimulation. Polyphenol oxidases (PPO) and peroxidases (POD) use phenolic compounds as substrates in oxidative browning. Apple browning leads to flesh color, taste, texture, and flavor degradation, representing a drawback for the variety and its’ market appraisal. This study was conducted to investigate the process of browning in 14 apple cultivars throughout post-harvest at three-time points: immediately (T0), one hour (T1), and 24 h (T2) after apples were cut in half. Color parameters L* (lightness), a* (red/green), b* (yellow/blue) were measured, and chroma (Δ In recent years, the attention of breeders, producers, and consumers alike have shifted towards ‘functional foods’, such as fruits high in biologically active phenolic compounds that are beneficial for human health ,2,3. PheBeyond their health benefits, phenolic compounds are involved in the genetically-regulated ripening process in climacteric fruit, particularly the physiological and biochemical pathways that involve flesh softening, pigment synthesis, and the production of volatile and aromatic compounds . These s2, the enzyme catalyzes the oxidation of phenols to quinones, which subsequently polymerize and generate brown and insoluble pigments known as melanin 1/2 and the chroma difference (ΔC) as ΔC = [(Δa)2 + (Δb)2]1/2 , and 24 h after that . The T2 measurement was established according to Kim et al. 1/2 , as theyΔb)2]1/2 . Three cm et al. , who shoSix flesh aliquots  were collected for each cultivar at T0, T1, and T2. Each aliquot ranged from 10 to 15 g for phenolic determination and from 1.5 to 2.0 g for enzymatic assays ,40. Each2EDTA (disodium ethylenediaminetetraacetic acid), and 0.1 g of PVPP (polyvinyl polypyrrolidone) . The samples were centrifuged at 10,000 rcf for 30 min at 4 °C , and the supernatant was collected and used for the activity analysis following the method described in Masia et al. [An aliquot of less than 2 g of frozen apple flesh, sampled from the equatorial region of the central portion of the mesocarp, was pulverized with liquid nitrogen to obtain a powder for enzymatic analysis. The frozen powder was added to 10 mL of an ice-chilled buffer made 200 mM, phosphate buffer (pH 7.0), 5 mM Naa et al. . The PODa et al. . The PPOa et al.  at 420 na et al. . The IAAa et al. .An aliquot of 15 g of frozen apple flesh chunks sampled from the equatorial region of the central portion of the mesocarp was homogenized in 20 mL of 70% chilled acetone  in a glass jar adapted for the Osterizer Sunbeam blender . The blender’s initial speed was set at ‘2’ to promote the breakdown of sizeable frozen apple pieces. After ca. 30 s, the speed was increased to ‘10’ and the sample was homogenized for 60 s. The homogenized sample and an appropriate volume of the extraction solution were added to ultracentrifuge tubes and rotated at 10,000 rcf for 20 min at 4 °C . The supernatant was collected, and the centrifugation steps were repeated twice more for 10 min each. The supernatants collected from the three centrifugation steps were mixed together. A 100 mL final volume was obtained by adding the extraction solution to the supernatant. All samples were filtered by syringe with 0.45 µm polytetrafluoroethylene (PTFE) filters , and a further speed vacuum rotation was performed to eliminate the organic components (acetone) and to obtain pellets. Pellets were freeze-dried and stored at −20 °C for a few days until further analyses.3CN (solution B) with 1 mL as total flux per minute. The following HPLC running settings were adopted for the two solutions: 0–11 min 9% B, 11–13 min 15% B, 13–20 min 17% B, 20–37 min 60% B, 37–38.5 min 100% B, 38.5–39.5 min 9% B. The injection volume equaled 20 µL, and the total running time for each sample was 39.50 min. Five minutes of reconditioning time were allowed between samples to stabilize the column. Empower™ 2 software  was employed to analyze the results.Samples for HPLC analysis were prepared as follows: each freeze-dried pellet was re-suspended in 500 µL of methanol and 500 µL of Milli-Q water and subsequently filtered through 0.22 µm polyvinylidene difluoride (PVDF) filters , into vials. The analysis was conducted in an HPLC apparatus made of a Waters 1525 binary pump, Waters Inline degasser AF, photodiode detector , auto-sampler (Waters 717 plus), and Waters Atlantis™ dC18  column . Two mobile phase solutions were adopted for all the samples and corresponded to 1% HCOOH + 0.5% MeOH (solution A); and 100% CHp-coumaric) and both retention time and ultraviolet (UV) spectrum were evaluated in the identification of the compound. Quantification was performed with the external standard method with the standards calibration curves . The reliability of the quantification method was assessed by the R2 value from the standards calibration curves. The chosen standards were catechin, epicatechin (for flavan-3-ols), chlorogenic acid, and p-coumaric acid , and phloridzin  since they are highly represented in apple flesh [The detection of compounds was carried out at 280 nm for flavan-3-ols  and dihydrochalcones  and at 320 nm for hydroxycinnamic acids  or Student Newman-Keuls (SNK) for post-hoc mean separation. Figures and tables reported different letters associated with the means when the model’s significance was at p < 0.05. Apple phenotypic and biochemical characteristics were also investigated via principal component analysis (PCA) performed in JMP . Data imagining was conducted in Prism v8.2.1 for Windows OS  and presented in the figures as means ± standard error. When error bars are not displayed in figures, Tukey’s least significant difference (LSD) is displayed.Flesh color change , phenolic compound concentration, and enzymatic activity were analyzed for cultivar and times of sampling. The analysis of variance was performed using Proc GLM in Statistical Analysis System software . The significance of the method was for C*), which expresses the saturation (brighter or duller) of that color [C*) is expressed as the total column for each cultivar , a* (red-green), and b* (yellow-blue) coordinates were captured to calculate color (E), along with the chroma  in Persic et al. [R2 = 0.06), suggesting that there may be other mechanisms involved with flesh oxidation and color changes. Tang et al. [Color changes were significant between T1 and T2, whereas chroma differences were more pronounced between T0 and T1, except for the severe browning cultivars A,B. Prevc et al. , along wg et al.  recentlyPeroxidase (POD) and polyphenol oxidase (PPO) activities were evaluated across time points and cultivars. Average POD activity levels across all cultivars remained stable between T0 and T1, although it increased significantly 41%) between T1 and T2 A. A simi% betweenPOD and PPO activity showed significant variability across the 14 cultivars regardless of sampling time . SimilarIn respect to PPO activity, ‘Mondial Gala’ and ‘Braeburn’ were amongst the highest across the cultivars, similar to the POD results . OverallR2 = 0.65) (data not shown). This trend underscores their similar behavior as defensive mechanisms [‘Delorina’ and ‘Topaz’ highlighted similar results for PPO activity and POD activity with the lowest levels overall, despite their more moderate/severe browning classifications . ‘Topaz’chanisms .R2 = 0.54, data not shown), perhaps suggesting an additional oxidative pathway that contributes to browning, especially later in post-harvest (24 h post-slicing).IAAox activity did not reveal significant differences across the cultivars evaluated ; althougAdditionally, similarities have been previously noted between ‘Topaz’ and ‘Jonagold’, a progeny of ‘Jonathan’, each containing high levels of quercetin glycosides, a flavanol primarily found in the skin, but also in the flesh, and may contribute to browning ,52. In tp-coumaric acid, phloridzin, and proanthocyanidins , appear to serve as a more efficient substrate for PPO [p-coumaric acid, catechin, and chlorogenic acid, browned the least  between these two factors (data not shown). Vrhovsek et al. [p-coumaric acid and lacking high levels of proanthocyanidins, may help illuminate the complexity involved in its’ enzymatic browning behavior (R2 = 0.32) and TPC:ΔE (R2 = 0.06), highlighting the vast dynamics involved in browning and the other potential mechanisms that contribute to enzymatic oxidation [R2 = 0.53), while Kołodziejczyk et al. [R2 < 0.40) at all across 22 apple cultivars. Overall, quantification of enzyme activity and phenolic concentration is not sufficient to predict browning. Instead, it is a comprehensive understanding of the phenolic compounds’ composition, as phenolic compounds are strong “manipulators” of enzymatic activity, whether inhibition or promotion [These four varieties that presented high QPC levels also reflect higher levels of proanthocyanidins . This wak et al.  also shok et al. . This hik et al. ,46. Convbehavior . Again, xidation . Furtherxidation  demonstrromotion .Two varieties further underscore the complexity of oxidative enzymatic browning in this present study: ‘Topaz’ and ‘Red Rome’.‘Topaz’ demonstrates the highest levels of proanthocyanidins amongst varieties  along wi‘Red Rome’ is characterized by an alternative narrative with similarly high levels of QPC as ‘Topaz’, but with moderate levels of proanthocyanidins, PPO, and POD , Table 2In sum, these exceptional cases of why ‘Topaz’ changed color dramatically, while ‘Red Rome’ changed minimally, given their phenolic compositions, enzymatic activities, and maturity status continue to underscore the narrative of a complex series of biochemical reactions that contribute to enzymatic browning in apples ,22.C*1, ΔC*2, PPO, POD, and IAAox (at 254 nm). Based on these factors, PC1 showcased nearly 44% of the variation, while PC2 described 28%, for a total of 72% of all variation explained by these factors across the cultivars evaluated  was conducted considering each cultivar’s biochemical, enzymatic, and color-changing parameters. This analysis included: QPC, ΔE1, ΔE2, Δvaluated . The varp-coumaric acid , and ric acid . Moderatubstrate , along wubstrate ,54. OtheLastly, the severe browning varieties appear to cluster and behave in two distinct groups/patterns, underscoring this process’s complexity . All of p-coumaric acid, chlorogenic acid, and proanthocyanidins are confirmed in these 14 apple cultivars. These phenolic compounds act as enzymatic modifiers, both as suppressors  and promoters . We noticed that polyphenol oxidase (PPO) and peroxidase (POD) increased in activity across the time post-harvest, as well as quantified phenolic concentration (QPC). Generally, cultivars with high QPC and high enzymatic activity led to more moderate or severe browning. Although oxidative browning appears to be more complicated than this simple formula, QPC’s composition seems to play an important role. In particular, the polymerization of individual phenolic compounds influences enzymatic activity differently. Furthermore, additional oxidative pathways may facilitate browning in apples, as some varieties such as ‘Topaz’ demonstrate high QPC, low PPO, and yet still brown severely after 24 h.The presence of the major phenolic compounds such as catechin, epicatechin,


