The cultivar Annurca is an apple that is cultivated in southern Italy that undergoes a typical redding treatment and it is appreciated for organoleptic characteristics, high pulp firmness, and nutritional profile. In this study, the effects of three different biostimulants , Protein hydrolysate (PEP), and Macro-algae mixed with zinc and potassium (LG)), with foliar application, on the quality parameters of Annurca apple fruits at the harvest, after redding, and at +60 and +120 days of cold storage were analyzed: total soluble solids (TSS) content, total acidity (TA), pH, firmness flesh, and red coloration of epicarp. Additionally, the polyphenolic quali-quantitative profile of pulp and peel was analyzed by UHPLC-Q-Orbitrap HRMS and Folin-Ciocalteu and the antioxidant capacity with the methods 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric-reducing antioxidant capacity (FRAP). The results obtained suggest that biostimulants are involved in the regulation of the secondary metabolism of the treated plants, acting positively on the quality of the Annurca fruits and their nutritional value. Fruits treated with PEP have shown, during cold storage, a significantly higher content of total polyphenols in flesh and a higher concentration of phloretin xylo-glucoside and phloridzin (350.53 and 43.58 mg/kg dw respectively). MA treatment caused, at the same time, an enhancement of flavonols between 0.6–28% and showed the highest total polyphenol content in the peel after 60 and 120 days of cold storage, with 2696.048 and 2570.457 mg/kg dw, respectively. The long-term cold storage (120 days) satisfactorily maintained phenolic content of fruits deriving from MA and PEP application, in accordance with data that were obtained for peel, showed an increase of 7.8 and 5.8%, respectively, when compared to the fruits cold stored for 60 days. This study represents the first detailed research on the use of different types of biostimulants on the quality of the Annurca apple from harvest to storage. Malus pumila Miller cv. Annurca, is one of the most important cultivars in Souther Italy, especially in Campania region, which accounts for 5% of Italian apple production. This cultivar is the only one to have received from the European Council the “Protected Geographical Indication”, an official designation related to the preservation of local and characteristics agriculture commodities . This frNumerous studies in the literature report positive effects on human health of the polyphenolic compounds that are present in the Annurca apple ,5,6,7.Annurca polyphenolic extracts avoid the damage of gastric epithelia in vitro and rat gastric mucosa in vivo . AnnurcaTogether with the containing of plant vigor , one of The redding process lasts 20–30 days after the harvest, depending on the weather conditions  and it iThe regulatory mechanisms of anthocyanin biosynthesis are important, because skin redness is an important factor in market acceptance for many apple cultivars. Bio-stimulants are composed of biological substances and microorganisms containing bioactive compounds as mineral nutrients, humic substances, vitamins, free amino acids, chitin, polysaccharide, and oligosaccharides ,19.According to a recent EU Regulation, plant biostimulants are defined mainly based on their activity, therefore biostimulants include various organic and inorganic substances (humic acids and protein hydrolysates), but also prokaryotes  and eukaryotes, such as mycorrhiza, N-fixing bacteria, and macroalgae ,21,22.Several literature studies highlight the important role of biostimulants in improving the efficiency of the plant’s metabolism, increasing plant tolerance to and recovery from abiotic stresses, facilitating nutrient assimilation, translocation and use, enhancing quality attributes of produce, including sugar content, color, rendering water use more efficient and enhancing soil fertility, particularly by fostering the development of complementary soil micro-organisms.Studies reported in the literature showed the important role of biostimulants also in improving the coloring efficiency of fruit peel , showingANOVA on L*, a*, and b* showed a high statistical significance between the biostimulants (B) and fruit exposure (E), less or not significant (ns) was for the interaction B × E .At harvest, after application with PEP and MA , there iThe greater efficiency of the treatments with the PEP and MA biostimulants is also evident in An increase in TSS from harvest to fridgeconservation of about 20% was observed; this increase is strengthened while using LG and PEP biostimulants to +120 days of fridge conservation with brix° of 14.10 and 13.27. At harvest  there is2, to then decrease after “melaio” around 3 kg/cm2 until reaching on average values equal to 2.5 kg/cm2 during the fridge-conservation at +120 days. Another parameter analyzed in the present study is the variation in pH which does not show particular differences in all 4 theses; the lower values are observed at harvest with an average pH of 3.36, which gradually increases up to the maximum average values of 3.80 at +120 days of fridge-conservation.At harvest, for all 4 theses, the firmness showed values of around 5 kg/cmThe influence of three different commercially available biostimulants, including microalgae (MA), protein hydrolysate (PEP), and macroalgae mixed with zinc and potassium (LG), on the quali-quantitative profile of polyphenolic compounds, in the flesh and skin of annurca samples, is included in Literature data were also used for the comprehensive evaluation of phenolic compounds. Single phenolic compounds were quantified while using calibration curves built with appropriate reference compounds. The investigated compounds were grouped in five classes, such as hydroxycinnamic acids , flavanols (catechin and epicatechin), flavonols , procyanidins , and dihydrochalcones .In this study, we have analyzed the UHPLC-HRMS profile of the polyphenols in both flesh and peel of ‘Annurca’ apple, in particular, taking into account the peculiar post-harvest storage of this fruit, the effects of the foliar application of the biostimulants were assessed at harvest time (unripe fruit), after reddening in “melaio”, and at different times of cold storage (60 and 120 days at 2 °C). The amount of total phenolics calculated from the data obtained by UHPLC-HRMS analysis was slightly lower and not well correlated from that estimated by the Folin–Ciocalteau method .For these reasons, we have considered Folin results and UHPLC-HRMS data to evaluate, respectively, quantitative and qualitative biostimulant effects. With regard to the peel, the concentration of the extractable polyphenols remained unchanged during the reddening–ripening process for control and for fruits deriving from the foliar application of the micro-algae biostimulant, whereas PEP and LG biostimulant treatments caused an increase equal to 2.4% and 7.4%, respectively. Therefore, the application of PEP and LG bistimulants determined a positive effect on polyphenolic total content of fruits ripened in melaio which was significantly higher than the control and microalgae treated fruits (+18% and +9% respectively).This result was mainly due to the large abundance of hyperoside, in PEP treated fruits, the concentration of which was higher than that found in the other samples . Moreover, the qualitative results showed that LG application caused a higher peel concentration of chlorogenic acid when compared to the other samples analyzed . Bearing in mind the lack of data on the effects of biostimulants on the nutritional quality of apples (flesh and peel) during cold storage, in this study we assessed how the qualitative and quantitative profile of polyphenols was influenced by the different treatments, during the storage period of fruits.In particular, the control fruits increased their polyphenolic content by 28%, while the fruits derived from biostimulant treatments showed an increase of 17.5% (PEP), 25% (LG), and 28.7% (MA), and not significative differences, in total polyphenolic content, were observed between control and biostimulant treated fruits. Finally, after 120 days of cold storage, it was observed a decrease of total polyphenol content of 5.8% and 17.2%, respectively, for the control fruits and for LG biostimulant treated fruits. On the other hand, fruits derived from the PEP and MA treatments showed an obvious preservation of the nutritional quality, in terms of polyphenol content, not showing significant variations as compared to the fruits cold stored for 60 days . Moreovep < 0.05) in extractable polyphenols, with the exception of the fruits deriving from the LG biostimulant treatment, which have a significant higher polyphenol content than the other analyzed samples (0.59 at harvest time and 0.92 after reddening), and also the highest concentration of hyperoside . In agreement with peel results, cold storage determined, after 60 days, an increase of polyphenol level for all of the samples, in particular there was an enhancement of 55%, 44.4%, 36.9%, and 37.8%, respectively, for control and PEP, LG, and MA biostimulant treated fruits. This finding was a consequence of a greater abundance of some individual polyphenols, such as phloretin xylo-glucoside, hyperoside, phloridzin, and kaempferol-3-O-glucoside, the concentration of which was two or three times higher than that found in the flesh of fruits after “melaio”. Furthermore, fruits derived from LG treatments, after 60 days of cold storage, showed a significant higher content of total polyphenols and a higher concentration of procyanidins (164.77 mg/kg dw) than those that were determined in the other samples. The long-term cold storage (120 days) satisfactorily maintained phenolic content and even fruits derived from MA and PEP application, in accordance with data obtained for peel, showed an increase of 7.8 and 5.8%, respectively, when compared to the fruits that were cold stored for 60 days.Furthermore, qualitative investigations carried out by HRMS Orbitrap showed that this is due to an increased level of certain compounds that are probably released during the cold storage from their polymeric forms or from dietary fiber to which they are linked. In particular, this release has been highlighted, in the case of PEP, for procyanidins, flavanols, and hydroxycinnamic acids, which underwent an increase, between 60 and 120 days of cold storage, accounting for 5–113%. Moreover, MA treatment caused, at the same time, an enhancement of flavonols between 0.6–28%. With respect to the flesh , the redThese findings were supported by HRMS-orbitrap data, which showed, between 60 and 120 days of cold storage, an increase of flavanols and procyanidins, for fruits deriving from PEP treatment, of 11–56%, while MA application determined, mainly, an increase of flavonols, especially hyperoside and isorhamnetin glucoside, which increased from 10 to 19.7 and from 0.371 to 1.58 mg/Kg dw, respectively.