Starch, α-polyglucan consisting of a large number of anhydroglucose units joined by α-1,4- and α-1,6-glycosidic bonds, seems to be characterized by a simple structure when compared to other natural polymers. Nevertheless, starches of various botanical origins have different physicochemical properties that are related to the differences in molecular and supramolecular structure of this polymer. In terms of the functional value of starch, the behavior of its macromolecules in solution is the most important result of its structural features. Extremely high molecular mass is the fundamental structural property of starch. Water, considered simply as a solvent for solubilization, does not provide molecular dispersion of starch without its degradation. The objectives of this study are to characterize the suitability of a new aqueous media (urea/NaOH) for enhancing the dispersion of native corn and potato starches and its effect on the consequent size-exclusion chromatography (SEC) analysis. The results were referred to other aqueous base solvents used for dispersing starch (NaOH and KOH). The samples were separated using SEC with triple detection and phosphate buffer (pH 8.0) with urea as the eluent. The characteristics of tested normal and waxy starches were compared. The results revealed that urea/NaOH did not degrade starch during the dispersion process. The recovery of starches, however, was not higher than 42%. These results prove that while the urea/NaOH solvent allows to obtain cold-water-soluble starch, the degree of disintegration of the intramolecular interactions of amylopectin chains is still insufficient. Starch is one of the most useful polymers in the food and non-food applications . It is a biopolymer that is biodegradable and renewable . It is a6 Da) is much lower than amylopectin which is known as one of the largest biopolymers (approximately 108 Da) [Generally, starch granules consist of amylose (AM) and amylopectin (AP) as well as minor components–proteins, lipids, and minerals . AP has  108 Da) . General 108 Da) .Starch is classified into four crystalline types  according to X-ray diffraction (XRD). The A-type crystalline pattern is typical for most cereal starches, such as corn, wheat, and rice starch. Tuber starches (potato starch) with their high amylose starch content belong to B-type crystalline type. The combination of A- and B-type crystalline pattern is typical for legume starches, classified as type C. A V-type crystalline pattern is observed when amylose complexes with fatty acids or alcohols . In geneProperties of starch are also determined by other compounds. Root or tuber starches contain little or no lipids while significant amounts of lipids are complexed with amylose in amylose-containing cereal starches. Cereal and root (tapioca) contain lower phosphate monoester content than tuber starches  . InteracIt is often required that the starch withstands high temperatures, low pH, and high shear forces . TherefoIt is a huge challenge that is driven by high demand to destroy the molecular hydrogen bonds in starch in order to extend the range of applications of and enhance the processability of this biopolymer ,24. HydrThere is no general agreement as to the best approach to the characterization of starch which is a huge and highly branched molecule. However, it is very important to determine not only the average molecular mass of starch but also its distribution. Samples with the same molecular mass but with very different distribution could manifest different rheological properties. The knowledge of the average molecular mass and radius of gyration of starch is important for its industrial application, for example: (i) Beverage thickeners—too high molecular mass makes the product too viscous with a chalky or slimy texture; (ii) resistant starch-control of digestive properties and retrogradation requires molecular mass in the correct range; (iii) enteral nutrition solutions–excessive molecular mass reduces energy supply and causes clogging of the tubing .The biggest problem in determining the molecular mass of starch is the necessity to fully disperse the sample (disperse as separate molecules) without its degradation . HoweverWhole starch molecules are usually separated using common techniques of size-exclusion chromatography (SEC), field-flow fractionation (FFF), analytical ultracentrifugation (AUC), and hydrodynamic chromatography (HDC) . SEC canwM) with static light scattering (SLS) is limited since light scattering intensity is dependent on the increment in the refractive index (dn/dc) [2O  [Unfortunately, dispersing starch in DMSO is also not straightforward, mainly because the required conditions  are not universal to all starches. Namely, amylose-rich corn starch is easier to disperse in DMSO than waxy starch. Excessive mechanical stirring, high temperature, excessive boiling (2 h or longer in water bath at 100 °C), autoclaving  can lead to an increased size of the apparent amylose peak as a consequence of amylopectin degradation . However (dn/dc) . Compari74 mL/g)  with dn/Summing up, the dispersion of starch can be obtained by the formation of hydrogen bodings with a protonic solvent (water) or dipolar aprotic solvent (DMSO), or by hydration of the ionized hydroxyl groups of starch in presence of a base (NaOH) . HoweverThe aim of this study is to determine the suitability of urea/NaOH aqueous media for enhancing the dispersion of starch for the purpose of SEC analysis. Normal and waxy starches from corn and potato were tested as the most popular examples for A-and B-type crystalline pattern. The samples were neutralized immediately after the dispersion process using phosphoric acid, then diluted directly in the alkaline mobile phase (phosphate buffer (pH 8.0) with urea). The idea was to verify the possibility to characterize starch in an aqueous solvent which is its natural plasticizer and which enables to reflect the conditions encountered in food matrices. The obtained results were then compared with the effectiveness of dispersion in aqueous solutions of NaOH and KOH.2HPO4, NaH2PO4, NaOH, KOH, H3PO4, sucrose were obtained from Avantor Performance Materials Poland S.A. .The study was performed with commercial native potato starch, Superior Standard , waxy potato starch, Eliane , corn starch , waxy corn starch . Pullulan and amylose from potato standards were purchased from Shodex  and Sigma–Aldrich Chemie GmbH , respectively. Urea, NaAn Abbe refractometer was used to determine dn/dc for sucrose dissolved in 50 mM phosphate buffer (pH 8.0) with 100 mM urea at concentrations in the range from 0 to 0.3 g/mL. Measurements were performed at the temperature of 50 °C. Methodology was adapted form Behrens et al. . The dn/3PO4 and diluted with the mobile phase used for SEC separation. Results for corn starch were compared with other alkaline dispersion methods which employed 0.5 or 1 M NaOH [3PO4 and diluted with the mobile phase used for SEC. The final concentration of all samples was 1.2 mg/mL. The samples were clarified using 5 µm PTFE filters . The injection volume was 50 µL. The experiments were conducted in triplicate.Starch samples of 30 mg were allowed to disperse in 4.5 mL of urea (2.3 M)/NaOH (1 M) aqueous solution  at 0 °C 1 M NaOH  or 2 M K1 M NaOH ,47 at 65SEC equipment  with triple detection (Viscotek 305 TDA) was used for the separation of samples. A conventional dual cell refractometer (RI), viscometer (Vis), and light scattering  detectors were employed. Aqueous SEC analysis was performed using two aqueous SEC columns (Shodex OHpak SB-800HQ series) with a guard SB-G type column . Total of 50 mM phosphate buffer (pH 8.0) with 100 mM urea was used as the eluent at a flow rate of 0.4 mL/min. The detectors and column were in the same oven operated at 50 °C. Calibration was performed with a pullulan standard (11.3 kDa). The refractive index of the solvent was 1.334. The calculations were performed using OmiSEC 4.7 software .area) is described by the following equation: RIarea = k′ × (dn/dc) × minj [k/n0; the refractometer response constant, k, the refractive index of the solvent, n0), minj is the total injected mass calculated from the injected concentration and the injection volume (minj = cinj × Vinj). The wM was calculated from the Rayleigh equation limited for low scattering angles (Kc/∆θ =R (1/Mw+ 2A2c)1/θP,) where: c is the concentration of the polymer; ∆θR the Rayleigh ratio , A2 the second viral coefficient (in the limit of zero angle and near-infinite dilution this coefficient can be neglected); K the optical constant K = (4π2n02/NAλ4)(dn/dc)2; n0 the solvent refractive index; λ the laser wavelength; and NA is Avogadro’s number), θP is a form factor related to the size of the molecule and the angle at which the scattering is determined, at θ = 0°, θP = 1 [gR because θP can be determined for samples that scatter anisotropically. This in turn can be used to estimate a value of gR assuming a structural model such as a random coil or a hard sphere [gR is the distance from the center of mass of a body at which the whole mass could be concentrated without changing its moment of rotational inertia about an axis through the center of mass [ηR) was calculated as the radius of a homogeneous sphere according to the formula η =R ((3IVwM)/(10πNA))1/3, where: IV is the intrinsic viscosity, wM the molecular mass, NA Avogadro’s number [RI signal (RI) × minj , where: , Pθ = 1 ,49,50. Ld sphere . Rg is t of mass . The vis0 was 1.334. The calibration of the RI detector was done in the next step; the refractometer response constant (k) was 1.05689 × 103.Calibration of the RI detector should be performed using the same solvent that is applied during SEC. Therefore, a reference sample with a known dn/dc in this solvent is required. Unfortunately, reference values for dn/dc cannot always be found in the literature, and an Abbe refractometer is usually not sensitive enough to determine dn/dc precisely in a low concentration range. It seems justified to use sucrose for the calibration of the RI detector since it dissolves in a wide range of concentrations in aqueous eluents and gives the opportunity to determine dn/dc precisely with an Abbe refractometer. The dn/dc determined for sucrose dissolved in the tested solvent is presented in nM is less than 103 Da), the chemical structure of the polymer. Chain branching, however, has no effect on dn/dc [k/n0)/Vinj. The calculated dn/dc values for pullulan and amylose from potato were 0.129 and 0.124 (mL/g), respectively.Since different polymers have unique RI responses it is necessary to precisely determine dn/dc for the tested types of polymers. The dn/dc parameter depends on the solvent, the temperature, the wavelength of light, the average molar mass  for other detectors  were calculated with the pullulan standard (11.3 kDa). The recovery and accuracy of molecular mass determination were calculated for standards of pullulan (6.15–2460 kDa). The results  showed tFollowing the dispersion in NaOH/urea at 0 °C, the starch samples were diluted and separated using SEC. Signals recorded with RI, Vis, and LALS detectors are presented in Light scattering (LS) signals b were prη), intrinsic viscosity, parameters of Mark–Houwink (M–H) parameters, and M–H plot. Low polymer concentration or low polymer recovery, wide range of sample elution, incompatibility of solvent are the possible reasons for the weak signal of the Vis detector. Comparison of the signals for pullulan P2500 (elution range 15–20 mL) and potato starch with the same load on the column showed that the RI signal for pullulan  was about 2.8 times lower than the signal for potato starch . It proves that the main reasons for the weak signals from the Vis detector for the starch samples were mainly low recovery and a broad elution range /NaOH (1 M) aqueous solution at 0 °C with other methods where base conditions are applied, i.e., using 0.5/1.0 M NaOH or 2 M KOH at 65 °C. Corn starch was used for this purpose. RI chromatograms of corn starch are presented in wM) was typical for this fraction as determined previously [gR) were calculated for potato and waxy potato starches. Nonetheless, the recovery of waxy potato starch (21%) was significantly lower than the recovery of potato starch (42%) since the latter contains amylose which disperses fully in urea/NaOH solvent. Analogical difference was noted for corn and waxy corn starches. These observations provide evidence that the aqueous urea/NaOH solvent does not allow to properly disperse amylopectin. The highest recovery (58%) was obtained with 2.0 M KOH and corn starch in which case also the molar mass was determined to be higher compared to the respective sample dispersed in urea/NaOH but lower compared to corn starch dispersed in 1 M NaOH. Nonetheless, it is not possible to conclude that urea/NaOH solvent degrades amylopectin of corn starch more than NaOH or KOH. With such small recovery values it is possible that each of the applied solvents disperses a slightly different fraction of the sample.Parameters calculated for the tested starches dispersed in urea/NaOH, 0.5/1.0 M NaOH, or 2 M KOH are presented in eviously . The higThe nutritional and industrial importance of starch is unquestionable but its application requires knowledge of its structure and molecular size distribution. SEC is the most common technique used to characterize this distribution . Howeverg values were consistent throughout repeated injections performed over 24 h. Nevertheless, the weak signals recorded by the viscometric detector did enable to calculate the viscosimetric radius and intrinsic viscosity or apply a universal calibration validated for a number of branched polymers, including starch [7 Da [7 Da). Despite the fact that the solubility of corn starch in urea (2.3 M)/NaOH (1 M) was found to be 99% up to a concentration of 1% and pose no serious degradation problems [3) [v/v) was used to obtain starch dispersion prior to aqueous SEC the recovery was also not improved (41% recovery for corn starch) [The proposed SEC conditions yielded repeatable SEC separations. The determined molar masses and Rg starch . It was ch [7 Da . A similproblems , it was problems . Autoclalems [3) . When DM starch) . Dispers starch) .It was also reported that 20–30% of large amylopectin molecules may not pass the SEC column and the injection membrane filter (3.0 µm) . The samThe main problem that affects the analysis of the structure of starch using SEC is the poor solubility of this biopolymer in aqueous solvents. For this reason, DMSO is usually used as a solvent but its physical properties limit the sensitivity of RI and LS detectors. Moreover, utilization of this organic solvent does not allow to reflect the conditions in which starch is dispersed in the water phase of food. This justifies the investigation into the possibility of applying new aqueous solvents for SEC analysis of starches intended for food applications. The conducted experiments support previous conclusions that urea and NaOH are good plasticization and dissolving factors, respectively. However, the analysis of SEC data proves that urea/NaOH does not show as efficient dispersion of amylopectin as DMSO. At the same time the application of this aqueous solvent does not lead to the degradation of the sample which was also reported when aqueous solutions of NaOH were used.