The average δ13C and δ15N of C3 plants were −26.7 ± 1.5‰, and 3.9 ± 3.9‰, respectively, while the average δ13C and δ15N of C4 plants were −11.5 ± 0.8‰ and 4.6 ± 2.6‰, respectively. The δ13C and δ15N of plant-based processed foods were −21.8 ± 4.8‰ and 3.9 ± 2.7‰, respectively. The average δ13C and δ15N of meat, including beef, poultry, pork and lamb were -16.6 ± 4.7‰, and 5.2 ± 2.6‰, respectively, while the δ13C and δ15N of animal-based processed foods were −17.9 ± 3.3‰ and 3.3 ± 3.5‰, respectively. The average δ13C of beverages, including beer and wine was −22.5 ± 3.1‰. We verified that C-C4 constitutes a large proportion of fresh meat, dairy products, as well as animal and plant-based processed foods. The reasons behind this high proportion will be addressed in this study.Several previous studies on targeted food items using carbon and nitrogen stable isotope ratios in Brazil have revealed that many of the items investigated are adulterated; mislabeled or even fraud. Here, we present the first Brazilian isotopic baseline assessment that can be used not only in future forensic cases involving food authenticity, but also in human forensic anthropology studies. The δ Rapid industrialization and urbanization together with changes in lifestyles are among the factors responsible for changes in eating habits, especially the consumption of highly processed foods . The accOuro Branco” and “Carne Fraca” operations conducted by the Brazilian Federal Police revealed the addition of illegal substances in milk and beef, respectively, to increase business profits. The operations received public attention due to the health harm risks that those adulterations inflicted.Brazil is one of the world’s main food consumers which makes the country a vast market for food fraud and adulteration through the large quantity of foods produced, exported, or imported . In the 3 and C4 food sources in human and animal diets [4 photosynthetic pathway evolved in plants 24–35 million years ago as a response to the decrease in CO2 and increase in O2 atmospheric concentrations [Since sophisticated fraudsters have been generally capable of mimicking the physical and chemical compositions of targeted foods, there is a need to address and prevent food fraud and adulteration . Food saal diets . The pritrations . By the trations .4 plants became even more evident when humans started agriculture, since in this endeavor, C4 plants like maize (Zea mays L.), sorghum (Sorghum bicolor L.), pearl millet (Pennisetum glaucum L.) and sugarcane (Saccharum spp.) were among the species selected by humans to be cultivated [3 to a C4-based diet [4 plants were disseminated throughout the old and new continents [Several large terrestrial herbivores, like bovine, equid and other ungulates, followed by top carnivores, and, finally, hominids, have adapted to these new ecosystems . The preltivated . Maize hsed diet . Furtherntinents ,16,17.3 species, almost four times more than C4 species. The European Union (EU) and other temperate regions preferentially grow C3 rather than C4 plants, which modifies their food carbon isotopic composition. As an example, Martinelli et al. [4 grass or maize  have less negative δ13C values in comparison to countries with cattle preferentially fed with C3 plants .Even with this competitive advantage, there is still a large dominance of Ci et al.  observed2, nitrogen can be acquired from both the atmosphere through biological fixation and through the soil. After the Haber–Bosch process became a viable method at industrial scale, commercial crops also commenced to take up nitrogen from mineral fertilizers. Before the large-scale use of these fertilizers, crops had relied mainly on manures, organic sources of nitrogen generated by animals. Due to the multiple nitrogen sources for plant uptake, interpreting the nitrogen isotopic ratio (15N/14N) in plants is not an easy task [15N values around 0‰ can be distinguished from the use of manures with more positive δ15N values [15N of crops may be an indicative of the application of synthetic or organic fertilizers for improving plant growth .14N related to 15N along the way. Consequently, the organisms positioned higher in the food chain tend to be more enriched in 15N than the organisms positioned on the bottom of the chain, the so-called “trophic isotopic discrimination” [15N in comparison with omnivorous humans [After being incorporated into the plant tissue, nitrogen moves up along the food web with preferential loss of ination” . Therefos humans .Carbon and nitrogen isotopic ratios also have the potential to serve as tracers of dietary habits and migration in humans and animals ,27. Cons4 photosynthetic metabolism  constitute the major part of the diet in these large urban centers. Based on this fact, we hypothesize here that there is a pervasive presence of C-C4 in Brazilian food items although the staple foods in Brazil are rice (Oriza sativa L.), beans (Phaseolus spp. and Vigna sp.), and cassava (Manihot esculenta L.), plants that follow the C3 photosynthetic metabolism.Previous studies performed in Brazil have found that large urban populations have a relatively homogenous diet regardless of the geographic region of the country ,31. BaseIn this context, we developed the first Brazilian baseline assessment using carbon and nitrogen stable isotope ratios of about one thousand food items considered essential to test the above hypothesis. We also generated forensic data supporting food authenticity and traceability. As the assessment included in natura as well as processed foods, it might also become a powerful baseline for tracking human movement under the “supermarket diet” trend in Brazil.13C and δ15N values of every single sample of foods and beverages is available in the The complete dataset containing the δ3 photosynthesis metabolism and those using the C4 pathway (Ananas comosus L.), and their δ13C is intermediate between C3 and C4 plants (13C of C3 plants was −26.7‰ (n = 434), varying from −30.7 to −22.5‰, while the average δ13C of C4 plants was −11.5‰ (n = 47), varying from −14.2 to −10.2‰ (p < 0.01). The values in the CAM plants varied from −15.3 to −14.1‰, with an average of −14.8‰ . On the other hand, δ15N values of oilseeds (−0.3‰) and pulses (2.4‰) were significantly less positive (p < 0.01) in comparison with other the groups. In addition, nuts (17.3‰) had more positive δ15N values (p < 0.01) than vegetables, tubers, fruits, and C3-cereals, whose average δ15N values varied between −4.4‰ and 15.9‰  among different types of meat, close to the δ13C of C4 plants. The δ13C of lamb, pork, and poultry were also more negative than that of beef (p < 0.01) . In contrast, δ13C values in farmed-raised tilapia (Oreochromis niloticus) were similar to those values of pork and poultry  than pork and poultry due to the reasons which will be discussed later.Beef had the least negative δ < 0.01) a. On the poultry a. In add15N of animal protein consumed in southeast Brazil varied considerably among different products ; however, δ15N of farmed raised tilapia was only more positive (p < 0.01) than pork and poultry  and cassava, noodles and pasta. Alongside these products, cocoa (Theobroma cacao L.) powder and bitter chocolate had also δ13C values resembling C3 plants, as well as vegetal fat  have different δ13C values according to the contents of cocoa, milk and sugar. Chocolate powder for cooking, with an average δ13C of −19‰, represents a mixture of chocolate and C-C4 sugar.The isotope values of processed foods are summarized in the tables due to the large number of food items . Plant-bC values . Food itetal fat . Convers4 plants . Other p15N values of plant-based processed foods varied from 2‰ to 6‰ in most of the cases , which differed greatly from their feedstock, suggesting a high degree of isotopic discrimination in these products  related to other types of meat  is a bidimensional space, giving information about resources consumed by an organism and also the bioclimatic context in each such organism developed, the so-called “isotopic niche” as defined by Newsome et al. [4 and C3 plants in variable proportions as ingredients  with a wide range of values, from a minimum of −28.4‰ to a maximum of −14.8‰ (13C of beer samples was −20.9‰ (n = 78), having a bimodal frequency distribution, with values concentrating around −27‰ and around −19‰  tend to decrease, leading to an increase in the plant δ13C of crops in relation to forest leaves. This difference is obvious in 13C values of C3 crops in this study in relation to δ13C of tree leaves of the Brazilian Atlantic forest . There is not much overlap between δ13C of crops and forest besides a difference of ~6‰ between them  have less positive δ15N values compared to other domesticated plants in this study [There was large variability in the δ‰ to 18‰ b. This tse to 0‰ . Therefois study .15N of vegetables under the influence of mineral fertilizers was 3.1‰, increasing to 6.8‰ and 9.8‰ in vegetables fertilized with organic and animal-derived manure, respectively. The average δ15N of vegetables found here was 4.2‰, which is more positive than the value found by Huelsemann et al. [15N values of tropical soils relative to temperate soils [Huelsemann et al. , based on et al.  for plante soils ; and . Poultry and pork in Brazil are raised intensively and fed by a feed composed of maize and soy (Glycine max (L.) Merrill) that have the following isotopic composition: δ13C = −16.7‰, and δ15N = 0.6‰ [4  seeds, alfalfa (Medicago sativa L.) and oats (Avena sativa L.) are also used to feed dairy cattle in the former country [4 content in comparison with dairy products from other countries. The same is true for processed pork meats like ham, which, in Brazil, has a larger proportion of C-C4 than dry cured ham raised in Spain [C country . As milkin Spain  and Italin Spain . A similin Spain ,52.4 in animal proteins was even observed in farm-raised fish  and domesticated ones since bushmeat had a more positive δ15N (p < 0.05) than any domesticated animal , therefmal feed .4 is present in variable amounts in several products, including those for which C-C4 occurrence is somehow unexpected , saffron (Crocus sativus L.), and garam masala (an India-style blend of ground spices), which are mixed with a fine maize flour (termed in Brazil as fubá) (4 due to the use of sugarcane as an adjunct in the grape (Vitis vinifera) fermentation process [4, among them, sauces like mustard and ketchups. Perhaps the most iconic example of the heavy presence of C-C4 in Brazilian processed foods is soy sauce (shoyu), which has a dominant proportion of maize rather than soy or wheat  [Among plant-based processed foods, excluding those that are wheat-based like pasta, noodles, crackers, and snacks, C-Cexpected c. For inas fubá) b. Maize as fubá) . Most Br process . Other p recipe) .4 by the Brazilian food industry. We mention here that the cost of C-C4 in Brazil is overall cheaper than C-C3. Brazil is mostly a tropical country where the growth of C4 plants is favored by a high light incidence [4 plants and it seems that this distribution can be tracked by certain types of foods. This is the case of hamburgers made by a global fast-food company. The country-level δ13C of patties grouped by latitude clearly showed that there was an inverse correlation between the proportion of C-C4 and latitude, meaning that countries in the tropical belt had more C-C4 than countries located in higher latitudes [A question arises about the reasons for such pervasive use of C-Cncidence ,12,59. Tatitudes .4 plants are very important: sugarcane and maize. Brazil is one of the largest producers of sugar from sugarcane in the world. Brazil was the top sugar producer in 2014, producing ~37 million Mg of sugar [In Brazil, among agricultural commodities, two Cof sugar . Brazil of sugar . Therefoof sugar .4 forage grasses, mainly of the genus Brachiaria. Therefore, although raw meat is a relatively expensive item for Brazilians, any meat included in their diets would be a source of C-C4  instead of C3-derived plants in products such as wine [The price of maize grain in Brazil is lower than rice, soy, and wheat  and cons of C-C4 a,b. On tn region . Coupled as wine  and soy  as wine .3 metabolism , C-C4 plants have a higher degree of pervasiveness in the Brazilian food system , and Worcestershire sauce. Finally, in the group defined as “Others”, the following items were included: powder spices, like cumim, urucum, saffron, and sweeties like marshmallow, Japanese soy-based products like miso paste, instant misoshiro powder, baby formula, and pre-cooked polenta.We analyzed the stable carbon and nitrogen abundances in 1619 samples . Of the total, 1245 and 374 were food items and beverages, respectively. Most of the samples were bought during 2015–2019 in grocery stores located in Piracicaba, a municipality in the Southeast Region of Brazil with a population of ~400,000 inhabitants. In natura animal samples include the following species: cattle, chicken, pig, lamb, freshwater and marine fish, and bushmeat . In natura vegetal samples include cereals, fruits, pulses, tubers, and vegetables grouped according to the FAO classification. For processed food, the classification proposed by Monteiro et al.  was follSolid foods were oven-dried at 60 °C to a constant weight. Subsequently, samples were finely ground to facilitate homogenization. Processed foods were composed of several ingredients and cookies with chocolate chips and cookies stuffed with cream were ground together. After homogenization, 1–2 mg of sample was transferred to a tin capsule for further elemental and isotopic analysis. Beverages like soda, beer, and wine were directly placed in tin capsules.13C/12C) and nitrogen (15N/14N) of each sample were determined using a continuous-flow isotope ratio mass spectrometer  coupled to an elemental analyzer  at the Laboratory of Isotope Ecology of the Center for Nuclear Energy in Agriculture, University of São Paulo.The isotope ratios of carbon  thiophene  as an international standard while fine-milled sugarcane leaves were used as a laboratory standard.Carbon and nitrogen isotope compositions were calculated as13C and δ15N values of food and beverage samples. To test the differences between food items, ANOVA was used because carbon and nitrogen isotope values followed a normal distribution. The post-hoc Tukey test was used to pinpoint specific differences between food items. Statistical analyses were performed in R  and RStudio  using the “multcomp” package [Descriptive statistics  were used to report the δ package .