®, was employed as a positive control for both essays. In the case of DPPH (p = 0.05), for peels after ripening, when PEP and LG treatment were applied.The radical scavenging of the apple extract  was determined using two well-known spectrophotometric assays by determining DPPH free radical-scavenging activity and ferric reducing antioxidant capacity; the results are reported in  of DPPH , the antp = 0.05) increase of peel antioxidant capacity after long-term cold storage (120 days). With respect to the flesh, the antioxidant capacity, did not show neither remarkable changes related to the ripening process or positive effects associated with the application of biostimulants. On the contrary, during cold storage, in accordance with total polyphenolic content measured by Folin assay, all of the apple samples, showed a significant increase in antioxidant capacity (p = 0.05), without any significant effect of the treatments compared to the untreated sample.During the cold storage, a decrease in the antioxidant capacity was found for all of the samples. However, it should be noted that the MA treatment led to a significant (p = 0.05) increase in the antioxidant capacity after ripening with the notable exception of the sample treated with microalgae based biostimulant, for which, was observed a decrease of 16.5% compared to the treated unripe fruit. In accordance with the DPPH data, after ripening, PEP and LG treatments led to a significant ameliorating effect on the antioxidant capacity when compared to the untreated fruit (61.98 vs. 54.40 mmol trolox/kg dw). Unlike the DPPH data, a significant (p = 0.05) increase in the peel antioxidant capacity was observed during the cold storage. However, after 60 days, no significant effect linked to the application of biostimulants was observed. The long-term cold storage (120 days) caused a significant decrease of antioxidant capacity for the control and for fruits produced by the plants that were treated with LG biostimulant. On the other hand, PEP treatments showed no significant changes, while MA based biostimulant contributed to a significantly higher antioxidant capacity with respect to untreated peel fruit (89.91 vs. 78.61 mmol trolox/kg dw), in accordance with DPPH data. With regard to the flesh  increase DPPH and FRAP antioxidant capacity in the skin tissue of annurca apples, during reddening in “melaio”, whereas microalgae based biostimulant enhanced the antioxidant potential, especially at the skin level, when long-term storage was considered.These data were in accordance with the total polyphenolic content that was determined by Folin assay and with DPPH data. The FRAP values remained roughly constant during storage without remarkable variation at four months. Finally, we point out that LG and MA biostimulants significantly enhanced the FRAP values after 120 days of cold storage . Therefore, on the basis of our data, treatments with PEP and LG were able to significantly  found between the total polyphenols content and the antioxidant capacity measured with both DPPH and FRAP.Principal component analysis (PCA) was conducted to characterize each biostimulant treatment with respect to antioxidant activity and total polyphenolic content. Three principal components (PCs) with eigenvalues greater than 1 and explaining 99.6% of the total variance of the data were obtained with PC1, PC2, and PC3, accounting for 50.8%, 29.8%, and 19.0%, respectively (data not shown). The plot describes the correlation between the variables highlighting that LG treatment was well related with flesh total polyphenols after redding, FRAP, DPPH, and Folin results after 60 days of cold storage and with the FRAP results obtained from flesh cold stored for 120 days. On the other hand, PEP treatment was found to be related to FRAP, DPPH, and Folin results obtained from apple flesh at harvest and with total phenols measured on cold stored peels for 120 days. Finally, MA treatment was well related to FRAP and DPPH peel results at harvest and interestingly after 120 days of cold storage .Apple represents an important source of positive substances for human health and the beneficial activity of these compounds is due to the antioxidant capacity of these compounds that belong to the class of polyphenols. The main classes of polyphenols in apple are flavonoids, dihydrochalcones, and other polyphenolic compounds, such as chlorogenic acid. The annurca apple is a widespread apple cultivar of southern Italy, which generally undergoes a reddening treatment after the harvest. To encourage the development of eco-sustainable agriculture, fruit and vegetable operators are increasingly applying organic agricultural techniques. Frequently, the adoption of this production system determines a significant reduction in production yield, making it necessary to use natural substances that, in some way, promote productivity and, at the same time, determine an improvement in the nutritional quality of the fruits. In the present study, the effects of three different biostimulants , Protein hydrolysate (PEP), and Macro-algae mixed with zinc and potassium (LG)) on primary apple quality traits and potential nutraceutical compounds at harvest and during storage were evaluated. The application with biostimulants has positive effects on the peel and fruits coloring, as reported in the literature . SoppelsThe total soluble solids (TSS) content is an important character that indicates the evolution of post-harvest maturation and in this study , in accov/v) mixture employing an ultrasound bath.The classes of polyphenolic compounds that were found in the apple extracts were common to all investigated apple type and in agreement with those reported in previous studies on this cultivar ,31,32. PThe main compounds are chlorogenic acid, hyperoside, catechin, epicatechin, procyanidins, phoridzine, and phloretin xylo-glucoside. The total phenolic amounts obtained from the UHPLC-HRMS analysis was slightly lower and not well correlated from that estimated by the Folin–Ciocalteau method. This result is abundantly explained in the literature  and, in A. nodosum extract treatment to spinach (Spinacia oleracea L.) showed that the treatment improved postharvest storage quality and, at the same time, enhanced flavonoid content in spinach leaves [The application of seaweed extracts, or their components, to food crops is widely used and provides benefits to the health consumers. Seaweeds are rich sources of macro- and micro-elemental nutrients, amino acids, vitamins, and compounds, which may have effects on enhancing the nutritional value of the treated plants. Studies on the effect of h leaves . Lola-Luh leaves  indicateh leaves  reportedh leaves  attributh leaves  investigh leaves ,38,39. Oh leaves , which sIn the literature, it was also reported that the health promoting effect of microalgae treatments on peel polyphenolic content of Jonathan apples , in our One of the major objectives in agricultural production is the introduction of value-added qualities, especially nutritional characteristics. On this account, biostimulants have been described as a powerful tool for enhancing the nutritional properties of food crops . In morePolyphenolic standards, including rutin, catechin, epicatechin, chlorogenic acid, caffeic acid, procyanidin b1, procyanidin b2, phloridzin, kaempferol 3-O-glucoside, apigenin glucoside, and phloretin, were purchased from Sigma–Aldrich Chemical Co. . For the antioxidant tests, gallic acid, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,3,5-triphenyltetrazolium chloride (TPTZ), anhydrous ferric chloride, hydrochloric acid, and sodium acetate were purchased from Sigma–Aldrich . Methanol (MeOH) and water (LC-MS grade) were acquired from Carlo Erba reagents , whereas formic acid (98–100%) was purchased from Fluka .−1, the cv Sergente was used as a pollinator. The experiment was carried out on medium-textured soil with an adequate content of macro and micro elements. Irrigation was provided using a drip system equipped with two self-compensating drippers for plant, delivering 8 L/h. The orchard received standard horticultural cares and the treatments against the main parasites have been established in accordance with the regulation governing integrated production.The experiment was conducted in 2019 in Vitulazio (CE)  at an apple orchard of Giaccio Frutta Cooperative Society, on cv Annurca plants, aged eight years, grafted on M9 rootstock. The plants were trained to spindle training systems and spaced 4.5 m between the rows and 1.5 m on the row with a planting density of 1481 trees haThe experiment set up was organized as a completely randomized block design with four replications per treatment and 10 trees per replicate, per a total of 40 pants per treatment. To avoid any contamination between treatments, replicates on the same row were separated by an interval of 10 untreated trees. The trees were selected according to uniformity of fruit load and vegetative activity. For treatments were used a backpack atomizer with internal combustion engine, model Geotech Pro MDP 500, the quantity of water used for each treatment was 50 L. The foliar applications were carried out starting from the fruit set with a time interval of about 10 days between one application and another. In particular, the treatments started on 4/07/2019, when the fruit diameter was 43 mm, and they were repeated on the following dates: 18/07, 6/08, 19/08, 29/08, 9/09, and 18/09. The execution of the manual thinning of the fruits was prior to the treatments.Four treatments were compared:Control (Control), plants not treated with biostimulant but only with water.Protein hydrolysate (PEP), the product used was Peptone 85/16 from A.I.CHEM company , authorized in organic farming, a biostimulant with a high concentration of amino acids. The product was applied by foliar application with 150 g/50 L of water.Meaweed mix
(Ascophyllum nodosum and Laminaria digitate), a zinc-based formulation (ZINC 10 LG S) and a liquid potassium-based fertilizer (Red Skin LG). These products were mixed together and used by foliar application. The first two treatments were carried out with LG 201 + ZINC 10 LG S, while the following with Red Skin LG + seaweed mix with a dosage of 100 mL/50 L for LG 201 and LG74 while 150 mL/50 L for LG 347. The overall treatment was called LG.