Bacillus thuringiensis crystal toxins (Bt) are not effective against them. One approach is to produce fusion protein toxins for the control of these pests. Two protein toxins, Hvt (ω-atracotoxin from Hadronyche versuta) and onion leaf lectin, were translationally fused to evaluate the negative effects of fusion proteins on Phenacoccus solenopsis , a phloem-feeding insect pest. Hvt was cloned both N-terminally (HL) and then C-terminally (LH) in the fusion protein constructs, which were expressed transiently in Nicotiana tabacum using a Potato Virus X (PVX) vector. The HL fusion protein was found to be more effective against P. solenopsis, with an 83% mortality rate, as compared to the LH protein, which caused 65% mortality. Hvt and lectin alone caused 42% and 45%, respectively, under the same conditions. Computational studies of both fusion proteins showed that the HL protein is more stable than the LH protein. Together, these results demonstrate that translational fusion of two insecticidal proteins improved the insecticidal activity relative to each protein individually and could be expressed in transgenic plants for effective control of sucking pests.Sucking pests pose a serious agricultural challenge, as available transgenic technologies such as  However, the excessive use of chemicals causes destruction of non-target insects and development of resistance, along with environmental contamination. Since 1960, bio-insecticides have received considerable attention, due to being environment friendly and highly desirable alternatives for the control of insect-pathogenic microbes, viruses, and eelworms6. This typically allows the one-step expression of transgenic lines, unlike the gene stacking with recombinant traits, multiple T-DNA and promoter insertions, and thereby coordinated expression of different recombinant proteins in plants. A number of fusion proteins have been designed and tested for the control of pests and pathogens. Usually, fusion constructs comprise δ-endotoxins from B. thuringiensis (Bt) and/or other plant defense-related proteins9. Bt is the most widely used bio-insecticide for the control of insect pests. These toxins specifically destroy the intestinal lining of target insects to kill them10. More than 35 million hectares are being cultivated with Bt crops globally in at least 13 different countries. There are many crops have been transformed genetically with Bt genes, including cotton, maize, eggplant, tobacco, potato, rice, and tomato. However, on a commercial scale, only cotton and maize have been transformed to have insecticidal activity11. Bt toxins are quite effective for the control of insects belonging to the family Lepidoptera and Coleoptera12. Bt toxins are considered to be most effective for controlling stem borers and root worms13. They are quite specific in their mode of action and are ineffective against many sucking pests including African rice green hoppers, rice thrips, white backed bugs, and green leaf hoppers15. Recently, targeted mutagenesis of the Cry51 protein and a fusion complex of Cyt2Aa with aphid gut binding peptide were found to make Bt toxins effective against Lygus spp.16 and aphids17, respectively. Nevertheless, there is a need to discover and study new proteins with insecticidal activity that is specific for the control of sucking pests, in particular phloem feeders.Expression of two different proteins as a single translationally fused protein under the control of a single promoter has also gained much attention in the recent years18. It contains a variety of toxic polypeptides that paralyze the prey prior to feeding. These polypeptide neurotoxins have ligand binding activity for neuronal ion channels and, to a lesser extent, neuronal receptors and presynaptic membrane proteins20. ω -Atracotoxin from Hydronyche versuta (Blue Mountains funnel web spider) is a calcium channel antagonist. It binds to insect calcium channels and blocks nerve impulses, ultimately leading to paralysis and death of the insect22. Multiple compounds from different spider venom have already been proved effective against insect pests belonging to order Lepidoptera and Hemiptera25.Spider venom is a complex of many toxins that causes numerous neurological and biochemical changes in animals including mammals27. As plant defense proteins, lectins play a biological role in the recognition of different proteins, and exhibit their insecticidal, fungicidal and anti-microbial properties28. At the laboratory scale, lectins are used to study different cellular mechanisms, including apoptosis, proliferation, cell arrest, cell metastasis, and recognition of different glycans in microarray30. Galanthus nivalis agglutinin  is a plant lectin that shows its limited insecticidal activity for lepidopteran larvae. It has been shown in artificial diet and transgenic plant studies that GNA reduces larval weight gain and slows down the developmental rate of first instar Lacanobia oleracea larvae32. This is achieved by binding of the lectin to the gut epithelium surface glycoproteins33. However, the exact mechanism by which GNA effects insect pests is still unclear. The ability of GNA to cross the gut epithelium makes this protein a potential carrier to deliver fused peptides to the circulatory system of target insect species.Lectins belong to a superfamily of proteins that recognize and bind the carbohydrate moieties without altering their covalent structure. Some plants, in particular legumes and cereals have high concentrations of lectins because of their interaction with storage proteinsin planta37. To serve as expression vector, the PVX genome was cloned behind a T7 RNA polymerase promoter or a 35 S promoter derived from Cauliflower mosaic virus (CaMV)17.Transient expression studies were carried out using the potato virus X (PVX) expression system. This is a quick and reliable system for studying different genes 25. Here we have explored the significance of translational fusion of Hvt and lectin that was transiently in tobacco using the PVX expression vector. Both computational tools and insect bioassays using Phenacoccus solenopsis  as a model were used to assess the stability and insecticidal potential of fusion proteins.We have shown previously that the expression of Hvt and lectin in transgenic plants from phloem-specific promoters protects these plants against multiple sucking pestsNicotiana tabacum (tobacco) plants infiltrated with PVX carrying protein constructs, along with the control plants, were kept in cages and exposed to mealybug feeding. Initially, mealybugs took time to acclimatize with the new environment, and during the first 48 h we did not observe any lethal effects. After two days, mealybugs appeared lethargic, with very slow movements. Nymphs in particular showed early effects of the toxins. They started to lose body mass and had jerky and shaky movements. After three days, 35% of nymphs were found dead on the plants expressing HL (Hvt-lectin) toxin, and 26% on LH (lectin-Hvt) plants. In contrast, plants expressing the parent toxins, Hvt and lectin, caused only 5 and 7% mortality, respectively. Higher mortality was recorded after 6 days for all transgenes: HL (83%), LH (65%), Hvt (42%), and lectin (45%)  Figs  and 2.FiAs some insects were found to escape from the plants, we could not get 100% mortality data. Some of the adult individuals multiplied on the plants, but the nymphs could not grow or move to the next instar due to the effect of the toxic proteins. The ovisac of the multiplying adult mealybugs was found to be normal. The dead insects showed the symptoms of brown discoloration of their bodies along with reduction in body mass.In contrast, on healthy control and PVX control plants, the mealybugs grew and multiplied normally. Young nymphs developed into adults and adult individuals multiplied with true ovisac. All the phenotypes were normal, including gain of body mass and multiplication.in-silico studies.Computational studies were used to investigate the conformational changes in the fusion proteins and to validate the experimental results that the fusion protein expressing Hvt at the N-terminus (HL) is more stable and lethal to the insects compared to the protein expressing Hvt at the C-terminus (LH). Moreover, significant factors affecting the biological properties of the fusion proteins were also predicted by these in silico tools were utilized to predict the most suitable 3D model for both parent and fusion proteins. Initially, a 20 ns simulation was performed for each