Scientific Reports 10.1038/s41598-019-56904-y, published online 14 January 2020Correction to: This Article contains an error in the Methods section under the subheading ‘Randomization and intervention’ where,20,21,22.”“(a) Doses of cholecalciferol  60,000IU  to be taken per orally) was given once per week for eight weeks to intervention group and Lactose Granules as placeboshould read:20,21,22.”“(a) Doses of cholecalciferol  60,000IU  to be taken per orally) was given once per week for eight weeks to intervention group and Lactose Granules as placebo


The plant rhizosphere interfaces an array of microbiomes related to plant growth and development. Cultivar-specific soil microbial communities with respect to their taxonomic structure and specific function have not been investigated explicitly in improving the adaptation of lentil cultivars under rice-fallow ecology. The present study was carried out to decipher the rhizosphere microbiome assembly of two lentil cultivars under rice-fallow ecology for discerning the diversity of microbial communities and for predicting the function of microbiome genes related to nitrogen (N) and phosphorus (P) cycling processes deploying high-throughput whole (meta) genome sequencing. The metagenome profile of two cultivars detected variable microbiome composition with discrete metabolic activity. Cyanobacteria, Bacteroidetes, Proteobacteria, Gemmatimonadetes, and Thaumarchaeota were abundant phyla in the “Farmer-2” rhizosphere, whereas Actinobacteria, Acidobacteria, Firmicutes, Planctomycetes, Chloroflexi, and some incompletely described procaryotes of the “Candidatus” category were found to be robustly enriched the rhizosphere of “Moitree”. Functional prediction profiles of the microbial metagenomes between two cultivars revealed mostly house keeping genes with general metabolism. Additionally, the rhizosphere of “Moitree” had a high abundance of genes related to denitrification processes. Significant difference was observed regarding P cycling genes between the cultivars. “Moitree” with a profuse root system exhibited better N fixation and translocation ability due to a good “foraging strategy” for improving acquisition of native P under the nutrient depleted rice-fallow ecology. However, “Farmer-2” revealed a better “mining strategy” for enhancing P solubilization and further transportation to sinks. This study warrants comprehensive research for explaining the role of microbiome diversity and cultivar–microbe interactions towards stimulating microbiome-derived soil reactions regarding nutrient availability under rice-fallow ecology. Plant roots interface diverse groups of microbiotas including bacteria, fungi, algae, viruses, and archaea in soil ,2, whichLens culinaris Medikus subsp. culinaris) is a globally important cool-season food legume which plays a major role in household nutritional security in developing countries. This protein-rich crop is an ideal candidate for rice-fallow areas aimed at its intensification to meet the protein demand of vegetarian society. In South Asia, about 14 million ha area remain fallow after the rice harvest covering India, Bangladesh, Nepal, and Pakistan, with India alone accounting for 79% of area [Lentil  and Po (organic) into available forms through overexpression of phosphatase. Therefore, microorganisms impart a pivotal role in maintaining Po and Pi pools in the soil [Lentil plants maintain symbiotic association with diverse microbial populations for improving soil health and crop production through BNF . The hosn lentil . Bacterin lentil  encoded atalysis ,21. Moremunities ,23. Lentmunities ; secondlmunities ,26; and munities . Additiomunities  by intenthe soil .Rhizobium colonies and mildly tuned rhizosphere microbiome assembly [The abundance and diversity of a rhizospheric microbiome among the plant species are highly influenced by the plant genetic traits ,31 as weassembly .Recently, Next Generation Sequencing (NGS)-based OMICS technologies provided better insight into the crucial roles of rhizosphere microbiomes towards soil and plant genetic traits under varying conditions ,36,37. Cp = 0.034) manner under rice-fallow ecology. The varied performance of lentil cultivars under rice-fallow ecology was attributed to their divergent grain nutrient content, shoot–root ratio, and root characteristics. Grain nutrient content of lentil cultivars varied significantly (2), and root diameter (0.7 mm) than “Farmer-2” (Both the lentil cultivars responded in significantly different (ficantly . Grain Narmer-2” .p-nitrophenol/g soil/hr), alkaline phosphatase (36.9 μg p-nitrophenol/g soil/hr), and phytase (2.32 mg P/g soil/hr) activity in soil as compared to “Moitree”  . The remaining sequences matched Archaea (2% for both “Farmer-2” and “Moitree”) and Eukaryota (0.02% in “Farmer-2” and 0.2% in “Moitree”) or were uncategorised (0.006% in “Farmer-2” and 0.06% in “Moitree”). Enrichment of the phyla Actinobacteria and Proteobacteria was found at maximum in the samples of “Farmer-2” and “Moitree”, respectively. A large proportion of the sequences were unclassified at the phylum level (approximately 22.4% of sequences within “Farmer-2” and 23.77% for “Moitree”). The most abundant top 50 bacterial taxa within microbial communities between “Farmer-2” and “Moitree” were identified, and a comparative heat tree was generated . A princoitree”) .Paenibacillus was found only in “Farmer-2”. Moreover, most predominant genera exhibited differences between “Farmer-2” and “Moitree”. Specifically, the relative abundances of Sphingopyxis and Novosphingobium (belonging to Sphingomonadaceae), Lysobacter (belonging to Xanthomonadaceae), and Luteimonas (belonging to Xanthomonadaceae) were more in “Farmer-2” than in “Moitree”, but the relative abundances of Streptomyces (belonging to Streptomycetaceae) and Pseudomonas (belonging to Pseudomonadaceae) were higher in “Moitree”. The estimated bacterial diversity did not vary considerably between two lentil cultivars (p > 0.05) in alpha diversity were found for Shannon index among the observed species and Chao 1 under different cultivars, though microbiome composition of soils associated with “Moitree” reflected relatively high diversity in comparison to “Farmer-2”. Species richness can be viewed as the number of different species on the chart, and species evenness was derived from the slope of the line that fits the “Rank Abundance Plot”. A steep gradient in rank abundance curve exhibited by “Moitree” indicated low evenness, whereas in “Farmer-2”, it reflected that a few phylotypes comprised a major proportion of the rhizosphere microbiome where more communities were seen in “Moitree” , followed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein families (Pfam) databases in both samples. In the case of “Farmer-2”, 102,466 genes from the total of 186,216 genes exhibited hit at least in one database, whereas in “Moitree”, 122,803 genes were identified showing hit at least in one database out of 206,155 genes. The GO project provides controlled vocabularies of defined terms representing gene product properties. GO functional analysis assigned a total of 147,839 genes in “Farmer-2” and 165,424 genes in “Moitree” while KEGG functional analysis revealed that 120,946 genes have been assigned with 4300 KEGG classes in “Farmer-2” and that 140,995 genes have been assigned with 4336 KEGG classes in “Moitree”. Relative abundances of the top 50 KEGG Orthology (KO) groups which were common in both samples were identified, and a comparative heat tree was generated . The maxCOG (Clusters of Orthologous Groups of proteins) functional analysis revealed that 96,574 genes have been assigned with COG functions in “Farmer-2” where 11,364 genes fall in the category of general function prediction and 10,253 genes belong to the amino acid transport and metabolism category. However, in “Moitree”, COG functional analysis revealed that 116,357 genes have been assigned with COG functions, where maximum genes  fall in the category of general function prediction and 12,868 genes belong to amino acid transport and metabolism. Pfam functional annotation was used to assess protein families and domain. Pfam functional analysis showed that 109,486 gene sequences have been assigned, with a total of 3889 Pfam domains in “Farmer-2”, whereas in ‘Moitree’, 124,850 gene sequences have been assigned, with a total of 3813 Pfam domains. FIGfams (Fellowship for the Interpretation of Genomes protein families) are sets of protein sequences that are similar along their full length. Thus, all the proteins within a single FIGfam are believed to implement the same function. In other words, FIGfam is a set of functional homologs. FIGfam functional analysis revealed that 61,058 genes have been assigned, with a total of 6933 FIG (Fellowship for the Interpretation of Genomes) classes in “Farmer-2”, whereas in “Moitree”, 72,588 genes have been assigned, with a total of 7060 FIG classes .The functional profiles of samples from “Farmer-2” and “Moitree” were further analyzed with special emphasis on genes predicted to be linked with N cycling based on KO group assignments  and are GS, glutamate dehydrogenase gdh, cysteine desulfurase nifS, nitrite reductase (NO-forming), nitrite reductase (NAD(P)H) large subunit nirB, putative pyruvate-flavodoxin oxidoreductase nifJ, nitronate mono oxygenase nmo, MFS transporter, and NNP family nitrate/nitrite transporter narK were the most abundant gene families of the bacterial communities associated with both cultivars. The samples from “Moitree” had a relatively high abundance of genes related to denitrification processes , cytochrome c-type protein napB, nitrate reductase 2 alpha subunit narG, and nitrate reductase 2 beta subunit narH). In both cultivars, low abundance was detected for nitric-oxide reductase protein  responsible for denitrification of NO2− to N2. Our results revealed higher abundance of nitronate mono oxygenase nmo, urease alpha subunit ureC, glutamate dehydrogenase gdh, and especially glutamine synthetase GS in “Moitree”, which are involved in metabolism of organic N. Genes related to BNF, such as nitrogenase iron protein nifH, nifZ, nifU, and related proteins; ribosomal-protein-serine acetyl transferase nifP; putative pyruvate-flavodoxin oxido reductase nifJ; homocitrate synthase nifV; cysteine desulfurase nifS; nitrogenase molybdenum-iron protein alpha chain nifD; nitrogenase molybdenum-iron protein beta chain nifK; and nitrogenase molybdenum-iron protein nifN were also identified. The nifH gene is required for functional nitrogenase in almost all diazotrophs, and it was observed in equal frequency in both cultivars. An abundance of nifZ, nifJ, and nifS genes was higher in “Moitree” as compared to “Farmer-2”, whereas the nifD, nifK, and nifN genes were present only in “Farmer-2”.Among the gene families, glutamine synthetase p = 0.0002). The abundance of almost all genes involved in phosphate esterase activity , phytase activity , phosphonate degradation , inorganic phosphate solubilization , and phosphorus transportation  were more in the case of “Moitree” in comparison to “Farmer-2”, whereas