® , a high quality biological biostimulant that was obtained from micro-algae. The product was applied by foliar application with 200 mL/50 L of water.MicroAlgae (MA), the product used was AgriAlgae Biologico Original from AgriAlgaeEach sample consisted of 40 fruits per treatment taken at the harvest on 13/09, after “melaio” and after 60 and 120 days of refrigeration at +2 °C, the parameters analyzed were: weight, firmness pulp, epicarp coloring , iodine-iodide test for the determination of starch index, total soluble solids (TSS) content, pH, and titratable acidity (TA). The weight was determined with an electronic scale, while the pulp firmness with an EFFEGI manual penetrometer with an 8 mm tip on two sides opposite the fruit. The TSS content was determined with a HI 96,814 digital refractometer of Hanna instruments. TSS or Brix represents the percentage by mass of total soluble solids of a pure aqueous sucrose solution . The pH Fruit epicarp coloring was determined with a colorimeter  that was capable of quantifying colors according to international standards and expressed in defined color spaces. The instrument was calibrated with “white” managed by the light source on a white tile, before each measurement. The L * a * b * (CIELAB) color space is the most common for measuring the color of an object or materials of different origins and it is widely used in all sectors. In this color space, L * indicates brightness, while a * and b * the chromaticity coordinates: +a * is the direction of red, −a * is the direction of green, +b * is the direction of yellow, and −b * is the direction of blue . The meav/v), the mixture was vortexed intensively for 1 min. and sonicated in the dark, at room temperature, for 30 min. After centrifugation (3000 rpm/min.) for 10 min. the supernatants from both extractions were combined and made up to a final volume of 5 mL. The extracts were filtered through 0.22 µm nylon filters , prior to injection into the UHPLC-Orbitrap MS. The same extracts were used for antioxidant capacity and total polyphenolic content determinations. Chromatographic analysis was performed through an UHPLC system , equipped with a Dionex Ultimate 3000 Quaternary pump and a thermostated (25 °C) Kinetex 1.7 µm biphenyl (10 × 2.1 mm) column , with the following analytical conditions: solvent A, water/formic acid (99.9:0.1); solvent B, methanol/formic acid (99.9:0.1). Flow rate, 0.2 mL/min; injection volume, 2 µL. The autosampler and column temperatures were set at 10 °C and 25 °C, respectively. A gradient elution program was applied, as follows: 0 min, 5% of phase B; 1.3 min, 30% of phase B; 9.3 min, 100% of phase B; 11.3 min, 100% of phase B; 13.3 min, 5% of phase B; 20 min, 5% of phase B. As some standards were not available, quantitation for some polyphenols was calculated employing calibration curves of structurally related substances that belong to the same chemical group and with a similar response.Polyphenols were extracted according to the method of Petkovska et al. 2016 , with fem/z 200, whereas the AIF scan conditions were: microscans, 1; AGC target, 1e5; maximum injection time, 200 ms; mass resolution, 17,500 FWHM at m/z 200; HCD energy, at 10, 20 and 45. In both cases, the instrument was set to spray voltage, 3.5 kV; capillary temperature, 275 °C; sheath gas, 45 (arbitrary units); auxiliary gas, 10 (arbitrary units); m/z range, 80–1200; data acquisition, profile mode. The accuracy of MS analysis was ensured by calibrating the detector using the commercial calibration solutions that were provided by the manufacturer. Mass tolerance was kept at 5 ppm in both fullscan MS and AIF modes. Xcalibur software v. 3.1.66.10  was used to perform data analysis and processing.The mass spectrometry analysis was facilitated by a Q Exactive Orbitrap LC-MS/MS  that was equipped with an electrospray (ESI) source operating in negative ion mode . The acquisitions were conducted by setting a Full MS/AIF mode that uses a full MS scan (without HCD fragmentation), followed by an all ion fragmentation (AIF) scan (with a fragmentation energy applied). Full MS experiments were carried out with settings: microscans, 1; AGC target, 1e6; maximum injection time, 200 ms; mass resolution, 35,000 FWHM at v:v) was mixed with 500 µL of deionized water and 125 µL of the Folin–Ciocalteu reagent for 6 min. at room temperature.The fruit content of total phenolics was determined according to a Folin–Ciocalteu procedure , with slR2 > 0.995) that was computed over a dynamic range 0.05–2.5 g/L gallic acid. Each extract was analyzed in triplicate.Subsequently, 1.25 mL of 7.5% of sodium carbonate solution and 1 mL of deionized water were added in the mixture. The absorbance at 760 nm after 90 min. of incubation in the dark was measured. Concentrations of total phenolic were expressed in terms of mg of gallic acid equivalents (GAE) per gram dry weight (DW), based on a standard linear curve (. (4 mg in 10 mL) was diluted with methanol to an absorbance value of 0.90 (±0.02) at 517 nm to obtain a DPPH radical working solution. The scavenging activity of the apple extracts was determined by adding l mL of DPPH radical working solution and 200 µL of suitably diluted apple extract. The decrease in absorbance of the resulting solution was monitored at 517 nm after 10 min. of incubation at room temperature in the dark. The results were corrected for dilution and expressed in TEAC . All of the determinations were performed in triplicate.The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of apple extracts was determined using the procedure that was described by Brand-Williams et al. , with miFerric-reducing antioxidant capacity (FRAP) assay was conducted based on the method of Benzie and Strain , with mip < 0.05) were performed while using the XLSTAT, version 2013, statistical software package .Analysis of variance (ANOVA) on the complete randomized block design on the data and mean separation by Duncan’s multiple range test (This study represents the first detailed research into the use of different type of biostimulants on annurca apple quality at harvest and during storage. In the literature, there have not yet been reported data on the effects of biostimulant applications on annurca apple quality; therefore, the results obtained in this study appeared essential when considering that the biostimulant action can vary, depending on species/cultivar .These results suggest that selected biostimulants are involved in regulating the secondary metabolism of treated plants, leading to an improvement of annurca fruit quality and nutritional value. These beneficial qualities are also combined with their key role, in dealing sustainability challenges, reducing dependency on chemical fertilizers that are increasingly expensive due to resource depletion, and growing global demand and dangerous to human health and the ecosystem.The qualitative/quantitative improving effect on polyphenolic profile of fruits as well as the enhancement of their antioxidant capacity following biostimulant application are relevant factors to further improve its well-known nutraceutical potential, conservation, and commercialization.


Identifying the critical genes that differentiate gall bladder cancer from a normal gall bladder and the related biological terms was the aim of this study.The molecular mechanism underlying gall bladder cancer (GBC) trigger and development still requires investigations. Potential therapeutic biomarkers can be identified through protein-protein interaction network prediction of proteome as a complementary study.Here, a literature review of proteomics studies of gall bladder cancer from 2010 to 2019 was undertaken to screen differentially expressed proteins in this cancer. A network of 27 differentially expressed proteins (DEPs) via Cytoscape 3.7.1 and its plug-ins was constructed and analyzed. Ten proteins were introduced as hub-bottlenecks among which four were from DEPs. The gene ontology analysis also indicated that positive regulation of multi-organism process and regulation of response to biotic stimulus are the most disrupted biological processes of GBC considering their relationships with the DEPs. ACTG, ALB, GGH, and DYNC1H1, and relative biological terms were introduced as drug targets and possible diagnostic biomarkers. Introducing valuable biomarkers of gallbladder malignancy as the most known frequent type of the biliary tract cancer is essential for early diagnosis and treatment approaches. This type of cancer is recognized as the fifth frequent malignancy of the gastrointestinal tract . While iThe studies of gall bladder cancer proteomics were searched through Google Scholar and PubMed sources. The keywords used for our search were “Proteomics” and “Gall bladder Cancer”. These investigations had been published from 2010 to 2019. The proteins highlighted by these studies were gathered and then chosen for further analysis. Cytoscape v.3.7.1 and String db were used to construct a network of GBC, while the topological features were analyzed by Network Analyzer , 12. TheCytoscape via String db interaction analysis of the integrated DEPs based on Addition of 50 proteins from STRING database to the main network of 27 DEPs leads to participation of the isolated proteins in the interactome. The resultant network with centrality values, analyzed and visualized via Network Analyzer, is shown in MUC13 and RSU1 remain as individuals after addition of 50 neighbor proteins to the query proteins. Some nodes show larger centrality values in terms of degree and betweenness, suggesting that the network is scale free. Centrality analysis of the second network conducted by the Network Analyzer, the Cytoscape plug-in as the hub-bottlenecks, is tabulated in According to Gene ontology analysis via ClueGO has been conducted for DEPs in terms of biological process identifications. The pie chart summarizes the terms in groups of BPs. Single asterisk (*) shows p<0.05 and Double asterisk (**) implies p<0.01. Groups without stars are not statistically significant which have been omitted in this analysis by setting the query to show only groups with P value less than 0.05, see Eleven groups have been identified using ClueGO, among which one has one star suggesting it as significantly lower than 0.01. Positive regulation of multi-organism process and regulation of response to biotic stimulus are the most highlighted groups in the BP query of DEPs based on the number of gene participations. These two groups claim around 18% of all groups in terms of protein participations. Both of these groups are statistically highly significant based on the Group P value Corrected with Bonferroni step down as 0.005 and 0.00002. The second ranked groups in this query are positive regulation of prostaglandin secretion involved in immune response and neutrophil degranulation claiming 16% of all groups. The list of proteins participating in the first and second groups include CTSH, DMBT1, HMGB2, MIF, MUC13, NUCKS1, PSAP, S100A8, HTR1A1, and CTSH, DMBT1, HMGB2, HTRA1, MIF, MUC13, PSAP, S100A8 , respectively. The third and fourth groups also consist of proteins including MIF, CTSH, S100A8, MUC13, HTRA1, PSAP, DMBT1, HMGB2, and MIF, S100A8, CTSZ, CTSH, GGH, PSAP, GM2A, ANXA3, respectively. On the other hand, some proteins reveal more associations with the bp groups than with the others. These proteins are CTSH, PSAP, and S100A8 where the first two are linked to six groups while the latter one is associated with five groups.GBC is the most fatal fast growing type of cancer of the biliary tract  where onAs the analysis continued with the centrality identification of GBC, hub-bottlenecks, i.e. key proteins were introduced. Among the ten hub-bottlenecks, four proteins belonged to the differential expressed set of GBC. These proteins were DYNC1H1, ACTG, GGH, and ALB with the first one being down-regulated while the rest up-regulated in GBC. It can be inferred that up-regulation is dominant among the DE hub-bottlenecks in this study. Although MAPK1 does not belong to the list of DEPs in GBC, its phosphorylation has been mentioned in one proteomics study . This prLysozyme, the next ranked hub-bottleneck of the GBC network, has been reported with high expression in the sera of patients with malignancies including cancers of lung, melanoma , and breA panel of biomarkers could be more trustworthy than only one assigned biomarker. In this regard, proteins that have been reported by proteomics studies for GBC were gathered and those with greater importance in terms of interactions were introduced as ACTG, ALB, GGH, and DYNC1H1. This panel could be suggested as a promising therapeutic target for screening of GBC once confirmed by complementary evaluations. The follow up of patients and evaluation of treatment are the two important features of application of this finding if more investigation validates our outcomes.