P ≤ 0.05) reduced the lipid, protein, and amylose contents of the QPM. While the native starch had 0.72 g/cm3 bulk density, modified starches were between 0.59 and 0.88 g/cm3; chemical modification reduced the bulk density and physical modification increased it. In addition, all the modifications except oxidation significantly (P ≤ 0.05) increased water absorption capacity. The oil absorption of the starch samples was increased by modification techniques used with the exception of physical modification. Chemical modification reduced the viscosity of QPM starch while physical modification increased it. The reducing sugar content of the starch was reduced by both the physical and chemical starch modification techniques. Acetylated sample exhibited the highest swelling power while acid‐thinned sample had the least. The major functional groups identified via FTIR were OH, C‐H, C=H, and C≡H. Modifications did not affect the functional groups as all the native and modified starches (except oxidized sample) all have similar spectrum and bands stretch.Quality protein maize (QPM) is a biofortified maize rich in lysine and tryptophan, essential amino acids required in human nutrition. This research therefore characterizes native and modified starches from QPM by evaluating the physicochemical properties, Fourier transform infrared spectra (FTIR), and pasting properties. The native QPM starch was modified by oxidation, acetylation, pregelatinization, and acid thinning techniques. The starch yield of native QPM was 43.80%, while that of modified starches were from 88.22 to 98.34%. The moisture content of the native and modified starches was from 4.56 to 9.20 g/100g. Modifications significantly (The study contributes to existing knowledge on valorization of modified starch from quality protein maize. Profiling the chemical attributes of modified starches is especially valuable in novel food processing techniques. It also serves as an energy reservoir which is readily converted into useful chemicals products, hence, it has found numerous application in many industries  glycosidic linkages, while the amylopectin fraction of the starch is of a highly branched polymer which has one of the highest molecular weights recognized among naturally occurring polymers , Ibadan Oyo State, Nigeria. All chemicals and reagents used were of analytical grade.2.2All starch samples were prepared according to AOAC  method a2.3g for 20 min. The white sediment was dried at 40 °C in a convection oven  for 24 hr, ground with an attrition mill, and hurled past a 100‐mesh screen and stored in sealed polyethylene bag at room temperature (25 to 30 °C) prior to further analyses , washed and centrifuged  at 3,000 × 2.4The method of Lawal and Adebowale  with sli2.5The method adopted by Awolu and Olofinlae  was empl2.6The method of Lawal and Adebowale  was empl2.7The method of Yousif, Gadalla, and Sorour  was empl2.8The starch yield was evaluated as described by Awolu and Olofinlae . It is t2.9The proximate composition, including moisture, ash content, crude protein, crude fat, and crude fiber of the starches, were determined using AOAC  methods.2.10The methods of Lawal and Adebowale  were emp2.11The method of Williams, Wu, Tsai, and Bates  was used2.12The pH meter was calibrated with KOH buffer solutions of pH 7.0 and 4.0 before the measurements. The modified and native starch samples (5 g) were weighed in triplicate into a beaker, mixed with 20 mL of distilled water. The resulting suspension was stirred for 5 min and left to settle for 10 min. The pH of the water segment was determined using the calibrated pH meter (pH–107 Sinotester)  equipped with a 700 µg sensitivity cartridge was used for this study. The temperature of the slurry was raised from 30 to 95 °C, and kept at this temperature for 30 min, before cooling down to 50 °C. A constant rotational velocity of 75 rpm was maintained and the heating or cooling rate was 1.5 °C/min throughout the process.2.16The starch suspension of 5% w/v was heated to 90 °C for 30 min in a temperature controlled digital electric water bath (Model DK–420) with continuous stirring. The paste was transferred to a rotatory viscometer  using the rotor No. 1. Paste viscosity was measured from 90 to 30 °C cooling paste.2.17The native and modified QPM starches were dried to constant weight and the samples were labelled and measured to equal weights. The starch samples were then treated with equal quantity of potassium bromide (KBr) salt, then each sample was pressed in KBr Salt Plates and transferred to the Fourier transform infrared spectrometer . The instrument was on and kept to optimized and self‐set for about 15 min before usage. The scan button was pressed to start the process and various spectra for each functional group was displayed on the screen and a recording device notes the results.2.18L*, a*, and b* coordinates of the CIE scale.The color of the starches was measured using a Color Meter PCE–CSM 2 (Deutschland GmbH) connected to a CQCS3 software. The spectrophotometer was calibrated against a white plate before the reading was taken. The parameters recorded were 2.19α = 0.05.The results obtained was analyzed statistically using SPSS . The means and standard deviation of the samples analyzed were computed while comparison was performed using one‐way analysis of variance (ANOVA). Conversely, the statistical significant difference of all the samples analyzed were performed at 33.1The starch yields of the modified QPM starch are presented in Table For the modified starches, the yields were 98.34% for pregelatinized sample, while acetylated, oxidized, and acid thinned starches were 94.39, 90.06, and 88.22% respectively. Awolu and Olofinlae  reportedThe chemical composition and amylose content of native and modified quality protein maize starch are presented in Table P > 0.05) in the ash contents of the native and pregelatinized QPM starch.The moisture content value of 6.95 to 13.24% for native and modified potato starches was reported by Nadir et al.  while a Several authors have studied starches from different botanical sources and all obtained varying levels of ash contents  reduced the level of amylose content of acetylated, acid‐thinned, and oxidized starches. Cisse et al.  of acetylated quality protein starch were 0.022 and 0.07, respectively. It was reported by Lawal and Adebowale  that theThe DS should be less than 0.025 to be applicable for industrial use  was observed among the starch samples in terms of their bulk density. The coarseness of the starch samples was reduced during chemical modification while the physical method of modification employed for this study enhanced it, as reported by Awolu and Olofinlae .The viscosity of the starch samples ranged from 2.00 to 2.75 cP. Chemical modification reduced the viscosity of the starch samples except in pregelatinized starch which had an increase (2.99 cP). The reduction in viscosity may be due to the method of modification adopted for the process. Modified starches are used widely as thickeners and stabilizers in products such as gravies, sauces, soups, sausage fillings, milk‐based foods, and puddings.The pH of any starch is a significant asset when it comes to industrial applications, because pH is being used generally to indicate the acidic or alkaline properties of liquid media , closely followed by native starch. According to Rafiq et al. , modific3.4The swelling power for the native and modified starches at temperatures ranging from 50 to 90 °C is shown in Figure The solubility of native and modified QPM starch is shown in Figure 3.5Charts showing the effect of pH (2 to 12) on swelling capacity and solubility of native and modified QPM are shown in Figures 3.6The results for least gelation concentration for the native and modified QPM starch are presented in Table 3.7L* values of 85.52, a* value of 3.07, and b* values of 14.99 were observed. The values for pregelatinized quality protein starch was significantly (P ˂ 0.05) different from the native and chemically modified starches. The low L* value could be a result of the high temperature of pregelatinization of the sample.Summary of the color parameters of the native and modified starches are presented in Table L* value at 96.29 but it was not significantly (P > 0.05) different from the results obtained for acetylated, acid‐thinned, and native QPM starches. The high value of oxidized modified starch could be due to the interactions between the bleaching agent (hypochlorite) and starch granules. Oxidized QPM starch would be better suited and it can then be suggested that the pregelatinized QPM starch would be better for products requiring very clear starch raw material.Oxidized QPM starch had the highest 3.8The pasting properties of the native and modified QPM starches are presented in Table This increment may be as a result of the modification tending to increase the region of crystallinity owing to the reorientation of the starch granules as a strong intragranular bond permits the starch to require more heat before structural disintegration which will in turn lead to the formation of paste. Final viscosity is the ability of starch material to form a viscous paste or gel after cooking or cooling. It is a measure of a particular sample's quality  is sometimes referred to as shear thinning, holding strength or hot paste viscosity, is a period when the starch is exposed to a period of continuous heating and application of mechanical shear stress  into their respective FTIR spectrum. The major functional groups as obtained from FTIR spectrum in pregelatinized QPM starch are presented in Table The summary of FTIR spectrum for the oxidized QPM starch are presented in Table The functional groups identified in acid‐thinned sample were hydroxyl, alkane, alkyne, and alkene Table  as also The functional groups in the acetylated QPM starch Table  were alcThere were changes in wavenumbers of the native and modified samples which caused shifts in bands but did not in the total removal or alteration of the functional groups. The changes only indicated that the bands in the modified starch samples were marginally excited  reduction in the amylose contents of the modified starches with the exception of pregelatinized starch. Also, there were significant (P ≤ 0.05) reduction in the viscosities of modified starches except in pregelatinized starch. However, acetylated starch had highest water and oil absorption capacities. While oxidized starch had the best peak, trough, and final viscosities, acid‐thinned starch had the best breakdown and setback viscosities. The results of the FTIR indicated that the major functional groups identified in the starches includes alkanes, alkenes, alkynes, and hydroxyl. Modifications, therefore, improve the physicochemical characteristics of the QPM starch.Modification affected the properties examined in this study, though, individual modification technique performed best in different properties studied. While modifications increased QPM starch yield by over 100%, there were significant (Awolu Olugbenga conceived the research, carry out the experimental design, and supervised the research and manuscript preparation; Joshua Wisdom Odoro collected data and drafted the manuscript; Jumoke Bukola Adeloye proof read the manuscripts; while Oluranti Mopelola Lawal tested the data, and corrected the manuscript.The authors declare there are no conflicts of interest.