phosphodiesterase/alkaline phosphatase phoD, 3-phytase, guanosine-5′-triphosphate 3′-diphosphate pyrophosphatase ppx, phosphonoacetaldehyde hydrolase phnX, phosphonate transport system proteins viz. phnC and phnD, and Phno protein phnO showed lower abundance in “Moitree” as compared to “Farmer-2”.The combined abundance of predicted genes involved in P cycling was quantified through KO group assignments among the samples collected from both the cultivars  and presThe relationship between plants and their rhizospheric microbiota is gaining importance for proper understanding of the mechanism underlying microbial involvement in boost plant growth as well as subsequent tailoring of microbiome composition aiming towards improvement in agricultural productivity. Plant roots secrete a plethora of exudates which vary with plant species , ecotypeIn the present study, the tested lentil cultivars, Moitree and Farmer-2, exhibited differential response regarding N and P content in the tissues as well as enzymatic activities in relation to BNF and P cycling. P is the major limiting factor in the soil as about 30–80% of the total P is present in organic forms . EfficiePaenibacillus was found only in “Farmer-2”. This group is known as Plant Growth-Promoting Rhizobacteria (PGPR), which can solubilize phosphate [Paenibacillus, a number of other well-described phosphate solubilizing plant growth promoting bacteria (PGPB) including Pseudomonas and Lysobacter have been identified to improve various growth promoting activities including phosphate solubilization [In our study, Proteobacteria, Cyanobacteria, Bacteroidetes, Actinobacteria, and Acidobacteria were the most dominant bacterial phyla detected in rhizosphre of two lentil cultivars. Previous studies also reported a plethora of Proteobacteria, Acidobacteria, Bacteroidetes, and Actinobacteria in legumes like soybean and peanut ,11. Alphhosphate . Besideslization ,69. The lization ,34,35. Clization ,70. The p = 0.06) among the two tested cultivars though their nutrient foraging pattern varied owing to their differential root systems. Denitrifying microbial diversity (both classified and unclassified) as well as the abundance of denitrification process-related marker genes (napA and narG) were more in “Moitree” in comparison to “Farmer-2”  was detected only in “Farmer-2” and confirmed the differences in predicted abundance of genes related to BNF [Diversification in the composition of root exudates among different plant species or cultivars within the same species plays a pivotal role in communication and further recognition that ultimately tailors distinct colonization of microbiota as per the need of the host and vice versa . Lentil armer-2” . Gene dilization . Thus, “lization  and pea lization . Most ofd to BNF . This fid to BNF .p = 0.0002) in the P cycling functional genes of the microbiome. It can be assumed that “Moitree” with a profuse root system enables better nutrient foraging as well as improved efficiency to recruit more microbiota as compared to “Farmer-2”; the P solubilizing capacity of “Farmer-2” was greaeter, but microbial count was less. Limited information impedes our understanding towards the influence of heteroginities of root architecture on microbiome composition and diversity in the plant rhizosphere [phoX was higher in “Moitree”, in ‘’Farmer-2’′, the number of phoD transcripts was higher. The latter is a class of alkaline phosphatase which plays an important role in the release of plant-available inorganic P from organic P in soil [gcd, ppk, and ppa) and P transporter-related genes (Pst and Pit) have been detected in higher abundance in “Moitree”, which empowered the soil microbial communities to efficiently utilize and internalize P into their metabolic processes so that the unavailable form of P is converted into available forms to the plant [In legumes, P is the major yield-determining nutrient. Rhizosphere-associated microbes impart a crucial role in mobilization of recalcitrant forms of P . Functiozosphere . A recenzosphere . It was zosphere . Though  in soil . Among t in soil . In the he plant ,83. Ther2O5:K2O = 20:40:40 kg/ha) for normal crop growth. Finally, two cultivars, namely “Moitree” (WBL-77) and “Farmer-2”, were selected based on their differential performance under rice-fallow ecology for further analyses. “Moitree” is a well-adapted variety in rice-fallow ecology of Gangetic delta evolved from a cross between ILL 7723 and BLX 88176 with small seed size (microsperma) and having 115–120 days duration. “Farmer-2” is a landrace of the Mediterranean region and a newly introduced line to Indian ecology by ICARDA  with seed size larger than “Moitree” and 130–135 days crop duration.The present experiment was carried out at the Regional Research Sub-Station (RRSS) of Bidhan Chandra Krishi Viswavidyalaya located at Chakdah , Nadia district, West Bengal, India during the winter season of 2018–2019, where a long-term rice-based food legume cropping system has continued for the last six years. The soil of the experimental plot is sandy clay loam having pH: 7.05, organic C: 6.8 g/kg available N: 154 kg/ha, available P: 16 kg/ha, and available potassium:129 kg/ha. Genetically, pure seeds of 130 lentil cultivars were sown under rice-fallow situation after the harvest of monsoon rice, leaving 20 cm standing stubbles in the field by opening a narrow furrow in between two rows of rice using a manual furrow opener. The crop was raised following agronomic practices with mineral fertilizers (fertilizer dose: N:Paugar at the maximum vegetative stages (45 days after sowing/seeding) and cleaned thoroughly following the method described earlier [Three representative plants with complete root system from both cultivars were excavated manually using  earlier . After wi released by hydrolysis using sodium phytate as the substrate [Alkaline and acid phosphatase activities of the collected soil samples from both cultivars were estimated using standard protocol, as described by Tabatabai . Soil phubstrate .i from phytic acid , as per the standard protocol [Plant samples of both cultivars were first washed with running tap water followed by washing with double distilled water. Nodules were collected from each root sample of both cultivars for quantification. Roots and shoots were separated, and the fresh weight of each part was recorded. Further, shoot–root ratios were estimated by appraising the dry weight of shoot as well as root after drying inside a paper envelope in the oven at 70 °C until constant weight was recorded. The dried samples were ground into powder by using stainless steel grinder for further analysis. The total N content of the plant samples was analyzed by the Kjeldahl method using a Kel-Plus analyzer , while the P content was analyzed through the Vanadomolybdate yellow color method . Phytaseprotocol . The aciprotocol . The legprotocol . Finallyprotocol , in whic® 2.0 Fluorometer. The paired-end sequencing libraries were prepared using New England Biolabs® NEB Next Ultra DNA Library Prep Kit  for Illumina . After obtaining the Qubit concentration for the library and the mean peak size from Bioanalyser profile, the library was loaded onto the Illumina platform for cluster generation and sequencing. Paired-end sequencing allowed the template fragments to be sequenced in both the forward and reverse directions. The average size of the libraries were 447 bp and 395 bp for “Farmer-2” and “Moitree”, respectively. The libraries were sequenced on Illumina NextSeq 500 sequencer  to generate about 4–5 GB data for each sample. Read count summaries and assembly statistics are provided in Soil DNA was isolated from both cultivars by Xcelgen Soil DNA isolation Kit. The quality of DNA was checked on a 0.8% agarose gel, and the concentrations were determined using Qubithttp://kaiju.binf.ku.dk/server) [e-value of 1 × 10−5, run mode-Greedy, minimum alignment length of 11, and maximum alignment length of 75 and allowed mismatches of up to 5. For identification of read belonging to 16 S sequences, Parallel-meta tool was executed on FASTQ files for both samples followed by mapping on the SILVA database. The FASTA files for 16 S sequences were converted into FASTQ files using inhouse-scripts for use as input for QIIME  analysis to get taxonomic summaries integrating Python and R scripts and Qiime v.1.9.1 [−5 having a minimum identity of 80%.Paired-end raw sequences were quality filtered to remove adapter sequences, following earlier literature . The sca/server) , where r v.1.9.1 . The Kro v.1.9.1 . All seq v.1.9.1 . For com v.1.9.1  was usedExperimental results regarding plant and soil samples were expressed as mean ± standard error of mean considering three replicates for all measurements. Independent sample “t-test” was carried out to analyse individual plant traits, enzyme activity, as well as number of predicted microbiome genes related to N and P cycling processes. All statistical analyses were performed in R version 3.5.0 . Alpha dOur results revealed that lentil cultivars, Moitree and Farmer-2, exhibited trivial differences in diversity and function of microbiomes related to N metabolism in their rhizosphere. A higher abundance of genes associated with denitrification process was detected in “Moitree”; however, genes related to BNF exhibited almost equal distribution in both cultivars. Contrarily, the microbiome P cycling genes exhibited significant differences among the cultivars. Thus, it can be concluded that the availability of P is the major determining factor towards better performance of the genotype under rice-fallow ecology. Both the tested cultivars exhibited differential mechanisms to use the native P under rice-fallow ecology. The cultivar Moitree with profuse root system and high shoot–root biomass exhibited better “root foraging capacity” towards improved acquisition of native P coupled with better N turnover under rice-fallow ecology. Most of the rice-fallow is deficient in available P due to high moisture content in soil. Hence, “Moitree” could perform better under nutrient poor soil in rice-fallow ecology. On the contrary, the newly introduced “Farmer-2” from the Mediterranean region into the Indian subcontinent exhibited greater P content in roots as well as in grains along with good phosphatase and phytase activity in both plant and soil. This could shed some lights on the fact that the genetic makeup of this cultivar enabled better “P mining capacity” for solubilization of the fixed P into plant available form followed by efficient transportation of P from source to sink though its availability at the root/soil interface was poor due to retarded expression of the microbiome-associated P cycling genes in the rhizosphere. Our efforts for a single factor explanation regarding cultivar specific microbiome recruitment followed by nutrient availability exerted limited impact towards explaining the complex natural system. Comprehensive research is further needed regarding nutrient interaction integrating the role of the rhizosphere-associated microbiome for a better understanding of nutrient availability under rice-fallow ecology.