of the above-mentioned proteins to generate a refined structure, which was followed by additional analysis to predict the core factors responsible for the dynamics and stability of one construct relative to the other.The change in orientation of Hvt and lectin in the fusion constructs caused large differences in the tertiary structure of both parent proteins, Hvt and lectin Fig. . Therefo41.In this study, we dealt with the proteins having ligand binding properties. The ligand binding sites of the parent and fusion proteins were predicted by I-TASSER (COACH) tool. COACH is a meta-server approach that combines multiple function annotation results from the COFACTOR, TM-SITE, and S-SITE programsIn case of Hvt, I-TASSER-COACH server predicted P7, C23 and Q21 as the crucial residues involved in the ligand binding site, with a cluster size of 138 Å. For lectin, the Q28, D30, N32 Y36, P41, A44, and G48 residues were found to be a crucial part of the ligand binding site, with a cluster size of 34 Å. In the case of fusion proteins, the ligand binding site of HL consists of, Q105, D107, N109, Y113,, A119, A123, and N124 with a cluster size of 37 Å, while for LH protein, it consists of Q59, D61, N63, Y67, A73, A76, and R80 with a cluster size of 24 Å  plots Fig.  revealedWith the aim of evaluating a stable orientation of the parent protein from the two constructs, the compactness of all four proteins was studied. In this regard, the size of the protein was calculated via a radius of gyration plot, which determined the mean positional distances of atoms from the center of mass. Figure For better understanding the underlying basis for the greater stability of the HL construct relative to the LH construct, the total number of hydrogen bonds formed and deformed during the entire simulation period was calculated. HL and LH showed a similar number of hydrogen bonds throughout our simulation, as plotted in Fig. To analyze the mean atomistic motions of separate residues, root mean square fluctuation (RMSF) of Cα atoms in both LH and HL fusion constructs were calculated using final trajectories. High RMSF values denote more flexibility , while low values show less fluctuations in the structure. As shown in Fig. For the estimation of structural stability and fluctuations of residues in the formation of secondary structures, average secondary structure plots for both the HL and the LH fusion construct systems were calculated. The average secondary structure content and its position is presented in Fig. The ambiguous picture developed by analyzing hydrogen bond and secondary structure plots suggested further visualization of trajectories to explore the key players responsible for the structural differences between the HL and LH constructs. The molecular visualization of the trajectories brought forth an interesting finding, predicting a crucial role of the Hvt domain in the stability of the whole protein. Superimposition of post-MD structures of the HL and LH constructs showed that the structure of the lectin domain remains rigid and stable through the entire simulation, while the Hvt domain continues changing. The Hvt domains of both constructs were observed in different conformations having different secondary structures. The Hvt part of the HL protein contains both bends and turns, while the LH has just bends with very few turns and helices in its Hvt domain. We then calculated RMSF and hydrogen bonds found in this region of both constructs to predict whether the Hvt domain has any role in the stability of the fusion proteins Fig. . Our anast and 2nd instar) depend mainly on immediate feeding for their growth. Nymphs suck phloem sap to fulfill their dietary needs, while adults have the capability to store some food in their body and are not entirely dependent on feeding during the experimetn. This likely explains the delayed effect of toxin proteins on adult mealybugs compared to nymphs.Mealybugs feeding on plants expressing Hvt-lectin fusion proteins showed interesting symptoms. Nymphs were the first to suffer efects of the toxin proteins. This happens because nymphs (1Two different proteins Hvt (a spider venom toxin) and onion leaf lectin were fused together to get broad-spectrum resistance against insect pests specifically mealybugs. Both possible fusion constructs were prepared and expressed transiently in a model plant system, tobacco. Both of the single proteins have their own potential applications, but the fusion proteins are more toxic to mealybugs. The plants expressing the fusion protein HL were more effective against mealybugs  than the fusion protein LH , while only 42 and 45% mortalities were recorded on plants expressing parent proteins Hvt and lectin, respectively.From the proposed models, drastic conformational differences were observed between tertiary structures of both fusion constructs Fig. . Just chMyzus persicae (green peach aphid) individually and also as fusion proteins was tested. Fusion proteins proved to be more toxic to aphids than the parent proteins6. The Pl1a/GNA fusion protein killed all the individuals of 4106 A strain in 7-days when fed at 1 mg/mL concentration in the diet. For the Hv1a/GNA fusion protein, 100% mortality was observed after 6-days at the same protein concentration6. Promising results were collected when fourth instar larvae of L. oleracea were fed on the SMI1/GNA fusion protein. Larvae injected with a low concentration of SMI1/GNA (18–25 µg/g insect) were paralyzed and 40% mortality was recorded after 72 h, while 100% mortality was recorded after 48 h at high conc. (60–90 µg/ g insect). In the case of oral intake, 100% mortality rate was found in fifth instar tomato moth larvae when fed on 2.5% fusion protein containing diet42. The Butal T/GNA fusion fusion protein proved to be toxic against Nilaparvata lugens (rice brown plant hopper). After 6-days of fusion protein uptake 92% mortality was recorded, compared to 64% mortality when feeding on GNA alone5.To cope with the issue of sucking pest control, it is important to discover a broad-spectrum solution. In other recent research, different translationally fused proteins were designed for two reasons (i) to control the insect pest and (ii) to enhance the effect of a single protein. The effect of HVIa, Pl1a, and GNA toxicity against MD simulations were carried out to predict the core factors responsible for the stability and structural variations found between HL and LH constructs. The RMSD and RMSF plots identified the HL construct as the most stable structure among the tested proteins. The radius of gyration plots confirms the HL protein as a stable and compacted construct with very few fluctuations in its overall structure. In contrast, the LH construct undergoes major conformational changes along the time steps, with greater structural entropy in its structure. These enhanced fluctuations in the LH construct might be attributable to the instability of this protein compared to the other tested proteins.The calculation of electrostatic interactions of both constructs displays a vague picture regarding the stability of one construct relative to another. However, the detailed visualization and superimposition of post-MD structures of both constructs highlights the Hvt domain as the crucial part responsible for protein stability. Further investigation of the interplay between rigidity and flexibility in terms of stable electrostatic interactions verified the role of the Hvt domain in mediating compactness of the HL construct relative to the LH. The Hvt domain of LH constructs loses its stability because of a smaller number of stable hydrogen bonds among its residues, as compared to the HL construct, which establishes a greater number of electrostatic interactions with more than 50% occupancy through the entire simulation. All of these findings give a glimpse of a strong connection between stability, dynamics and electrostatic forces among a majority of the Hvt domain residues of both fusion constructs.Based on the above findings, we propose development of transgenic plants with fusion protein having Hvt at the N terminus and evaluation of resistance against multiple insects  by using either constitutive or phloem-specific promoters.Two translationally fused proteins were designed to check their effectivity against sucking insect pests. In one construct, Hvt was kept at the N-terminus and lectin at the C-terminus, removing the stop codon of Hvt while in another construct lectin was kept at the N-terminus and Hvt at the C-terminus by