To investigate the relation of child dietary diversity and household food insecurity along with other socio-demographic with child anthropometric indices in north-central Ethiopia, an area with a high level of food insecurity and inadequate diet quality.A community-based cross-sectional study was used.The study was conducted in Dessie and Combolcha towns of north-central Ethiopia from April to May 2018.Randomly selected 512 mother-child pairs with child’s age range of 6–59 months.Z-scores were 1.35 (± 2.03), − 1.89 (± 1.79), 0.05 (± 1.54), and 1.39 (± 2.06), respectively. From all anthropometric indicators, stunting and overweight/obesity remained the severe public issues hitting 43% and 42% of the children, respectively. In the model, mothers’ age and education and child’s age, sex, and dietary diversity were significantly related with child height-for-age Z-score while place of residence, sex of household head, child’s age, and dietary diversity score were the predictors of child BMI-for-age Z-score in the urban contexts of the study area. Nevertheless, food insecurity was not related to any of the child anthropometric indices.The mean (± SD) scores of weight-for-height/length, height/length-for-age, weight-for-age, and BMI-for-age The double burden of malnutrition epidemics (stunting and obesity) coexisted as severe public health concerns in urban settings. Anthropometric statuses of children were affected by multidimensional factors and seek strong integration and immediate intervention of multiple sectors. Currently, Africa is experiencing rapid demographic changes which are partly driven by the increasing migration of individuals to urban areas and rapid socio-economic transitions –3. NearlIn the early years, diet-related poor child health outcomes in urban areas are increasing and have negative ripple effects on the timing of entry into school educational attainment which ultimately resulting in intergenerational poverty and malnutrition . Thus, tEven though the prevalence of stunting and acute malnutrition (wasting or low weight-for-height) in Ethiopia has decreased over the past decade, it remains high, with 38% of children under 5 years stunted and 10% wasted . StuntinWe employed a cross-sectional study in Dessie and Combolcha towns from April to May 2018. Dessie and Combolcha cities are found in South Wollo Zone, north-central Ethiopia, with elevations between 1842 and 2550 m above sea level, as Combolcha takes the lower elevation. There is more than 58% of the total rainfall in the summer season, while 18% falls in spring and less than 5% of the total occurring during winter. The uneven distribution of rainfall gives rise to a serious shortage of water during the dry season in the area . Most ofAll children aged between 6 and 59 months that have resided in the study area for the last 6 months were included in the study sample, while any child with a severe medical problem, lack of household head or caregivers, or physical deformity was excluded from the study. The largest sample (512 mother-child pairs) was taken from a study conducted in Ethiopia by considering maternal education as a predictor for child stunting and overweight  with theA predesigned and pre-tested questionnaire was used to interview the study participants to elicit information on family and child socio-demographic characteristics like residence, religion, type of family, education, occupation of parents, socio-economic condition , household food insecurity, child feeding characteristics, and anthropometric measurements.The questionnaire was standardized to assure the quality and validity of the data and translated into the local language (Amharic) and was re-translated to English. All assessment team members were able to administer the questionnaires properly; a total of 5 days of rigorous training of enumerators and supervisors was given by the three authors. Before the actual data collection work, data collectors and supervisors carried out role-play practices and they filled the pre-test activities in the community other than target areas. Data collectors were responsible for filling out the data using mobile devices while supervisors checked the completeness and correctness of the filled data before sending it to the researchers. At the end of every data collection day, each questionnaire was examined for completeness and consistency by the supervisors and finally cross-checked by the researchers. A regular adjustment has been made for anthropometric measurements in each circumstance.The household socio-economic status (SES) was parameterized by the principal component analysis (PCA) method using house properties confirmed by the questionnaire: property owned, source of drinking water, type of toilet facility, and type of flooring, wall material, and roof material. The score in the first PCA component was used as an asset index of SES status for each household , and houChild dietary assessment was done based on the procedure recommended by the Food and Agriculture Organization (FAO) . MothersChild weight and length/height were taken by following critical and meticulous procedures. Ages were also recorded from immunization cards, direct probing of mothers, or birth certificates. The weight of children was taken using an electronic digital weight scale and recorded in kilograms to the nearest 0.1 kg  and withZ-score (WHZ), height/length-for-age Z-score (HAZ), weight-for-age Z-score (WAZ), and BMI-for-age Z-score (BAZ). Child nutritional status was determined using the above indices where each of the indices < − 2 SD is categorized as wasted, stunted, underweight, and thin. The child overnutrition was also defined when BAZ score between + 2 and + 3 SD and greater than + 3 SD reflecting the presence of overweight and obesity, respectively. We omitted outliers for WHZ and BAZ when less than − 5 and greater than + 5 and for HAZ and WAZ when the score less than − 6 and greater than + 6, respectively [The data were cleaned and prepared for analysis, and STATA version 15  was used to present the summary results and inferential statistics. Exploratory data analyses were done to identify missing values, influential outliers, and normality of data for both outcome and explanatory variables. Anthropometric data were exported to WHO Anthro Software version 3.2.2 to generate anthropometric indices for weight-for-length/height ectively . We fittDuring data collection, a letter of ethical clearance was collected from the Wollo University, College of Medicine and Health Sciences. Particularly, the institutional health research ethics review committee was consulted about the importance of the research to the community and the harms that would occur during data collection. An official letter has been written for each city administrator and health office where the data were taken. In addition, informed verbal consent was obtained from each client, and confidentiality was maintained by giving codes for each respondent rather than recording their names. Data collectors were informed that clients have full right to discontinue or refuse to participate in the study.A total of 506 mothers with child pairs were provided a complete response that gives a response rate of 98.8%. The mean age of the household head was found to be 32.2 (± 8.5 SD) years, and 355 (70.2%) of the study subjects were male household headed. Regarding the marital status of the participants, majority (459 (90.7%)) of them were married Table .Table 1About the sources of drinking water, majority (445 (87.9%)) of the respondents had a private pipeline in their compounds while the rest used from communal pipe and spring water sources. On the other side, about two fifth 43.1%) of the study participants used a latrine outside their home that might be from communal latrine or open defecation (Table % of the p < 0.0001) .The study reported that from the total of study subjects, 168 (33.1%) of them were found in households that experienced any form of food insecurity. The extent of food insecurity was much worse among households who are female-headed than male-headed  of them had good dietary diversity who consumed four and above food groups. Of the 24 h recall, the most common or staple food group consumed by children was pulses and nuts followed by cereals. However, consumption of iron-, zinc-, and vitamin A-rich animal source foods was eaten by less than 50% of the children , while it was higher among children from food-secure households compared to an insecure one . Additionally, children from educated mothers, married women, and male-headed households had at least twofolds of better dietary diversity as compared to those from non-educated mothers, unmarried women, and female-headed households, respectively  of them were wasted and boys seemed to be more affected than girls (5.8% vs 4.3%). Similar results were found between WHZ and BAZ to illustrate the burden of acute malnutrition among children. By BAZ scores (< − 2 SD), 5.2%  of children were acutely malnourished. Regarding the degree of chronic undernutrition from 340 children, about two of every five children were stunted , and of which 21.4%  and 25.2%  were moderately and severely stunted, respectively. On the other hand, the proportion of children who had either wasting or stunting or both was 10.5% . The co-existence of a double burden of malnutrition in the area was observed. Of all children, 42.2%  of them were overweight and/or obese. With the BAZ score of greater than 3, and between 2 and 3 of SD, 27.9%  and 15.2%  of children were categorized as having obesity and overweight, respectively.From a total of 316 children who had complete data for weight-for-height r = 0.18, p = 0.02), child dietary diversity , and animal source food score  were positively correlated with child WHZ score, but the food insecurity score  was negatively correlated, while HAZ score was positively correlated with age of household head  but negatively correlated with child dietary diversity scores , child age , and animal source food .The mean of WHZ, HAZ, BAZ, and WAZ scores (SD) was 1.35 (± 2.03), − 1.89 (± 1.79), 1.39 (± 2.06), and 0.05 (± 1.54), respectively. The presence of a linear relationship between different anthropometric indices and many covariates was examined  HAZ and BAZ scores were − 1.89 (± 1.79) and 1.39 ± 2.06), respectively, a significant number of children were categorized as stunted and overweight/obese. In the urban settings, the rate of malnutrition among under-five children was persistently higher, and the nutrition transition phenomenon impacted the health of children in which the rate of obesity has been radically progressed and that ultimately attributed to many life-threatening non-communicable diseases including diet-related cardio-metabolic risk and cancer , respect, 24. TheIn the generalized linear model, after adjustments, child’s various anthropometric indices in the study area had been influenced by socio-demographic features, socio-economic status, and child feeding practices. But it was not affected by the urban food insecurity status of the household. Child linear growth  were significantly associated with mothers’ or caregivers’ age and education, sex and age of the child, child DDS, and ASF, while the child’s mean BAZ score was affected by the sex of household head, age of the child, and child DDS. Furthermore, children with a higher score of animal source food were less likely to have shorter statures.Child diet diversity was inversely and significantly associated with child HAZ score, and it was contradicted with the findings in rural areas of Kenya and East and West Gojjam Zones of Amhara region, Ethiopia, implying that DDS was a proxy measure of childhood stunting , 43. HowThe child mean HAZ score was negatively affected as a child became aged which was compatible with shreds of evidence from China , Gojjam The child BAZ score had shown a significant relation to child dietary diversity score and supported by studies conducted in Bahir Dar  and SoutChildren from male-headed households and reside in Dessie  were negFood insecurity and