Peroxisome proliferator-activated receptor (PPAR) β/δ belongs to the family of hormone and lipid-activated nuclear receptors, which are involved in metabolism of long-chain fatty acids, cholesterol, and sphingolipids. Similar to PPAR-α and PPAR-γ, PPAR-β/δ also acts as a transcription factor activated by dietary lipids and endogenous ligands, such as long-chain saturated and polyunsaturated fatty acids, and selected lipid metabolic products, such as eicosanoids, leukotrienes, lipoxins, and hydroxyeicosatetraenoic acids. Together with other PPARs, PPAR-β/δ displays transcriptional activity through interaction with retinoid X receptor (RXR). In general, PPARs have been shown to regulate cell differentiation, proliferation, and development and significantly modulate glucose, lipid metabolism, mitochondrial function, and biogenesis. PPAR-β/δ appears to play a special role in inflammatory processes and due to its proangiogenic and anti-/pro-carcinogenic properties, this receptor has been considered as a therapeutic target for treating metabolic syndrome, dyslipidemia, carcinogenesis, and diabetes. Until now, most studies were carried out in the peripheral organs, and despite of its presence in brain cells and in different brain regions, its role in neurodegeneration and neuroinflammation remains poorly understood. This review is intended to describe recent insights on the impact of PPAR-β/δ and its novel agonists on neuroinflammation and neurodegenerative disorders, including Alzheimer’s and Parkinson’s, Huntington’s diseases, multiple sclerosis, stroke, and traumatic injury. An important goal is to obtain new insights to better understand the dietary and pharmacological regulations of PPAR-β/δ and to find promising therapeutic strategies that could mitigate these neurological disorders. Peroxisome proliferator-activated receptors (PPAR) belong to the family of hormone and lipid-activated nuclear receptors, which are involved in metabolism of cholesterol, sphingolipids, and fatty acids. The transcriptional activity of PPARs is known to engage in a variety of cellular functions including cell differentiation, proliferation, and development   which is tightly connected with long-chain fatty acid (LCFA) homeostasis  evoked by the endotoxin lipopolysaccharide (LPS) may contribute to neurodegenerative disorders Brown . PPARs aIn our previous studies, acute SIR evoked by LPS administered i.p. to mice-induced memory impairment showed alterations of transcription of pro-oxidative, inflammatory genes, and genes engaged in cells death signaling  is the most prevalent, progressive, and irreversible neurodegenerative disease that leads to dementia. There are many underlying mechanisms towards the pathogenesis of AD, including the widely known amyloid pathogenesis with liberation and oligomerization of amyloid beta peptides (Aβ), and hyperphosphorylation of the microtubule-associated tau protein, and its polymerization into insoluble, neuronal fibrillary tangles (NFTs). These alterations lead to astrocytes and microglia cells activation, and consequently, inflammatory response (Selkoe and Hardy Previous studies have shown that downregulation of PPAR-β/δ could be linked to both neuroinflammation and insulin resistance in the brain (de la Monte and Wands A new PPAR-δ/γ agonist (T3D-959) with 15-fold higher PPAR-β/δ selectivity/potency (comparing to PPAR-γ) is in an exploratory phase II clinical trial on thirty-four mild-to-moderate AD patients. , the PPAR-β/δ agonist GW0742 could decrease parenchymal Aβ deposits, although intraneuronal Aβ was not affected  , a disease involving demyelination of central nervous system and affecting nearly one million people worldwide  at 1 and 6 h after SCI, significantly reduced inflammation, nitric oxide synthesis, nitrotyrosine formation and activation of apoptotic signaling. Moreover, this agonist protected against edema and showed positive effect on motor recovery score. The study of Esposito et al. (In the recent review article, Gamdzyk et al.  comparedo et al.  indicateo et al.  showed ao et al. , cultureo et al. . It is po et al.  oral admo et al. .Despite of the recognition of PPAR-β/δ in metabolic and inflammatory diseases, there is increasing interest in developing appropriate ligands/antagonists towards treatment of cancer (Wagner and Wagner Considering their anti-inflammatory, neuroprotective, and anti-tumors properties, PPAR-β/δ agonists are promising treatments of AD and other neurodegenerative disorders. A list of the natural and synthetic agonists for PPAR-β/δ is shown in Table


Caenorhabditis elegans. We discovered that “T+S” significantly stunted the overall growth in wildtype nematodes, an effect that was exacerbated by concurrent exposure to heat stress. “T+S” also negatively impacted fecundity as measured by increased germline apoptosis, a decrease in egg-laying, and fewer viable offspring. Lastly, exposure to “T+S” resulted in degenerative changes in nicotinic cholinergic neurons in wildtype nematodes. As a whole, these findings demonstrate widespread toxic effects of neonicotinoids to critical functions in nematodes.Neonicotinoids, a class of insecticides structurally similar to nicotine that target biting and sucking insects, are the most widely used insecticides today, in part due to their supposed low toxicity in other organisms. However, a growing body of research has found that even low doses of neonicotinoids can induce unexpected negative effects on the physiology and survival of a wide range of non-target organisms. Importantly, no work has been done on the commercial formulations of pesticides that include imidacloprid as the active ingredient, but that also contain many other components. The present study examines the sublethal effects of “Tree and Shrub”™ (“T+S”), a commercial insecticide containing the neonicotinoid imidacloprid as its active ingredient, on  Neonicotinoids, a relatively new class of insecticides, are the most widely used insecticides today . They ac50 for non-target organisms  ), ced-1::GFP (transgenic apoptosis reporter), bus-17 (cuticle defective), bus-5 (cuticle defective), and vit-5 (egg yolk protein). Nematodes were incubated at 20º C on nematode growth media (NGM) petri plates with small lawns of bacterial strain OP50 ), which express GFP in all cholinergic neurons, were allowed to lay eggs for one hour on control plates with a lawn of OP50 bacteria, and the eggs were then transferred to 2 cm plates either treated with 1X “T+S” or left untreated. All plates contained a bacterial lawn of OP50 bacteria. Worms were allowed to develop for 90 hours at 20ºC before adult hermaphroditic worms for each of 4 independent trials were transferred to a 3% agarose pad and immobilized in a drop of buffer containing 2.5 mM NaN3. Images were taken using a Nikon PCM2000 confocal laser-scanning microscope equipped with an argon laser.A total of 113 neurons from 12 different worms exposed to either “T+S” or control conditions were examined. For each neuron analyzed, images were collected using a 40X lens. The percent of cholinergic neurons along the nerve cord that exhibited blebbing and rounding, morphological changes observed in the progression of neuronal degeneration, was quantified for both treated and control worms by blinded participants. Neurons that were of undeterminable classification were discarded from analysis.ced-1::GFP expression were used to score apoptosis in response to exposure to “T+S”. AO is pH-sensitive and fluoresces in response to a pH change which occurs when the cells are apoptotic [ced-1::GFP worms contain a transgene which is expressed when the sheath cell engulfs a cell which has been tagged for apoptosis due to physiological stress or damage to DNA [Acridine orange (hereafter “AO” staining”) and poptotic . ced-1::e to DNA ,50. Briee to DNA .ced-1:GFP, bus-17, and vit-5 worms were staged as L4 larvae and picked 1 worm per plate. Brood size analysis was conducted as described in [To assess fertility, wild-type N2, ribed in . BrieflyAnimals were staged as L4s and dissected 48 hours later. Removal of gonads was performed in egg buffer supplemented with 0.1% Tween, followed by fixation in 5% paraformaldehyde. Dissections were performed as described in  with thet-tests with Bonferroni correction. For developmental assays, individual worms were measured, and data were analyzed by one- way (6 concentrations of T+S) ANOVA for “T+S” impaired growth and by two-factor (concentration x treatment) ANOVA for studies examining the synergistic effects of “T+S” and either heat or paraquat exposure. When the ANOVA detected statistical significance, Tukey’s multiple comparison test was used to determine where the significance lay.All statistical analyses were conducted using Prism . A p-value of less than 0.05 was considered statistically significant. For survival assays, the percent surviving for ten independent trials at each concentration was analyzed was analyzed using a 2-way ANOVA and pairwise To determine the effects on “T+S” on locomotion, body bends of individual worms were counted and pair-wise t-tests were used to evaluate significance. To analyze the effects of “T+S” exposure on egg-laying behavior, eggs from individual worms were counted and analyzed using one-way ANOVA. To quantify the effect of “T+S” exposure on cholinergic neurons, the percentage of swollen neurons was determined for both control and treated worms, and a one-way ANOVA was used to determine statistical significance.Two-tailed Mann Whitney tests were used to analyze germline apoptosis and brood size. A Fisher’s exact test was used to assess presence of chromosomal abnormalities in DAPI-stained germ lines.