removing the stop codon of lectin. This strategy was planned to determine which construct/gene orientation is more effective against sucking pests.Sal I and Sma I endonucleases. The individual genes, Hvt and lectin, were also cloned in the PVX vector to compare the effect of the parent toxins with translationally fused toxins.To avoid frame shift of the proteins, both of the gene constructs were synthesized by GenScript using the pUC57 vector backbone. The constructs were then cloned in the expression vector (PVX) using Agrobacterium tumefaciens strain GV3101 competent cells. Electro-competent cells of GV3101 were prepared and constructs were transformed via electroporation. Colonies were confirmed for the presence of the construct by PCR using gene-specific primers. PCR-positive colonies were picked and cultured in 50 mL LB containing 25 µg kanamycin, 50 µg rifampicin, and 10 µg tetracycline and incubated at 28 °C and 180 rpm for 48 h. This culture was further used for plant infiltration.The final constructs were transformed into 2. After re-suspension acetosyringone was added in at a concentration of 100 mM/mL. Plants were infiltrated with the cultures following incubation of 2–3 h at room temperature. Nicotiana tabacum was used to carry out all the transient assays. Plants were grown in a plant room at 26 ± 2 °C and 16 L/8 D. plants were infiltrated with a syringe when they reached the 3–4 leaf stage. After infiltration, plants were again kept in the plant room under controlled conditions.After 48 h, cultures were centrifuged in 50 mL polypropylene tubes at 12,000 × g for 10 min. The supernatant was discarded and the pellet was re-suspended in 10 mM MgClMealybugs were collected from the field and reared in a separate glass house on cotton plants. Mealybugs multiplied readily on cotton plants and were used afterwards for bioassays to check the effects of fusion protein toxins.N. tabacum plants used as experimental controls.As a result of chlorosis, PVX symptoms appear as mosaic pattern in plants. Usually, it takes 10–14 days for the PVX vector to give visual symptoms in plants. Plants were kept in the cages and mealybugs were allowed to feed at 12 dpi, when plants had clear PVX symptoms. For this purpose, six large cages were used, each carrying 10 plants. The first cage had plants inoculated with the HL construct, the second carried LH-inoculated plants, the third carried Hvt and the fourth carried lectin infiltrated plants. The remaining two cages were used for control plants with which to compare the effects of fusion protein toxins. PVX-inoculated plants were used to see whether the vector impacts the mortality of insects, while healthy i.e. 26 ± 2°C and 16 L/8 D. Plants were observed every 24 h to evaluate mealybug performance and collect the mortality data.All of the plants in six cages were subjected to feeding by an equal number of mealybugs under controlled conditions of the plant room, 43.ANOVA and Tukey’s SD test were used to assess significance in the mortality data using the statistical software program SPSS-20The sequences of the proteins were subjected to analyses using computational biology tools. This study was carried out primarily to observe conformational/structural modifications produced as a result of the fusion of two proteins.41. For further structure validation in detail, 3D coordinates of the best selected models were analyzed. In this regard, RAMPAGE44 Ramachandran Plot Assessment was utilized to calculate the Ramachandran plots for each of the four proteins i.e. Hvt, Lectin, HL and LH.The tertiary structures of fusion and parent proteins were predicted by using I-TASSERi.e. Hvt, lectin, HL and LH proteins, were submitted for extensive MD simulation using AMBER1445. All four systems were prepared using xleap module in explicit solvent under periodic boundary condition via application of an FF99SBildn force field. The proteins were solvated in a cubic box of TIP3P water molecules46 with a distance of 10 Å between the solute and edge of water box. The perturbed and unperturbed charges of the system were neutralized by adding appropriate number of counter ions.For the structural refinement and to predict dynamic stability, four validated predicted models, 47. Particle Mesh Ewald Method (PMEM)48 was employed to compute long-range electrostatic interactions with a cut off distance of 8 Å, integration time step of 2 fs and the SHAKE algorithm49 was utilized to restrain all bonds involving hydrogen atoms.Initially, energy minimization of each system was performed to remove the steric clashes between protein-protein and protein-solvent molecules. A total of 35,000 steps of minimization was carried out involving 15,000 steps each for steepest decent and for conjugate gradient minimization by gradual decreasing the straining force applied on all atoms of protein. Finally, another 5,000 steps of minimization were performed without any restraint to relax the system. It was followed by gradual heating of each system from 0 K to 300 K under NVT conditions for a period of 500 ps. After heating, each system was equilibrated under NPT ensemble (P = 1 atm) for another 500 ps. Finally, a 20 ns production run was conducted for each system at constant temperature and pressure50 incorporated in AMBER suite was used for trajectory analysis according to their RMSD, RMSF, radius of gyration, hydrogen bonds and secondary structure plots. During entire simulation period plots presented the comparative stability of fusion constructs over one another.The CPPTRAJ module


Sorghum bicolor) genotypes. Eight sweet sorghum genotypes were evaluated at five different locations in two growing seasons of 2014. The aim was to determine the interaction between genotype and environment on cane, juice, and ethanol yield and to identify best genotypes for bioethanol production in Kenya. The experiments were conducted in a randomized complete block design replicated three times. Sorghum canes were harvested at hard dough stage of grain development and passed through rollers to obtain juice that was then fermented to obtain ethanol. Cane, juice, and ethanol yield was analyzed using the additive main effect and multiplication interaction model (AMMI) and genotype plus genotype by environment (GGE) biplot. The combined analysis of variance of cane and juice yield of sorghum genotypes showed that sweet sorghum genotypes were significantly (P < 0.05) affected by environments (E), genotypes (G) and genotype by environment interaction (GEI). GGE biplot showed high yielding genotypes EUSS10, ACFC003/12, SS14, and EUSS11 for cane yield; EUSS10, EUSS11, and SS14 for juice yield; and EUSS10, SS04, SS14, and ACFC003/12 for ethanol yield. Genotype SS14 showed high general adaptability for cane, juice, and ethanol yield. The genotype and environment interaction influences the selection criteria of sorghum ( Sweet sorghum is gaining popularity for ethanol production due to its high sugar level in their stem juice. It is widely grown for food, feed, and fuel in semiarid tropics of Asia, Africa, America, and Australia  due to iStudies of adaptability and stability provide information about the behaviour of each genotype under different environmental conditions. The phenotypic performance of each genotype is influenced by abiotic and biotic factors; some genotypes may perform well in one environment but fail in several others . These fGlycine max L. Meril) [Ipomoea batatas) [Capsicum annuum) [Eleusine coracana) [Triticum aestivum) [Oryza sativa) [The genotype, environment, and the genotype by environment interactions impact crop performance. Genotype by environment interaction (GEI) complicates breeding, testing, and selection of superior genotypes . The GEI. Meril) , sweet pbatatas) , pepper  annuum) , finger oracana) , wheat (estivum) , grain sestivum) , and ric sativa) . GGE andSweet sorghum field experiments were carried out in Kisumu, Siaya, and Busia Counties of Kenya. The specific sites were Sinyanya  at 1168 m above sea level (ASL), Masumbi  at 1370 m ASL both in Siaya County, Mundika  at 1222 m ASL in Busia, Nyahera  at 1387 m ASL, and Sagam  at 1216 m ASL both in Kisumu County.All sites fell within the same agroecological zone, lower midland (LM); the difference in yield was due to difference in sub agroecological zones as depicted in Eight sweet sorghum genotypes were grown in a randomized complete block design (RCBD). The genotypes were EUSS10, EUSS11, and EUSS17 as candidates with the controls being ACFC003/21, SS04, SS14, SS21, and