socio-economic status of households in the urban context of the current study did not show association with any of the child anthropometric indicators which were similar to the reports from rural Cambodia , rural cThis study has several limitations. It does not consider potential mediator variables such seasonal variability that may affect food insecurity and feeding practices, environmental factors, host factors, physical activity, and other lifestyle factors that cumulatively impact child stature and fat body composition of children. We cannot also discount the effect of nutrient intakes and details of food frequency for processed foods and fruits and vegetables. We excluded some data due to missing anthropometric measurements that may have introduced a selection bias (if not missing at random), and may thus have affected both the internal validity and the representativeness of the findings in the broader Ethiopian context.The study concludes that the co-existence of the double burden of malnutrition epidemics in urban settings. The presence of different forms of malnutrition is characteristics, rapid urbanization, population growth, and high inequalities. Both forms of malnutrition are calling for immediate interventions due to their severe public health concerns. Mothers’ age and education, child’s age, sex, and dietary diversity were the potential determinants of child’s linear growth while the area of residence, sex of household head, child’s age, and dietary diversity were the predisposing correlates of a child’s body fat composition. The dual epidemic of undernutrition and overweight/obesity requires differential policy inputs in metropolitan areas. Very little obesity prevention interventions targeting children have been effective and a comprehensive, multifaceted strategy tackling diet, physical inactivity, coupled with psychosocial support and local food environment change may prove more effective. Nutrition policies tackling child obesity must promote household nutrition security and healthy growth, decrease overconsumption of nutrient-poor foods, better shield children from the increasingly pervasive marketing of energy-dense, nutrient-poor foods and sugar-sweetened beverages as well as reduction of growing physical inactivity . The cos


Fixed sample entropy (fSampEn) has been successfully applied to myographic signals for inspiratory muscle activity estimation, attenuating interference from cardiac activity. However, several values have been suggested for fSampEn parameters depending on the application, and there is no consensus standard for optimum values. This study aimed to perform a thorough evaluation of the performance of the most relevant fSampEn parameters in myographic respiratory signals, and to propose, for the first time, a set of optimal general fSampEn parameters for a proper estimation of inspiratory muscle activity. Different combinations of fSampEn parameters were used to calculate fSampEn in both non-invasive and the gold standard invasive myographic respiratory signals. All signals were recorded in a heterogeneous population of healthy subjects and chronic obstructive pulmonary disease patients during loaded breathing, thus allowing the performance of fSampEn to be evaluated for a variety of inspiratory muscle activation levels. The performance of fSampEn was assessed by means of the cross-covariance of fSampEn time-series and both mouth and transdiaphragmatic pressures generated by inspiratory muscles. A set of optimal general fSampEn parameters was proposed, allowing fSampEn of different subjects to be compared and contributing to improving the assessment of inspiratory muscle activity in health and disease. Alternatively, inspiratory muscle force and activation can be measured by surface mechanomyography (MMG) [lic and sEMGlic respectively) [para and sEMGpara respectively) [Measuring respiratory muscle function is a key step in the assessment of many respiratory diseases, such as chronic obstructive pulmonary disease (COPD) . Respiray muscle , and deplectrode . Howeverhy (MMG) ,6 and elhy (MMG) ,8,9,10, Inspiratory muscle EMG and MMG signals are, however, contaminated by cardiac noise corresponding to the electrical (ECG) and mechanical (MCG) activity of the heart, respectively. Conventional approaches to analyse EMG and MMG signals, based on amplitude estimators, such as the average rectified value (ARV) or the root mean square (RMS) ,12, are lic [lic [N (referred as window length), the length of the sequences to be compared, m (embedded dimension), and the tolerance for accepting that two sequences are similar, r. In SampEn, tolerance is set for each time-series as r times the standard deviation (SD) of the time-series analysed, so that SampEn is not influenced by differences in amplitude. However, fSampEn is intended to track both complexity and amplitude variations, and therefore it is determined by calculating SampEn using a fixed tolerance for different time-series. In this way, amplitude variations of a single time-series can be tracked by calculating fSampEn within a moving window and using a fixed tolerance of r times the SD of the whole time-series.As an alternative, our group has recently proposed fixed sample entropy (fSampEn) as a method to estimate respiratory muscle function from sEMGlic ,9 and sMlic [lic ,15 signalic [lic , fSampEnTherefore, fSampEn is not only sensitive to changes in signal complexity, but also to changes in signal amplitude . In myogm and r. Previous studies [m either at 1 or 2, r ranging from 0.1 to 0.5 and window length ranging from 0.25 to 1 s. A recent study evaluated the influence of window length, m, r and the sampling frequency on the estimation of respiratory activity from sEMGlic signals using fSampEn [r are the most critical parameters determining the shape and magnitude of fSampEn time-series. However, fSampEn time-series were calculated only for sEMGlic signals recorded from one healthy subject and compared to non-invasive measurements of mouth pressure (Pmo). Therefore, there is need for further research on the performance of fSampEn in different myographic respiratory signals related not only to the level of inspiratory muscle activation in healthy subjects, but also to that in patients with impaired respiratory mechanics.Despite the potential for using fSampEn to analyse respiratory muscle EMG and MMG signals, there is no consensus standard for optimum fSampEn parameters, and several values have been suggested for window length,  studies ,18,19,25 fSampEn , demonstr parameters to ensure an optimal performance of fSampEn in oesEMGdi, sEMGlic, sMMGlic, sEMGpara and sMMGpara signals recorded in healthy subjects and COPD patients.The principal aim of the present study is, therefore, to provide an in-depth evaluation of the performance of fSampEn in myographic respiratory signals, which lead us to propose a set of optimal general fSampEn parameters for inspiratory muscle activity estimation. We analysed different combinations of window length and 2, forced expiratory volume in 1 second/forced vital capacity 81.9 (74.1–83.9)%), with no history of cardiorespiratory or neuromuscular disease, and from fourteen stable COPD patients  years, body mass index 25.5 (19.4–28.0) kg/m2, forced expiratory volume in 1 second/forced vital capacity 38.2 (30.2–46.5)%). This study was approved by the NHS Health Research Authority (NRES Committee London–Dulwich 05/Q0703) and the experiments conformed to the standards of the Declaration of Helsinki. All subjects were fully informed of any risk associated with the study and provided their written consent before participation.Measurements of inspiratory muscle force and activation were obtained from twelve healthy subjects  years, body mass index 22.2 (20.6–24.2) kg/mlic, sEMGpara, sMMGlic, sMMGpara, respiratory airflow and Pmo measurements were obtained from all participants  placed on the skin over the seventh or eighth intercostal spaces, between the mid-axillary and the anterior axillary lines [para was recorded using two surface electrodes positioned in the second intercostal space bilaterally [lic was recorded using two triaxial accelerometers . The accelerometers were attached bilaterally to the skin with adhesive rings as close as possible to the sEMGlic electrodes along the seventh or eighth intercostal space, over the anterior axillary line [para was recorded using another triaxial accelerometer placed on the right side in the second intercostal space, between the right sEMGpara electrode and the right border of the sternum. In COPD patients, sEMGlic and sMMGlic were recorded only on the right side for patients’ convenience. Respiratory airflow was measured using a pneumotachograph  connected to a differential pressure transducer . Pmo was measured from a side port on the pneumotachograph using a second differential pressure transducer .Non-invasive sEMGicipants . sEMGlicry lines ,9,27. sEaterally ,28. A grary line ,19. sMMGdi and oesEMGdi measurements were obtained from healthy subjects only , as previously described [di was recorded using a multipair oesophageal electrode catheter , consisting of nine consecutive recording electrode coils, which formed five pairs of electrodes [Invasive Pcts only , since tescribed ,30. Crurectrodes ,31. The The EMG signals were amplified (gain 100), high-pass filtered at 10 Hz, and AC-coupled before acquisition . All signals were acquired using a 16-bit analogue-to-digital converter  and displayed on a laptop computer running LabChart software  with analogue to digital sampling at 100 Hz (airflow and pressures), 2000 Hz (MMG) and 4000 Hz (EMG).Maximal static inspiratory pressure (PImax)  was measAll participants performed an inspiratory threshold loading protocol at five inspiratory threshold loads set at 12% (L1), 24% (L2), 36% (L3), 48% (L4) and 60% (L5) of the subject’s PImax. Inspiratory threshold loads were generated using an electronic inspiratory muscle trainer  attached to the distal end of the pneumotachograph. Subjects were seated and breathed through the pneumotachograph via a mouthpiece with a noseclip in place. Baseline measurements were recorded during a minimum of 2 minutes of quiet tidal breathing, following which the inspiratory muscle trainer was attached to the pneumotachograph and the series of threshold loads was imposed. Subjects were not provided with any specific instructions to adopt a certain duty cycle and were free to choose their own breathing frequency. Subjects were, however, informed that effort was needed to overcome the threshold loads, and they were therefore encouraged to focus on using their diaphragm, to perform quick deep inspirations and to ensure that expiration was complete before making their next inspiratory effort. Each load consisted of 30 breaths at most followed by a resting period to allow all respiratory measures to return to baseline.LabChart data were exported as MATLAB files, and analysed offline using our fSampEn algorithms developed in MATLAB . di, sEMGlic and sEMGpara signals were resampled at 2000 Hz, and filtered with an 8th-order zero-phase Butterworth band-pass filter between 10 and 600 Hz and with a 2-Hz bandwidth notching comb filter to remove the power line interference at 50 Hz and all its harmonics up to 1000 Hz. Two 10th-order zero-phase notch filters were also applied to the EMG signals of COPD patients to remove additional interferences that appeared at 64 and 192.5 Hz. sMMGlic and sMMGpara signals were resampled at 500 Hz and filtered with an 8th-order zero-phase Butterworth band-pass filter between 5 and 40 Hz. After filtering, the total acceleration measured by each accelerometer was arithmetically calculated as the norm of the vector formed by its three sMMG signals .oesEMGmo signal. After segmentation, all cycles were visually inspected and those either containing artefacts within the EMG and MMG signals or having an unusual pressure pattern were rejected.All signals were segmented into inspiratory and expiratory signal segments by means of a zero-crossing detector on the Pdi signals , another containing sEMGpara and