Acetylated starch possessed higher solubility and swelling power than native starch under the temperature regimes considered. Addition of acetylated pigeon pea starch as a stabilizer in yoghurt had positive influence on the water holding capacity (7.7% to 10.4% compared to 13.3% in yoghurt stabilized with native pigeon pea starch) and whey syneresis  at the end of a 28-day cold storage period. In addition, pea starch-stabilized yoghurt possessed an enhanced sensory attribute (firmness), and compared favourably with gelatin-stabilized yoghurt in terms of overall acceptability. Thus, acetylated pigeon pea starch exhibited improved physicochemical properties and showed usefulness as a stabilizer in yoghurt because it enhanced the physicochemical, storability, and sensorial quality, while improving the body and texture of the product.The behaviour of graded acetylated pigeon pea starch during heat processing was evaluated in addition to the corresponding effect of their incorporation at 1.5% ( Yoghurt is a product of the lactic acid fermentation of milk and it is among the most common fermented dairy product widely consumed in many parts of the world. Syneresis (expulsion of whey) is a common technological defect in cultured semi-solid products, especially during refrigerated storage . ControlNative starches have some limitations preventing their use for industrial applications; these limitations can be overcome by techniques developed to modify, and thus, improve the physicochemical properties of starch. Modified food starches, specifically from cassava and corn, have been used as stabilizers in food systems to confer specific functions, such as increased viscosity, improved mouthfeel and creaminess, and reduced syneresis of yoghurt ,9. SeverCajanus cajan) is a legume that forms a good portion of the human diet in many African, Asian, and South American countries [Pigeon pea , PPAS2 (acetylated pigeon pea starch DS 0.09), and PPAS3 (acetylated pigeon pea starch DS 0.14) were obtained and used in this study. Starter culture DANISCO Yo Mix 495 LYO (containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) was obtained from the Dairy unit, University of Manitoba, Winnipeg, Manitoba, Canada. Commercial homogenized and pasteurized milk  was purchased from Maizube farms, Minna, Niger State, Nigeria; skimmed milk powder (SMP) and sucrose were purchased from Ceci Supermarkets, Alagbaka, Akure, Ondo State, Nigeria. All reagents were of analytical grade and procured from Fisher Scientific  and Sigma Chemicals .Native and acetylated pigeon pea starches obtained from a previous study  in our lw/w) of starch were prepared in a flask, heated to 50, 60, 70, 80, and 90 °C for 30 min, with shaking every 5 min, and left for cooling to room temperature, thereafter, being centrifuged for 15 min at 3000× g. The supernatant was decanted, and the residual volume was determined. The solid part was dried in an oven for 2 h at 130 °C.The solubility and swelling power of starch was determined, following the method of Gani et al. . Suspensw/v db) was heated in a water bath at 90 °C for 30 min with constant agitation to ensure complete pasting. The starch paste was cooled to room temperature (around 25 °C) and the light transmittance of pastes was measured at 650 nm. The samples were stored for 5 days at 4 °C in a refrigerator and percentage transmittance was determined every 24 h using a UV/visible spectrophotometer .Paste clarity/light transmittance of starch paste was determined, as described by Craig et al. . An aqueThe pasting property was determined using a Rapid Visco Analyser (RVA) , as described in AACC 44-31A . About 2w/v) skimmed milk powder (SMP); 2% (w/v) sucrose was added as sweetener, followed by addition of 1.5% (w/v) acetylated or native pigeon pea starch as the stabilizer, and then, mixed thoroughly. The mixture was pasteurized at 85 °C for 30 min, cooled to 40–43 °C, and starter culture  per 100 L) was added, then, distributed into sterile 250 mL containers to allow easy collection of sample for analysis. Fermentation was carried out at 40 °C for 6 h to develop proper acidity. Fermentation was terminated and the yoghurt samples were stored at 4 °C for 24 h prior to chemical analysis, sensory evaluation, and storage stability test. The prepared yoghurt was stored in the refrigerator (4 °C) for 28 days and samples (three independent containers of 250 mL) were analysed for each of the parameters evaluated at 7-day intervals throughout the refrigerated storage period. A commercial yoghurt sample was used as a control, while laboratory control was prepared with the above stated materials  and 0.2% gelatin as the stabilizer.The yoghurt was prepared in triplicate, as described by Cui et al. . Two litpH of the yoghurt was measured at 23 °C using a pH meter  after calibrating with fresh pH 4.0, 7.0, and 10.0 standard buffers.TA expressed as percent of lactic acid was determined using titrimetric method, as described by Schmidt et al. . YoghurtYoghurt samples were mixed thoroughly using a glass rod and about 1 g of sample was oven-dried for 2 h; the percentage Total Solids (% TS) was calculated using the equation below:g and 4 °C. The amount of supernatant whey was recorded. The percentage syneresis was calculated using the equation below:Syneresis of yoghurt samples was measured according to the modified method of Guzman-Gonzalez et al. . Approxig at 4 °C for 10 min. The supernatant was collected and weighed, and WHC was calculated according to the following equation:The water holding capacity (WHC) of yoghurt is defined as its ability to hold all or part of its own water. WHC of the samples was determined using method reported by the authors in reference . YoghurtAssessment of the sensory attributes was carried out as described by Soukoulis et al. . Thirty Data were generated in triplicate and subjected to Analysis of Variance (ANOVA) using Statistical Package for Social Sciences (SPSS) V. 17.0 . The mean values were separated using Duncan’s Multiple Range Test (DMRT) at 95% confidence level.3) also had the highest starch solubility (5.7% at 50 °C to 34.6% at 90 °C). Similarly, acetylation increased the swelling power, which is a measure of the hydration capacity of the starch granules. The swelling power showed direct proportionality to temperature and degree of acetylation.The effect of temperature on solubility of native and acetylated pea starches was progressive A. The pe1, PPAS2, and PPAS3, respectively.The effect of acetylation and storage duration on paste clarity of pigeon pea starch is shown in p < 0.05) reduced PT (87.80 to 85.60 °C). Pasting time (Pt) ranged from 4.93 to 5.07 min. The acetylated starches required less time to form a paste when compared to the native starch. Peak viscosity (Pv) ranged from 314.70 to 588.70 RVU and values reduced with an increase in level of acetylation. Fv of starches followed similar trend as Pv and Tv with PPNS showing highest Fv and the value decreased with increase in DS of starch. Similar to other pasting parameters, native pea starch showed highest breakdown viscosity (BD) and setback (SB), which was significantly (p < 0.05) reduced by 24% to 49% and 38% to 44%, respectively, when the starch was acetylated.Pasting properties of pigeon pea starch as influenced by acetylation are summarized in p < 0.05) in percentage syneresis, with yoghurt stabilized with native pea starch (YoPN) exuding the highest amount of liquid from the yoghurt matrix, thus, having the highest degree of syneresis (46%). The degree of substitution showed an inversely proportional relationship with degree of syneresis; the yoghurts containing acetylated pea starch had lower syneresis (18.6% to 22%) compared to YoCN and YoPN. However, the market yoghurt (CTR) had a far lower level of syneresis at 10.8%.Acetylated pigeon pea starch was employed as the stabilizer in set-type yoghurt and the physicochemical properties presented in Water holding capacity (WHC) and whey syneresis of the yoghurt during the 28 days was evaluated at 7-day intervals throughout the refrigerated storage period A,B. Commp > 0.05) for colour (7.1 to 7.6), whereas firmness of the yoghurt was significantly different (p < 0.05) from one another (5.8 to 7.2).The sensory attributes of the laboratory control (gelatin-stabilized yoghurt) compared favourably with the market control , except for aroma, flavour, and overall acceptability . GeneralIncreased solubility in acetylated starches can be attributed to the increased hydrophilicity of the starch . SolubilThe swelling behaviour of starch is dependent on the amylose content, as well as the presence of amylopectin and non-carbohydrate substances, especially lipids, which may inhibit swelling. Swelling commenced at 60 °C earlier than observed in native starch and the highest swelling capacity was observed at 90 °C for all starches (2.5 to 4.0 g/g), which indicates that the penetrating power of water into starch granules can be increased at high temperature. The increase in swelling power may be a result of starch gelatinization, which causes disruption of the granular membrane and weakening of the internal starch structure that favoured increased water absorption. Acetylation conferred a 1.5 to 2.1-fold increase in swelling of starch paste, an effect that progressed with temperature. Introduction of the bulky acetyl group promotes structural reorganization, resulting in repulsion between starch molecules, which facilitates increased water percolation within amorphous regions of starch granules with a consequent increase in swelling capacity .The high solubility and swelling capacities of acetylated pigeon pea starch may also be due to the introduction of hydrophilic substituting groups that allows the retention of water molecules, owing to their ability to form hydrogen bonds. This ensures a high retention of the water that enters the granule, thereby increasing the swelling capacity. This is a useful property in the manufacture of some confectionery products. A similar trend showing increased swelling capacity has been reported for chickpea  and blac2 and PPAS3 exhibited higher light transmittance than PPNS and PPAS1. More so, PPNS and PPAS1 displayed significant decreases in light transmittance up until the fifth day, unlike PPAS2 and PPAS3 that exhibited lower rates of decrease in light transmittance during the storage period. Low light transmittance observed in native pigeon pea starch may be a consequence of turbidity development during storage caused by granule swelling and intermolecular bonding that enhanced retrogradation of amylose chains [PPASe chains . In addie chains . Higher e chains . Substite chains , where ae chains  also repp < 0.05) reduced PT (87.80 to 85.60 °C). The high pasting temperature observed in PPNS indicate native pigeon pea starch granules exhibited higher resistance to swelling and membrane rupture, while lower pasting temperature in acetylated starches may be as a result of the introduction of the acetyl group to the amorphous region, which made swelling of the acetylated starch easier than in the native starch. The decreased pasting temperature may be a result of weakening of starch structure due to interrupted hydrogen bonds upon introduction of the acetyl group. This property is important in systems where thickening of gels is required at low temperature in order to reduce energy cost during production [Pasting temperature (PT) is an important indicator of the minimum temperature required to achieve cooking in the starch