Our results indicate that atom doping may significantly modify the electronic properties. Surprisingly, doping Cr into a holey site of C6N6 monolayer was found to exhibit a narrow band gap of 125 meV upon compression strain, considering the spin–orbit coupling effect. Also, a C atom doped in C6N8 monolayer shows semi-metal nature under compression strains larger than −2%. Our results propose that Mg or Ca doped into strained C6N6 may exhibit small band gaps in the range of 10–30 meV. In addition, a magnetic-to-nonmagnetic phase transition can occur under large tensile strains in the Ca doped C6N8 monolayer. Our results highlight the electronic properties and magnetism of C6N6 and C6N8 monolayers. Our results show that the electronic properties can be effectively modified by atom doping and mechanical strain, thereby offering new possibilities to tailor the electronic and magnetic properties of C6N6 and C6N8 carbon nitride monolayers.In this work, by performing first-principles calculations, we explore the effects of various atom impurities on the electronic and magnetic properties of single layers of C 6N6 and C6N8 monolayers.Using first-principles calculations, we explore the effects of atom doping and strain on the structural, electronic, and magnetic properties of C Depending on the composition of C and N atoms in the atomic lattice, these allotropes can show diverse electronic properties, ranging from semiconducting to half-metallic. 2D-CN monolayers exhibit considerable structural, electronic and magnetic properties17–21 with diverse electronic properties, originating from their unique and different atomic lattices, made from strong covalent bonds.22–25Recently, two-dimensional carbon nitride (2D-CN) nanomaterials have attracted remarkable attention, not only because of astonishing experimental advances concerning their synthesis but also due to their exciting physics2N, C3N, C6N8 and C6N6 through bottom-up procedures, has motivated researchers to consider possible approaches for tuning the band gap and electronic response of these attractive nanosheets.22,26–28 Some 2D-CN nanosheets have been experimentally realized using different approaches.1,3,29–33 C6N6 is a 2D-CN allotrope in which two hexagonal rings are separated by a C–C bond,1 and it has already been theoretically investigated.34–36 Recent theoretical work showed that C6N6 exhibits a topologically nontrivial band gap, which can be modulated to a topological insulator by doping.1 To search for a suitable spintronic material such as a diluted magnetic semiconductor, first-principles calculations of transition metal (TM) atoms doped in semiconducting 2D-CN sheets have been performed2,37,38 using density functional theory (DFT) calculations.39,40 The semiconducting nature and band alignment in this nanosheet are suitable for hydrogen generation via photo-catalytic water splitting.41–44The successful synthesis and fabrication of two-dimensional carbon nitride (2D-CN) nanomaterials, including C45 Recently, carbon nitride materials with stoichiometry of C6N8 have been synthesized experimentally.12,26,46,47 C6N8 are the prevailing building blocks that are joined together directly or by sp2-hybridized nitrogen/carbon atoms.12 The electronic and magnetic properties have subsequently been studied,48,49 and theoretical studies demonstrate that C6N8 is not a magnetic material; however, after replacing a nitrogen atom with a carbon atom in the unit cell of C6N8, it displays intrinsic half-metallicity.3 Hydrogenation can also induce magnetism in carbon-based nonmagnetic materials.50,51 It was predicted theoretically that hydrogen doping on the irradiation-induced carbon vacancy with hydrogen adsorption could induce a stronger magnetic moment than the bare vacancy.52 Moreover, first-principles calculations also show that semihydrogenated graphene sheets are ferromagnetic semiconductors with a small indirect gap.53 On the other hand, it is well-known that the application of mechanical strain can be an effective way to adjust the electronic structure of 2DM,54 and in previous studies strain has been proven as a common way to regulate properties. In order to enhance the application of a material in electronic and optoelectronic devices, it can be very useful to regulate the electronic properties. Atom impurities can tailor the electronic properties and can be used for a wide range of applications. Despite the fact that many 2D nanosheets have been synthesized recently, their tunable electronic properties have rarely been reported.55–90,108–110First-principles calculations indicated that topologically nontrivial electronic states near the Fermi level can be characterized by a ruby model.6N6 and C6N8 monolayers. Our results confirm that the electronic properties can be modified by doping with Cr atoms. In addition, by applying strain, we found that the magnetic moment can be modulated and a transition from semiconductor to metal can be achieved. Our results reveal that the band gap and magnetism can be modified or induced by various atom impurities, thereby offering effective possibilities to tune the electronic and magnetic properties of C6N6 and C6N8 monolayers.In this paper, using first-principles calculations, we systematically investigate the effect of different atom impurities in the holey sites of C291 Wave functions were expanded by the linear combination of multiple pseudoatomic orbitals (LCPAOs) created using a confinement scheme.92,93 The generalized gradient approximation method proposed by Perdew–Burke–Ernzerhof (GGA-PBE)94 was applied to deal with the exchange-correlation functionals, along with the norm-conserving pseudopotentials.95 A quasi-Newton algorithm was employed to find the energy-minimized structures using the OpenMX package. The convergence criteria for the structural optimization were set as a force of less than 1 meV Å−1 acting on each atom. After the completion of this step, we used a cutoff energy of 300 so that the total energies converge to below 1.0 meV per atom. The Brillouin zone was sampled by a k-point grid of 23 × 23 × 1 for the primitive unit cell and scaled according to the size of the supercells, on the basis of the Monkhorst–Pack scheme.96 The monolayers are modelled as a periodic slab with a large vacuum region (20 Å) in order to prevent interactions between adjacent layers. To describe the vdW interactions we use the empirical correction method described by Grimme (DFT-D2).97 The charge transfer was calculated using Mulliken charge analysis.98 Scanning tunneling microscopy (STM) images were simulated using the Tersoff–Hamann theory.99 The STM simulated images were plotted using WSxM software.100We report the results of our spin-polarized DFT calculations for the electronic structure as implemented in the OpenMX 3.8 package.33.16N6 and C6N8 monolayers are shown in 6N6 is calculated to be 7.11 Å and the corresponding C–C and C–N bond lengths are found to be 1.503 Å and 1.343 Å, respectively. The nanopore diameter is found to be 5.447 Å, which is equivalent to the distance from one N atom to the opposite N atom across the pore. These results are consistent with previous reports.36,101–103 The charge density difference, in which the blue and yellow colors show the charge accumulation and depletion, respectively, reveals that the positively charged C atoms are surrounded by negatively charged N atoms. Moreover, the simulated STM image indicates that the atoms around the C atom sites show bright spots. The electronic band structure, density of states (DOS) and partial DOS (PDOS) of C6N6 and C6N8 monolayers are shown in 6N6 monolayer is a semiconductor with a direct band gap of 1.5 eV located at the K-point, which is also supported by a previous report.36 From the PDOS, we found Fermi-level sharp peaks +2 eV below the Fermi level, suggesting that there are localized wave functions in C6N6 and indicating that sharp double peaks  are formed from the N s and px,y orbitals. In the VBM, the N atoms are in the sp2 configuration and their lone pairs are further exhibited in the rising band structure from the Γ to the K point near the Fermi-level. The lone pairs of N s and px,y lie in in-plane orbitals, suggesting the donor nature of the nanopore, although the N/C pz antibonding delocalized orbitals form the CBM above the Fermi-level, showing the acceptor nature of C6N6. The filling of all the in-plane bonding orbitals implies high stability of the C6N6 structure.The atomic structures, simulated STM images, and charge density differences of C6N8 is found to be 7.14 Å, with two inequivalent C–N bonds (1.414 and 1.455 Å). In addition, the ring is not a regular hexagon since the C–N–C angle is equal to 120°, while the N–C–N angles are 118° and 122°, respectively. It is clear that every six C–N hexagon rings enclose a nanopore, while the edges are surrounded by six N atoms, with a nanopore diameter of 4.773 Å  between the doped atoms and the C6N6/C6N8 sheets by the following formula:Etot is the total energy of the doped C6N6/C6N8 sheet, and Esheet and Eatom are the energies of the pristine C6N6/C6N8 sheet and isolated spin-polarized atom, respectively. According to this definition, a more negative value of Eb indicates that the system has stronger binding between the atom and the C6N6/C6N8 sheet. Our results show that the binding energies for C, Mg, Ca and Cr atom doping in C6N6 (C6N8) sheets are −3.46 eV (−3.38), −3.70 eV (−3.43), −3.95 eV (−3.87) and −4.65 eV (−3.65).The optimized structures of Mg@, Ca@ and Cr@Cvely see . The Cr 6N6 are shown in 6N6, the band structures are modified and give rise to localized mid-gap states arising from the atomic doping. Note that the doping of Mg and Cr atoms gives rise to some localized states in the band structure, which modify the electronic properties. Our results show that Mg@C6N6 exhibits direct band gap semiconducting behavior, with a small band gap of 100 meV where the VBM and CBM are located at the Γ point. Interestingly, we can see that the Ca@C6N6 monolayer exhibits metallic character. In contrast, Cr@C6N6 is a ferromagnetic metal with a net magnetic moment of 2.4 μB. Note that the energy bands of the ↑ and ↓ spin channels near the Fermi-level cross together near to the Dirac-point. On the other hand, monolayer C@C6N8 becomes a direct band gap semiconductor with a narrow band gap of 25 meV, with the VBM and CBM located at the M point. With Ca atom doping in C6N8, we see