Information about the postharvest physiological behavior of blood orange cultivars can provide comprehensive insight into the best period of storage to maintain the highest fruit quality during prolonged cold storage. In this paper, changes in nutritional quality, bioactive compounds, and antioxidant enzymes in the juice of four blood orange cultivars  stored at 2 and 5 °C were studied. Parameters were measured after 0, 30, 60, 90, 120, 150, and 180 days, plus 2 days at 20 °C for shelf life. Sucrose was the sugar found in higher concentrations and decreased during storage in all cultivars, as did glucose and fructose. Organic acids decreased at both temperatures, with the highest content found in “Sanguinello”, especially major (citric acid) and ascorbic acid. Total phenolics content (TPC), total anthocyanins (TAC), and individual cyanidin 3-glucoside and cyanidin 3- increased for all cultivars, with “Sanguinello” having higher concentrations. The antioxidant enzymes catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) were also higher in “Sanguinello” and increased during storage. Overall, these results together with the sensory analysis suggested that “Sanguinello” would be the best cultivar for prolonged storage. The results of this study could be useful to select the best storage duration and temperature for each cultivar and provide the presence of such a high-value commodity for fresh consumption or juice processing long after the harvest season. Citrus sinensis L. Osbeck) are one of four groups within the sweet orange species. The main difference between blood orange fruit and other oranges is the synthesis of anthocyanins pigment in the flesh and sometimes in the peel [Blood oranges  rootstock. All trees were grown under the same conditions and cultural practices. Fruit were selected based on uniformity of size, checked for no defects or rind injuries, disinfected with 2% sodium hypochlorite (NaOCl) solution, and rinsed with distilled water. Fruit were divided into sets of three replicates with five fruit in each replicate, placed in a polyethylene bag containing 16 holes, and stored for 180 days at 2 and 5 °C and 90% relative humidity (RH). Parameters were measured after 0, 30, 60, 90, 120, 150, and 180 days of cold storage, plus 2 days at 20 °C (shelf life). For each cultivar, sampling date, and replicate, the 5 fruits were cut into two halves for a total of 10 halves. Then, 5 halves were squeezed and the juice was extracted to be used for total phenolics, total anthocyanins, and antioxidant activity determinations, and the other halves were freeze-dried  for measurement of individual sugars and organic acids, individual anthocyanins, and antioxidant enzyme activities in the juice.Cultivars of blood oranges, “Moro”, “Tarocco”, “Sanguinello”, and “Sanguine”, were harvested from the commercial citrus orchard of Dashtenaz company in Sari , Mazandaran province, Iran, in mid-January 2018 and immediately transported to the postharvest laboratory. In this area of Iran, the climate is subtropical; the four cultivars reached commercial maturity in January and were harvested at the same time according to the total soluble solids (TSS) and titratable acidity (TA) ratio. The trees were seven years old and grafted on “C-35” citrange (2H3NaO2) buffer at pH 4.5 were added to the obtained juice (1:4 dilution) and the absorbance read at 510 and 700 nm  and TAC was calculated as mg L−1. The molecular weight and molar extinction coefficient of cyanidin-3-glucoside were 449.2 and 26,900, respectively. For measuring the concentration of total phenolics (TPC), the Folin–Ciocalteu method was used. A juice aliquot (32 µL) was diluted with 2% Na2CO3  and mixed for 3 min, and then, the Folin reagent was deposited in each tube and kept in darkness for 30 min. Different concentrations of gallic acid were used for preparation of the standard curve. Samples and the standard were measured at 750 nm and the results were reported as mg gallic acid equivalents (GAE) L−1 [The content of total anthocyanin (TAC) was evaluated using the pH differential method previously reported . In brieGAE) L−1 .The determination of total antioxidant activity (TAA) was based on the protocol of Brand-Williams et al. . Brieflyv/v/v). After centrifugation , the upper phase was subjected to filtration (0.45 µm filter) and an aliquot of 20 µL sample injected into a HPLC. The individual anthocyanins were separated in a 25 × 0.46 cm Luna C18 column equipped with a C18 guard pre-column . The concentrations of both anthocyanins ) were calculated (mg L−1) after detection at 520 nm and compared with known standards. HPLC chromatograms of individual anthocyanins are presented in For individual anthocyanins, the protocol of Martínez-Esplá et al.  was adap−1, pH = 7.8) was added to 0.5 g of freeze-dried tissue, and after homogenization and centrifugation , 10 µL of sample was injected into HPLC (Hewlett-Packard HPLC Series 1100) after filtration by using a 0.45 µm filter. The mobile phase was 0.1% phosphoric acid under isocratic conditions at 0.5 mL min−1 rate. For organic acids determination, the absorbance detector was set at 210 nm (UV), while sugars were detected by refractive index. HPLC chromatograms of individual sugars are presented in −1, with the exception of citric acid (g 100 g−1). HPLC chromatograms of individual organic acids are presented in −1; both standards were purchased from Sigma-Aldrich .In order to quantify organic acids and sugars, the method described by Martínez-Esplá et al.  was used3PO4) was added to the aforesaid solution and the volume reached to 1000 mL with distilled water. Then, 5 mL of Bradford reagent and 100 µL protein extraction were added in the test tube and shaken vigorously for a few seconds. The protein extraction was the same as the antioxidant enzyme samples. The reaction mixture was read using a spectrophotometer at 595 nm. Bovine serum albumin (BSA) was used to elaborate a standard curve [−1 protein.The protocol for enzyme activities was the same that for flavedo tissue . In brierd curve . The resBlood orange cultivars were submitted to descriptive sensory analysis for market acceptability by using ten trained panelists (five men and five women). In this case, fruit was peeled with a knife and segments were separated with hand, then placed in glass dishes with three-digit codes. Each combination of segments prepared from five fruit of three replicates from each cultivar and 3 scattered segments of fruit were tasted by the panelists. For each cultivar, the edible quality was evaluated by a 9-point scale, in which 1 was the poorest and 9 the maximum edible quality, in accordance with the method previously reported .p < 0.05) by using the SAS software package v. 9.4 for Windows. Linear regressions were performed with SigmaPlot software v. 11.A totally randomized experiment was conducted according to a completely randomized design (CRD) by using three replicates for each cultivar. Data were subjected to analysis of variance (ANOVA) based on three factors . Mean values with standard errors (SE) of the means were separated by least significant difference  were quantified by HPLC-refractive index detection  and wereIndividual organic acids  were quantified by HPLC-diode-array detector (HPLC-DAD) detection  of blood−1), while “Tarocco” showed the lowest (53.6 ± 6.4 mg L−1) at the end of storage at 5 °C. Two major individual anthocyanins were detected by HPLC-DAD for all cultivars: cyanidin 3-glucoside and cyanidin 3-, which was found at higher concentration. For both, and similarly to TAC, levels were higher at 5 than at 2 °C and “Sanguinello” reached the maximum contents for both cyanidin 3-glucoside and cyanidin 3-, while “Tarocco” had the lowest levels of these anthocyanins.−1, respectively, at the end of storage. Total phenolic content (TPC) was affected by cultivars, storage, and temperatures . TPC inc−1) and 5 °C (5.64 ± 0.46 mg 100 g−1), respectively.Ascorbic acid content at harvest was different among cultivars, with “Sanguinello” and “Moro” having the highest and lowest concentrations, respectively . For allTotal antioxidant activity (TAA) increased for all cultivars and then decreased towards the end of storage at both temperatures , althougThe activities of the antioxidant enzymes  in the flesh were affected by cultivars, storage times, and temperatures , althougThe activities of the antioxidant enzymes increased up to 30 or 60 days and then, decreased towards the end of storage at both temperatures, this reduction being greater at 2 than 5 °C. Among cultivars, “Sanguinello” had the highest level of antioxidant enzyme activities at both temperatures, while “Moro” and “Tarocco” showed lower antioxidant enzyme activities at both storage conditions.PAL activity in the flesh of the cultivars was affected at both temperatures during storage . PAL actAcceptability of the blood orange cultivars was analyzed during storage at both temperatures. The highest edible quality was scored immediately after harvest (9 points for both 2 and 5 °C). For all cultivars, edible quality was always higher at 5 than at 2 °C, although it decreased at both temperatures. At the end of storage, “Moro” and “Tarocco” showed the lower scores at 2 °C . “Sanguinello” was evaluated as the best cultivar at 5 °C, with scores of 6.3 ± 0.18 compared with those obtained at 2 °C (4.2 ± 0.15).Information about changes in nutrition, bioactive compounds, and antioxidant activity of the blood orange cultivars can provide a comprehensive insight for assessment of the best storage period as a result of maintaining the highest fruit quality during long-term cold storage. In this paper, a comparative study of four blood orange cultivars  stored for 180 days at 2 and 5 °C was carried out.Citrus fruit after harvest and during cold storage needs to provide energy by catabolism of organic acids and sugars as substrates for maintaining cellular metabolism. Accumulation and degradation of sugars and organic acids occur through glycolysis and the Krebs cycle. On the other hand, the reduction in organic acids and sugars in the flesh may be affected by biosynthesis and catabolism . Therefo+-ATPase pump on the vacuolar membrane, which can provide a large influx of H+ within tonoplast. This proposed mechanism can accumulate additional citric acid in citrus fruit [Besides sugars content, organic acids are important components of citrus fruit juice and their concentration depends on the fruit species and cultivars ,32. In ous fruit . The redus fruit . In addius fruit .Among the citrus species, blood oranges accumulate anthocyanin pigments which are considered as a quality index ,12. Low 2 = 0.65–0.79). The anthocyanins content followed the order “Sanguinello” > “Moro” > “Sanguine” > “Tarocco”, as did the PAL activity.In this study, all cultivars had a pale red color at harvest except “Sanguinello” and TAC increased during cold storage, the enhancement of TAC at 5 °C being significantly higher than at 2 °C. Accordingly, TAC increased in “Moro”, “Tarocco”, “Sanguinello”, and “Sanguine” up to 31-fold, 14-fold, 11-fold, and 20-fold, respectively at 5 °C, while at 2 °C it remained almost unchanged. Anthocyanin synthesis in blood oranges during cold storage depends on the activation of the enzymes involved in phenylpropanoid metabolism. Therefore, activation of these enzymes can be induced by low temperatures . MoreoveOn the other hand, stability and degradation of the anthocyanin molecule depend on PPO and POD activities during cold storage . However2 = 0.67–0.72). Similarly, TAA increased for all cultivars and then, decreased to the end of storage, the reduction being higher at 2 °C. However, ascorbic acid content was drastically reduced over storage for all cultivars and probably did not contribute to TAA in blood oranges. These results would confirm previous reports in which there was a positive correlation between TAC and TPC with TAA [In this study, TPC increased at both temperatures and then remained constant or decreased to the end of storage, although concentrations were higher at 5 °C. As occurred with TAC, the changes of phenolic compounds could be attributed to PAL activity since a close relationship was found  were enhanced, which is consistent with the increased PAL activity. Among cultivars, “Sanguinello” was the best one, since quality parameters were better retained during the 180 days of storage. Based on these results, we propose that a moderate cold temperature after harvest can increase bioactive compounds, especially anthocyanin and phenolic compounds, which are related to human health.