SS17. Sowing was done on 18th March in Sinyanya and Masumbi and 19th March 2014 in Mundika for first season. Sowing in the second season was done on 13 September 2014 for both Mundika and Sagam while Nyahera was planted on 24 September 2014. Genotypes were sown in plots measuring 4 × 2.5 m in RCBD with three replications. Each plot consisted of four rows of sorghum at a spacing of 60 cm by drill and the blocks were separated by 1.5 m path. Triple superphosphate fertilizer was applied uniformly to all plots at a rate of 17.2 kg per ha before sowing. Control of weeds was done manually using hoes, three weeks after seedling emergence, and sorghum were thinned to a spacing of 10 cm within the row. And then, calcium ammonium nitrate (25% N) was top-dressed at the rate of 20 kg N/ha. Birds guarding was effected soon after the panicles formed to prevent grains damage.Emergence was observed in all plots two weeks after planting, and stand counts were determined for all sorghum experimental units. Days to 50% heading were determined by calculating a number of days from sowing to when 50% of the sorghum heads in each plot had formed panicle. At dough stage of grain, plant height of each genotype was determined, and their panicles were harvested. Three randomly selected plants from each genotype in all replicates were used for recording plant height. Plant height was measured from base of the stem to tip of panicle and data averaged across three plants.Harvesting took place on 9 July 2014 at the three sites: Masumbi, Mundika, and Sinyanya for the first season while Sagam and Mundika were harvested on 12 December 2014 and Nyahera on 29 December 2014 for the second season. Eight different genotypes were harvested at hard dough stage of grain taking plants in two inner rows of each plot. The leaves were stripped off by hand from harvested stalk and panicles removed using secateurs. The harvested stalks were weighed with a weighing balance to get fresh cane weight and then transported to the laboratory for juice extraction. Juice from the stalk was extracted in one roller crusher  and strained through a sieve into a juice container. The volume of juice was measured, and Brix (%) was taken using hand refractometer. Saccharomyces cerevisiae, was added to juice and fermentation process carried out at 35°C for four days and then distilled to obtain ethanol whose volume was determined. Refractometer  was used to determine the concentration of ethanol. The refractive index of distillate was taken and then compared with that of a standard curve. Absolute ethanol was mixed with distilled water to give the concentration of 0, 5, 10, 15, 20, 25, and 30% ethanol whose refractive index was taken to obtain standard curve showing the relationship between the refractive index and percent of ethanol in the distillate.Juice was sampled from each plot taking 100 mL for fermentation. Yeast (1.5%),YGEr is mean yield of genotype G in the environment E for replication r; μ is grand mean; αG is deviation of the genotype G from the grand mean; βE is deviation of the environment E from the grand mean; λn is singular value for the interaction principal component axis (IPCA) n; γnG is the PCA score of a genotype for PCA axis n; δnE is the environmental PCA score for PCA axis n; ρGE is AMMI residual; and εGE is the error term when the experiment is replicated.Statistical computations were carried out using Genstat software version 15.1  for AMMI and GGE biplot analysis. The graphic representation of genotypes and environments by AMMI analysis results from a model of main additive effects and multiplicative interaction . This moAMMI stability value (ASV) was calculated by AMMI analysis was used to determine the stability of genotypes across locations using principal component axis (PCA) scores and ASV. Genotypes having the least ASV were considered as widely adapted genotypes. Similarly, IPCA2 scores close to zero revealed more stable genotypes, while large values indicated more responsive and less stable genotypes. GGE biplot analysis was used to visualize the relationship between testers and entries and to determine “which-won-where” portion. GGE biplot also reveals stability of genotypes; genotypes located near the biplot origin are considered as widely adapted genotypes while genotypes located far are considered as being specifically adapted.The effects of genotype were significant on days to 50% heading, with SS21 being early maturing genotype across environments . GenotypPlant height differed among sweet sorghum genotypes and across locations. The tallest and shortest plant height were recorded by genotypes in Sagam and Sinyanya, respectively . GenotypGenotypes varied with environments for cane yield . Among tJuice yield differed significantly among the sweet sorghum genotypes; there were high and low performers . Among tGenotypes EUSS11 recorded consistent high Brix values across environments with the controls SS04, SS14, and ACFC003/12 except in Sagam . Among tGenotypes varied within environments for ethanol yield . GenotypThe test locations vary in latitude, rainfall, soil types, and temperature. The three environments with high yielding potential, Masumbi, Mundika, and Sagam, are characterized by high bimodal rainfall patterns as compared to lowest yielding environments, Sinyanya and Nyahera. LM1 and LM2 agroecological zones can be utilized for commercial production of sweet sorghum. Genotypes showed satisfactory yields in the most favorable environments (LM1) such as Sagam and Masumbi, the reason being the ability of genotypes to respond advantageously to a higher amount of rainfall in LM1 compared to LM3 agroecological zones. High temperatures and low precipitation are some of contributing factors to poor performance in LM3 AEZ. The slightly better performance of genotypes in Mundika during season one compared to the second season could be due to the difference for rainfall during early growth stages of sorghum plants. Since the fluctuation of ethanol yield of SS14 was smaller than other genotypes, it was suggested that its ethanol yield would be superior to other genotypes in unfavorable environments.The combined analysis of variance of cane and juice yield of sorghum genotypes showed that sweet sorghum genotypes were affected by environments (E), genotypes (G), and genotype by environment interaction (GEI) . HoweverA large sum of squares shows that environments were diverse, influencing yields differently which was in harmony with the findings of Reddy et al.  in sweetThe first interaction principal component (IPCA 1) and the second (IPCA 2) accounted for 8.53 and 6.47%, respectively, of the cane's IPCA sum squares. The IPCA1 accounted for 8.22 and 8.26% of juice and ethanol yield interaction sum of squares, respectively, while IPCA2 accounted for 6.97 and 5.07% . The firEnvironments and genotypes with least ASV scores are considered as they are the most stable. Accordingly, genotypes SS14, SS17, and ACFC003/12 had a general adaptation for cane yield while SS14 was the most stable for juice and ethanol yield . On the Furthermore, the IPCA2 scores of genotypes in AMMI analysis indicate stability of genotypes across locations; high IPCA2 scores (either negative or positive) are unstable while those with low scores are stable . Table 9Genotypes or environments located on the right-hand side of the midpoint of the axis (IPCA1) have higher yields than those on the left-hand side . In thisThe polygon view of GGE biplot for cane yield  indicateThe GGE biplot for juice yield  indicateBiplots were divided into four sectors in In Figures A study by Abubakar and Bubuche  in NigerCane yield was found to be highly correlated with plant height. Environmental effects, as well as GEI, had strong effect on yield of sweet sorghum genotypes. The significant GEI for cane and juice yield observed from analysis of variance in this study shows that sweet sorghum genotypes respond differently when grown in different environmental condition. The results from this project indicate that SS14 was most stable and best genotype across environments whereas EUSS10 has excellent potential for ethanol in areas with high yield potential. The best-performing genotypes were EUSS10, ACFC003/12, and SS04 while average performers were EUSS11, EUSS17, and SS14. The genotypes SS21 and SS17 were poor performers for ethanol yield located outside limits of any environments. It is evident that performance of sweet sorghum is attributed to both genetic make-up and environment.