a spin-polarized ferromagnetic metal with impurity levels crossing the Fermi-level and inducing a net magnetic moment of 1 μB per formula unit. The atomic contributions to the electronic states can be further seen by the PDOS, which are presented in 6N6 mostly contribute to the deep valence and conduction states. On the other hand, Cr in C6N6 and C in C6N8 also contribute to the states around the Fermi-level. The optimized structures and electronic structures of C@C6N6, Mg@C6N8 and Cr@C6N8 are shown in Fig. S2(a–c),2-hybridization and form two σ bonds with the neighboring N atoms of C6N6. The C@C6N6 structure shows semiconductor behavior with an indirect band gap of 1 eV, where the VBM and CBM are located at the K and M points, respectively 6N8 and forms six σ bonds, while the neighboring N atoms of C6N8 form four σ bonds with the Mg atom  and (b)6N8 turns into a metal, whereas the Cr@C6N8 structure becomes a half-metal and a magnetic moment of about 2.1 μB is induced.The electronic structures of Mg@, Ca@ and Cr@C46N6 and C6N8 monolayers. The strain is defined as:107ε = (a ± a0)/a0 × 100, where a and a0 are the strained and non-strained lattice constants, respectively, and the positive (negative) sign denotes tensile (compressive) strain. The band structures of Cr@, Mg@ and Ca@C6N6, and C@ and Ca@C6N8 as a function of the applied strain are shown in 6N6 displays dilute magnetic semiconductor behavior under +2% tensile strain and it is preserved up to +8% strain. In addition, the magnetic moment increases from 2.8 (at −2%) to 3 μB (at −8%) with increasing compressive strain, while it is almost constant under tensile strain. Interestingly, when the compression strain reaches −2%, Cr@C6N6 exhibits spin-polarized semi-metal characteristics.Strain engineering is a robust method to tune the electronic properties and topological nature. In the following, we investigate the effects of uniaxial tensile and compressive strains on the electronic properties of atom doped C6N6 in the range of −2% to −6% exhibits a decreasing band gap from 90 meV to 70 meV, respectively, while in the strain-free case the band gap is 110 meV. This situation differs under tensile strain, such that the band gap changes in the range of 130 meV to 115 meV. Interestingly, at a tensile strain larger than +8%, the band structure of Mg@C6N6 in both ↑ and ↓ spin channels becomes spin-polarized, leading to the transition from nonmagnetic to magnetic states  of the ↓ (↑) spin channel continuously shifts upward (downward) to the Fermi level, resulting in ferromagnetic metallic behavior with magnetic moments of 0.8 μB (+6%) and 1.8 μB (+8%). The electronic band structure of C@C6N8 under strain is shown in 6N8 is initially a direct semiconductor with a narrow band gap of 30 meV; however, under 0% ≤ tensile strain ≤ +8%, the band gap increases monotonically from 70 meV (at +2%) to 200 meV (at +8%), while the VBM and CBM of the direct band gap are located at the M point. Our results show that the semiconducting characteristics are preserved in the range of +2% to +8%. Interestingly, C@C6N8 under compression strains of −2, −4 and −8% exhibits semi-metal behavior. When it reaches −6%, it transforms to a metal due to the CBM decrease and VBM increase to the Fermi-level. The electronic band structure of Ca@C6N8 under uniaxial strain in the ↑ and ↓ spin channels is shown in 6N8 is initially a ferromagnetic metal with a magnetic moment of 1 μB in the ground state. Interestingly, when a strain larger than +2% to +8% is applied, the ferromagnetic metal state is lost and both the ↑ and ↓ spin channels become non-spin-polarized, leading to a transition from a ferromagnetic metal to a nonmagnetic-metal state. Note that by increasing strain up to +2%, its magnetic moment vanishes. Also, for strains of 2–4%, Ca@C6N8 transforms into a nonmagnetic metal, while for larger strains (>−6%) the structure transforms into a ferromagnetic metal with magnetic moments of 0.47 μB (−6%) and 0.53 μB (−8%).Our results show that Mg@Cates see . Ca@C6N6vely see , whereas6N6 without strain and with −2% strain, considering spin–orbital coupling (SOC) and without SOC, are shown in 6N6 upon −2% compression strain, without considering SOC, we can see that the ↑ and ↓ spin bands cross each other at the Fermi-level, which implies a magnetic semi-metallic nature. Our results show that when the SOC effect is considered, we see a touching point along the Γ and K paths at the Fermi level, and a narrow band gap of 125 meV forms in the proximity of the Fermi level, which implies a semi-metallic character. The effect of SOC correction on the evolution of electronic structure was found to be remarkable, such that degeneracies at the VBM are removed. Due to weak screening of the Coulomb interaction in the doping of transition metal atoms, the Hubbard U energy band structure may be expected to be weak. Since the accurate value of U has not been determined, we investigate the correlation effects by the value of the Hubbard U. To confirm that semi-metallicity is not an artificial result from the GGA+U method, we perform calculations for the doping of Cr atoms. The electronic band structures of Cr@C6N6 without strain and with −2% strain, without (left) and with consideration of the Hubbard U effect (right) are shown in Fig. S3(a) and (b),6N6 without and with strain are significant and cause changes in the spin polarization. In the Cr-doped C6N6 without strain and upon −2% compression strain, we can see that the ↑ and ↓ spin bands cross each other at the Fermi-level, which implies a spin-polarized semi-metallic nature, without considering Hubbard U. We can see that a nodal-line forms around the K and M points when U = 2 eV is considered. Our results show that the magnetic moment of Cr@C6N6 in U = 2 eV are 3.53 μB (without strain) and 3.63 μB (with −2% strain).The electronic structures of Cr@C6N6 monolayers. We consider two Cr atoms doped in one and two neighboring cavities for comparison. The spin density differences of Cr doped in one and two neighboring cavities of C6N6 are shown in Fig. S4.6N6 are considered, and only the most stable configurations are exhibited. The exchange energy Eex, defined as Eex = EFM − EAFM, is calculated to determine the most stable phase. Negative and positive Eex indicate the FM and AFM ground states, respectively. For the case of one neighboring cavity, it is found that the Cr atoms exhibit FM coupling (with Eex = 140 meV) in their most stable phases, and the corresponding spin density differences are shown in Fig. S4(a).Eex = 30 meV) with corresponding spin density differences.Here, we discuss the magnetic coupling between Cr–Cr doped in C56N6 and C6N8 monolayers. Our theoretical results indicated that with atom doping, C6N6 becomes a semiconductor with a narrow band gap (Mg-doped), semi-metal (Ca-doped) or ferromagnetic metal (Cr-doped). Additionally, we applied mechanical strain to both monolayers with atoms doped in their lattices. Our first-principles results revealed that the band gap and magnetism can be finely tuned by strain as well. Surprisingly, when the compressive strain reaches 2%, Cr-doped C6N6 displays a phase transition to a spin-polarized semi-metal without SOC; when SOC is considered, a narrow band gap of 125 meV is opened. Notably, in the case of Cr atom doped C6N6 upon compressive strain, considering SOC, an unusual semi-metallic character can be observed. In the C-doped C6N8 between applied tensile strains of 0–8%, the band gap was found to increase monotonically from 70 meV (at 2%) to 200 meV (at 8%), while it exhibits semi-metallic behavior under compressive strain larger than 2%. Our results show that a magnetic-to-nonmagnetic phase transition can occur under large tensile strain in the Ca doped C6N8 monolayer. In addition, Mg and Ca atom doped C6N6 can show topological insulator states under strain. The results provided by our extensive theoretical analysis highlight very promising electronic properties of C6N6 and C6N8 monolayers doped with various atoms and under mechanical strain, and will hopefully serve as a guide for future experimental and theoretical studies.In summary, using first-principles calculations, we have explored the effects of atom doping on the structural, electronic, and magnetic properties of CThe authors declare that there are no conflicts of interest regarding the publication of this paper.RA-010-D0RA04463F-s001


The impact of yellow pumpkin powder (YP) substitution  on wheat flour's physico-functional, pasting, gel texture, and dough rheology was studied. Moreover, the nutritional, organoleptic properties and bioactivity of the composite pan and pita bread were evaluated. An improved water holding capacity was noticed for the blended flour than for control. The pasting parameters were declined significantly (p < 0.05) with increasing YP. Composite flours presented a softer gel texture in the presence of YP. Reduced water absorption, increased dough development time, and lower stability for combined flour dough. The bread with YP depicted increased protein, fat, fiber, and mineral contents, while a reduced volume and specific volume were noticed for pan bread. YP incorporated 5% and did not compromise pan and pita bread's color and overall acceptability. Additionally, composite bread depicted higher total phenolics with enhanced antioxidant activities at the higher substitution of YP. Wheat bread is relished for its tempting organoleptic parameters, cost competitiveness, and ready-to-eat characteristics. Additionally, it is an evident source of macro and micronutrients, such as carbohydrates, proteins, fiber, vitamins, and minerals . HoweverCucurbita moschata is one of the cultivars under the pumpkin family called Cucurbitaceae. Among cucurbitaceous vegetables, pumpkin has been appreciated for its high yield, long storage life, and high nutritive value  were significantly improved, and a greater antioxidant activity was observed . Yildiz 22.1Cucurbita moschata, wheat flour, yeast, salt, sugar, shortening, and improver were bought from the local supermarket, Riyadh, Saudi Arabia.Yellow pumpkin fruits 2.2The cleaned yellow pumpkin fruits were cut, seeds were removed. Seedless fruits wear cut into 2-mm-thick slices and freeze-dried. The dried pumpkin was milled, sieved through a 60-mesh sieve, and stored in a refrigerator for further use.2.3To prepare Wheat flour and YP composites, the wheat flour was replaced