sample and depicts the temperature at which viscosity begins to increase during the heating process. In this study, PT ranged from 86 to 90 °C. PPNS had the highest pasting temperature, however, acetylation significantly  is a measure of the cooking time  and thisPeak viscosity is the maximum viscosity developed during or soon after the heating portion of the pasting period and it correlates to the final product quality. Generally, high viscosity of starch is desirable for industrial uses, for which a high thickening potential at high temperature is required and a high peak viscosity suggests more starch has been gelatinized during processing. The peak viscosity of native pigeon pea starch was higher than the modified counterpart, suggesting that the starch can be incorporated into food that requires high thickening such as custard, while lower viscosity of the acetylated starches could facilitate better flow of starch paste when introduced into food systems. Similar to the present finding, Wani et al.  reportedFinal viscosity (Fv) is a parameter used to indicate the ability of starch paste to form a viscous paste resultant from retrogradation of soluble amylose during cooling and gelling of amylose . High Fvp < 0.05) reduced this property by 24% to 49%. High BD infers less stability of starch during heating. This property was lower in acetylated starches, which suggests that they may exhibit better starch granule swelling with slow tendency to lose viscosity and might be able to withstand more heating and shear stress [3, will be more stable under high temperature processing or storage conditions than PPNS. High BD values reflect granular swelling that make starch granules more susceptible to shear [Breakdown (BD) measures the tendency of swollen starch granules to rupture when held at higher temperatures and subjected to continuous shearing; this is also indicative of the stability of the starch upon heating as well as a measure of the susceptibility of cooked paste to disintegration. Highest breakdown viscosity (BD) was observed in PPNS; acetylation, however, significantly  lower in the acetylated starches (202.4 to 222.80 RVU) compared to the native pigeon pea starch counterpart. Ashogbon [Set back (SB) of the starches was significantly  between them. This suggests that incorporation of pea starch had no significant effect on pH.Acidity of yoghurt is a result of fermentation of milk sugar (lactose) into lactic acid by the starter culture during incubation. The development of lactic acid in fermented dairy products is essential for the proper formation of a yoghurt gel network. Acidity is inversely related to pH value, as increased lactic acid production results in a corresponding pH drop. Nevertheless, pH is a valuable tool for measuring the extent of lactic acid fermentation, since acidity influences the perception of other attributes  and playThe acidity of commercial yoghurt (0.90%) was significantly different from those of developed samples (1.03% to 1.21%), and this may be due to differences in starter culture, milk type, fermentation time, and temperature. Acidity observed for LCTR was within the range (0.98% to 1.10%) in another report for gelatin-stabilized camel milk yoghurt , which mSolids content was higher in yoghurt with stabilizer  than the YoCN, whereas graded acetylation of pigeon pea starch showed no effect on the solid content of yoghurt. The high total solids content supports the firm, custard-like body of set-type yoghurt; values are significantly higher than the 13% to 17% range earlier reported by Smit  for comm3 in terms of retrogradation tendency, breakdown, and set back. The improved properties of PPAS3 may have contributed to the improved ability to bind the free water as well entrap the yoghurt gel during the coagulation process. Significant whey syneresis (46.5%) observed in YoCN may be attributed to the absence of a stabilizer and, hence, the expulsion of liquid could not be efficiently prevented. The yoghurt stabilized with acetylated starches had lower syneresis values (19–22%), although was significantly higher than the gelatin-stabilized yoghurt and market sample. However, acetylated starch with higher DS (PPAS2 and PPAS3) resulted in reduced whey expulsion when compared to 36.8% in the sample stabilized with native starch (YoPN). Overall, starch-stabilized yoghurts showed 53–60% reductions in whey syneresis when compared to control yoghurt without pea starch (YoCN). Syneresis is an important factor in set-type yoghurt, as it can lead to the accumulation of whey on the surface of the yoghurt, which can result in poor consumer acceptance [Syneresis ranged from 10.8% to 46.5%, and the market sample (CTR) exhibited the lowest value; this property significantly differed among the yoghurt samples. The loss of ability of the yoghurt gel to entrap the serum phase due to the weak gel network results in whey separation, which is also referred to as syneresis . The perceptance ; the prep < 0.05) higher than yoghurt without pigeon pea starch that had 57% WHC at day 0. More so, the WHC of YoPA1, YoPA2, and YoPA3 was not significantly different (p > 0.05) from each other and compared with no significance difference with LCTR. There were no significant changes in the WHC of each yoghurt sample during storage, except on the 28th day, where lower WHC for some of the samples was observed. The results suggest that introduction of acetylated pea starches as stabilizers was able to effectively bind water, thus, reducing water loss from the yoghurt gel matrix.The DS of the pea starch showed no significant effect on the WHC of yoghurt; however, native pea starch exhibited lower WHC (66%), which gradually reduced over the 28-day cold storage period. The WHC (85%) of yoghurts with stabilizer (acetylated starches) was significantly  showed no significant difference (p > 0.05) in syneresis percentage. Although the acetylated pigeon pea starches did not totally prevent serum separation during storage, they sufficiently reduced syneresis to levels that are similar to that of the commercial sample.The stabilized yoghurts showed no significant susceptibility to syneresis over the refrigerated storage period when compared to yoghurts with no stabilizer (YoCN) or with native starch (YoPN), which was initially stable for 14 days, after which progressive syneresis was observed. Nonetheless, YoPN had a lower degree of serum separation than YoCN, suggesting that native starch exhibited better ability to retain water within the yoghurt matrix than samples without stabilizer. Degree of substitution had no significant effect on the amount of syneresis, as yoghurts with the acetylated starches (YoPAp > 0.05) in their sensory attributes, as shown by the similar values obtained for YoPA1, YoPA2, and YoPA3. Moreover, the colour of all yoghurt samples was not significantly different (p > 0.05) from the commercial sample, thus, addition of pigeon pea starch had no effect on the colour quality. The introduction of PPAS3 in yoghurt showed the best stabilizing effect by enhancing firmness of the sample (YoPA3); regardless, this sample did not significantly differ (p > 0.05) from the others, except YoCN, which may result from the absence of any form of stabilizing agent in the product.The sensory attributes of the laboratory control (gelatin-stabilized yoghurt) compared favourably with the market control  except for aroma, flavour, and overall acceptability ; this ma3 also showed superior properties. The sensory ratings showed that the use of native starch was inadequate for yoghurt stabilization because YoPN exhibited poor firmness and high syneresis. YoPA3 had the highest overall acceptability among the experimental yoghurt samples, which might be attributed to the superior firmness. The lower level of syneresis may have contributed to the increased firmness of YoPA3, despite the lower viscosity of the modified starch ingredient. This is because texture  is one of the major determinants of yoghurt quality based on its influence on yoghurt appearance, mouthfeel, and overall acceptability [The sensory results correlate to the WHC and syneresis, where YoPAtability . Earliertability . Similartability  reportedThe higher sensory scores for firmness, consistency, syneresis, and overall acceptability showed that the use of acetylated pigeon pea starch as a yoghurt structure stabilizer has greater potential than the native starch with respect to adoption by the industry as a food ingredient.3 with 0.14 DS produced the most acceptable sample and could be considered a potential ingredient for industrial production of set-type yoghurt.Acetylation of pigeon pea starch enhanced the physicochemical properties , which promoted its suitability as a functional ingredient in yoghurt formulation. The use of acetylated starch as a stabilizer was comparable with gelatin and suitable for significant reductions in serum separation, while maintaining firmness, consistency, flavour, and overall acceptability of yoghurt. PPAS


An outbreak of infection caused by SARS-CoV-2 recently has brought a great challenge to public health. Rapid identification of immune epitopes would be an efficient way to screen the candidates for vaccine development at the time of pandemic. This study aimed to predict the protective epitopes with bioinformatics methods and resources for vaccine development.The genome sequence and protein sequences of SARS-CoV-2 were retrieved from the National Center for Biotechnology Information (NCBI) database. ABCpred and BepiPred servers were utilized for sequential B-cell epitope analysis. Discontinuous B-cell epitopes were predicted via DiscoTope 2.0 program. IEDB server was utilized for HLA-1 and HLA-2 binding peptides computation. Surface accessibility, antigenicity, and other important features of forecasted epitopes were characterized for immunogen potential evaluation.315–324, Spike333–338, Spike648–663, Spike1064–1079) exhibited high antigenicity score and good surface accessibility. Ten residues within spike protein  are forecasted as components of discontinuous B-cell epitopes. The bioinformatics analysis of HLA binding peptides within nucleocapsid protein produced 81 and 64 peptides being able to bind MHC class I and MHC class II molecules respectively. The peptides  were predicted to bind a wide spectrum of both HLA-1 and HLA-2 molecules.A total of 63 sequential B-cell epitopes on spike protein were predicted and 4 peptides (SpikeB-cell epitopes on spike protein and T-cell epitopes within nucleocapsid protein were identified and recommended for developing a protective vaccine against SARS-CoV-2. An outbreak of infection caused by a novel coronavirus has spread worldwide rapidly , 2. The The pathogen causing the new type of pneumonia was named as severe acute respiratory syndrome coronavirus 2  by the Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses . Its genThe purpose of this study is to predict B-cell epitopes on spike protein and T-cell epitopes within nucleocapsid protein of SARS-CoV-2 by applying the bioinformatics methods and immunoinformatic tools. The step-by-step procedure of in silico analysis is depicted in Fig. Protein sequences of SARS-CoV-2 were retrieved from the NCBI database . Clustal Omega is the current standard version of the Clustal family which was widely used for biological sequences alignment. The program uses seeded guide trees and Hidden Markov model engine to generate alignments . In thisABCpred  and BepiPrediction of discontinuous epitopes on spike protein (PDB ID: 6VSB chain B) was conducted via DiscoTope 2.0 server . The parWe used the free online service provided by IEDB to forecast T-cell epitopes within nucleocapsid protein binding to HLA-1  or HLA-2Key features including digestion, mutation, toxicity, allergenicity, hydro and physiochemical properties were analyzed via vaxijen 2.0 , proteinA map of the predicted open reading frames (ORFs) is depicted in Supplementary Figure To better understand the characteristic of SARS-CoV-2, we compared its protein sequences with other selected coronaviruses. All protein sequences were downloaded from NCBI database with accession IDs shown in Fig. 315–324 (TSNFRVQPTE), Spike333–338 (TNLCPF), Spike648–663 (GCLIGAEHVNNSYECD), Spike1064–1079 (HVTYVPAQEKNFTTAP) displayed good surface accessibility in spike trimer .104–112 (LSPRWYFYY) was predicted as both HLA class-I and class II binding peptides. Additionally, this peptide may excel in the capability of binding to a large number of HLA molecules as shown in Tables 66–74 (FPRGQGVPI) and the helper T-lymphocyte (Th) epitope Nucleocapsid67–75 (PRGQGVPIN), which suggests the sequence containing these two epitopes may initiate both CD4+ and CD8+ dependent immune response.For HLA-1 binding peptide prediction, the top 50% scoring peptides were retained for further analysis. A total of 81 nonrepetitive peptides with ANN_IC50 value not higher than 500, indicative of stronger than medium binding affinity, were identified , a universal Th epitope that activates CD4+ cells [To induce humoral and cellular immune response simultaneously, five peptides that contain four linear B-cell epitopes and three T-cell epitopes Fig. a were se4+ cells , was int4+ cells , 33.FigDifferent from the less harmful human coronaviruses continuously circulating in the human population, coronaviruses originated from animals could become fatal pathogens by crossing species barriers. SARS-CoV in 2003, MERS-CoV since 2012, and SARS-CoV-2 right now all have caused large-scale epidemic problems. Effective and economic preventive approaches are in need urgently at the current situation of pandemic.333–338 produces a random coil structure and has a high antigenicity score. Noticeably, this peptide sits in the receptor-binding domain (RBD) of spike protein of SARS-CoV-2, which has been proved to mediate the binding to ACE2 on the epithelial membrane of human lungs [648–663 predicted in this study is 12 amino acids upstream of the furin cleavage site which is critical for SARS-CoV-2 biogenesis [648–663 could have expanded therapeutic function.Compared to traditional vaccine development, potent epitopes can be predicted via bioinformatics analysis, which makes the vaccine design straightforward and fast . As the an lungs . Likely,ogenesis . We expe104–112 showed an immunogenicity score of 0.36, suggesting a capability to initiate a strong immune response. Moreover, the peptides Nucleocapsid104–112 and Nucleocapsid66–75 harbor experimentally determined epitopes that bind to a number of HLA-1 molecules. In this study, Nucleocapsid104–112 and Nucleocapsid66–75 are forecasted as multi-epitopes that bind to both HLA-1 and HLA-2 molecules, suggesting a potency of both CTL and Th-mediated immune response initiation. Though these peptides were predicted to be non-toxic, we noticed that a large number of these peptides could be allergens. Thus, special attention should be paid to potential allergic reactions during the pre-clinical and clinical trials. As nucleocapsid protein is highly conserved between SARS-CoV-2 and SARS-CoV, available information on nucleocapsid protein-based vaccine against SARS-CoV could be helpful.Besides B-cell epitope prediction on spike protein, we selected nucleocapsid protein as a target protein for T-cell epitope computation for the following reasons. First, nucleocapsid protein in SARS-CoV and MERS-CoV has been experimentally tested as a robust immunogen to induce cytotoxic T lymphocyte response , 27; SecThe final vaccine construct comprising of CTL, Th, and B-cell epitopes is predicted to initiate protective humoral and cellular immune response against SARS-CoV-2.496, Glu498, Pro499, Thr500, Leu1141, Gln1142, Pro1143, Glu1144, Leu1145, Asp1146) were forecasted by DiscoTope 2.0 program as components of conformational B-cell epitopes. IEDB server was used for T-cell epitopes prediction, which gave rise to 81 and 64 peptides with binding capability to class-I and class-II molecule respectively. Four B-cell epitopes: Spike315–324 (TSNFRVQPTE), Spike648–663 (GCLIGAEHVNNSYECD), Spike1064–1079 (HVTYVPAQEKNFTTAP), and Spike333–338 (TNLCPF) were selected from the list based on their antigenicity score and surface accessibility. The T-cell epitopes Nucleocapsid104–112 (LSPRWYFYY) and Nucleocapsid66–75 (FPRGQGVPIN) on nucleocapsid protein could bind a wide spectrum of both HLA-1 and HLA-2 molecules. The final vaccine construct consists of CTL, Th, and B-cell epitopes that potentially protect individuals against SARS-CoV-2 by inducing both humoral and cellular immune response, which should be successively validated within both in vitro and in vivo models.B-cell epitopes on spike protein and T-cell epitopes in the nucleocapsid protein were predicted and analyzed in the current study. A total of 63 linear B-cell epitopes on spike protein were forecasted by ABCpred and BepiPred servers. Ten residues within spike protein (GlyAdditional file 1. Supplementary file S1.Additional file 2. Supplementary file S2.Additional file 3. Supplementary figures and tables.Additional file 4. B-cell-epitope-animation.


The main aim of this study was to establish the relationship between strength, power characteristics, individual muscle stiffness, international tennis number (ITN), and stroke velocity (StV) in junior tennis players.2), with an ITN ranging from 2 to 4, performed measurements regarding muscle stiffness of selected muscles involved in tennis strokes. StV , strength (maximum isometric strength) and power  measurements were also performed .Twenty one junior male tennis players , and large positive correlations were observed between pectoralis majoris stiffness (PMStiff) , isometric wrist flexion  and ITN, respectively. PMStiff was moderately inversely correlated to forehand velocity (FV)  and gastrocnemius (GStiff) and infraspinatus stiffness (IStiff) positively to SV . No significant correlations were found regarding strength and power measurements. Moderate inverse correlations were found between serve velocity (SV) and ITN (Greater stiffness values may enhance StV, especially when transferring power from lower to upper body. On the other hand, high scores could interfere in technical parameters that are key for velocity production in complex tennis strokes. Strength and power values proved to correlate poorly to StV in this particular sample of junior tennis players, possibly due to the multifactorial nature of tennis strokes and the possibility that they become more important as age and level increase. Today’s tennis is considered as a fast-paced, explosive and highly dynamic sport . High hiBecause of the aforementioned characteristics, it is commonly been accepted that these strokes are affected by several parameters such as technique , b, anthFurthermore, the influence of complex neuromuscular factors has hardly been studied in relation to any tennis specific stroke. Enhanced values of mechanical stiffness, that can be defined as the resistance of an object or body to deformation or change in length , have beIn short, the influence of specific strength and power parameters on StV, especially groundstrokes, and how muscle mechanical properties affect dynamic actions seems of importance for professionals. Therefore, the aim of this study was to examine the relationship between strength and power characteristics, individual muscle stiffness values, international tennis number (ITN) and StV in competitive junior tennis players. Our working hypothesis was that a strong positive association will exist between strength and power characteristics, ITN and all strokes, as seen previously in SV . Also, d2) with an ITN ranging from 2 to 4 (advanced level) participated in this study. A priori power analysis for a Pearson correlation was conducted in G∗power to estimate a sufficient sample size. With the alpha level set at 0.05, using a large target effect size (ES) of 0.6, a power of 0.80 and two tails, it was determined that 19 subjects would be needed. The player’s ITN was established by the consensus of three coaches accredited with RPT  level 3, following the ITN Description of Standards , aerobic and anaerobic training , and strength training. Inclusion criteria for all subjects required each participant to have a minimum of 1 year experience in strength training and 5 years of tennis training and competition. Participants were excluded from the study if they had history of upper extremity surgery, shoulder, back or knee pain and/or rehabilitation for the past 12 months. All subjects were informed in advance about the characteristics of the study and, before their participation, the participants or their legal tutors, in the case of being underage, voluntarily signed an informed consent. The study was conducted following the ethical principles for biomedical research with human beings, established in the Declaration of Helsinki of the AMM (2013) and approved by the Ethics Committee of the Catalan Sports Council (26/2018/CEICEGC).Twenty-one junior male tennis players , three times in the StV  and five times in stiffness measurements in all tennis players. Two familiarization sessions of all the strength and power tests were completed during the 2 weeks prior to the application of the protocol.This was a cross-sectional laboratory study with uninjured participants. The collection of data took place in May during a normal in-season training week in groups of 4 or 5 players and on 4 separate testing sessions, executed from 8 a.m. to 2 p.m. approximately, before the player’s afternoon normal technical-tactical training. On session 1, participants were assessed for 1 repetition maximum (1RM) on the bench press exercise. On session 2, participants were assessed for individual muscle stiffness (Stiff) via muscle natural oscillation. On session 3, participants were assessed for maximal StV on the forehand, backhand and serve actions. On session 4, participants were assessed using strength tests including bench press peak power (Wmax), maximal isometric strength (IsoMax) in 5 different positions, squat jump (SJ), countermovement jump (CMJ), and medicine ball overhead throw (MBT), following that order. Sessions 2, 3, and 4 were separate 2 h apart and session 1 was executed 24 h before. Players performed one tennis technical-tactical training session of 90 min between sessions 1 and 2 and ceased activity for at least 14 h before resuming the testing protocol. The use of pain-relieving strategies  was not allowed during testing in order to avoid interferences with the results. Players were allowed to consume water –1 were calculated by means of a linear transducer   . This en, Spain) .In session 2, individual muscle stiffness was recorded on the dominant side of the body using a hand-held myometer . The dominant extremity or side of the body was established based on upper body dominance. Before the assessment, body marks were established for the nine measurement points using the SENIAM electrode placement guides . The mush–1).In session 3, subjects performed a standardized warm-up that included