CTDIvol values of 24, 18, 12, 6, 3, 2, and 1 mGy. Images were reconstructed at 2.5 mm thickness with filtered back‐projection (FBP); 20%, 40%, and 70% ASiR; and MBIR. The potential for dose reduction and low‐contrast detectability were evaluated from noise and contrast‐to‐noise ratio (CNR) measurements in the CTP 404 module of the Catphan. Hounsfield units (HUs) of several materials were evaluated from the cylinder inserts in the CTP 404 module, and the modulation transfer function (MTF) was calculated from the air insert. The results were confirmed in the anthropomorphic Kyoto Kagaku abdomen phantom at 6, 3, 2, and 1 mGy. MBIR reduced noise levels five‐fold and increased CNR by a factor of five compared to FBP below 6 mGy CTDIvol, resulting in a substantial improvement in image quality. Compared to ASiR and FBP, HU in images reconstructed with MBIR were consistently lower, and this discrepancy was reversed by higher pitch factors in some materials. MBIR improved the conspicuity of the high‐contrast spatial resolution bar pattern, and MTF quantification confirmed the superior spatial resolution performance of MBIR versus FBP and ASiR at higher dose levels. While ASiR and FBP were relatively insensitive to changes in dose and pitch, the spatial resolution for MBIR improved with increasing dose and pitch. Unlike FBP, MBIR and ASiR may have the potential for patient imaging at around 1 mGy CTDIvol. The improved low‐contrast detectability observed with MBIR, especially at low‐dose levels, indicate the potential for considerable dose reduction.The purpose of this study was to characterize image quality and dose performance with GE CT iterative reconstruction techniques, adaptive statistical iterative reconstruction (ASiR), and model‐based iterative reconstruction (MBIR), over a range of typical to low‐dose intervals using the Catphan 600 and the anthropomorphic Kyoto Kagaku abdomen phantoms. The scope of the project was to quantitatively describe the advantages and limitations of these approaches. The Catphan 600 phantom, supplemented with a fat‐equivalent oval ring, was scanned using a GE Discovery HD750 scanner at 120 kVp, 0.8 s rotation time, and pitch factors of 0.516, 0.984, and 1.375. The mA was selected for each pitch factor to achieve PACS number(s): 87.57.Q‐, 87.57,nf, 87.57.C‐, 87.57.cj, 87.57.cf, 87.57.cm, 87.57.uq With the introduction of computed tomography (CT) in the 1970s, complex analytical approaches were conceived to reconstruct the scanned image. Algebraic Reconstruction Technique (ART) was the first image reconstruction method proposed for CT.The latest developments in CT reconstruction techniques, adaptive statistical iterative reconstruction  and model‐based iterative reconstruction ,In this study, we have evaluated the performance of both ASiR and MBIR in a clinical setting for achieving potential patient dose reduction. We scanned the Catphan 600 modular phantom, supplemented with a fat‐equivalent oval ring, using standard clinical scan parameters at our institution for a medium adult, modified to evaluate image noise, contrast, material Hounsfield unit (HU), and resolution across seven dose levels and three pitch factors. To account for the dependence of tube‐current modulation and iterative reconstruction on the shape and size of the patient, the results were confirmed with a Kyoto Kagaku anthropomorphic abdomen phantom. The evaluation of FBP, ASiR, and MBIR was performed under controlled, reproducible and clinically relevant scan conditions.CTDIvol values. During the scans, each phantom module  was supplemented with a fat‐equivalent oval ring to better approximate an adult body shape and size, as shown in CTDIvol of 1, 2, 3, and 6 mGy. Images were reconstructed using the Standard kernel for FBP; 20%, 40%, and 70% ASiR, which represent a wide range of percentage blends of the ASiR with the FBP; and MBIR. CTDIvol and reconstructed using different algorithms. The display field‐of‐view (DFOV) was 36 cm for the reconstructed images of Catphan phantom and was 30 cm for the reconstructed images of Kyoto Kagaku abdomen phantom. These default DFOV sizes were selected based on the lateral dimensions of the phantoms from skin‐to‐skin. The image matrix was 512×512.Catphan 600 phantom  and Kyoto Kagaku abdomen phantom  images were acquired on a 64‐detector row CT scanner : 120 kVp, 0.8 s rotation time, 40 mm beam width, large scan field‐of‐view (SFOV), 2.5 mm image thickness, and three pitch factors . For the Catphan phantom, the mAs was selected for each pitch factor to achieve 1, 2, 3, 6, 12, 18, and 24 mGy To ensure that the data is sampled from images approximating clinical conditions, all quantitative image analysis was performed directly from the images with default DFOVs, without reconstructing to a small DFOV. To minimize statistical variations in noise for each scan condition, we analyzed images from 10 independent acquisitions at the same identical location in a phantom. ImageJ software 0.4 cm2 regions of interest (ROIs) located in the background material of the Catphan. Three ROIs increased the total area of data sampling (from 0.4 to 1.2 cm2). For the Kyoto Kagaku abdomen phantom image analysis, one 3.1 cm2 ROI was used , an ROI was placed in the center of the 1% contrast, 15 mm diameter supraslice target of the Catphan CTP515 module and three identical ROIs were placed in the immediate background. The CNR was defined as the mean target signal minus the mean background signal divided by the background standard deviation. For the Kyoto Kagaku abdomen phantom, the target organs used were the spleen, liver, pancreas, and kidneys. CTDIvol value was chosen to represent a very low‐dose target level and is consistent with several recent clinical CT imaging studies where CTDIvol levels near or at 1 mGy were evaluated.The difference in noise and CNR between the three techniques  was calculated. To evaluate changes in image quality with dose, a baseline reference value was defined as the noise or CNR achieved with MBIR at 1 mGy. The 1 mGy Visual inspection of the CTP528 high resolution module of the Catphan 600 was used to evaluate gross changes in spatial resolution, as typically performed during annual compliance testing. The air target in the CTP404 module was used to measure the modulation transfer function (MTF) of reconstructed images using the edge method as depicted in Eight cylindrical inserts in the CTP404 module of the Catphan were used to quantify changes in HU: air, PMP, LDPE, water, polystyrene, acrylic, Delrin, and Teflon. Slight variations from the estimated CT numbers in phantom manual were expected to result from use of the fat ring during scan acquisition.t‐test, the linear mixed model is a generalized form of paired t‐test. The interpretation of linear mixed model is the same as ANOVA. Dunnett's procedure was used to adjust for multiple pairwise comparisons against the reference level (MBIR at CTDIvol of 1 mGy). ANOVA was used to compare MTF between algorithms by pitch, CTDIvol, and spatial frequency. Pairwise comparisons against FBP, based on ANOVA estimates, were also adjusted using the Dunnett's procedure. All tests were two‐sided and adjusted p‐values of 0.05 or less were considered statistically significant. Statistical analysis was carried out using SAS version 9 . The values of statistical analysis  have been associated to the outcomes in Noise, CNR, and MTF were summarized using mean, standard deviation (SD), and range. Noise and MTF were transformed to the logarithmic scale prior to statistical modeling. For noise, a positive difference means higher noise (worse) and a negative means lower noise (better). For CNR, a positive difference means higher CNR (better), while a negative difference means lower CNR (worse). Logarithmic transformation is to reduce skewness in the data to better suit the underlying Normal assumption of linear mixed