with YP at 5%, 10%, and 15% w/w basis.2.4Water and oil holding capacities (WHC/OHC) were measured according to WHC/WOC (g/g) = (W1 − W0)/W0, where W0 is the initial weight (g) of the samples and W1 is the final weight (g) of samples.2.5A Rapid Visco-Analyser (RVA)  was used to study the pasting behavior of wheat flour-YP blends. The flour samples (3.5 g at 14% moisture basis) were weighed directly in RVA aluminum pans, and distilled water was added to 28 g as total weight. The suspension was loaded in RVA and stirred. Then speed was decreased. The obtained slurry was equilibrated after that heated; it was then held. The suspension was cooled and then grabbed. Thermocline® for Windows  was used for processing viscosity data .2.6The gel obtained from the RVA experiment was stored overnight at room temperature in a 25-mL beaker with a 35-mm height and 30-mm internal diameter. The gel was compressed using a Brookfield CT3 Texture Analyzer  in two penetration cycles at a speed of 0.5 mm/s over a distance of 10 mm using a 12.7-mm-wide and 35-mm-long cylindrical probe. Gel texture parameters such as hardness, springiness, cohesiveness, and adhesiveness were recorded. According to the method given by 2.7Micro-doughLab 2800  was used to determine the optimum water absorption of flour blends to reach a peak of 500 BU using 4.00 ± 0.01 g samples with a 14% moisture basis. The masses of the sample and water were corrected based on the moisture contents of the samples. The manufacturer's rapid mixing protocol was used: the composite flour was mixed at 30 °C for 10 min at a constant speed of 64 RPM. A mixing curve was obtained that reflected the dough's bending properties: the peak (FU), water absorption (WA %), development time (min), stability (min), softening (FU), and mixing tolerance index  .2.8The pan bread was prepared using the Approved Methods of the American Association of Cereal Chemists International  Method 12.9The pita bread was made according to 2.103/g) was calculated according to The baking bread samples were cooled to room temperature for 1 h and weighed. The volume of bread samples was determined by the rapeseed displacement method. Specific volume . The L* indicates lightness, a* indicates redness, and b* indicates yellowness.2.12%Total Carbohydrate = [100 − % (Protein + Fat + Moisture + Ash + Fiber)].The moisture content, protein, fat, and ash for bread samples were conducted using 2.13Sample extraction: one gram of bread samples was mixed with 25 mL of ethanol in a shaker. The blend was centrifuged وcollected and filtered. According to ferric reducing antioxidant power (FRAP) and radical scavenging activity DPPH  was performed according to radical scavenging activity ABTS 2,2′-azino-bis  was used with the PASW® 18 software to compare the mean values.33.1The WHC of powdered material is given by the amount of water that can be retained under centrifugal or compressive forces. 3.2The pasting properties of the composite flours are presented in 3.3The texture of the composite flour gel was estimated according to parameters such as hardness, cohesiveness, adhesiveness, springiness, and gumminess 3.4Optimizing the rheological properties of the dough guarantees bread with a good volume, low density, and uniform grain structure. To reach the stage of optimum development, water was added to the optimal absorption level. The water absorption (%) for the corrected peak (500 FU) decreased with increasing YP substitution. The effect was significant (p < 0.05) with increasing YP substitution 3.5The bread volume indicates a foamy crumb structure, which appeals to consumers. The gluten network mainly provides the bread volume, which holds the gas during the final baking, and starch generally acts as filler. The bread volume, weight, specific volume, and water activity data are presented in 3.6The colors of the crust layer and internal foamy crumb for both the pan and pita bread were measured as a function of the L, a, and b parameters 3.73.8Pumpkin is rich in various bioactive fractions such as carotenoids, ascorbic acid, tocopherols, flavonols, and phenolic acids . In this3.9Sensory analysis is crucial to establishing a correlation between consumer preferences and the microstructure of the composite bread. The bread's sensory attributes and nutritional composition are greatly dependent on the composition, methods used for dough development, proofing, baking, and other additives. Bread organoleptic properties are generally divided into two sections due to the bread crust and crumb attributes. 44.1The presence of relatively high amounts of carbohydrates in the form of soluble and insoluble fibers in the YP may have increased the WHC of the composite flours. In general, soluble fibers have a higher WHC than insoluble fibers . The OHC4.2The viscosity reduction might be related to the lower total starch content, lower swelling rate, and higher proteins and fibers in the composite flour with YP substitution . Flour wThe setback viscosity is the viscosity of a paste cooled to 50 °C and held there for some time before the final viscosity is determined. The setback viscosity is correlated with the gelation process of the flour due to the recrystallization of the leached amylose from starch when the flour suspension is heated. A lower setback viscosity indicates less starch retrogradation. In the current study, the presence of YP increased the setback viscosity of the composite flour, although it was non-significant (p > 0.05). However, increasing the YP substitution led to the opposite trend, with a substantial decrease in setback viscosity at 15% YP substitution. Similarly, adding pumpkin seed cake to wheat flour has reduced the setback viscosity .The final viscosity represents the ability of composite flour to form a gel after cooking and cooling. The final viscosity data followed the peak viscosity trend and ranged between 2940 and 3607 cp. Increasing the YP substitution significantly reduced the final viscosity (p < 0.05) and gelling ability of the composite flour. The reduced final viscosity may be correlated with the dilution of starch, a lower degree of starch recrystallization, and lower gel hardness. Thus, a product with YP may have a softer and less brittle gel.The composite flours showed significant reductions in the pasting temperature (p < 0.05) compared with the control wheat flour. The reduced pasting temperature suggests the extent of cooking at lower temperatures, which is favorable for minimizing energy consumption during food processing. 4.3The hardness reduction suggests that the gels were more viscous. In a previous study, adding okra pod extract was found to reduce the firmness of wheat flour gels . SimilarHowever, the cohesiveness remained statistically the same for all gels regardless of the amount of YP substitution. Springiness represents the ability of a gel to retain its geometric shape after the external stress is removed. The least springy gel was with 10% YP. Gumminess is the product of hardness and cohesiveness. Thus, a reduction in the hardness would reduce the gumminess of the composite flour gels. Only the highest YP substitution level (15%) significantly reduced the gumminess (15.6) compared with the control gel. 4.4Also, 4.5YP addition may have hindered the stretching of the thin-walled gluten cells during baking and reduced the volume. 4.6The quick and uniform formation of Maillard pigments in the baked layer may explain the relative darkness of the crust. In contrast, In line with the crust data, the crumbs of the composite bread also showed reduced redness and yellowness, especially with increasing YP substitution. Conversely, the crust color was significantly darker for the composite bread than for the control bread (p < 0.05). It was reported that the addition of quinoa flour to the flatbread reduced the lightness, although the redness and yellowness were increased . The com4.7The most significant variation was observed with 5% and 10% YP substitutions, whereas increasing the substitution to 15% did not improve the prepared bread's proximate compositions. The increases in the fat and protein contents may be attributed to the higher fat and protein fractions of YP compared with those of wheat flour. In line with the current study, they enhanced protein, fat, and ash contents for their wheat-quinoa composite bread .4.8The formation of Maillard reaction products in the control bread facilitates bioactivity in vitro that may augment the wheat phenolics and flavonoids ; this ma4.9Incorporating YP did not compromise the crust color of both the pan and pita bread. However, significant modifications in the symmetry and uniformity were observed. The control pan bread had the highest break shred, whereas the lower volumes of the composite bread did not provide enough oven spring to break and shred the final loaf. The composite bread with 15% YP had the lowest break shred. The control and 5% YP bread for pita bread showed comparable break shreds. However, further increasing the YP substitution reduced the break shred. For the crumb, the composite pan and pita bread were significantly less liked by the panelists than the control bread. The higher WHC and relatively soggy crumb may have resulted in the less positive reception. The crumb grain develops by the uniform mixing and distribution of gas bubbles within the gluten network. The dilution of gluten in the composite flour retarded the OHC and uniformity of the grains. A poor loaf volume was observed with higher YP content, which may be an indirect indicator of an inconsistent and non-uniform grain structure for both types of bread.Bread with a softer texture and flexibility is preferred; however, increasing the YP substitution reduced the volume, which indirectly increased the firmness of the composite bread. The pan and pita bread with 15% YP substitution had the least preferred textures. 5With the increasing popularity of ready-to-eat bakery products, enriching them with underutilized fruits and vegetables high in fiber and bioactive ingredients is an attractive approach to improving public health. This study blended YP with wheat flour to develop nutritionally rich pan and pita bread. The composite flours showed better WHC and softer gels. The composite flour pastes provided lower consistency and viscoelasticity. The presence of YP reduced the dough strength and stability, which lowered the pan bread volume. However, the nutritional quality of the composite bread was greatly improved with enhanced antioxidant activity. Additionally, a low YP substitution (5%) maintained the overall acceptability of the composite bread. Thus, underutilized pumpkin-based products can be suitable for enriching bakery products with enhanced nutritional quality and bioactivity.