right and left sEMGlic signals (only right for COPD patients), and one more containing |sMMGpara| and right and left |sMMGlic| signals (only right for COPD patients).In order to track amplitude changes evoked by inspiratory muscle EMG and MMG activity during the inspiratory threshold loading protocol, fSampEn requires a single tolerance value to be fixed for each subject and group of myographic respiratory signals. Based on signal nature, the following three groups of signals were defined for each subject: one containing the five oesEMGr times the SD of the signal analysed, a unique individual SD was calculated for each subject and group of signals. Firstly, SD of all inspiratory signal segments during resting breathing and threshold loading was calculated for each signal. Then, SD values of the five oesEMGdi signals of each healthy subject were averaged to obtain a unique individual SD oesEMGdi. In the same way, SD values of sEMGpara and right and left sEMGlic signals (only right for COPD patients) of each subject were averaged to obtain a unique individual SD sEMG. Finally, SD values of |sMMGpara| and right and left |sMMGlic| signals (only right for COPD patients) of each subject were averaged to obtain a unique individual SD |sMMG|. In this way, fSampEn time-series of a group of signals were calculated using the same tolerance value for all signals and all moving windows throughout resting breathing and the inspiratory threshold loading protocol.Since tolerance is usually set as It seems clear that, for a given subject and group of signals, a unique individual SD is required to make fSampEn time-series of resting breathing and threshold loading comparable. Moreover, a unique global SD is required in order to compare fSampEn time-series of different subjects. The question is, however, whether a global SD represents well the variation of a given group of signals in all subjects. To study the effect of using a global SD on fSampEn time-series, a unique global SD was calculated for each group of signals and separately for healthy subjects and COPD patients, as the mean of individual SDs.di, three sEMGlic and three sMMGlic in healthy subjects, and two sEMGlic and two sMMGlic in COPD patients) acquired during resting breathing and the inspiratory threshold loading protocol were calculated using m equal to 2, window length ranging from 0.1 to 0.5 s in increments of 0.05 s and tolerance set as r times SD, with r ranging from 0.05 to 0.6 in increments of 0.05 and SD equal to individual SD and global SD. A 90% overlap between adjacent windows was used. As a result, a total of 14,256 and 5184 fSampEn time-series were calculated for each healthy subject and each COPD patient, respectively.For each subject, fSampEn time-series of all myographic respiratory signals .Individual and global SDs are shown in di was calculated only for healthy subjects (0.0121 V). The effect of using a global instead of individual SDs is analysed in the next two sections, separately for healthy subjects and COPD patients.Interestingly, despite the intra- and inter-subject variability of individual SDs (black boxes), global SDs (blue boxes) of healthy subjects were very similar to those of COPD patients, for both sEMG  and |sMMG|  signals. A global SD oesEMGmaxc values for healthy subjects. First, mean maxc of each group of signals during resting breathing and threshold loading was calculated for each subject and combination of fSampEn parameters . Then, mean maxc values of all healthy subjects were averaged.maxc values were obtained for oesEMGdi, sEMG and |sMMG| using individual and global SDs, suggesting that global SDs could reasonably be used in order to make fSampEn time-series of different subjects comparable.Very similar maxc values in all cases were obtained for a global window length of 0.5 s. However, optimal global r varied among groups of signals: 0.05 for oesEMGdi, 0.35 and 0.25 for sEMG, and 0.45 and 0.4 for |sMMG|.Using global SDs, the highest maxc, between the maxc value obtained using global parameters and that obtained using individual parameters . Then, mean maxc values of all COPD patients were averaged.maxc values were obtained for sEMG and |sMMG| using individual and global SDs in COPD patients. Using global SDs, the highest maxc values in all cases were obtained, again, for a global window length of 0.5 s. Regarding optimal global r, it varied from 0.2 for sEMG to 0.55 and 0.5 for |sMMG|.As in healthy subjects, very similar maxc value obtained using global parameters and that obtained using individual parameters , 0.3 × 0.0022 (sEMG) and 0.5 × 0.0060 (|sMMG|).In light of the very similar global SDs in healthy subjects and COPD patients , and theAs observed in In this study, we conducted an evaluation of the performance of fSampEn for inspiratory muscle EMG and MMG analysis, which led us to propose, for the first time, a set of optimal general fSampEn parameters adapted to each type of myographic signal.Evaluating the amplitude of myographic signals recorded from different respiratory muscles using conventional amplitude estimators is difficult due to the cardiac muscle interference. In order to reduce crosstalk from the heart in myographic respiratory signals, the use of fSampEn was adopted in our previous studies ,15,17,19m, r and window length. However, even for the same application, such as respiratory muscle activity estimation, several values have been suggested for fSampEn parameters. A previous study by our group demonstrated that r and window length are the most critical parameters, influencing the magnitude and shape of fSampEn time-series [m equal to 1 and r ranging from 0.1 to 0.64 or m equal to 2 and r ranging from 0.13 to 0.45 could be suitable values for respiratory muscle activity estimation using fSampEn in sEMGlic signals. However, some limitations were acknowledged in this previous study. Firstly, only sEMGlic signals were analysed for a healthy subject. However, other myographic signals, such as sMMGlic or oesEMGdi, could require different fSampEn parameters. Moreover, impaired respiratory mechanics  may affect the amplitude and complexity of myographic respiratory signals [mo, which was used as an approximation of the overall mechanical output of the inspiratory muscles during breathing. However, Pmo was conditioned by the threshold loads imposed by the inspiratory muscle trainer, which was attached to the distal end of the pneumotachograph in series with a mouthpiece.Depending on the application, fSampEn requires an adjustment of its input parameters e-series . It was  signals , and thedi, sEMGlic, sEMGpara, sMMGlic and sMMGpara. Moreover, the participants in the present study cohort were twelve healthy subjects with an age range from 21 to 44 years and fourteen COPD patients with an age range from 53 to 80 years. This heterogeneous population covers a wide age range and both normal and impaired respiratory mechanics. Furthermore, the inspiratory threshold loading protocol provided the potential to acquire inspiratory muscle EMG and MMG signals under a wide range of loads on the inspiratory muscles, allowing the performance of fSampEn to be evaluated for a variety of inspiratory muscle activation levels. The performance of fSampEn was assessed by means of the cross-covariance of fSampEn time-series and both non-invasive measurements of Pmo and the gold standard invasive measurements of Pdi. While Pmo assesses global inspiratory muscle strength, Pdi is more specific for the diaphragm.In this current study, we have thoroughly investigated the performance of fSampEn, for the first time, in different types of myographic respiratory signals, including oesEMGr times the SD of the signal analysed. When calculated within a moving window using a fixed tolerance for all windows, fSampEn can quantify amplitude variations on a signal. In this study, for a given subject and type of myographic respiratory signal, choosing a unique individual tolerance, and therefore a unique individual SD, allows fSampEn to track amplitude variations during the inspiratory threshold loading protocol and optimises the performance of fSampEn, mainly in terms of cardiac noise attenuation, for that subject. On the other hand, a unique global SD is required in order to compare fSampEn time-series of different subjects. Due to differences in myographic respiratory signal amplitude among subjects, using a global SD, however, may not be optimal in all subjects and may compromise the performance of fSampEn in some subjects.The kernel of the fSampEn algorithm is the search for similar sequences within a signal, so that more similar sequences indicate lower complexity and yield lower fSampEn values. Two sequences are similar if the maximum difference of their corresponding values is less than or equal to the tolerance parameter, which is usually set as di, sEMG and |sMMG|) using different r values (0.05–0.6) and both individual and global SDs. Intra- and inter-subject variability of the SD of myographic respiratory signals was firstly analysed  values of 0.05 × 0.0121 (oesEMGdi), 0.3 × 0.0022 (sEMG) and 0.5 × 0.0060 (|sMMG|) to be used in healthy subjects as well as in COPD patients. The use of these general SDs allows fSampEn time-series to be optimally calculated and compared in different subjects.In light of these results, we have proposed general tolerance (Different combinations of window lengths between 0.1 s and 0.5 s were tested, and a window length of 0.5 s maximised the performance of fSampEn in all study participants and myographic respiratory signals. In general, the longer the window, the smoother the fSampEn time-series . Long wiIn this study, fSampEn time-series were compared with pressure signals, which are low-frequency signals and vary slowly over time, and therefore longer windows performed better than shorter windows. On the other hand, a lower threshold of 0.5 s was applied to respiratory phase durations during segmentation of myographic signals. Therefore, in order to focus the analysis of myographic respiratory signals on inspiratory activity, window lengths longer than 0.5 s were not tested. Yentes et al. proposed a minimum window length of 200 samples for the correct performance of fSampEn . The winm parameter is typically set at 1 or 2 [m = 1 and m = 2 for fSampEn calculation in sEMGlic signals [m = 2 has been properly used for the analysis of different myographic respiratory signals [m = 2 has also been used in the present study for the analysis of myographic respiratory signals.The t 1 or 2 ,37. In r signals , and m = signals . AccordiSampling frequency is another parameter to be taken into account in fSampEn calculation. It has been reported that higher sampling frequencies yield lower fSampEn values . Howeverr and window length, for a proper estimation of inspiratory muscle activity from EMG and MMG signals. The evaluation of fSampEn performed in a heterogeneous study population, including healthy subjects and COPD patients within a wide age range, allowed us to propose general values for fSampEn parameters that permit optimal calculation and comparison of fSampEn time-series of different subjects, thus contributing to improving the assessment of inspiratory muscle activity in health and disease.In summary, we performed a thorough evaluation of the most relevant parameters of fSampEn, that are