model or ANOVA. Linear mixed model was used to estimate and compare noise and CNR between algorithms. Linear mixed model can account for correlations between measurements from the same experimental unit. As ANOVA is a generalized form of two‐sample CTDIvol, the improvements observed with MBIR were within experimental error to those of 70% ASiR. Noise reduction with MBIR was constant among the three pitch factors and similar in magnitude to the improvements observed with CNR  70% ASiR reached the baseline CNR at CTDIvol of 6 mGy and it yielded significantly better CNR at higher doses ; c) 20% ASiR and 40% ASiR CNR values at 12 mGy were comparable to MBIR CNR at 1 mGy and didn't yield significantly better CNR until CTDIvol reached 24 mGy ; d) FBP needed to reach 18 mGy CTDIvol to yield similar CNR as MBIR at 1 mGy CTDIvol, and it didn't yield significantly better CNR even at CTDIvol of 24 mGy.Estimated noise and CNR differences from a baseline, reference level were calculated to quantify changes in image quality with increasing chniques . From thchniques , it can ferences , the folCTDIvol. At pitch 1.375, the dose threshold for significant improvement in MTF with MBIR was 2 mGy. Seventy percent (70%) ASiR showed significantly higher MTF than FBP only at 3 mGy for pitch 0.516, 3 and 6 mGy for pitch 0.984, and at 2 and 6 mGy for pitch 1.375. Twenty percent (20%) ASiR and 40% ASiR MTF were not significantly different from FBP MTF at any dose level for all pitch factors.To evaluate spatial resolution, an initial inspection of bar patterns demonstrated an improvement with iterative reconstruction techniques; this observation was supported by line profiles drawn across the 7 lp/cm bar pattern . The MTF−200 HU), LDPE (−100 HU), polystyrene (−35 HU), acrylic (120 HU), Delrin (340 HU), Teflon (990 HU), and air (−1000 HU) — the material HU values in MBIR images were closer overall to those listed in the Catphan 600 user manual than the HU values in ASiR and FBP images. CTDIvol, the measured material HU values differed greatly from the HU values of the FBP images at 24 mGy CTDIvol level.Given their impact on clinical diagnosis, the effects of iterative reconstruction on HU were evaluated. For all eight materials in the sensitometry module of the Catphan 600 phantom — background material (100 HU), PMP (60 years±15), Leipsic et al.Since adaptive statistical iterative reconstruction has become available for clinical patient imaging, the potential for dose reduction with ASiR has been extensively studied in the literature. The reported dose reduction ranges from 30%–60%. In pediatric imaging (aged 1‐year‐old to adolescence), 100% ASiR was estimated to reduce dose by 82% compared to FBP in a phantom study.et al.CTDIvol of 4.38−23.35 mGy, an increase in CNR  and an 80% decrease in noise with MBIR compared with FBP were observed. Shuman et al.The modest reduction in dose previously reported confirms that ASiR was not intended to result in marked dose reduction, but was rather a balanced approach to maintain image quality with reasonable dose savings.A phantom study provides the opportunity for performing in‐depth evaluations, by adjusting one scan/reconstruction parameter at a time, while other conditions remain unchanged, so that appropriate comparisons are conducted. Such work is especially valuable when a phantom is scanned repeatedly at many different dose levels for assessing the potential for dose reduction. In contrast with other phantom studies, the phantoms used in our work mimicked patient size/ shape, the base scan protocol was a routine clinical protocol used for patient abdominal imaging, and the phantom images were reconstructed with clinical parameters. We therefore performed an analysis of image quality under various conditions of image reconstruction and at a number of radiation dose levels. The quantitative image analysis included noise, CNR, material HU, and spatial resolution. Our results show that the largest improvement in noise reduction and contrast with MBIR (threefold to fivefold) occurred at the lowest dose levels, demonstrating the potential feasibility of low‐dose patient imaging where the appropriate dose levels depend on clinical applications as well as sizes of patients. Conversely, noise, CNR, and material HU for three ASiR blends are less dependent on dose level and provide for a more modest reduction in dose, 15%–20%.However, image analysis based on noise and CNR does not completely capture the differences in image texture, which may affect the outcomes of patient diagnosis. This limitation of the study could account for the differences in results presented here compared to published clinical studies. Additionally, the use of clinical scan parameters, the size/shape and attenuation of the phantoms (added a fat ring to the Catphan 600 for approximating a medium size patient and the anthropomorphic abdomen phantom), the data sampling directly from the large DFOV, plus additional validation with an anthropomorphic phantom might also account for the discrepancy in results. Overall, this study is an initial step in the systematic evaluation of iterative reconstructions and is limited by the use of phantom data and objective ROIs. Depending on clinical applications, future task‐based image quality assessment will be conducted to evaluate overall image quality including, but not limited to, texture characteristics and spatial resolution at various contrast levels.CTDIvol, 120 kVp, and 1.375 pitch, Mieville et al.Regarding if/how iterative reconstructions affect material HU, there exist a small number of publications. Using the Catphan 600 phantom at 1 mGy It must be emphasized that the results presented here must be considered alongside the limitations of the methodology used to evaluate the reconstruction approaches. Although contrast and noise are basic properties of image quality, there are other metrics, such as the Fourier‐based noise power spectrum (NPS), that may provide characterization of additional dimensions of image quality by taking into account the multifaceted properties of noise and image texture. However, it can also be argued that the application of FFT to FBP and iterative reconstructed images, such as for the purposes of MTF or NPS analysis, is not appropriate because the basic assumptions of linearity and shift‐invariance are violated, especially for MBIR images. In the case of FBP, the assumptions come reasonably close and the scientific community has adopted the use of FFT methods.Therefore, a perceptional reader study is perhaps a more appropriate option for making a direct comparison of images reconstructed using FBP and iterative reconstruction methods. There are reports that, even though the apparent difference in the texture of the MBIR images makes it difficult to conduct a blinded reader study,We performed an objective comparison of FBP, ASiR, and MBIR using both modular and anthropomorphic phantoms over a wide range of image acquisition conditions: 3 levels of ASiR, 3 pitch factors, and 6–7 dose levels. Our results show that iterative reconstruction produced low‐dose images that are equivalent in image quality to that of conventional FBP images acquired at higher dose levels. In addition, HUs in MBIR images are highly sensitive to low‐dose levels, requiring careful attention for quantitative assessment of anatomy. A combined effort by clinical staff, radiologists, and medical physicists will be needed to integrate these findings into the clinical workflow and establish new CT protocols and standard operating procedures.This work was performed in collaboration with General Electric Healthcare Technologies at the Center for Advanced Biomedical Imaging, University of Texas MD Anderson Cancer Center.Creative Commons Attribution 4.0 International License.This work is licensed under a Supplementary Material FilesClick here for additional data file.