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Technological breakthroughs such as high-throughput methods, genomics, single-cell studies, and machine learning have fundamentally transformed research and ushered in the big data era of biology. Nevertheless, current data collections, analyses, and modeling frequently overlook relative specificity, a crucial property of molecular interactions in biochemical systems. Relative specificity describes how, for example, an enzyme reacts with its many substrates at different rates, and how this discriminatory action alone is sufficient to modulate the substrates and downstream events. As a corollary, it is not only important to comprehensively identify an enzyme’s substrates, but also critical to quantitatively determine how the enzyme interacts with the substrates and to evaluate how it shapes subsequent biological outcomes. Genomics and high-throughput techniques have greatly facilitated the studies of relative specificity in the 21st century, and its functional significance has been demonstrated in complex biochemical systems including transcription, translation, protein kinases, RNA-binding proteins, and animal microRNAs (miRNAs), although it remains ignored in most work. Here we analyze recent findings in big data and relative specificity studies and explain how the incorporation of relative specificity concept might enhance our mechanistic understanding of gene functions, biological phenomena, and human diseases. For a gene to fulfill its functions, the gene product, i.e., a protein, RNA, or enzyme, must interact with proteins, RNAs, DNA elements, or other molecules, so characteristics of those interactions underline the mechanism. Use the enzyme:substrate relationship for analogy, the enzyme and substrate entities referring to interacting partners with the former being the party inducing conformational, catalytic, and/or functional changes in the latter. It is clear then that an enzyme seldom has only one substrate, and it is also intuitive that if an enzyme has multiple substrates, it will not interact with them equally. This phenomenon has been defined as relative specificity and generalized by the relative specificity hypothesis, which proposes that quantitatively different enzyme:substrate reactions have functional significance through impacting the substrates selectively and, hence, regulating the underlying biological processes and phenotypes .2, CO2, and CO, and CO associates much tighter than O2 with hemoglobin, which has a profound physiological implication. On the other hand, biology is dominated with complex enzymes and systems, such as transcriptional factors, RNA-binding proteins, protein kinases, which have hundreds or more substrates and interacting partners, and the ribosome and RNA polymerases with thousands or tens of thousands of substrates. Studying their relative specificity has been traditionally hampered by technological limitations and the ensuing avoidance of the subject  of the differential expression of over 4,000 RNAs underscores the importance of relative specificity.RNA-binding proteins regulate RNA metabolism and functions. YTHDF proteins bind m6A sites . Likewis6A sites . Another6A sites . HuR tar6A sites . Critica6A sites . That a S. pombe , e.g., the breasts and ovaries? And how do mutations in genes, e.g., TP53 and MYC which have a lot of targets, cause cancers? To the first question, an organ-specific factor may relay signals from germline mutations to trigger disease in that particular organ. Consistent with this view, a new study identified Pax8, a kidney-enriched transcription factor, as a downstream effector of VHL mutations in renal carcinogenesis (VHL in those organs. Furthermore, Pax8 is a transcription factor and counts MYC as a target (TP53 and MYC have been identified and studied for decades, but not a single one mediates or recapitulates the effects of TP53 or MYC mutations. From the relative specificity viewpoint, mutations in TP53, MYC, or other genes might induce unbalanced changes in the targets, and it might be the targets’ unbalanced changes that cause the diseases. So a new research angle is to quantify the relative levels or changes in many targets: the identity of individual genes still matters, just more must be considered together.Below we use three fundamental subjects to explain the importance of pursuing relative specificity. The first pertains to gene functions in general and in disease. While a gene, e.g., ogenesis . Tissue-a target . Thus, tS. cerevisiae, from which sequence-to-expression models were built (S. cerevisiae genes in 1,011 S. cerevisiae isolates and comparing gene expression changes (The second subject is transcription. A recent study cloned tens of millions of random 80-basepair-long DNA sequences into the promoter region of a yellow fluorescence protein reporter gene and measured reporter expression in re built . The aut changes . This paS. cerevisiae genes, and incubated the mRNAs consisting of up to 122-nucleotide-long 5’ untranslated regions in front of at least 24-nucleotide-long coding sequences with yeast extract for in vitro translation initiation reactions (in vivo? It was not documented in the paper, so we analyzed the correlation of RRS to protein expression in yeast (The last subject is translation. Differential translation has been investigated using bioinformatics and reporter libraries, but direct, large-scale validation in endogenous protein expression has rarely been performed. A latest study constructed a library of thousands of natural mRNA sequences from 4,252 eactions . The autin yeast . Figure in yeast , and Figin yeast . Both inAltogether, the above examples suggest that more evidence of relative specificity remains to be unearthed, and that we need a deeper understanding of its biological relevance. In pathways that involves multiple enzymes or steps, e.g., translation initiation, elongation, and termination, is there a step whose relative specificity is the most critical? As there is often a rate-limiting reaction in metabolism and cell signaling, is there a rate-limiting relative specificity? Lastly, if relative specificity is a general principle, is it evolutionarily conserved? Studies in animal miRNAs have begun to address these questions.miRNAs are a family of approximately 22-nucleotide-long RNAs that are widely expressed in metazoans, and they regulate biological processes by binding to complementary sequences, usually in the 3’ untranslated regions, in target mRNAs to repress their expression . miRNA bin vitro, which correlates with mature miRNA expression in vivo, revealing a role of Drosha’s relative specificity in regulating miRNA biogenesis (in vitro, as does human Exportin5 bind pre-miRNAs, yet there is no significant impact on miRNA expression associated with Dicer or Exportin5 action (We found that human Drosha cleaves hundreds of pri-miRNA substrates at different rates ogenesis . Human D5 action . A ratio5 action . Seconda5 action . Differe5 action , suggestin vivo (miRNA function similarly exhibits relative specificity. In cell culture models miRNAs such as miR-124 inhibit reporter genes containing the 3’ untranslated regions of approximately 200 target mRNAs to different degrees, and such differential inhibition correlates with the expression levels of the corresponding, endogenous target mRNAs in relevant human tissues . This rein vivo .in vitro, but in both animals only the preference of Drosha correlates with global, differential miRNA expression in vivo, just as in humans. Hence, relative specificity of the enzymes is evolutionarily conserved, so is, crucially, their relative contribution to the regulation of global miRNA production.Lastly, is relative specificity conserved through evolution? To answer this question cleavage of zebrafish and fruitfly pre-miRNAs and pri-miRNAs by Dicer and Drosha, respectively, were examined . Both DiStudies of miRNAs and other, completely different systems have converged on similar findings. Both Drosha and fission yeast Cdk1 have essential targets and perform essential functions, and neither acts like production-line robots. By intrinsic biochemical properties, Drosha ensures that miRNAs are generated at varying amounts, thereby regulating miRNAs, miRNA targets, and processes downstream , and CdkRelative specificity and its physiological importance have been documented in a tiny number but wide range of complex biochemical systems that have been sampled, and we have gained novel and unexpected insights into how biological processes are regulated. What is the origin of relative specificity? As it describes the interactions between molecules, the same physics and chemistry principles apply. In the earliest days when there were only one enzyme and one substrate, no relative specificity existed. But as the biological world increased in diversity, more substrates with different sequences and structures emerged, which naturally diverged in their interactions with the enzyme. Relative specificity might highlight a new layer of controlling mechanisms and complexity, as natural selection could act on relative specificity in addition to the more visible, sequence space to maintain stability while fostering evolution at the same time.In vitro studies cannot capture the intricacy that the levels of enzymes and substrates may fluctuate temporally and spatially, between and within cells (As important as relative specificity is, it works alongside with other regulatory mechanisms. in cells . For exain cells . Many biin cells . These cin cells , but togIn summary, new technologies and studies in the 21st century have generated massive data, now further aided by artificial intelligence to study complex systems and diseases. But these efforts have pitfalls because in most cases the information about relative specificity is missing or insufficiently utilized. Fortunately, relative specificity can be studied with current technologies and the right mindset, and its incorporation in the big data era will help us better understand and model complex systems.