mobility exercises, 5 min of free rallies and 5–10 progressive serves. Each subject executed a series of 6 flat services on each side of the court with 60 s of rest between sets and 12 forehands and 12 backhands (crossed-court) without alternating strokes. Only the serves that were in the serve box and the groundstrokes that landed in the court were counted. StV was determined using a hand-held radar gun  and peak velocity was registered for further analysis. The radar was positioned in the center of the baseline, 2 m behind the line and at an approximate height of 2 m for the serves and behind the player following the trajectory of the ball for groundstrokes. Hitting as hard and precise as possible to the “T” was indicated when serving and cross-court when hitting groundstrokes. Immediate feedback was provided to the subjects to encourage maximum effort. To avoid variability performing groundstrokes, balls were fed by a ball-throwing machine  at a constant speed . The participants sat in a position with a 90° hip flexion and the back resting on a bench. All the participants were fastened with a harness on the chest to avoid unwanted movements. Only the dominant extremity was evaluated. The maximum isometric force peak was recorded using a strain gauge sampling at 80 Hz . Positions were established before each test using a goniometer. Subjects performed three maximal voluntary contractions for 3–6 s and spaced by 1-min rest between attempts. Regarding upper body, peak power (Wmax) was assessed with a linear transducer  and analyzed with Chronojump Software (v1.8) using a load based on each participant’s 1RM. Each subject performed eight repetitions on the bench press exercise without verbal encouragement given. Following the literature . Each participant executed three maximum jumps spaced by 45 s of passive rest. The best trial  was used for the subsequent analysis.In session 4, the participants were asked to perform five maximum isometric tests following a protocol similar to that offered by terature  the loadterature . Regardip ≤ 0.05. All statistical analyses were performed using IBM SPSS Statistics 23.0 .The values presented are expressed as mean ± SD and 95% confidence intervals (95% CI). The normality of the distributions and homogeneity of variances were assessed with the Shapiro–Wilk test, all variables showed normal distributions except for ITN. The reliabilities of test measurements were assessed using intraclass correlation coefficients (ICCs), the standard error of measurements (SEM), and the coefficient of variation (CV). All of stiffness, strength, power and serve, forehand, and backhand velocity measurements reached an acceptable level of reliability and are presented in r = −0.43; p = 0.05), and large positive correlations between ITN and pectoralis majoris stiffness (PMStiff)  and isometric wrist flexion (IsoWrF) . Also, moderate inverse and positive correlations were observed between PMStiff and forehand velocity (FV)  and between gastrocnemius/infraspinatus stiffness (GStiff/IStiff) and SV , respectively. Regarding strength and power values, no significant correlations were found between upper or lower body values and hitting velocities.The correlation coefficients between strength, power and StV), and ITN are presented in This study aimed to analyze strength (maximum isometric strength) and power  characteristics alongside ITN as possible determinants of StV, including serve and groundstrokes, in junior tennis players. The main finding was that an increased gastrocnemius (GStiff), infraspinatus (IStiff), and decreased pectoralis majoris stiffness (PMStiff) may have some positive influence over performance in serve (SV) and forehand velocity (FV) respectively. Also, SV was inversely correlated to ITN. Moreover, strength and power values proved to be weak predictors of StV in this particular sample of junior tennis players. These results indicate that players of these characteristics that are able to reach higher velocity production in the serve and groundstrokes don’t specifically rely on the assessed strength and power characteristics.In other investigations it has been shown that physical aspects such as strength and power are determinant for producing ball velocity , also whStudies focused on SV have generally established positive results between MBT and velocity production in young tennis players . MBT havRegarding maximal isometric values, no correlations with any of the actions measured were found besides results indicating a positive association between maximal isometric wrist flexion and a higher ITN score. Previous studies have positively correlated isometric strength values with throwing  or even Lower body power variables analyzed  did not correlate with any of the StV variables. Although the contribution of the legs is considered widely as one of the main parameters supporting the effectiveness of the kinetic chain , these rRegarding muscle stiffness, results indicate moderate positive correlations between GStiff, IStiff and SV, PMStiff, and ITN, and inverse correlations between PMStiff and FV. As far as we know, no previous studies have attempted to correlate individual stiffness values and StV, although in other investigations, some findings indicate the importance of stiffness when we refer to actions that rely on the SSC . GeneralThis study showed some limitations. Maximal speed measurements, especially in groundstrokes, don’t take into account technical and tactical aspects on which skilled players may rely on in order to reach greater performance .As specific values of stiffness remain unclear, this study suggests practitioners include control and evaluation of stiffness as it may have influence in performance or injury risk. Moreover, due to multiple aspects affecting StV, designing programs that include technical and tactical assessment alongside strength and power enhancement, coordinative training and biomechanical aspects seems essential to enhance velocity production. Performance in these actions are affected by several aspects and the influence of them over StV may vary depending on the athlete’s age and level.In conclusion, an increased GStiff and IStiff seem to correlate to greater SV and high values of PMStiff affect negatively the player’s FV. Greater stiffness values of the gastrocnemius may enhance StV, possibly supporting power transfer from lower to upper body. On the other hand, enhanced levels in muscles surrounding the shoulder complex could interfere in technical parameters that are key for velocity production in complex tennis strokes. Also, SV is inversely correlated to ITN, indicating that players with a higher number in this rating seem to serve faster. Moreover, strength and power values proved to correlate poorly to StV in this particular sample of junior tennis players. Results indicate that athletes of these characteristics that are able to reach higher velocity production in the serve and groundstrokes don’t specifically rely on the assessed strength and power characteristics, possibly due to the multifactorial nature of tennis strokes and the possibility that they become more important as age and level increase.All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by Catalan Sports Council Research Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JC, EB, and FC contributed to the conceptualization, methodology, and review. EB contributed to the statistical analysis. JC contributed to the writing of the original draft preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


NOTCH1, CD133, CD44, CD24, and ALDH1A1, positively and significantly correlated with tricarboxylic acid cycle (TCA) enzymes. OVCAR3-OCSCs  exhibited enrichment of the electron transport chain (ETC) mainly in complexes I, III, IV, and V, further supporting reliance on the oxidative phosphorylation (OXPHOS) phenotype. OVCAR3-OCSCs also exhibited significant increase in CD36, ACACA, SCD, and CPT1A, with CD44, CD133, and ALDH1A1 exhibiting positive correlations with lipid metabolic enzymes. TCGA data show positive correlations between OCSC markers and glutamine metabolism enzymes, whereas in OCSC experimental models of GSE64999, GSE28799, and CCLE, the number of positive and negative correlations observed was significantly lower and was different between model systems. Appropriate integration and validation of data model systems with those in patients’ specimens is needed not only to bridge our knowledge gap of metabolic programing of OCSCs, but also in designing novel strategies to target the metabolic plasticity of dormant, resistant, and CSCs.Ovarian Cancer is the fifth most common cancer in females and remains the most lethal gynecological malignancy as most patients are diagnosed at late stages of the disease. Despite initial responses to therapy, recurrence of chemo-resistant disease is common. The presence of residual cancer stem cells (CSCs) with the unique ability to adapt to several metabolic and signaling pathways represents a major challenge in developing novel targeted therapies. The objective of this study is to investigate the transcripts of putative ovarian cancer stem cell (OCSC) markers in correlation with transcripts of receptors, transporters, and enzymes of the energy generating metabolic pathways involved in high grade serous ovarian cancer (HGSOC). We conducted correlative analysis in data downloaded from The Cancer Genome Atlas (TCGA), studies of experimental OCSCs and their parental lines from Gene Expression Omnibus (GEO), and Cancer Cell Line Encyclopedia (CCLE). We found positive correlations between the transcripts of OCSC markers, specifically CD44, and glycolytic markers. TCGA datasets revealed that  Ovarian cancer (OvCa) is the fifth most common cancer in females and remains the most lethal gynecologic malignancy in the present day . The fivhighCD24low cell surface markers isolated from metastatic tumors were able to self-renew and re-establish tumors when injected in immune-deficient mice [SNAI1/SNAIL), Snail Family Transcriptional Repressor 2 (SNAI2/SLUG), Twist-related protein 1 (TWIST1), and Zinc Finger E-Box Binding Homeobox 1 (ZEB1) concomitant with down-regulation of the epithelial marker E-cadherin and upregulation of mesenchymal markers, N-cadherin and vimentin [ALDH1A1, has been recently identified as a useful CSC marker for further enrichment of subpopulations expressing one or more of the aforementioned stem cell markers [CSCs have been long established in hematologic malignancies; the first reports of CSCs in solid tumors were published in 2003 , demonstvimentin . Since tvimentin . Interesvimentin . Transcrvimentin . Moreove markers . It is n markers .OCSCs were first identified by Bapat and colleagues in 2005 in an experimental model system as a small subset of the cancer cells associated with sustained self-renewal, ability to drive tumor growth, metastatic dissemination, and chemo-resistance . OCSCs hin vitro phenotypic behavior, specifically their ability to form spheroids that express one or multiple CSC markers, and their resistance to the standard of care therapy. Chemo-resistant OvCa cells were also reported to exhibit the unique ability to form spheroids in vitro and express CSC markers as CD24, CD44, cKit/CD117, PROM1/CD133, ALDH1A1, SOX2, NANOG, POU5F1/OCT4, and NOTCH1 as well as multi-drug resistance markers [in vitro cell culture conditions, along with the expression of distinctive transcriptomic signatures [Most reports of OCSCs relied on  markers ,35,36,37 markers  as evidegnatures .Several studies reported the signaling pathways implicated in the maintenance of cancer cells stemness. However, the metabolic pathways associated with the regulation of stemness are in infancy. CSCs were believed to exhibit the same metabolic programing as non-cancer stem cells, however, recent reports indicated that CSCs rely on multiple metabolic pathways depending on the cancer type, environmental cues, and the experimental model system that induces and/or supports the CSC phenotype ,39,40. TGSE28799 [GSE64999 [GSE28799 included OVCAR3-derived spheroids and their parental OVCAR3 in triplicates. GSE64999 included undifferentiated spheroids and their parental differentiated spheroids in quadruplicates. Studies were selected using keywords: ovarian cancer and stem cells. Only data from studies with 3–4 biological replicates were used for analysis.Gene expression profiles of two studies of OCSC GSE28799  and GSE6GSE64999  with plat-test with Holm–Sidak method, and each gene was analyzed individually between the two groups, without assuming a consistent SD with p ≤ 0.05. Data of the OvCa cell lines were downloaded from the Broad Institute Cancer Cell Line Encyclopedia (CCLE) portal  and were similarly analyzed. The transcripts of OCSC markers and metabolic enzymes were correlated in the OCSC populations using Pearson’s correlation. All analyses were performed using GraphPad Prism 7.0 . Correlations of the genes from The Cancer Genome Atlas (TCGA) data were performed using Gene Expression Profiling Interactive Analysis (GEPIA) web tool (http://gepia.cancer-pku.cn/) [The differential expression of the OCSCs markers and the enzymes involved in the metabolic pathways in OCSCs and their parental cells was analyzed by the multiple pku.cn/) . Bar graCD44 transcripts significantly correlated with other putative OCSCs markers SOX2, NOTCH1, OCT4/POU5F1, ALDH1A1, but not with CD24, CD117/KIT, CD133/PROM1, or NANOG . In this study [ALDH1A1 and SOX2 compared to parental undifferentiated spheroids cultured in stem cell media. Further analysis of the associated GSE64999 dataset confirmed the significant upregulation of ALDH1A1 and SOX2 but not the other putative OCSCs’ markers , and pyruvate dehydrogenase kinase 1 (PDK1) were significantly upregulated in OVCAR3 spheroid-derived OCSCs compared to their parental OVCAR3 cells (GSE64999 (CD44 and CD24 exhibited significant positive correlation with HK1 and phosphoglycerate kinase 1 (PGK1). NOTCH1 exhibited significant positive correlation with HK2. None of the other stemness markers exhibited significant positive correlation with glucose transporters or glycolysis . ALDH1A1 and SOX2 positively correlated with SLC2A3, whereas POU5F1/OCT4 and NANOG positively correlated with SLC2A2. However, NOTCH1 did not exhibit any significant correlation with glycolytic enzymes or glycolysis , HK1 and HK2, the rate limiting enzymes in glycolysis, and nine out of thirteen mapped enzymes involved in glycolysis. Similarly, CD44 was significantly positively correlated with three out of the six glucose transporters, hexokinases as well as nine out of thirteen enzymes involved in glycolysis. CD117/KIT was positively correlated with SLC2A1-4, HK2, and two out of thirteen mapped glycolytic enzymes, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and phosphoglycerate dehydrogenase (PHGDH); CD133/PROM1 correlated with HK1 and HK2, and two out of the thirteen enzymes in glycolysis. ALDH1A1 correlated with HK1, SLC2A1, and seven out of the thirteen enzymes involved in glycolysis. SOX2 correlated positively with SLC2A3 and four out of the thirteen enzymes in glycolysis. Other cell stemness markers as CD24, OCT4, and NANOG did not significantly correlate with glucose transporters, but positively correlated with only two of the glycolytic enzymes.Analysis of patients’ tumors, from TCGA data revealed that  enzymes . NOTCH1 CD44 positively correlated with a glycolytic signature with significant positive correlation with HK2, lactate dehydrogenase A (LDHA), and ENO1, but a negative correlation with SLC2A4 (CD117/KIT exhibited a positive correlation with SLC2A4 and fructose-bisphosphatase (FBP1) which catalyzes hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate, the rate-limiting step in gluconeogenesis. CD133/PROM1 expression significantly negatively correlated with SLC2A3, PFKFB3, LDHA, and triosephosphate isomerase 1 (TPI1), and positively correlated with FBP1 and PHGDH. ALDH1A1 negatively correlated with SLC2A4 and SLC2A6. SOX2 expression is negatively correlated with PFKFB3 and positively correlated with SLC2A2 and SLC2A4. POUF5F1/OCT4 was negatively correlated with HK1, SLC2A3, and PHGDH and positively correlated with SLC2A1. NANOG negatively correlated with PGK1, LDHA, TPI1, and positively correlated with PHGDH. NOTCH1 was negatively correlated with SLC2A5 and positively correlated with LDHA  inhibitor used in the OCSC model in GSE64999 [in vitro culture models lack vital extracellular matrix components including immune cells, stromal cells, and structural matrices, all of which are known to influence OCSC expression, growth, and differentiation. The absence of biophysical properties including interstitial flow, oxygen partial pressure, and surrounding environmental stiffness as well as the lack of biochemical cues are all factors that must be considered when comparing cell culture systems to in vivo CSC properties [CD44 positively and significantly correlated with most glycolytic phenotype in OCSC model systems, patients’ tumors as well as non-stem OvCa cells. This finding is consistent with earlier reports that CD44 is crucial for the regulation of glycolytic metabolism [Taken together, data from GSE64999  do not rGSE64999 . SpecifiGSE64999 . HypoxiaGSE64999 . Furtheroperties . In CCLEtabolism .GSE28799. We found that aconitase 1 (ACO1), isocitrate dehydrogenase (IDH1), IDH3A, succinate-CoA ligase GDP-forming subunit beta (SUCLG2), and malate dehydrogenase 2 (MDH2) were significantly upregulated in OVCAR3-stem cells, whereas malic enzyme 2 (ME2) was significantly downregulated , IDH3B, and SUCLG1. NANOG negatively correlated with seven enzymes with significant negative correlation with MDH2; while NOTCH1 negatively correlated with six enzymes with significance with ACO1. CD24, CD117, and CD133 were all not found to be significantly correlated with enzymes involved in the TCA cycle . These reactions facilitate the generation of an electrochemical proton (H+) gradient, which subsequently drives the synthesis of energy rich adenosine triphosphate (ATP) by ATP synthases [OXPHOS is a process by which NADH and FADHynthases .+/CD117+ OCSCs isolated from patients’ ascitic fluid exhibited enhanced glucose uptake with heightened OXPHOS. Upon glucose starvation, these OCSCs underwent complete quiescence and down-regulated most metabolic activities, while maintaining an OXPHOS profile [CSCs may exhibit highly glycolytic or OXPHOS phenotypes with plasticity of metabolic switch between phenotypes depending not only on cancer type and environmental cues, but also upon glucose starvation or OXPHOS blockade ,63. For  profile . Integra profile .GSE28799 revealed significant upregulation of most of the transcripts of complex I  in OVCAR3-OCSCs compared to parental cells , a key enzyme in lipogenesis correlated with poor disease outcome [FASN reversed platinum resistance in resistant OvCa cells [Lipid-associated pathways are essential for the maintenance of CSCs . Lipid mcultures . Consistnolayers ; an effe in vivo . Moreove outcome . AccordiCa cells .GSE28799 dataset, revealed that compared to parental OVCAR3, OVCAR3-OCSCs exhibited significant increase in the transcripts of fatty acid transporter (CD36), acetyl CoA carboxylase A (ACACA) that catalyzes the first committed step in fatty acid (FA) synthesis from acetyl CoA to malonyl CoA [stearoyl-CoA desaturase (SCD), that is also involved in FA biosynthesis, mainly unsaturated FAs, as well as carnitine palmitoyltransferase 1A (CPT1A) that catalyzes the first committed step in FAO [GSE64999 dataset revealed a significant increase in SCD in the undifferentiated OCSCs with an increase, though insignificant, in the enzymes involved in FA synthesis and oxidation as well as CD36  , ACACA, FASN, and SCD1 and positively correlated with CPT1A/B. NANOG was positively correlated with CPT2. NOTCH1 was not significantly correlated with the enzymes investigated (Correlation of the putative OCSC markers and lipid transporters and enzymes in OVCAR3-OCSCs in  (FABP4) . In GSE6th ACACA . TCGA da by SOX2 . Conversstigated . These dalanine, serine, cysteine transporter 2  and is subsequently hydrolyzed to glutamate and ammonia by glutaminase (GLS). Glutamate has dual roles: while it can be combined with cysteine and glycine to form the reduced glutathione (GSH), a major antioxidant that regulates oxidative stress [glutamate dehydrogenases (GLUD) to provide TCA cycle intermediates and, hence, energy production [Glutamine metabolism provides the carbon and amino-nitrogen that are necessary for biosynthesis of amino acids, nucleotides, and lipids ,75,76. Ae stress , it can oduction . Furtheroduction . Additiooduction . Targetioduction , suggestGSE28799 revealed that the glutamine transporter SLC1A1 and the enzyme GLS, were significantly upregulated in OVCAR3 spheroid-derived OCSCs compared to their parental OVCAR3 cells (GSE64999 (, very few correlations between stem cell markers and glutamine transporters were noted. In GSE28799, both CD24 and CD44 positively correlated with SLC1A1, while CD133 positively correlated with GLS and negatively correlated with GLUD2. ALDH1A1 positively correlated with SLC1A2 while OCT4 positively correlated with SLC1A3 (GSE64999, CD133 positively correlated with GLS. CD24 negatively correlated with GLUD1. ALDH1A1 and SOX2 both negatively correlated with SLC1A6. NOTCH1 negatively correlated with SLC1A1 (Analysis of the expression of the enzymes involved in glutamine metabolism in OCSCs and their parental cells in R3 cells A. In conGSE64999 B. In both SLC1A3 . In GSE6h SLC1A1 .CD44, ALDH1A1, and NOTCH1 exhibited significantly positive correlations with enzymes involved in glutamine metabolism. CD44 positively correlated with SLC1A1-5, GLS, and GLUD1/2. Similarly, ALDH1A1 exhibited significant positive correlation with four out of the seven glutamine transporters, along with GLS and GLUD1/2. NOTCH1 significantly positively correlated with five out of the seven glutamine transporters, along with GLS and GLUD1/2. CD133 positively correlated with five out of the seven glutamine transporters, while CD117 correlated with three out of seven glutamine transporters, as well as with GLS and GLUD1/2. SOX2 and OCT4 positively correlated with three and two glutamine transporters, respectively. OCT4 also positively correlated with GLS and GLUD1/2. CD24 positively correlated with one glutamine transporter, as well as SLC1A1, but no other significant correlations with other glutamine transporters. NANOG was not positively correlated with any of the glutamine transporters yet was correlated with GLS and GLUD2 (CD44 and OCT4 both positively correlated with SLC1A1 and GLS; whereas OCT4 negatively correlated with SLC1A2 and SLC1A5. CD117 positively correlated with SLC1A3, while CD133 positively correlated with SLC1A5. Additionally, ALDH1A1 negatively correlated with SLC1A7, while NANOG negatively correlated with SLC1A1 (GSE64999, GSE28799, and CCLE, the number of positive and negative correlations observed was significantly lower implying that