Near-threshold tactile stimuli perception and somatosensory temporal discrimination threshold (STDT) are encoded in the primary somatosensory cortex (S1) and largely depend on alpha and beta S1 rhythm. Transcranial alternating current stimulation (tACS) is a non-invasive neurophysiological technique that allows cortical rhythm modulation. We investigated the effects of tACS delivered over S1 at alpha, beta, and gamma frequencies on near-threshold tactile stimuli perception and STDT, as well as phase-dependent tACS effects on near-threshold tactile stimuli perception in healthy subjects. In separate sessions, we tested the effects of different tACS montages, and tACS at the individualised S1 μ-alpha frequency peak, on STDT and near-threshold tactile stimuli perception. We found that tACS applied over S1 at alpha, beta, and gamma frequencies did not modify STDT or near-threshold tactile stimuli perception. Moreover, we did not detect effects of tACS phase or montage. Finally, tACS did not modify near-threshold tactile stimuli perception and STDT even when delivered at the individualised μ-alpha frequency peak. Our study showed that tACS does not alter near-threshold tactile stimuli or STDT, possibly due to the inability of tACS to activate deep S1 layers. Future investigations may clarify tACS effects over S1 in patients with focal dystonia, whose pathophysiology implicates increased STDT. Previous neurophysiological and neuroimaging studies have shown that the abilities to perceive single tactile stimuli and to temporally discriminate paired electrical tactile stimuli are mainly encoded in primary somatosensory cortex (S1)10 and are influenced by alpha and beta cortical rhythms16.The ability to perceive a single tactile stimulus can be measured with near-threshold tactile stimuli detection20. While a previous study found that tACS modulates near-threshold tactile stimuli perception21, another study showed no effect22. Only one study has investigated the effect of tACS on STDT, and this study found that tACS delivered at alpha frequency did not affect STDT23. Therefore, the effects of tACS applied over S1 on near-threshold tactile stimuli perception and STDT are still unclear. Studies on the interference exerted by tACS on ongoing S1 activity may clarify the mechanisms underlying tACS applied over S1 in healthy subjects.Cortical rhythms can be modulated with transcranial alternating current stimulation (tACS), which is a non-invasive brain stimulation (NIBS) technique that allows neuronal oscillations to be entrained by inducing coherent changes in the firing and timing of neuron populations24.These studies are important for possible therapeutic neuromodulation protocols in neurological conditions whose pathophysiology implicates abnormal sensory processing in S1 , as demonstrated by the increased STDT12 and near-threshold tactile stimuli perception13. Possible phase-dependent effects of tACS on near-threshold tactile stimuli were also investigated. In a separate experiment we targeted S1 according to a modelling-based montage and tested the effects of tACS on the same parameters. Finally, since tACS effects are maximal when stimulation frequency matches the endogenous rhythm19, we recorded the individual EEG activity of S1 and investigated the effects of tACS delivered at the individualised μ-alpha frequency peak.The aim of this study was to investigate the effects of tACS delivered over S1 on STDT and near-threshold tactile stimuli perception. For this purpose we tested the effects of tACS delivered at various frequencies  on STDTp = 0.31; STDT: F = 0.319, p = 0.73). The non-significant effect of the factor “FREQUENCY” was confirmed by the analysis performed on STDT values normalised to sham  = 0.231, p = 0.79). Additionally, tACS did not modify near-threshold stimuli perception, either phase-independently  and “PHASE”  = 2.935, p = 0.07), and the lack of a “FREQUENCY” × “PHASE” interaction in rmANOVA  = 0.601, p = 0.72).Sensory thresholds and STDT values Fig.  were como sham . The analysis performed to evaluate whether near-threshold stimuli perception changed over time within each block showed a non-significant effect of the factor “TIME”  = 0.024, p = 0.87) and the lack of a “TIME” × “FREQUENCY” interaction  = 0.361, p = 0.78).Analysis of sham-normalised near-threshold stimuli perception values also confirmed no effect of the factor “FREQUENCY”  = 0.24, p = 0.19; STDT: F = 0.433, p = 0.52) and the lack of a “MONTAGE” × “FREQUENCY” interaction  = 0.15, p = 0.89; STDT: F = 0.159, p = 0.8) in rmANOVAs. Additionally, neither sensory thresholds nor STDT changed during alpha , beta , gamma , or sham tACS , as shown by the non-significant factor “FREQUENCY”  = 0.295, p = 0.68; STDT: F = 0.344, p = 0.65). The non-significant effect of the factor “FREQUENCY” was further confirmed by the analysis performed on STDT values normalised to sham  = 0.237, p = 0.7). Similarly, near-threshold tactile stimuli perception was not influenced by the montage used  = 3.082, p = 0.09; “MONTAGE” × “PHASE”: F = 0.537, p = 0.65; “MONTAGE” × “FREQUENCY” × “PHASE”: F = 0.2423, p = 0.96). Accordingly, tACS did not modulate near-threshold tactile stimuli perception in a frequency- or phase-dependant manner even in this experiment  = 1.04, p = 0.35; for the factor “PHASE”: F = 0.59, p = 0.62; and for the “FREQUENCY” × “PHASE” interaction: F = 1.167, p = 0.3317). Analysis of sham-normalised near-threshold stimuli perception values also confirmed no effect of the factor “FREQUENCY”  = 0.19, p = 0.74). The analysis performed to evaluate whether near-threshold stimuli perception changed over time within each block showed a non-significant factor “TIME”  = 0.962, p = 0.34) and the lack of a “TIME” × “FREQUENCY” interaction  = 0.196, p = 0.9).Sensory thresholds and STDT values were comparable between experimental sessions 1 and 2, as suggested by the non-significant factor “MONTAGE”  = 1.87347, p = 0.23) and “PHASE”  = 2.106, p = 0.11), and the lack of a “FREQUENCY” × “PHASE” interaction  = 0.288, p = 0.83) in rmANOVA. Moreover, tACS delivered at the individualised μ-alpha frequency peak did not determine phase-dependent effects on near-threshold tactile stimuli perception  = 1.146, p = 0.3497).Sensory threshold and STDT were comparable between individualised  and fixed μ-alpha stimulation , as shown by paired t-tests. The effects of tACS on near-threshold tactile stimuli perception did not differ when tACS stimulation was performed at a fixed frequency of 12 Hz (experiment 1) or at the individualised μ-alpha frequency, as shown by the non-significant factors “FREQUENCY”  = 1.552, 23, but even at beta and gamma frequencies. Moreover, our study showed that the phase of tACS stimulation over S1 did not affect near-threshold tactile stimuli perception. In addition, we demonstrated that tACS delivered using a modelling-based montage did not modify STDT or near-threshold tactile stimuli perception. Finally, tACS did not modify near-threshold tactile stimuli detection and STDT, even when delivered at the individualised μ-alpha frequency peak.In the present study we demonstrated that tACS failed to modify STDT and near-threshold tactile stimuli perception, not only when delivered at alpha frequency23. Our finding that tACS did not affect overall near-threshold tactile stimuli perception contrasts with that of Sliva et al.21, who showed a decreased performance in near-threshold stimuli perception during alpha tACS. However, this effect was present only with baseline-corrected detection rates, but not for absolute detection rates, as was used in our study and in that by Gundlach et al.22.Our first result demonstrating that tACS does not modulate STDT regardless of stimulation frequency is in line with previous evidence showing no effect of 10 Hz tACS applied over S1 on STDT25.To avoid possible confounding as an explanation for the lack of effect of tACS stimulation on both STDT and near-threshold tactile stimuli perception, our procedure adopted several precautions. First, in order to reduce task interference and ensure a proper blinding of participants, we lowered tACS intensities to avoid uncomfortable skin or visual sensations. In addition, sham tACS was adopted as a control condition. We randomized the four tACS frequencies and stimulation phases for each subject in order to avoid a learning effect on STDT or near-threshold tactile stimuli perception. To ensure that subjects maintained an appropriate attention level throughout the task, we included two “no tactile stimulus” trials after every 10 stimuli in our procedure investigating near-threshold tactile stimuli perception. STDT bias attributable to a lack of attention was excluded by using “catch trials”22. In addition, although we did not specifically localise S1 with TMS26 or magnetic resonance imaging techniques for each participant, the size of tACS electrodes  was large enough to ensure that the current reached S1. Furthermore, tACS stimulating electrodes were applied to the same region from which we recorded μ-alpha during task execution, a rhythm which is known to correlate with STDT performance and tactile stimuli perception12, as shown by previous studies with magnetoencephalography (MEG) and EEG12.One possible methodological reason which may explain the lack of tACS effect on near-threshold tactile stimuli perception and STDT relates to the suboptimal targeting of S1. However, we believe this is unlikely for several reasons. First, we adopted the same montage used in previous studies showing significant effects of tACS over S1A further possible explanation for the lack of tACS effect on STDT and near-threshold tactile stimuli perception is that the frequency used did not correspond with the oscillation within S1. To verify this issue, we adjusted the stimulation protocol to the individualised μ-alpha frequency peak in experiment 3. However, we again found no effect on near-threshold tactile stimuli perception. Thus, since stimulation was performed at a functionally relevant endogenous frequency, our results exclude that tACS over S1 failed to modulate STDT and near-threshold stimuli perception due to the targeted neuronal population specificity.31. In addition, when tACS matches endogenous frequency, as in experiment 3, entrainment occurs even at very low stimulation intensities32. Recent studies have found that scalp-applied currents-compared to the conventional tACS approach-should exceed 4–6 mA to achieve measurable effects on transmembrane potential spikes35. Although we used the highest tolerable intensity in our study, we cannot exclude the possibility that higher current intensities of alpha and beta tACS S1 stimulation may be effective in modulating STDT and near-threshold tactile perception. Nevertheless, since the mean intensity of gamma tACS was 1 mA, we may exclude that the lack of gamma tACS effect on STDT and near- threshold tactile perception was due to stimulation intensity.To explain the lack of tACS effect on STDT and near-threshold tactile perception we considered the possibility that the stimulation intensity used was too low to affect behavioural tasks. Previous studies in humans using current intensities of 0.5–0.7 mA have demonstrated brain oscillation entrainment in occipital areas and the primary motor cortex (M1)37. Similarly, STDT underlies interneuronal inhibitory circuits in L5 and L6 layers in S138. To date, the specific features and location of neurons entrained by tACS are not fully understood, but recent data has suggested that candidate elements are interneurons in L2 or L3, or cells located in L1 such as single-bouquet and elongated neurogliaform cells, which are reciprocally connected with L2 and L3 interneurons40. Thus, we may speculate that tACS is not able to reach deeper layers within S1  where neurons that are functionally involved in STDT and near-threshold tactile stimuli encoding lie. Hence, we suggest that intrinsic features of tACS, together with the peculiar architecture of S136 may explain why tACS failed to modulate STDT and near-threshold tactile stimuli perception. It is also possible that tACS entrains oscillatory neurons in S1, but the amount of this effect was not sufficient to determine significant modifications in the somatosensory behavioural task.Alternatively, we may theorise that the lack of S1 tACS effects on STDT and near-threshold stimuli perception depends on S1 architecture. Evidence in animals has shown that the electrical activity evoked by a sensory stimulus simultaneously involves specific layers of sensory cortices, namely