The mechanism of atypical hyperplasia of the ovarian epithelium and ectopic endometrium caused by iron overload remains unclear. Accordingly, we investigated possible effects on the human ovarian ectopic endometrium and ovarian epithelium by producing a high-iron environment with rat ovaries.  Human ovarian ectopic endometrium with atypical hyperplasia was collected, and the correlation between transferrin receptor GPR30 and Pi3K protein expression was studied by immunohistochemistry staining. Twenty SPF Sprague–Dawley female rats were microinjected with iron into one side of the ovary once a month, and the other ovary was used as the control. After 10 months of microinjection, the iron histological analysis was examined by Prussian blue staining, and ovarian endometrium morphology was assessed by HE staining. Abnormal lesion changes were measured by Pi3K staining. Evaluation of GPR30 was performed using reverse transcription PCR (RT-PCR) and western blotting, and the interrelationship between GPR30 and Pi3K was also assayed. P = 0.001).  GPR30 was significantly increased and correlated with the transferrin receptor and Pi3K in atypical human ovarian ectopic endometrium. Iron overload was confirmed in the 20 microinjected ovary cortexes, epithelial hyperplasia was observed in 12 ovaries, and papillary atypical hyperplasia was noted in eight ovaries. The RNA and protein levels of GPR30 were significantly increased in atypical hyperplasia compared to hyperplasia tissue samples. A positive relationship between GPR30 and Pi3K was found ( The results suggest that persistent iron exposure may be a potential stimulus for ovarian endometriosis with atypical changes, and the abnormal increase in the new estrogen receptor GPR30 is closely related to this process. Endometriosis is a very common gynecologic disorder in reproductive-age women that is defined as the implantation of endometrium-like glandular and stromal cells outside the uterus. Although benign in nature and clinical behavior, endometriosis is associated with an increased risk of malignant transformation. Molecular and epidemiological studies have also provided a strong association between atypical endometriosis and ovarian cancer , 2. It hEndometriomas, also called chocolate cysts or endometrioid cysts, repeatedly bleed during the menstrual cycle and are gradually filled with thick, viscous, and decomposed blood that is often referred to as “old blood,” which appears dark, brownish red, or chocolate-like. After decomposition, the old blood forms and accumulates a large number of iron metabolites. Iron is the most abundant heavy metal in mammals and is an essential element and critical component of the molecules involved in energy production, the cell cycle, and intermediate metabolism. However, excess of iron results in toxicity and is linked to pathological disorders. Nonprotein-bound “free” or “catalytic” iron functions to damage biomolecules with a hypothetical “site-specific” mechanism . AdditioIn recent years, GPR30, a 7-transmembrane G protein-coupled receptor family member, has been reported to be related to rapid and transcription-mediated responses to estrogen in certain circumstances , 9, whicA total of 20 atypical ectopic endometrial samples were obtained from the Pathological Diagnosis Center of Chongqing Medical University, and this study was approved by the ethics committee of Chongqing Medical University. The inclusion of atypical endometriosis cases was performed by two pathologists with a double evaluation . In addiAll animal experiments were conducted in accordance with the approval and guidelines of the Animal Care and Use Committee at Chongqing Medical University. All strategies were made to minimize the number of animals used and their suffering.Twenty SPF Sprague–Dawley female rats that were two months of age (weighing 160 g to 180 g) were provided by the animal center of Chongqing Medical University  and housed in a 12-hour light/dark cycle under controlled temperature and humidity with free access to food and water.μl  of iron dextran for injection was microinjected into the subenvelope of the ovary and superficial ovarian tissue. The cut was then sutured with a 4/0 nylon thread. The other side, the left-side ovary, was the control, and glucose water was microinjected  using Prussian blue staining. Paraffin sections were dewaxed, rehydrated with water, stained with liquid Perl's solution for 20–30 min, thoroughly washed in distilled water after 0.5% basic fuchsin solution for 30–50 s, and followed by rapid differentiation with 95% alcohol, dehydration with anhydrous alcohol and clearing with xylene. Sections were observed under an inverted optical microscope after neutral gum cementing.μm thick serial sections were stained with hematoxylin and eosin (HE stains) for histological evaluation. Next, phosphoinositide 3-kinase (Pi3K) immunohistochemical (IHC) staining was carried out. Briefly, after inhibition of endogenous tissue peroxidase, unmasking of the antigen and inhibition of nonspecific reactions in a blocking solution containing 10% normal goat serum and 1% bovine serum albumin were conducted, and sections were kept overnight in a 1/500 dilution of monoclonal Pi3K antibody . Staining was performed with peroxidase-labeled secondary antibody and liquid diaminobenzidine as the chromogen. The twenty control ovaries were treated in the same way.Twenty ovaries (iron microinjected) were necropsied, put into 4% buffered paraformaldehyde, and embedded in paraffin. Then, 10 Σ (i + 1) × pi, where i represents the intensity of staining, pi is the percentage of positive cells stained/total number of test cells, and the correction factor is 1.As above, immunohistochemical staining at 1 : 200 for GPR30 (sc-48525) and transferrin receptor  was performed using avidin-biotin-peroxidase methods. The entire tissue section was evaluated by objective Hscore to measure the staining intensity, and the percentage of stained tumor cells was positive. Five fields of view of each section were randomly selected, the magnification was 400×, and the number of positive cells was counted per 100 cells per field. Staining intensity was classified as negative (<5% positive tumor cells), 1+ (mild), 2+ (medium intensity), or 3+ (greater than the intensity of the positive control). Two pathologists at the Pathological Diagnosis Center of Chongqing Medical University used double-blind methods to evaluate the sections to determine the immunohistochemical results. The formula was Hscore = β-actin, 95°C, 5 min; 95 and 59.5°C, 30 s; 72°C, 30 s; and 72°C, 10 min.Total RNA was extracted from microdissected ovarian epithelium  stored at -80°C using the RNAiso plus  extraction method, as described in the manufacturer's directions. Briefly, the total RNA was extracted with RNAiso plus, precipitated with isopropanol, washed in ethanol, and resuspended in RNase-free water. The quantity and quality of RNA were determined by spectrophotometry. Two micrograms of total RNA was used for reverse transcription (RT) with RT kits  as described in the manufacturer's directions. The specific primers for GPR30 mRNA were F1 5′-CACCCCTCCGCCTGGAGAGC-3′, R1 5′-CACAGTCCCGCGTGGAGC-3′, and the product size was 346 bp; the specific primers for the beta-actin gene were F1 5′-CTCGTCATACTCCTGCTTGCTG-3′, R1 5′-CGGGACCTGACTGACTACCTC-3′, and the product size was 546 bp. For amplification of both GPR30 and the reference gene beta-actin, the following PCR protocol was applied: GPR30: 95°C, 5 min; 95°C, 30 s; 56°C, 30 s; 72°C, 30 s; 72°C, 10 min; μg per sample). Anti-β-actin antibody was obtained from Sigma-Aldrich . The antibody used was directed against rabbit anti-human GPR30 IgG . The secondary antibody was an anti-rabbit immunoglobulin horseradish peroxidase-linked F (ab) 2 fragment (Amersham Biosciences) from donkey. Western blotting reagents were obtained from an electrochemiluminescence kit (Amersham Biosciences).Total protein extracts from the ovary tissues (the samples were the same as RT-PCR) were stored at -80°C, and equal amounts of the protein obtained using lysis buffer were analyzed by immunoblotting  was analyzed with contingency tables.The outcome of IHC staining and RT-PCR was carried out by semiquantitative analysis with Image-Pro Plus 6.0 and SPSS 16.0 software. Data are expressed as the mean ± SEM. To compare means between the two groups, we used an independent-samples P <0.05)  .P = 0.001, Tables For the atypical ectopic endometrium, the HScore analysis suggested that the expression levels of GPR30 and Pi3K were both significantly increased. The pairwise correlation between the expression of GPR30 and iron metabolites and Pi3K was examined, and the expression of GPR30 significantly correlated with TfR and Pi3K immunopositivity in the atypical ectopic endometrium group . The relative expression levels of GPR30 mRNA and protein in the atypical group were 0.831 ± 0.003 and 0.838 ± 0.001, respectively; those in the hyperplasia group were 0.132 ± 0.025 and 0.127 ± 0.003, respectively; and those in the control group were 0.056 ± 0.012 and 0.047 ± 0.003, respectively,  and in only 1 of the 10 hyperplasia specimens examined. The perspective that Pi3K is closely related to the occurrence and development of tumors indicates that persistent iron overload may lead to precancerous lesions or early malignant transformation of the ovarian epithelium.To further study and confirm the expression of GPR30 and iron metabolites in atypical ovarian endometriotic cysts, we established an ovarian model of sustained iron overload, and iron-induced histomorphology changes were observed in the ovarian epithelium. For the 20 ovary models, iron overload was identified by Prussian blue staining, epithelial hyperplasia was observed in 12 ovaries, and atypical papillary hyperplasia was observed in 8 ovary samples. We also characterized the difference in Pi3K staining between hyperplastic and atypical hyperplastic epithelium. We found that Pi3K was significantly expressed in all atypical hyperplasia specimens (GPR30 gene has not been well studied or evaluated in endometriosis lesions, such as atypical changes. To achieve this objective, our previous study described the difference in GPR30 expression between benign and malignant ovarian endometriosis cysts. We found significant GPR30 expression in malignant ovarian endometriotic cysts compared to similar benign cysts, indicating that GPR30 may produce a wide range of responses to estrogen in endometriosis-associated ovarian carcinoma.In recent years, GPR30, the G protein-coupled estrogen receptor, has been shown to mediate both rapid and long-term transcriptional events against estrogen under certain circumstances. GPR30 has also been involved in estrogenic signaling in malignant cells, such as breast cancer and endometrial cancer , 21, sugGPR30 at the gene level in iron-induced atypical ovarian epithelium. We found that the mRNA and protein expression levels of GPR30 were significantly higher in the atypical papillary hyperplasia group than in the typical hyperplasia and control groups. This finding suggests that there was transcriptional activation of GPR30 gene expression in atypical hyperplasia, which led to a significant increase in the secreted expression of GPR30, as detected by western blotting, which is consistent with the changes in GPR30 expression levels in human atypical ovarian endometriosis tissues. Moreover, our results also demonstrated that GPR30 protein upregulation was significantly correlated with Pi3K in the iron-induced atypical ovarian epithelium. Increased Pi3K activity is associated with a variety of cancers and plays an extremely important role by mediating the carcinogenic processes of tumorigenesis, invasion, and metastasis, especially in the proliferation of normal epithelial cells to cancer cells [Additionally, in the present study, we further investigated a possible sequential progression from endometriosis through typical to atypical endometriosis by assessing er cells . In addier cells , 25.Our previous study first hypothesized that GPR30 may be involved in the major pathologies of atypical changes in human ovarian endometriosis tissues and in the rat ovarian epithelium with persistent microinjection of iron. The significant association between GPR30 and the iron metabolites TfR/Pi3Ks suggests that GPR30 may be related to atypical hyperplasia of the ovarian epithelium under iron overload, which sheds light on the molecular mechanisms of precancerous transformation associated with ovarian endometriosis. However, the sample size should be expanded, and the relationship between GPR30 and iron metabolism in ovarian endometriosis should be further studied.