glutamine metabolism is indispensable for OCSCs and OvCa cells grown in normal conditions, whereas in their complex TME, OvCa cells utilize glutamine metabolism in an anaplerotic reaction for generating energy and metabolic intermediates to fulfill their increasing demands.Further analysis of patients’ tumors from TCGA data indicated that nd GLUD2 . Of noteh SLC1A1 . In summOCT4, ALDH1/2, CD44, and LGR5/Ki67+ markers and non-proliferating CSCs expressing SSEA4+/Ki67− or ALDH1/2+/Ki67− markers. Other subsets of OCSCs include CD133+ and CD44+/CD117+ all of which can be influenced by tumor microenvironmental cues, upregulation of signaling pathways, and tumor grade, making it difficult to confine OCSCs to a standardized list of markers [While our findings are based on OCSC transcript levels, it is necessary to clarify that the transcript level and proteome levels may not necessarily correlate due to post transcriptional modifications, including splice variation. Alternative splicing accounts for the major difference between the number of protein-coding genes and the number of proteins that are ultimately produced from translation . Through markers .In this study, we sought to investigate the predictive significance of the gene expression of OCSCs in correlation with transcripts of metabolic pathways that are upregulated in HGSOC, that could inform about the metabolic plasticity of tumors, predict the behavior of recurrent or chemo-resistant disease, and ultimately may guide on therapeutics targeting the enriched pathways in combination with standard of care therapy. Comparison of transcripts of OCSC markers with those of enzymes involved in metabolic pathways in OCSC spheroid model systems with patients’ data revealed several distinctive differences in the expression of the stem cell markers. Correlations with energy metabolic pathways, not only between the two model systems investigated, but also with patients’ tumors and cumulative behavior of OvCa cell data from CCLE were elucidated. While these differences between spheroid model systems could be attributed to technical variability in establishing and maintaining of OCSCs as spheroids, nevertheless, they highlight the significance of OvCa heterogeneity, and the influence of micro-environmental cues on metabolic plasticity of OCSCs that may influence their survival and self-renewal capabilities.It is noteworthy that the limitation of the present study is the paucity of data from established model systems of OCSCs with adequate biological replica that can be used for systematic biological, metabolic as well as transcriptomic analyses to bridge the gap of metabolic programing in OCSCs.


This article explores the role of narratives as drivers that guide the institutional change associated with globalization and deglobalization. For China’s Belt and Road Initiative (BRI) to succeed as a driver of institutional change in favor of globalization, it must pass the narrative “virality” test and successfully contend with competing narratives. Rival narratives will be launched by firms and organizations worldwide that expect to win or lose from deglobalization or from new forms of globalization. This study develops a useful framework for establishing the extent to which China’s BRI is a genuine narrative or just a story. In this regard, four testable propositions are put forth to ascertain whether the BRI is values-based, extends an invitation to participate, is open-ended, and is associated with economic performance for both Chinese and non-Chinese participants. The analysis of the BRI-related institutional change that leads to globalization applies a theoretical lens centered on the narrative economics perspective and on the institution-based view and political economy perspective. Implications for BRI stakeholders, international business practitioners, and international business scholarship are outlined. This article addresses two research questions. First, how are the institutional changes associated with globalization and deglobalization influenced by the contexts of narratives in general and the Belt and Road Initiative (BRI) in particular? Second, what factors drive the virality of the BRI as a narrative and the impact on international firm strategy? These questions are relevant for scholarship and policymaking and have important practical implications for the conduct of international business and for firms’ strategies.Researchers and practitioners alike have wondered exactly how the present economic, geopolitical, political, and technological disruptions will reshape institutional arrangements. Executives, thought leaders, and policy-makers in China, Russia, Switzerland, and the United States, with whom we have engaged in discussions, view 2020 as an inflection point for globalization. They note that many outcomes are still possible, including both greater integration and greater fragmentation. For instance, China could decide to deemphasize the BRI and focus its resources inward on domestic recovery. Alternatively, China could seek ambitious institutional change with European partners through the EU-China Comprehensive Agreement on Investment (CAI) finalized in December of 2020 or in digital cooperation  in relation to the BRI. It employs the theoretical lens of narrative economics, along with an institution-based view and, to a lesser extent, a political economy perspective to address conceptual problems associated with social, political, geopolitical, economic, and other aggregate forces that disrupt international business practices and force the reevaluation of generally accepted approaches.Narratives are coherent desciptions with structures built arround sets of circumstances, actions and actors: moreover, they point to outcomes and can be employed “to predict the new state of the world.” Abell : 295. ToScholarly work has identified multiple drivers or antecedents of de/globalization and its institutional changes. We conceive of these drivers as either narratives, such as nationalism, that develop into institutional forms  or as exogenous forces that narrativize . Examining how exogenous drivers are cast as narratives along the globalization to deglobalization spectrum is helpful.The contextual drivers of deglobalization are usually thought of as being economic, political, and sociocultural Meyer . They arA narrative’s plausibility can change in accordance with the changing attitudes toward de/globalization Jones . Table 1As a general principle, the narrative attached to an institutional, political, geopolitical, or technological force is a key determinant of whether such a force drives further institutional change. Many of the forces and facts described in Table When the narrative associated with a set of drivers goes viral and manages to win in the market for narratives, institutional change and even institutional reversal can occur fairly quickly, as shown by the rapid resurgence of the deglobalization sentiment. In this vein, Braga  asks wheInstitutions are “humanly devised constraints that structure political, economic and social interaction” North : 97. PenWhereas states appear to set “the rules of the game” Wilkins : 640, inInstitutional arrangements favoring globalization  or deglobalization  reflect the interests of those who have prevailed in domestic policy disputes. Wilkins : 640 detNarratives, as understood from the literature reviewed in this article  driving institutional change (dependent variable), it must have the ability to go viral. Four testable propositions are suggested in this regard and are summarized in Table telling of a story”  hosted by the Cyberspace Administration of China (CAC) and the NDRC. Countries that have signed DSR-specific MOUs include Cuba, Saudi Arabia, and the United Kingdom  Reconnecting Asia Project notes that, although the BRI claims to endorse universal values, only approximately 11% of Chinese-funded transportation projects tracked have been won by non-Chinese companies Hillman . Is thatthe BRI globalization narrative to go viral, its matching institutions and rules must provide sustained perceived benefits to both Chinese and non-Chinese business interests and society.For The BRI as a globalization narrative that goes viral will impact other variables, first and foremost institutional change and the globalization institutions. The viral BRI narrative will also affect firm strategy and international firm performance levels. At the same time, the BRI is now in the realism space of geopolitics, where the success or failure of narratives with international business relevance is decided in part on the relative power of nation states.An adaptation of Mearsheimer’s realism theory of international relations  will determine their performance. Narrative capital becomes essential to international business strategy.. Oliver : 707 disWalk the “thin line.” Geopolitical monitoring is becoming essential as firms navigate their selections between contending narratives and develop their own narratives. The dilemma for foreign multinationals in Shanghai, for Hong Kong investors in the United States, or for participants in natural resource projects in Russia is that firms’ narratives must be acceptable to China, the United States, and other relevant countries. International firms must carefully tread a very thin line. The alternative is the persistence of regionally constrained international strategies  of Chinese and non-Chinese stakeholders alike. In this regard, such an observatory would establish virality and related patterns and then determine the confluences of the BRI with relevant phenomena, such as de/globalization, foreign direct investments (FDI), or firm strategies.Relationship between the BRI narrative and dependent variables. This study suggests that the nature and strength of the relationship between the BRI viral narrative of globalization as an independent variable and the selected dependent variables is a fundamental future research direction. The dependent variables would first include institutional changes in globalization. Concreate research questions would be numerous, such as whether BRI and RCEP are “complimentary in design” (Ji sign” Ji  or whethsign” Ji  and centThis article aims to elicit directions for future research such as:Academics, executives, and entrepreneurs all view the rollout of BRI institutions, the reconfiguration of global value chains, technology disruptions, the integration of Eurasia, trade wars, and regionalization with trepidation and concern. Will the BRI narrative bring about meaningful institutional change and increased opportunities for improving firm performance? Its success, virality and impact will depend on its pull with Chinese domestic political and economic coalitions and with international businesses and broader social interests. The four propositions provide a framework for forecasting how that will go. The framework incorporates the insights gained by addressing the first research question on the factors that drive institutional change, as well as the second question on the implications for international business practice and scholarship of the BRI narrative. More generally, however, this article nudges the decision-maker occupied with international strategy towards a deep and inclusive understanding of narratives in order to position the organization for global performance. This article has aimed to supply novel ideas and a stimulating framework for management scholars, decision makers, and leaders in the policy and corporate worlds.