CNC-FS = 21.1 GPa, ECNC-OP = 28.7 GPa) were statistically larger than those obtained from CNFs . Additionally, the FS analytical approach provided statistically significant lower estimates. Thermal stability of CNFs and CNCs was investigated using thermogravimetric analysis. Significant differences were found between CNF and CNC onset temperatures , decomposition temperatures , and residues . This research enriches the information on thermal stability and nanomechanical performance of cellulose nanomaterials, and provides increased knowledge on understanding the effect of CNs as a matrix or reinforce in composites.Wood-based cellulose nanomaterials (CNs)  and cellulose nanocrystals (CNCs)) are environmentally sourced low-impact materials with remarkable thermal, mechanical, and physical properties. This uniqueness makes them great candidates for creating nanocomposite materials with a wide range of attributes. Investigating the morphological, thermal, and nanomechanical properties of CNs becomes crucial to intelligent development of novel composite materials. An atomic force microscope equipped with a nanoindenter was used to investigate the compression modulus of CNFs and CNCs using two analytical approaches (denoted as Oliver Pharr (OP) and Fused Silica (FS)). The CNC modulus values (E Reducing energy consumption, decreasing the dependency on unstable and uncertain petroleum sources, and an increase in environemental awareness produced and motivated researchers to work on developing new products using bio-based, eco-friendly, and sustainable polymers. Due to it’s nature, wood and its components are great candidates to develop ecofriendly products, since they have a very low environmental impact. Wood is an abundant renewable raw material, consisting of cellulose, hemicelluloses, lignin, and extractives in its structure. A crucial compound—cellulose—can be found as individual fibrils or as bundles in the cell walls, “glued” together by hemicelluloses and lignin . CelluloThe overall goal of this study is to provide introductory information to academic and industrial researchers working on developing composite materials using nanocellulose. Knowledge of the mechanical performance of nanoscaled materials is crucial to the development of new composites. Additionally, the thermal stability of reinforcements is required to select appropriate processing and use conditions.Et (transverse elastic modulus) is increased, which was found 17.7 ± 5 GPa [The lack of information on the nanomechanical performances of cellulose nanomaterials motivated many researchers to work on investigating the nanomechanical properties of CNs. Nanoindentation is a well established method to determine the mechanical performances of nanomaterials. A recent study on determining the mechanical performances of microcrystalline cellulose and investigating the effect of acid hydrolysis using nanoindentation provided modulus values 3.5–6 GPa for MCC [ ± 5 GPa . AnotherTM, was used to investigate the nanomechanical performance of CNFs and CNCs. The nanoindentation behavior was analyzed using two different approaches; the Oliver-Pharr (OP) [This study measured the nanomechanical behavior and thermal stability of two classes of cellulose nanomaterials (CNFs and CNCs). An Asylum Research MFP-3D Atomic force microscope, (AFM) equipped with nanoindenter (NI) and MFP Instrumented Nanoindenterarr (OP)  equationIn this study two types of cellulose nanomaterials  were used. The cellulose nanofibrils (3.0–4.0 w% aqueous gel) and cellulose nanocrystals (11.5–12.5 w% aqueous gel) were obtained from The Process Development Center (PDC), University of Maine. The CNFs were produced applying mechanical forces, using the double disk refiner system. The CNCs used in this study were produced using sulfuric acid hydrolysis to break down the amorphous regions in the structure, and to leave the crystalline cellulose to have a uniform, well designed structure with high crystallinity.h) to film thickness (t) ratio (h/t) equal or smaller than 25%, to minimize the errors in nanomechanical measurements that occur due to the substrate effect. For this purpose, CNF and CNC suspensions were dripped on the aluminum stubs with thicknesses varying from 8 μm to 15 μm, using partial oven drying (5 min at 40 °C) and air drying (till completion of the drying at 23 °C). The h/t ratio used in this research was kept under 5%.Starting materials: CNF/water suspensions were obtained at 3.4% solids by weight, and CNC/water suspension was obtained at 11.9% solids by weight. The solid contents of the suspensions were determined using a moisture content analyzer  before preparing the samples. The samples (films in this case) need to have smooth surfaces for AFM and NI applications. Film thickness has been shown to significantly impact nanomechanical response. Hay and Crawford  investigThe morphologies  of CNFs and CNCs films were examined using an Asylum Research MFP-3D atomic force microscope . For imaging, tapping mode is as known as non-contact or AC mode, and was performed using an Asylum Research AC240TS-10 cantilever tip with a 9 ± 2 nm radius  = 2 × (0.5–4.4)).The previously developed sampling method by Yildirim and Shaler (2016) was applied for each sample. For increased accuracy, 64 nanoindents (8 × 8 array over a 20 μm × 20 μm scan area) were applied to each group  for nanoindentations. A))—which goes into the equations directly—was calculated through two (2) different approaches. The approaches used in this research were Oliver Pharr (OP) and Fused Silica (FS).The calibration is a very critical step for measurements in nanoscale. For the evaluations, Oliver-Pharr  equationA) created on the surface after nanoindentation was assumed equal to the perfect Berkovich tip geometrical area (1). This is a strong assumption, which not only assumes that the area of the tip has a flawless geometrical Berkovich shape, but also assumes that the created area on the film surface will be identical to the tip’s geometrical area. The mathematical area calculation of the Berkovich shape, which was used for the OP approach area calculations, is given below (1).H = depth of indentation.In the OP approach, the projected area  specimen with 68 GPa–72 GPa  actual m−1 indentation rate. The slope of the unloading curves were used to calculate the stiffness of the samples. The stiffness value (S) of CNs was determined using the unloading curve slopes (n = 64), obtained from the force—indentation data.The nanoforces were applied to the CNs’ surfaces at a 125 μmsA) was calculated at 60 nm to a couple of hundred nm (H range) using the two different approaches previously described (OP and FS). The reduced modulus (ER) was then calculated (2) using the measured stiffness and area information from actual testing.The indented area .Equation (3) was then used to calculate E value of the diamond Berkovich tip (Eind) and the Poisson’s ratio (νind) were assumed to be 865 GPa and 0.2, respectively. The Poissons ratio (νCNx) of the sample CNs was assumed to be 0.3 [In this study, the o be 0.3 ,12,13.The thermal stability of CN’s was determined using a TA Instrument Q 500 thermogravimetric analyzer . Evaluations were performed according to ASTM E1868–10. Five (5) samples from each group were evaluated with a 10 °C per minute ramp, ranging from 20 °C to 500 °C. Initial sample weights varied from 10 mg to 15 mg.E), onset temperature, decomposition temperature (DTGA), and residue were analyzed and compared by conducting a one-way Means/ANOVA to check if there was a significant overall difference  = 0.01) due to difference in the type of cellulose nanomaterials (CNF vs. CNC). A two-way ANOVA  analysis was used on the nanomechanical data to assess whether the measured response was significantly impacted by material type or analytical method, or if an interaction effect was present. Significant differences between groups were evaluated by use of a Tukey-Kramer Honestly Significant Differences (HSD) test, with alpha = 0.05.The modulus  films were produced by air drying from each material. However, airdrying created imperfect (non-flat) films, due to heteregoneus drying (as expected). These non-flat samples were not suitable for imaging, so the flattest film was picked from each group and investigated. The produced CNF film included fibrils with diameters ranging between 20 and 100 nm, and lengths of the order of several micrometers. The CNC film was composed of whiskers with diameters less than 100 nm, and lengths with less than 300 nm .Due to heterogenous distribution, the imperfections on the surface and high height differences in samples effected the trace-retrace quality through topogrophical imaging, and resulted in lower quality 3D images.The nanoindentations were applied by centering the small scan grid in the filmed samples to avoid miscaluclations and evaluations. Also, researchers reviewed each force-indentation curve to see if there was skipping, cracks, and any other kind of failure through nanoindenting. Only, succesfull and accurate nanoindentations were analyzed. Results of the nanoindentations are presented and discussed in the following subsections.A fused silica quartz (FSQ) sample was obtained from Asylum Research for calibration. The mean modulus was calculated and statistically compared  for two The significant overestimation of the FSQ modulus, using the OP method, is primarily due to differences between the area of the perfect Berkovich tip and the projected area created on the sample surface.The tip geometry changes over the course of multiple uses, due to damage and surface contamination. Due to humidity in the chamber particles, the material surface may bind to the tip, and scratches may be created