The isochore theory, which was proposed more than 40 years ago, depicts the mammalian genome as a mosaic of long, homogeneous regions that are characterized by their guanine and cytosine (GC) content. The human genome, for instance, was claimed to consist of five compositionally distinct isochore families. The isochore theory, in all its reincarnations, has been repeatedly falsified in the literature, yet isochore proponents have persistently resurrected it by either redefining isochores or by proposing alternative means of testing the theory. Here, I deal with the latest attempt to salvage this seemingly immortal zombie—a sequence segmentation method called isoSegmenter, which was claimed to “identify” isochores while at the same time disregarding the main characteristic attribute of isochores—compositional homogeneity. I used a series of controlled, randomly generated simulated sequences as a benchmark to study the performance of isoSegmenter. The main advantage of using simulated sequences is that, unlike real data, the exact start and stop point of any isochore or homogeneous compositional domain is known. Based on three key performance metrics—sensitivity, precision, and Jaccard similarity index—isoSegmenter was found to be vastly inferior to isoPlotter, a segmentation algorithm with no user input. Moreover, isoSegmenter identified isochores where none exist and failed to identify compositionally homogeneous sequences that were shorter than 100−200 kb. Will this zillionth refutation of “isochores” ensure a final and permanent entombment of the isochore theory? This author is not holding his breath. For exad, e.g., , most ofThe isochore theory in all its reincarnations has been repeatedly falsified, e.g., ,9,10,11.Many research papers published before genome sequencing became available used the GC content at third-codon positions (GC3) in protein-coding genes as a proxy for genomic GC content ,15. The Many analyses of the first-draft sequence of the human genome  cast serCohen et al., analyzed the GC composition of the human genome  by usingCohen et al.’s  segmentaAs an alternative to Cohen et al.’s  algorithA “new-and-improved” algorithm by the isochore proponents, isoSegmenter, was put forward by Cozzi et al. . isoSegmOne-hundred simulations were generated for each category of equal-length and variable-length domain sequences, for a total of 1100 equal-domain-length and 900 variable-domain-length sequences. The simulated sequences generated were segmented using isoPlotter and isoSegmenter, and the predictions of each algorithm were compared to the ground truth and scored. A summary of the overall performance metrics of both algorithms is shown in t-test. Overall, isoPlotter had a 10–20-fold higher median sensitivity, precision rate, and Jaccard index compared to isoSegmenter, and the differences in these performance metrics were all highly statistically significant.I note that the medians of all three performance metrics for isoSegmenter were much closer to zero than to the mean, implying that all the performance metrics for isoSegmenter were skewed upwards by high-performance outliers. The medians for isoPlotter, on the other hand, were much closer to the mean, indicating more consistent performance. Because of this skew in isoSegmenter, the Wilcoxon test was used instead of the standard two-mean Let us now examine how domain size influences the performance of isoPlotter and isoSegmenter. With very large domain sizes (above 300 kb), isoSegmenter exhibited high performance metrics . As the To better understand the performance changes across domain lengths, the average predicted domain lengths for both isoPlotter and isoSegmenter on the equal-length datasets were plotted against the true domain sizes . IdeallyIn this study, I used a series of controlled, randomly generated simulated sequences to conduct a benchmark comparison of isoPlotter and isoSegmenter, two compositional segmentation algorithms that deliver highly contradictory predictions. The main advantage of using simulated sequences is that, unlike real data, the exact start and stop point of the isochore or homogeneous compositional domain is predetermined. This enables me to directly compare the domain predictions of the two algorithms.The performance of isoSegmenter against the benchmark sequences was dismal. In comparison, isoPlotter exhibited a much higher performance because of its ability to deal with a wider range of domain lengths. It did, however, exhibit a systematic tendency to predict domain sizes that were slightly smaller than the ground truth.Cozzi et al., criticized algorithms such as isoPlotter as having “no biological relevance” . HoweverThese systematic failures of isoSegmenter raise questions regarding the validity of the findings of , as wellDrosophila genome [Interestingly, this algorithm has an inbuilt self-delusionary routine that discovers “isochores” in random sequences, in artificial sequences, and in literary masterpieces. For example, isoSegmenter and its parent algorithm  were usea genome , which aMoby-Dick into DNA by using the method in [As an exercise in satire, I translated Herman Melville’s 1851 novel ethod in  and applWill the isochore theory be finally put to rest? We are not optimistic; brain-eating zombies are notoriously hard to kill.For my first analysis, I generated 1 Mb-long DNA sequences. Each sequence was divided into equal-length segments. Several divisions were used: 1, 2, 4, 5, 10, 20, 25, 40, 50, 80, and 100. If, for example, a simulated sequence was divided into 10 domains, it was composed of ten 100-kb equal-length domains. Each domain was randomly assigned to one of the five isochore families  with GC contents of 22.8%, 33.2%, 22.7%, 11.2%, and 3.01% . The prexmin = 10,000 bp and α = −2.55 using the inverse cumulative distribution function. Afterwards, the domain lengths were normalized so that their total sequence length was 5 Mb.For the second analysis, a more realistic segmentation method was used. This segmentation method partitioned the initial genomic sequence into domains of varying lengths according to a power-law distribution with an exponent of α = −2.55, as stipulated in . For thiExamples of GC profiles for equal-length and variable-length simulated segments are shown in Both isoPlotter and isoSegmenter were run on each of the simulated sequences. Following Elhaik et al. (2010), a predicted domain was deemed a true positive (TP) if both its boundaries matched the actual boundary positions of the domain within ± 5% of the predicted domain size. A false positive (FP) was a predicted domain for which one or both predicted boundaries did not match the ground truth boundaries. A false negative (FN) domain was a ground-truth domain that was not successfully predicted by the algorithm .Three key performance metrics, i.e., sensitivity, positive predictive value (or precision rate), and the Jaccard index, were compared side by side on identical benchmark simulations for isoPlotter and isoSegmenter as follows:


In order to contribute to the reduction of nutritional deficiencies in Morocco, this study was undertaken to develop a healthier tortilla with higher iron and protein, while maintaining adequate technological and sensory qualities. Composite durum wheat flour enriched with 20, 25, 30, and 35% chickpea flour was assessed for nutritional, functional, and technological properties. Then, we selected two composite blends of 75:25 and 70:30 of durum wheat and chickpea flours for making tortillas to study nutritional, technological, and sensorial qualities. In addition, we studied the effects of making and cooking process and storage time. Kruskal–Wallis and Mann—Whitney tests were used for data analysis, and GraphPad Prism was used to create graphs. The results showed that composite tortilla had significantly higher nutritional value than durum wheat tortilla, and the best ratio was 30% chickpea flour. At this ratio, the results showed the best cooking time and the best yellowness, but tortilla fluffiness and puffiness decreased. Tortilla processing significantly increased protein at 30% chickpea flour, while minerals except sodium, weight, and diameter decreased. Adding 30% chickpea flour to durum wheat tortilla improved flavor. Then, storage decreased the weight resulting in decreased flexibility, and sanitary quality was lost early for 30% chickpea flour. In conclusion, adding 30% chickpea flour to durum wheat flour results in a healthier and tastier tortilla, which should be consumed fresh. Triticum durum Desf.) is one of the most important food crops in the Mediterranean region because of its various end-products such as bread, pasta, macaroni, and couscous. In Morocco, durum wheat is one of the oldest cultivated cereals [Cicer arietinum L.) is nutritionally rich in terms of protein, dietary fibers, essential minerals, and vitamins [Durum wheat  variety “Farihane”, listed in the official national catalog, were milled into whole meal flour using UDY Cyclone and mini–Hammer mill equipped with a 1 mm and 0.5 sieves respectively. Durum wheat flour (control) was substituted for four combinations of 20, 25, 30, and 35% of chickpea flour. Nutritional, functional, and technological properties were evaluated, in triplicate, for each composite compared to the checks, durum wheat and chickpea flours. Then, nutritional, technological, and sensory qualities were assessed for tortilla made from such durum wheat-chickpea blends.Flours and composite flours were analyzed for quality traits including physico-chemical composition  following American Association of Cereal Chemists (AACC) approved methods . Total cBulk density was determined using  method. Swelling capacity was determined using  method. Water/oil absorption capacity was determined using  method. Foaming capacity was determined using  method wFoaming stability was determined using  method ww/v) were prepared in distilled water, adjusted to pH 7.0, and mixed in a Waring Blender at the highest speed for 2 min. About 5 mL each, of dispersions were poured into three test tubes and heated to 100 °C in a water bath for 1 h and cooled to 4 °C in an ice bath. The lowest concentration at which all dispersions in triplicate formed gels that did not collapse or slip from inverted tubes was reported as least gelation concentration.Least gelation concentration was determined using  method. Gelatinization temperature was determined using  method. Color measurements were carried out using a calibrated Minolta Color Reader CR 400   based onGluten strength was evaluated using SDS Sedimentation Test according to the Moroccan Standard 08.1.217 . This teDurum wheat tortilla and composite durum wheat tortilla using 25 and 30% chickpea flours were produced adopting a traditional homemade procedure with the following ingredients: Flour 100 g, salt 1 g, olive oil 20 mL, water 50 mL (variable), and baking powder 0.6 g.Technological process: Tortilla doughs were prepared based on  process Moisture, crude ash, and crude proteins were determined following AACC approved methods . Mineral3. Specific volumes were determined by dividing volumes by corresponding weights (cm3/g) [Amount of water added to flours was measured for each test. Tortilla weights were determined , and the (cm3/g)  using thDiameter and thickness were measured after cooking, and color measurement was carried out using a calibrated Minolta Color Reader CR 400 .Protein and mineral values in tortillas were compared with those in flours, and their weight, diameter and thickness values were compared with those of doughs.Tortillas were stored under the same conditions at room temperature, wherein weight was measured, and appearance, odor, and flexibility were observed, each day.Tortillas made with 0, 25, and 30% chickpea flours were subjected to sensory assessment by an untrained panel of 50 people accustomed to eating tortillas. Sensory characteristics namely, dark spots, flexibility, puffiness, layering, weight, mouth feel, aroma, and flavor were rated as 1 = excellent, 2 = high, 3 = medium, 4 = poor, and 5 = very poor .p-value ≤ 0.05. The GraphPad Prism software  was used to create graphs. Kruskal–Wallis’s test was used to show if there are statistically significant differences between groups, followed by Mann–Whitney test for comparing means; expressed by mean ± SD, using the SPSS software . Statistical significance was defined as p ≤ 0.05) increase in ash (8.16–26.53%) and proteins (19.88–41.60%) in all combinations, and in fat (22.22%) and energy (0.27%) at 30 and 35% ratios, respectively. In contrast, a significant (p ≤ 0.05) decrease in carbohydrates (3.20–7.14%) was observed in all combinations.Chickpea flour had more ash, proteins, fibers, fat, and energy, and less moisture and carbohydrates than durum wheat flour . Adding Similar trends were obtained for ash, proteins, fibers, fat, carbohydrates, and energy in  study. Ap ≤ 0.05) increase in both iron (3.84–6.41%) and magnesium (6.03–8.03%) contents at 25% ratio (p ≤ 0.05) decrease was noticed in zinc (13.26%) at 35% ratio  increase in total phenolic compounds (5.88–9.80%), condensed tannins (2.80–4.80%), total flavonoids (66.67–133.33%), and antiradical activity (24.04–50.12%), in all combinations.Furthermore, chickpea flour had more phenolic compounds, condensed tannins, total flavonoids, and antiradical-activity than durum wheat flour . ComposiThese results are in line with the study of . An incrp ≤ 0.05) increase in foaming capacity (8%) and foaming stability (28.20%) at 30% ratio, least gelation concentration (100%) and gelatinization temperature (1.13–1.59%) in all combinations. In contrary, a significant (p ≤ 0.05) decrease was revealed in bulk density (3.75–6.25%) in all combinations, swelling capacity (0.83%) at 35% ratio, water absorption capacity (15.30%) at 30% ratio, and for oil absorption capacity (9.55–13.38%) in all combinations.The results showed higher foaming capacity, foaming stability, least gelation concentration and gelatinization temperature values in chickpea flour than durum wheat flour, and lower values of bulk density, swelling capacity, and water and oil absorption capacities . ComposiThese results are in accordance with the results of bulk density, water absorption capacity, oil absorption capacity, foaming properties, least gelation concentration, and gelatinization temperature reported by ,40,41,42p ≤ 0.05) increase in redness (3.49%) at 35% ratio and in yellowness (3.93–7.03%) in all combinations.Durum wheat flour showed more redness, yellowness, and lightness than chickpea flour . ComposiSimilar results were reported in  study. Tp ≤ 0.05) gluten strength.Sedimentation test values  ranged fThis decrease in gluten strength is unequivocally due to the replacement of durum wheat gluten by chickpea proteins. Considering quality classes of durum wheat as proposed by , 100% dup ≤ 0.05) more moisture, ash, protein, iron, copper, manganese and potassium, and less zinc than sole durum wheat tortilla. Moreover, tortilla enriched with 30% chickpea flour revealed significantly (p ≤ 0.05) more moisture, protein, iron, manganese and potassium, and less zinc than tortilla enriched with 25% chickpea flour  . An incrp ≤ 0.05) the high cooking time and low weight values compared to tortilla enriched with chickpea flour. The lowest cooking time and the highest weight values were recorded significantly (p ≤ 0.05) for tortilla enriched with 30% chickpea flour, whereas volume and specific volume values were significantly (p ≤ 0.05) higher for durum wheat tortilla, and lower for tortilla enriched with 30% chickpea flour. Concerning the parameters of color, enriched tortilla showed significantly (p ≤ 0.05) more yellowness  increased protein for tortilla enriched with 30% chickpea flour, while minerals except sodium significantly (p ≤ 0.05) decreased in all combinations. Baking process significantly (p ≤ 0.05) decreased weights and diameters in all combinations.Tortilla processing significantly (An increase in protein value is probably caused by the loss of minerals, soluble fibers, and sugars during cooking . An incrResults presented in The weights obtained on the 2nd day are likely because gluten has the strongest imbibition power compared to protein from other sources . The decBased on the previous results, tortillas were prepared with enriched chickpea flours of 25 and 30% ratios. Assessment of sensorial quality traits  revealedThis study aimed to produce enriched tortilla from durum wheat-chickpea flours. Based on the quality analysis of durum wheat-chickpea composite flours using different combinations, 25 and 30% ratios of chickpea flour were chosen for making enriched tortillas. Nutritionally, many improvements were obtained especially higher ash, protein, iron, copper, manganese, and potassium, being beneficial for malnourished people. Technologically, 30% ratio yielded better results for yellowness and cooking time, and weights were not negatively influenced. In addition, tortillas making and cooking improved protein content, while weights and diameters were not negatively influenced. During storage, no negative impact on tortillas weights was observed, but their decrease led to a decrease in flexibility, and sanitary quality was lost early for 30% chickpea flour. Sensorially, the choice of enriched tortillas differs with consumers based on the evaluated sensorial characteristics. Overall, durum wheat-chickpea-enriched tortilla is feasible, providing an innovative cereal product with added nutritional value, while keeping or even improving many parameters for other aspects of quality. So, the amalgamation of legumes-based products into our daily diet is recommended, and further research in novel durum wheat-pulses-based products with high nutritive value should be strengthened.