L4, L5, and L645. It is important to consider that the effectiveness of an externally applied field may be influenced by a variety of factors, including neuronal density and architecture, alignment of dendrites and axons relative to the induced field, type and distribution of ion channels in the neurons, degree of myelination, and glia density. These factors, which change in different brain regions, probably differ between S1 and M148 thus explaining, at least in part, the different effectiveness of tACS on these two cortical areas.One remaining question is why an ample body of studies using tACS applied over the primary motor cortex (M1) showed an effect of tACS on motor neurophysiological parameters encoded in M1 in healthy subjectsIn conclusion, we have demonstrated that tACS applied over S1 does not alter behavioural tasks that rely on neural processing in S1, such as STDT, or near-threshold tactile stimuli perception. These findings may suggest that S1 tACS only entrains superficial cortical layer neurons that do not play a major role in STDT or near-threshold tactile stimuli encoding. However, it cannot be ruled out that higher tACS intensities, smaller electrodes or multifocal modalities of stimulation may allow to modulate these tasks. Finally, although our study showed that in healthy subjects tACS applied over S1 fails to modify sensory performance, future studies are needed to investigate possible tACS effects on perception in neurological conditions implying abnormal sensory processing in S1, such as focal dystonia. It is possible that in dystonia, which is characterised by an increased STDT, tACS delivered over S1 at specific frequencies may normalize or improve STDT processing.49. All participants except two were right-handed, as evaluated by the Edinburgh Handedness Inventory. All experimental procedures were performed in accordance with the Declaration of Helsinki. The study was approved by the ethics committee of Sapienza, University of Rome, and informed consent was signed by all study participants.Seventeen healthy volunteers  were enrolled in the study. Participants had no history of neuropsychiatric disorders, were not taking drugs acting on the central nervous system at the time of the experiments, and had no contraindications to NIBS according to the latest international guidelines50. Electric suprathreshold stimuli were applied to the index finger of the dominant hand through surface electrodes, with the anode located 0.5 cm distally from the cathode, using a constant current stimulator (Digitimer DS7AH) while participants looked at a central point in front of them. Participants were required to verbally report whether they had perceived a pulse. The rater manually set the intensity, and the threshold for the main experiment was defined as 120% of the sensory threshold.We first determined each participant’s individual sensory threshold, defined as the intensity at which the participant reported feeling 50% of tactile electrical stimuli delivered according to the staircase method25. Paired electrical stimuli consisted of square-wave electrical pulses delivered with a constant current stimulator (Digitimer DS7A) through surface electrodes placed with the anode located 0.5 cm distally to the cathode. The surface electrodes were applied on the distal phalanx of the index finger of the dominant hand. Stimulation intensity was defined for each subject by delivering a series of stimuli at an increasing intensity, starting at 2 mA and increasing in increments of 0.5 mA. The intensity used for STDT was the minimum intensity perceived in 10 out of 10 consecutive stimuli25. Before STDT testing started, subjects familiarised themselves with the task to achieve a stable performance. Subjects were asked to verbally report whether they had perceived a single stimulus or two temporally separate stimuli. The first of three consecutive ISIs at which participants recognised the stimuli as temporally separate was considered the STDT. To maintain a constant attention level during the test and to minimise the risk of perseverative responses, the experimental procedure included “catch” trials consisting of a single stimulus delivered randomly. Each session included four separate blocks. STDT was defined as the average of the four STDT values and was entered into data analysis.STDT was investigated by delivering paired stimuli starting with an interstimulus interval (ISI) of 0 ms (simultaneous pair) and progressively increasing the ISI by 10 ms using a previously described experimental procedureThe ability to detect near-threshold tactile stimuli was investigated by delivering a single stimulus on the index finger of the dominant hand at an intensity of 120% of the sensory threshold, and subjects were asked to report whether they had perceived the stimulus. Tactile stimuli consisted of square-wave electrical pulses delivered with a constant current stimulator (Digitimer DS7A) through surface electrodes placed with the anode located 0.5 cm distally to the cathode. The surface electrodes were applied on the distal phalanx of the index finger. Before the perception task started, subjects underwent a training block to familiarise themselves with the task. Each block consisted of 120 tactile single stimuli, separated by an ISI of 10 s. Perception task performance was measured as the ratio between the number of stimuli perceived/the number of stimuli delivered during each tACS frequency stimulation. A rater manually recorded participant responses and analysis was performed offline.51, in the main experimental session (see below) the electrodes were placed over CP3 and CP4, according to the 10–20 international system 52, we estimated the current density distribution produced by stimulating with various montages at 1 mA intensity. We then identified the montage that most precisely targeted S1. As a result, the electrode connected to the positive pole of the stimulator was placed over CP3, while the other was positioned over Pz, and both electrodes were specifically oriented  was delivered through conductive rubber electrodes (5 × 5 cm in size) enclosed in saline-soaked sponges using BrainSTIM . In order to ensure targeted S1 stimulation, two different tACS montages were used. Consistent with previous S1 tACS studies42. Since cortical processing of peripheral electrical stimuli starts 20 ms after stimulus onset, stimuli were delivered 20 ms before the targeted phase (30 stimuli for each phase) in a randomized order using a custom-made script in Signal. Moreover, since the duration of one tACS cycle differed based on the stimulation frequency, we delivered electrical stimuli according to the four tACS phases tested, taking into account the duration of each cycle. The rater manually recorded stimuli perceived by the subjects online. Data were analysed offline in order to measure the number of stimuli perceived in each phase of tACS stimulation.To investigate possible effects of tACS phase on near-threshold stimuli perception, tactile stimuli were delivered at four different phases of the tACS waveform during alpha, beta, gamma, and individualised μ-alpha stimulation. Following the methods described in Guerra et al., 2016, the tACS signal was streamed to a battery-operated interface connected to the current stimulator , recorded in real time using Signal software (Cambridge Electronic Design). The instantaneous phase of the tACS waveform was calculated using an in-house written script. Four specific tACS phases were targeted (0 degrees (°), 90°, 180°, and 270°)51. EEG was sampled at 5 kHz  from channel CP3 or CP4 (10–20 layout), which were online referenced to POz channels, in right- and left-handed participants respectively. The Fpz channel was used as the ground. Impedance was kept below 5 kΩ. Subjects were fully relaxed with their eyes open. During continuous EEG recording we delivered 120 electric single stimuli with an ISI of 10 s using the same methodological procedure as previous experimental sessions. EEG was pre-processed and analysed immediately after the recording using custom script with EEGLAB53 and Fieldtrip54 toolboxes in Matlab . Continuous EEG down-sampled to 1 kHz, detrended, notch filtered (48–52 Hz), bandpass filtered from 1–95 Hz, and segmented into 150 3-s epochs (− 1500 to 1500 ms) around each electric stimulus. EEG epochs were then used to calculate the time–frequency power spectrum between 5 and 35 Hz using time-domain complex Morlet wavelet convolution with 0.1 Hz resolution and using five cycle long wavelets. The time–frequency power spectra for each epoch were decibel-converted with respect to the average value of the baseline period (− 600 to − 300 ms) and averaged across epochs. For each participant, we defined the individual μ-alpha rhythm frequency peak as the frequency with the maximum ERD (i.e. the maximum decrease in power) in the 8–14 Hz frequency band between 200 and 600 ms poststimulus. The average μ-alpha rhythm frequency peak was 11 ± 1.95 Hz (mean ± SD).We identified the individual μ-alpha rhythm frequency peak of each participant by measuring somatosensory event-related desynchronization (ERD) using a method similar to that described by Gundlach et al. (2017)All 17 participants underwent three experimental sessions that took place at least one week apart in order to exclude any possible carry-over effects. In each session, tACS was applied at different frequencies in separate and randomised blocks. The tACS inter-block interval was twice as long as the duration of the previous stimulation in order to avoid habituation with repeated stimulation and to exclude after effects. In the first experimental session (experiment 1: effects of tACS on STDT and near-threshold tactile stimuli perception), we studied the effects of tACS on STDT and near-threshold tactile stimuli perception during alpha, beta, gamma, and sham tACS delivered over S1 contralateral to the dominant hand by using the CP3/4 montage. During alpha, beta, and gamma tACS, the effect of tACS phase on near-threshold tactile stimuli perception was also investigated.In the second experimental session (experiment 2: effects of tACS using a modelling-based montage), we investigated the effects of tACS on STDT and near-threshold tactile stimuli perception using a modelling-based montage.In the third experimental session , we recorded cerebral activity with EEG to identify the individual μ-alpha rhythm frequency peak of oscillatory activity within S1 using the same montage as experiment 2. Subsequently, STDT and tactile stimuli perception were investigated during tACS applied over S1 at the individualised μ-alpha frequency. In addition, in experiment 3 tACS phase was recorded and phase-dependent effects were tested Fig. .Figure 455 with a sample of 16 subjects and an alpha error probability of 0.05, study power was 0.9.G* Power software was used for power analysis. Based on previous results on tactile perception modulationp < 0.05 for all analyses. Data were analysed using SPSS Statistics for Windows .Repeated measures analysis of variance (rmANOVA) with “FREQUENCY” as a factor  was used to investigate possible effects of tACS on sensory threshold and STDT. A further rmANOVA with “FREQUENCY” as a factor  was performed on STDT and near-threshold stimuli perception values normalised to those obtained in the sham session. To evaluate whether possible effects of tACS changed over time during each block, we also performed a rmANOVA with “TIME” as a factor considering the first and second half of each block separately . To evaluate the effects of tACS on near-threshold stimuli perception we performed rmANOVA with “PHASE”  and “FREQUENCY”  as factors. To verify whether tACS effects depended on different electrode montages, we also compared the effects produced by tACS stimulation using CP3/4 to those obtained using the modelling-based montage. We first performed rmANOVA with “MONTAGE”  and “FREQUENCY”  as factors to contrast sensory thresholds and STDT between experiment 1 and 2. We then performed rmANOVA with “MONTAGE”, “FREQUENCY” , and “PHASE” as factors of near-threshold stimuli perception. To compare the effects of tACS delivered at the individualised μ-alpha frequency to those obtained at the fixed alpha frequency of 12 Hz, we used rmANOVA with “FREQUENCY”  and “PHASE” as factors. We also specifically tested whether tACS delivered at the individualised μ-alpha frequency modulated near-threshold tactile stimuli detection by using rmANOVA with the factor “PHASE” on data recorded in experiment 3. The level of significance was set at