Knee osteoarthritis (KOA), with a high incidence in old-age population, adversely affects their life quality. The valgus knee bracing is an important physical therapy for KOA, but its clinical effects on pain release and functional improvement remained unclear. This meta-analysis is to systematically evaluate the clinical outcomes of valgus knee bracing in patients with KOA.A meta-analysis of clinical randomized controlled trials (RCTs) on pain and functional changes in patients with KOA after using valgus knee braces. The search period was ranged from the inception of the database to May 2020. The enrolled research databases included PubMed, Embase, and Web of Science databases. Two investigators independently formulated inclusion criteria and exclusion criteria and screened and determined the final enrolled literature. Then the outcome indicators were extracted and organized from the included literature, and the risk of bias was assessed by Cochrane Handbook 5.0.1.P = 0.20; four articles were related to the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) function score, and the results showed that RR = − 0.15, 95% CI , P = 0.26; two articles were related to the Knee Injury and Osteoarthritis Outcome Score (KOOS), and the results showed that RR = 0.58, 95% CI , P = 0.81; and three articles were related to the KOOS Activities of Daily Living (KOOS-ADL), and the results showed that RR = 0.04, 95% CI , P = 0.91. These results indicated that the valgus knee bracing has no statistical significance in pain and functional activity improvement of patients with KOA. The subgroup analysis showed that the follow-up time was the source of the heterogeneity of the VAS pain score.A total of 10 articles were included in this study, including 739 patients. Eight articles were related to the visual analog scale (VAS) pain score, and the results showed that RR = − 0.29, 95% CI − 0.73, 0.15], Our current evidence suggests that valgus knee bracing may not improve pain release and function activates in KOA patients in the long-term period, but only being beneficial to the short-term rehabilitation. Osteoarthritis (OA) is a worldwide degenerative joint disease, being one of the main causes of joint disability . The incValgus knee bracing is found to effectively correct the lower limb force lines, release pain feelings, and improve joint functions of KOA patients by optimizing their biomechanical axis –13. KirkPubMed, Embase, and Web of Science databases were searched by computer. The search period was from the inception of the database to May 2020. To supplement the literature, manually enter the references included in the study if necessary. The search terms include “Osteoarthritis,” “knee,” and “brace OR bracing OR Valgus brace OR Unloader brace.”The inclusion criteria included the following: (1) RCTs of using valgus knee bracing to treat KOA; (2) in RCTs, the experimental group was given valgus knee bracing treatment, and the control group was given non-bracing and other conservative treatments ; (3) the age, sex, race, nationality, and course of disease in the included studies are not limited; (4) RCTs have one or more of the following outcome indicators: WOMAC function score , VAS pain score , KOOS , KOOS-ADL score .The exclusion criteria included the following: (1) non-clinical randomized controlled trials; (2) the conference included literature, reviews, and published the same research literature repeatedly in different languages; and (3) the outcome indicators data in RCTs cannot be used.Two researchers screened the literature separately, including the first step of screening after browsing the titles and abstracts of the literature, as well as reading the full text and determining whether it was finally included. After the data were included, the two researchers checked each other. If the opinions were not uniform, the third researcher should judge whether they were included. The relevant information extracted includes (1) basic information of the literature, including title, year, first author, and course of treatment, (2) incorporate the basic characteristics of RCTs and patient baseline data, (3) intervention measures of the two groups, (4) outcome indicators, and (5) key factors for evaluating the quality of literature. Microsoft Excel was used to record the relevant information. Besides, EndNote was used for document management.The bias risk of the included studies was evaluated according to the Cochrane Handbook 5.0.1 RCT bias risk assessment tool. The quality of the literature is assessed by whether the included literature is a random method, whether there is allocation concealment, whether the blind method is used, whether the result data is complete, and whether the research results are selectively reported. Each result is divided into low risk, unclear, and high risk. The quality of the methodology was evaluated by two researchers, and if there were different opinions, the third researcher would participate in the discussion and resolved.χ2. The size of heterogeneity is judged by I2. When I2 < 50%, a fixed-effect model should be used; when I2 > 50%, a subgroup analysis of the causes of heterogeneity may be performed; and when the difference between the two studies was not statistically significant, a random-effects model can be used for analysis. When P < 0.05, it means that the difference of the research results was statistically significant.Revman 5.3 software was used for data analysis. Heterogeneity analysis: The heterogeneity of the research results was tested by The literature search process was shown in Fig. A total of 739 patients were included in the 10 studies, including 412 patients in the experimental group and 414 patients in the control group. The minimum sample size was 10 and the maximum sample size was 86. Table Using the Cochrane Handbook 5.0.1 RCT bias risk assessment tool to evaluate the quality of 10 studies, the results are shown in Figs. I2 = 88% > 50%, the random effect model was selected. The results showed that RR = − 0.29, 95% CI , and the combined effect test Z = 1.28, P = 0.20  are applied in clinical practices, such as weight control, exercise, physical therapy , oral analgesics, and joint cavity injection drugs , 29. EmeP = 0.29), WOMAC function score (P = 0.26), KOOS (P = 0.81), and KOOS-ADL (P = 0.91). It is indicated that valgus knee bracing cannot improve the pain, activity function, and quality of the daily life of KOA patients. Due to the high heterogeneity of the VAS pain score, we conducted a subgroup analysis of the follow-up time. The results showed that valgus knee bracing enable to improve the VAS pain score of KOA patients if the follow-up period was less than 24 weeks, (P = 0.03). In contrast, a negative results were obtained if the follow-up time was between 24 and 48 weeks (P = 0.17), indicating that valgus knee bracing could not improve the pain symptoms of KOA patients in a long term. When the follow-up time was greater than 48 weeks, the result was P = 0.0001. But VAS pain score of the experimental group was found to be greater than that of the control group, and it is still shown that valgus knee bracing cannot improve or even increase the pain symptoms of KOA patients.Our findings demonstrated that valgus knee bracing did not improve the VAS pain score  due to the small amounts of RCTs enrolled, publication bias is unavoidable in our project, and (2) the valgus knee braces used in the testing group of RCTs are created in different shapes and construction by different manufacturers. The intervention time of valgus knee bracing in the control group are usually not the same. These above factors lead to clinical heterogeneity; (3) it is impossible to enroll all relevant studies that have been reported whereas our search filed covered PubMed, Embase, and Web of Science databases; (4) the RCTs included in this study did not provide complete outcome indicator data, which led us to use statistical methods to determine the outcome indicators data based on the information provided. But after we excluded this study, the results were not affected.Taken together, our current evidence shows that valgus knee bracing can only improve knee joint activities and relieve pain feelings of KOA patients in a short-term therapy, but showing no contribution to the long-term outcomes. More high-quality clinical RCTs with longer follow-up time are needed to further verify our conclusions.


Streptococcus agalactiae strain isolated from a dairy cow with mastitis. In addition, TS can inhibit the biofilm formation ability of this strain by down-regulating the transcript levels of biofilm-associated genes including srtA, fbsC, neuA, and cpsE. Hence, TS might be a potential alternative herbal cure for bovine mastitis.Tea saponin (TS), an inexpensive and easily-available plant extract, exhibited antibacterial activity against a Streptococcus agalactiae (GBS) is a highly contagious pathogen which not only can cause neonatal meningitis, pneumonia, and septicemia but is also considered to be a major cause of bovine mastitis (BM), leading to large economic losses to the dairy industry worldwide. Like many other pathogenic bacteria, GBS also has the capacity to form a biofilm structure in the host to cause persistent infection. Tea saponin (TS), is one of the main active agents extracted from tea ash powder, and it has good antioxidant and antibacterial activities. In this study, we confirmed that TS has a slight antibacterial activity against a Streptococcus agalactiae strain isolated from dairy cow with mastitis and inhibits its biofilm formation. By performing scanning electron microscopy (SEM) experiments, we observed that with addition of TS, the biofilm formed by this GBS strain exhibited looser structure and lower density. In addition, the results of real-time reverse transcription polymerase chain reaction (RT-PCR) experiments showed that TS inhibited biofilm formation by down-regulating the transcription of the biofilm-associated genes including srtA, fbsC, neuA, and cpsE. Staphylococcus aureus, Streptococcus agalactiae, commonly known as Group B Streptococcus (GBS), is a major causative agent of bovine mastitis [Bovine mastitis (BM) is the most significant disease of dairy cattle and it can cause substantial decrease in the milk yield and quality, as well as an increase in death rates of cows, bringing about major economic losses in the dairy industry worldwide ,2,3. In mastitis . GBS canmastitis . Transmimastitis .Formation of a biofilm is a common strategy for pathogenic bacteria to adapt to the host environments. Biofilm is defined as a complex structure formed by microbial cells that adhere to media surfaces and are surrounded by a self-produced extracellular polymeric matrix. Under the protection of the extracellular matrix, the microbial cells become resistant to host defenses and tolerant to antibiotic treatment, and usually cause chronic infections which were difficult to treat ,8,9. GBSS. aureus and coagulase-negative staphylococcus (CNS) strains have been found to possess penicillin resistance. [Streptococcus uberis and Streptococcus dysgalactiae, which were considered to be the important mastitis causative agents [Due to excessive and inappropriate use of antibiotics, drug-resistant bacteria have spread throughout the world. The antimicrobial resistance of mastitis pathogens has drawn much attention around the world ,13. For istance. . Studiesistance. . In addie agents . Generale agents ,18.S. agalactiae and is associated with bacterial biofilm formation has not been reported. In this study, we explored the antibacterial effect of TS on a GBS strain isolated from bovine mastitis and performed biofilm assays to determine whether TS can affect the biofilm-formation capacity of this strain.In recent years, some plant-derived bioactive compounds, which have definite biological functions, have been considered as alternatives to conventional antibiotics . SaponinS. agalactiae strain GBS2 (hereafter referred as GBS2) was isolated from milk samples in cows with mastitis and was identified by 16S rDNA sequencing. The cells of GBS2 were cultured in tryptic soy broth  medium at 38 °C. The GBS2 serotype was confirmed to be type III, and it was shown to have susceptibilities to norfloxacin, oxacillin, doxycycline, ampicillin, ciprofloxacin, penicillin G, amoxicillin, and ofloxacin and resistance to erythromycin, clindamycin, chloramphenicol, and gentamicin. In addition, this strain possesses several known virulence genes including fbsA, spb1, hylB, cylE, and cspA.In this work, the Growth curves of strain GBS2 were measured as follows: The overnight cultures were diluted to an OD600 of approximately 0.03 in 100 mL of fresh TSB medium without or with different concentrations of TS , which was extracted from tea seeds and purified by HPLC. Subsequently, the cultures were