The effects of ultraviolet-B (UV-B) radiation on the physiological properties, phenolic acid accumulation, and dough-making quality of wheat during germination were investigated. UV-B radiation inhibited the wheat sprout length and reduced the dry matter loss. As phenolic acids were principally present in the kernels' bran, UV-B radiation could promote their accumulation in the interior of germinated wheat (GW). The total phenolic compounds, ascorbic acid, and antioxidant activity were also enhanced significantly during germination with UV-B. UV-B improved the development time, stability time, rheological properties, and viscosity of GW, and inhibited the α-amylase activity, the destruction of the amorphous region of starch particles, and the proteins degradation process during germination, and thus the deterioration of dough-making quality caused by germination was inhibited. Therefore, UV-B radiation could be a potential approach to enhance the nutritional and dough-making quality of germinated whole wheat flour. Whole grains contain unique bioactive ingredients that promote health, presenting a more complex and beneficial nutritional profile than refined grains. A moderate daily intake of whole-grain foods has been linked to lower blood pressure, which in turn reduces the risk of cardiovascular disease . Among wThe processing of whole wheat flour can affect the composition and nutritional value of whole wheat products . CompareGermination is the process of enhancing the bio-accessible minerals and nutrient content absorbed by the human body in plant foods . It can Ultraviolet-B (UV-B) radiation on wheat kernels during germination may weaken the adverse effects of germination on the processing quality of whole wheat flour. The effect of UV-B radiation, which is considered as stress on plant seed germination and growth, is a hot area of research at present, and the impact mechanisms of plants in response to UV-B stress are also different. UV-B stress will affect plant growth, from the morphology of plant parts to physiological and biochemical nutrients, and other aspects. UV-B radiation makes a difference to plant morphology and structure, physiological metabolism, and growth state . So far,Although UV-B radiation has been proven to exert influence on plant growth, the mechanism of the effects of germination and UV-B radiation on the physiological activity, phenolic acid content, and dough-making quality of whole wheat flour is not clear. To obtain higher nutrition and better processing quality of whole wheat flour, we studied the evolution of the nutritional composition and quality characteristics of germinated whole wheat flour during UV-B radiation.Triticum aestivum L. cv. Zhongmai 998) was cultivated by the Hebei Woyu Agriculture Technology Co., Ltd. .Wheat (2) for 3 h every 12 h period  for 6 h, then placed on a tray lined with wet gauze in a constant temperature and humidity incubator  for germination . Pure water was sprayed every 6 h to keep the gauze moist and the wheat kernels germinated for 0, 12, 24, and 36 h, respectively. Some soaked kernels were germinated under dark, and others were radiated by UV-B  at different germination and radiation time were measured using a vernier caliper with three repeats. Dry matter losses were calculated according to the difference in dry matter weight between dried UW and GW, with three repeats, and 100 granules were measured for each repeat.Wheat kernels were milled in an MLU202 test mill  to obtain 1B, 2B, 3B, 1M, 2M, and 3M coarse bran and fine bran flours. 1B, 2B, and 3B streams belonging to the break system were the flour obtained by milling the wheat 1, 2, and 3 times with the break roll of the above test mill, respectively. 1M, 2M, and 3M streams belonging to the milling system were the flours milled 1, 2, and 3 times with the milling roll from the fractions that could not be sufficiently milled with the break roll, respectively. 1B, 2B, 3B, 1M, 2M, and 3M coarse bran and fine bran flours of all samples were mixed as whole wheat flour. The flours were placed at −18°C for subsequent analysis.Different layers of flours were divided into three groups . Different groups of wheat flour samples (2 g)  were extracted with 80% methanol (10 ml) for 3 times , respectively, under the dark . Then the extracted solutions were centrifuged  and the supernatants extracted three times were mixed and rotatably evaporated to dry at 40°C. Next, they were dissolved in 50% methanol (10 ml) and used as free phenolic extracts at constant volume. After being filled with nitrogen, the samples were stored at −18°C for subsequent research.The precipitates obtained by centrifugation three times were mixed and hydrolyzed by 20 ml sodium hydroxide (2 mol/L) under dark . The pH of the hydrolyzed solutions was adjusted with a hydrochloric acid solution (6 mol/L) at a range of 1.5–2.0. Then 25 ml ethyl acetate was mixed with the solution for 15 min and centrifuged . The upper layers of ethyl acetate were rotatably evaporated to dry at 40°C and then dissolved with 50% methanol (10 ml). After being filled with nitrogen, all samples were stored at −18°C. Phenolic acid contents were determined by the high-performance liquid chromatography (HPLC)  and loaded on a Gemini C18 column  at 280 nm .The contents of AsA in the ungerminated and germinated whole wheat flour were measured using the method of dinitrophenylhydrazine . The extThe contents of TPC in UW and GW flour were analyzed using Folin–Ciocalteu's phenol reagent . The abs0min at 515 nm in a UV/Vis spectrophotometer. After 30 min of incubation, the absorbance of the sample was measured as A30min.The whole wheat flour was evaluated spectrophotometrically by their shoot. According to the method described by Pajak et al. , UW and The whole wheat flour was added into the farinograph equipped with a mixing bowl  in the corresponding quantity according to the water content (ICC 115/1). The thermostat with a circulating water pump kept the temperature of the bowl steady at 30°C. Development time, stability time, and degree of softening of wheat dough were determined.The dynamic rheological properties of whole wheat flour were measured using a dynamic rheometer . UW was used as control. The whole wheat flour with different radiation and germination time was mixed with water (64 g/100 g flour) to prepare dough. Dough samples (3 g) were placed between the plates after 10 min of wakening to eliminate the stress generated. Before the test was started, the edges of the excessive dough samples were scraped off with a plastic sheet and were applied a thin layer of liquid paraffin wax. The frequency sweep was carried out at a frequency of 0.1–100 Hz at 25°C, using parallel plate geometry (40 mm diameter and a gap of 2 mm).The gelatinization properties of whole wheat flour were measured by a Rapid Visco Analyzer . The suspensions were prepared from pure deionized water (25 g) and whole wheat flour (3.5 g), which were manually homogenized using a plastic paddle. The tests were performed in a programmed heating and cooling cycle.Thermal properties of whole wheat flour were analyzed to evaluate gelatinization temperature and enthalpy. Every flour sample (~4 mg) and deionized water with a water-sample ratio of 2:1 were precisely weighed and kept in a sealed DSC pan. All pans were equilibrated at 25°C for 12 h before being analyzed and then heated from 25 to 105°C.The X-ray diffractometer equipped with a divergence slit  was operated at 40 kV and 40 mA. All whole wheat samples were scanned from 4° to 40° (2θ) at a speed of 2°/min and a step size of 0.02°. The diffractograms were analyzed and the relative crystallinities were calculated using the software of MDI Jade 6.5. The relative crystallinity (RC) (%) was calculated using the percentage ratio of the crystalline and amorphous regions. The RC of the samples was calculated using the following equation:where Ac is the area of the crystalline peak and Aa is the area of the amorphous peak.g), the supernatants  of each sample were injected into a TSK G4000-SWXL column . The PBS solution was achieved as elution of the samples with a flow rate of 0.7 ml/min, at 30°C, 214 nm signal intensity  equipped with a UV detector. All whole wheat flour samples (15 mg) were extracted with 1 ml phosphate buffer saline  consisting of 1.0% SDS (w/v). All solutions were stirred with a constant temperature mixer  . After centrifugation  to obtain the supernatants (Whole wheat flour samples (1 g) were added to distilled water (50 ml) and the suspensions were adjusted to pH 6.0 by 0.1 mol/L HCl and NaOH. Next, the suspensions were shaken adequately and centrifuged . All data were means of at least three measurements. Significance was determined by one-way analysis of variance (ANOVA) and Duncan's test, and the As shown in The contents of free and bound phenolic acids in different layers of wheat flour are shown in As shown in p-hydroxybenzoic acid, vanillic acid, and syringic acid , the increase rate of gic acid  in the bl et al.  reportedl et al. . TherefoUV-B radiation and germination time interacted significantly on AsA accumulation . The conP < 0.05), and the effect was time-dependent. As shown in Germination generated an increase in TPC  was one of the important indexes to measure the quality of wheat gluten. The higher the value of tanδ, the higher the degree of deterioration of wheat gluten. Therefore, The α-amylase is commonly deemed as one of the principal factors for wheat flour production, which can hydrolyze large starch molecules, resulting in a dramatic reduction in viscosity . As showFurthermore, the influence of germination and UV-B radiation on gelatinization characteristics (RVA parameters) is shown in -amylase.As shown in Meanwhile, the results of thermal characteristics analysis showed that the onset temperature, peak temperature, and conclusion temperature decreased with the extension of germinating time, which indicated that wheat starch needed less energy to start gelatinization and had lower thermal stability to resist glass transition after germination. This was due to the destruction of amorphous areas during germination, which shortened the diffusion time of water in starch particles and accelerated starch gelatinization . The gelSize exclusion high-performance liquid chromatography is a simple and efficient method for protein molecular weight fractionation. As shown in p-coumaric acid, and sinapic acid), TPCs, AsA, and enhanced the DPPH radical-scavenging activity of GW. In addition, UV-B radiation inhibited the α-amylase activity, protein degradation process, and dough weakening and improved the mixing stability, so the dough-making quality of whole wheat flour during germination was enhanced. Therefore, as a benign inducer in plants, UV-B radiation could be an alternative approach to produce whole wheat flour with higher nutritional value and better dough-making quality.Germination and UV-B radiation exerted a clear influence on the physicochemical properties, the accumulation of phenolic substances, and the dough-making quality of wheat. Under UV-B radiation, germination and dry matter loss of wheat were inhibited. Meanwhile, UV-B radiation improved the contents of free and bound phenolics , Young Elite Scientists Sponsorship Program by CAST (2019QNRC001), the Program for Youth Science Innovation in Colleges and Universities of Shandong Province (2020KJF005), Advanced Talents Foundation of QAU (1120038), China Scholarship Council (201806850075), and the Open Project Program of China-Canada Joint Lab of Food Nutrition and Health, Beijing Technology and Business University (KFKT-ZJ-2106).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.