incubated at 38 °C for about 24 h with shaking. The OD600 value of each sample was then measured at 2 h intervals by using a UV/Vis spectrophotometer .S. agalactiae strain GBS2 were transferred into 3 mL of TSB medium and incubated with shaking (180 rpm) for about 16 h at 38 °C. The cultures were transferred into fresh TSB medium and diluted to an optical density of 0.03 (OD600 = 0.03), and then the dilutions were dispensed into the 96-well plates  with addition of TS at final concentrations ranging from 0.002 to 2 mg/mL (diluted with sterile water). The bacteria were cultured at 38 °C for about 10–12 h, and then the cultures were 10-fold serial diluted with TSB medium, spread onto the TSB plates, and cultivated at 38 °C for about 16–24 h. After cultivation, viable colony-forming units (CFUs) on every plate were counted, respectively, and the results were compared between the test groups and the control groups. All experiments were repeated at least three times with four parallels.Colonies of The experimental method of biofilm assays was according to a previous study  and modiThe biofilm structures of the GBS2 were investigated by SEM XL20 scanning electron microscopy . For biofilm formation, the overnight GBS2 cultures were diluted (1:50 ratio) into the fresh TSB broth . Sterile coverslips (18 × 18 mm) were placed into the bottom of the wells of the 12-well plates, then the dilutions were transferred onto the coverslips, and the coverslips served as the bacterial attaching surfaces. The cells in the 12-well plates were cultured for about 40–48 h at 38 °C, and then the coverslips were picked out and washed at least two times with PBS buffer. For SEM observation, the samples were prepared according to a previous study , BiofilmThe dilution (1:100 ratio) of the GBS2 cells were transferred into the fresh TSB broth (with or without addition of 2 mg/mL TS), and when the cultures grew to the late exponential phase, bacteria cells were enriched by centrifugation and incubated with Tris-EDTA (TE) buffer (pH 8.0) and 10 g/L lysozyme for 30 min at 37 °C, and then total RNA was extracted by using the Trizol method . DNaseI  was used to remove the residual DNA. The PrimeScript 1st Strand cDNA synthesis kit and the SYBR Premix ExTaq  was used for RT-qPCR assays, and the RT-qPCR assays were performed by using the StepOne Plus real-time PCR system . The 16S cDNA abundance was used to normalize to the quantity of the target genes. All experiments were repeated at least three times with four parallels. The primers used for RT-qPCR assays in this work are listed in t-test method was used for statistical comparisons between groups.The Statistical Product and Service Solutions (SPSS) software  and a one-way ANOVA method were used to analyze the raw data, and the paired To determine the antibacterial effect of TS on strain GBS2, the growth curves of the cells were measured in the presence of different concentrations of TS. As shown in As shown in Additionally, scanning electron microscopy (SEM) experiments were performed to explore the effect of TS on biofilm integrity. As shown in srtA, fbsC, csrR, neuA, cpsE, and luxS) have been reported to be involved with the biofilm formation in GBS [csrR and luxS were not significant changed, but the transcript levels of srtA, fbsC, neuA, and cpsE were decreased upon the addition of TS. According to these results, we suggested that TS might inhibit GBS2 biofilm by down-regulating biofilm-associated genes including srtA, fbsC, neuA, and cpsE.To investigate the potential mechanism of how TS affects biofilm formation ability of GBS2, RT-qPCR experiments were performed, and the transcript levels of genes associated with biofilm formation in GBS were measured. According to previous studies, several genes (including n in GBS ,18,25. TS. agalactiae was first identified in 1887 as a pathogen causing the mastitis. Although today, GBS has been well recognized as a leading cause of the meningitis in neonates and can cause severe invasive disease in the elderly and in immuno-compromised adults, it is still considered as a major causative agent of bovine mastitis in addition to S. aureus [Bovine mastitis has been considered to be a great challenge to the dairy industry around the world because it usually results in the reduction of yield and milk quality, and is also associated with the deaths and high treatment costs . S. agal. aureus ,26. Oste. aureus . In thisS. aureus is usually proved to be capable of forming biofilms [Among the common causative agents of bovine mastitis, biofilms . Althougbiofilms . In the TS is a natural components of plants which has a series of practical uses. It has been reported that TS has antibacterial effects, and has modified rumen fermentation by reducing the number of rumen protozoa and reducing the loss of intestinal methane ,31. SuppStreptococcus agalactiae isolated from dairy cow with mastitis. Results showed that TS had a slight antibacterial activity against GBS2 and inhibited its biofilm formation. We observed that the biofilm of the test group in the presence of TS had a looser structure and lower density compared with the control group without TS. Besides, the results of RT-qPCR indicated that TS inhibited biofilm formation by down-regulating the transcription of the biofilm-associated genes srtA, fbsC, neuA, and cpsE.This work explored the effect of tea saponin on the growth and biofilm formation in


Twelve new azole compounds were synthesized through an ene reaction involving methylidene heterocycles and phenylmaleimide, producing four oxazoles, five thiazoles, and one pyridine derivative, and ethyl glyoxylate for an oxazole and a thiazole compound. The twelve azoles have a stereogenic center in their structure. Hence, a method to separate the enantiomeric pairs, must be provided if any further study of chemical and pharmacological importance of these compounds is to be accomplished. Six chiral stationary phases were assayed: four were based on macrocyclic glycopeptide selectors and two on linear carbohydrates, i.e., derivatized maltodextrin and amylose. The enantiomers of the entire set of new chiral azole compounds were separated using three different mobile phase elution modes: normal phase, polar organic, and reversed phase. The most effective chiral stationary phase was the MaltoShell column, which was able to separate ten of the twelve compounds in one elution mode or another. Structural similarities in the newly synthesized oxazoles provided some insights into possible chiral recognition mechanisms. Heterocyclic fragments or moieties are present in the majority of marketed active pharmaceutical ingredients (APIs) . The reaN-containing heterocycles with a six-membered ring with a single N atom, five-membered rings with both O and N heteroatoms, and S and N heteroatoms, respectively [N-containing heterocyclic compounds reacting N-phenylmaleimide or ethyl-glyoxylate with methylidene oxazolines or thiazolines. Upon the addition reaction, carbon 3 of the substituted N-phenylsuccinimide, or the secondary alcohol obtained with ethyl-glyoxylate, became stereogenic centers. In the present iteration, the reaction produced racemic mixtures of the N-containing heterocycles. The long-term goal of this investigation is to develop asymmetric variants.Pyridines, oxazoles, and thiazoles are aromatic ectively ,4. The cectively . Recent ectively ,7. A subectively  has beenectively . FollowiToday, it is difficult to submit a new racemic API to regulatory agencies due to possible differences in the biological effects of the two enantiomers . In drugHeptane, hexane, methanol, ethanol, and acetonitrile were the HPLC grade solvents obtained from Fisher Scientific  and used as received. Acetic acid, triethylamine, and ammonium formate were purchased from Sigma-Aldrich .N-phenylmaleimide or ethyl glyoxylate delivered the corresponding ene adducts I-V in modest to good yields [N-phenylmaleimide and ethyl glyoxylate, which afforded the corresponding adducts in generally good yields.As outlined in d yields . The thid yields ,7 and thThe liquid chromatography system used was the 1220 Infinity II set from Agilent (Santa Clara. CA. USA) including a quaternary pump, mobile phase degasser, 96 vial sample injector, column thermostat, and diode array UV detector. A personal computer drove the chromatographic system and handled data with the OpenLab CDS ChemStation software (Agilent). Acetonitrile solutions of all racemic samples were made at a concentration of 2 mg/mL. One microliter of each individual solution was injected for each analysis.N-phenylmaleimide or ethyl glyoxylate reacted with the corresponding oxazoline [A degassed solution of toluene containing xazoline  and thiaxazoline  methylidxazoline . Ten comSeparations of the azole enantiomers were investigated with six different chiral stationary phases (CSPs) as listed in k1 and k2, using the column dead time, o,t determined as the first UV detector variation after injection:The chromatographic parameters considered were the retention times of each enantiomer, allowing one to calculate the corresponding retention factors, k2/k1, and the resolution factor, Rs, is defined asiW is the peak width of peaks 1 and 2 at peak base, respectively. A resolution factor of 1.5 corresponds to the signal returning to baseline between separated peaks. A Rs higher than 1.5 corresponds to fully separated peaks with space between, and a Rs smaller than 1.5 corresponds to partially merged peaks. If Rs is equal or smaller than 0.4, the two peaks are not distinguishable and only a broadened single peak is observed.The enantioselectivity factor, α, is the ratio of the two enantiomer retention factors, Rs > 1.5), blue indicates a partial separation (0.4 < Rs < 1.5), and no color is for no visible separation (Rs < 0.4). The heat map or score card shows that the MaltoShell chiral stationary phase provides a baseline separation for 10 of the 12 azole enantiomers followed by the NicoShell, with 6 separated out of 12, and the VancoShell (with 5 of 12). In the normal phase mode alone, the MaltoShell column separated 9 azoles or 75% of the set of 12. It could separate seven pairs in the polar organic mode. The IA-3 column also could separate nine enantiomeric pairs but with a lower resolution, although the PO and RP mode were not assayed with this column.The TeicoShell column could separate in one mobile phase mode or another, at least partially, 11 of the 12 compounds tested. Similarly, the VancoShell and the NicoShell columns could separate, respectively, 9 and 7 compounds in the different mobile phase modes . The Tag3 hybridized carbon atom, which is referred to as the stereogenic center  by one column or another (Thiazoles VII to XI also have similar structures, but very different results were obtained under similar or even identical chromatographic conditions. No definitive conclusions on chiral recognition of thiazoles can be obtained with these results, but all six chiral thiazoles could have their enantiomers fully separated ( another .Twelve newly synthesized racemic azole compounds have not been resolved previously by any means, until now. Additionally, such compounds are known to be pharmacologically active. Such enantiomeric separations are most expeditiously accomplished by direct chiral separation techniques. Using six chiral stationary phases with different selectors, it was possible to fully separate the enantiomers of the entire set of azole compounds. Some insights into the azole chiral recognition mechanism could be obtained by comparing the separation results obtained under identical chromatographic conditions with compounds having similar structures. The oxazole recognition ability by the MaltoShell chiral selector was very sensitive to the presence of an electron-withdrawing group on its phenyl substituent, even though this group was far from the stereogenic center. Indeed, both retention and resolution factors of the enantiomeric pairs of the rigid chiral oxazoles were affected. Such an observation was not possible in the limited case of the chiral thiazoles examined.