through the indentation; also, potential residues can be bound through mechanical interlocking, after the indentation. This nanoscale geometry change is dependent on the medium, material property, and adhesion, etc., so it is a random change that can be limited but not controlled. Our first assumption is: the area of the tip is accepted, perfectly and geometrically, in the same way, with the area of the Berkovich shape. However, this is almost impossible after the couple measurements. This overestimation can be lowered using new tips.A major limitation of the OP method is the assumption that the area of the Berkovich tip will be equal to the projected area created on the surface. This is mostly a material-related assumption, and brittle materials can increase overestimation, due to potential nanocracks through nanoindentation. The errors in the projected area calculations could easily increase, due this nanocracks or microcracks.Consequently, the required correction function  ) was appNanoindentations were performed on CNF and CNC films, and their nanomechanical performances were evaluated using two different approaches, to understand the importance of the chosen approach on the final result accuracy.A total of 64 nanoindents, created on different locations on a CNF film, were evaluated and statistically compared using OP and FS Approaches. The CNFs modulus and the average contact depth values are summarized in The difference between the reported modulus values in 64 nanoindentations were created on the CNC film samples using the same procedure as the CNF samples and represented in ECNF-FS = 12.4 GPa, ECNF-OP = 15.1 GPa–21.8% difference) and the CNC modulus value differences  between the two approaches were compared. The error (difference) for CNCs was found to be higher than the error in the CNFs. This showed that the error percentage in the results and failure in accuracy depend on the tested materials’ characteristics, and can easily go up. This increased error provided a good example to understand the importance of using the FS approach instead of the OP approach.As expected, a statistical difference was also found between the two approaches for the CNCs. The OP modulus values were again found to be significantly higher than modulus values calculated using the FS area approach. The CNF modulus value differences  was found to be almost two (2) times (47.4%) higher than CNF modulus . Also, the comparisons (according to the FS approach) showed that the CNC modulus  is 41.2% higher than the CNF modulus . The FS approach decreases the difference (error) between the CNF and CNC modulus, by decreasing errors through corrected area calculations.The CNF and CNC comparison, using both approaches, showed that the modulus differences in percentages (47.4% versus 41.2%) in both approaches are very similar. This finding shows that the OP approach can be used for comparison. However, it is strongly suggested to use the FS approach for investigating the properties of new materials, to obtain more accurate results.p < 0.0001) differ as a function of approach and material. Also, it differs as an interaction between approach and material. The effect of the approach differs according to the different type of material, and the material differs according to the different type of approach. The least square means were investigated, and CNC (on average) showed a higher response than CNF; the OP Approach showed a higher response than the FS Approach. The interaction showed that the chosen approach effect depends on the material, and also that the effect of CNC and CNF were identical.The results indicated that the modulus values significantly  values were found to be higher and statistically different than the CNF modulus . These high mechanical properties of cellulose nanomaterials created an attraction to use them as reinforce materials. Therefore, many studies were performed to investigate their feasibility to use as reinforce materials. A research on reinforcement of PVA (polyvinyl acetate) using NCC  showed that an addition of 1% NCC to PVA increases the MOE by 48% [As a result, the nanomechanical properties of CNF and CNC were investigated using two different approaches, with the coefficient of variation values less than 13.9%. In both approaches, the CNC modulus  for 48 h before testing. The initial weight loss, up to 10%  particles, which offer a higher surface area  .The residue of CNCs (34.4%) was significantly higher (50.9% higher) than the residue of CNFs (22.8%) at 500 °C. The literature studies showed similar results with this research; Lu and Hsieh  determinThe nanomechanical and thermal stability performances of the cellulose nanomaterials (CNs)  and cellulose nanocrystals (CNCs)) were determined and compared in this study. The nanoindentation technique, using two different approaches (the Oliver-Pharr and Fused Silica approaches) was performed to investigate CNs’ nanomechanical properties for increased accuracy. CNCs showed higher mechanical performance in both approaches, which can be related to CNCs’ potential higher crystalline structure . Contrary to the positive effect of the crystallinity on the nanomechanical performance of cellulose nanomaterials, it lowered the thermal stability properties of CNCs. CNCs are introduced to a higher amount of sulfate groups through the production process, which produces a pathway that has a fast heat transfer capability. This pathway decreases the thermal degradation temperatures of CNCs. Also, higher surface areas of CNCs, exposed to the heat source, lower the thermal stability.This research provides increased knowledge about the thermal stability and nanomoechanical performance properties of cellulose nanomaterials. Future work will include investigating the crystallinity and surface area of cellulose nanomaterials, and developing a relationship between their nanomechanical and thermal properties, theoretically.


MSTO1 gene: compound heterozygous mutations were identified in two sisters and in an unrelated singleton case, who presented a multisystem complex phenotype mainly characterized by myopathy and cerebellar ataxia. Human MSTO1 is a poorly studied protein, suggested to have mitochondrial localization and to regulate morphology and distribution of mitochondria. As for other mutations affecting genes involved in mitochondrial dynamics, no biochemical defects typical of mitochondrial disorders were reported. Studies in patients’ fibroblasts revealed that MSTO1 protein levels were strongly reduced, the mitochondrial network was fragmented, and the fusion events among mitochondria were decreased, confirming the deleterious effect of the identified variants and the role of MSTO1 in modulating mitochondrial dynamics. We also found that MSTO1 is mainly a cytosolic protein. These findings indicate recessive mutations in MSTO1 as a new cause for inherited neuromuscular disorders with multisystem features.We report here the first families carrying recessive variants in the  Mitochondria are highly dynamic organelles, which undergo constant fusion and fission events, and cristae remodeling. Mitochondrial dynamics influences several homeostatic and execution pathways involving the organelle and other compartments of the cell, and is in turn controlled by intraorganellar and extraorganellar metabolic requirements and signals, including the availability of nutrients, cell cycle, and calcium homeostasis. Mitochondrial fusion allows the exchange of damaged mitochondrial DNA (mtDNA) and proteins between impaired and “healthy” mitochondria, whereas mitochondrial fission facilitates their redistribution inside the cell  cause Charcot–Marie–Tooth disease type 2A (MIM# 609260), dominant optic atrophy (MIM# 165500), and severe infantile encephalomyopathies , respectively. Here, we report a family of four with two sisters, presenting with severe congenital myopathy and cerebellar ataxia associated with skeletal abnormalities and mild mental retardation as corollary symptoms of a multisystem disease. Whole‐exome sequencing (WES) analysis identified a compound heterozygous genotype of two missense mutations in MSTO1. Functional characterization of mutant cells demonstrated profound MSTO1 reduction in patients’ fibroblasts. Different compound heterozygous variants in MSTO1 were found by WES also in a singleton case presenting a dystrophic myopathy and cerebellar ataxia. Our results demonstrate that MSTO1 is a novel disease gene causing a mitochondrial morphology defect and leading to a muscular recessive disease  with multisystem involvement.Several mutations in genes‐encoding factors mediating mitochondrial fission or fusion have been associated with diverse human genetic diseases, with predominant neurological involvement. For instance, mutations in Detailed methods for biochemical assays, molecular genetics, structural and protein analyses, fluorescence microscopy, and functional studies in cells are reported in Supp. Information.Informed consent for genetic and biochemical studies was obtained from the parents of patients, in agreement with the Declaration of Helsinki and approved by the Ethical Committees of the Meyer Children Hospital, Florence, Italy, and of the Health Research Authority, NRES Committee East of England – Hatfield.rd percentile for both weight and height), had fine tremors and no autonomous walk, but normal muscle tone and normal cognitive development. At 7 years of age, a brain MRI disclosed global cerebellar hypotrophy, with reduction of the N‐acetyl aspartate peak at proton magnetic resonance spectroscopy ([1H+]‐MRS), and abnormal signal in the supratentorial peritrigonal white matter. These findings were confirmed at 16 years of age ; he has a healthy older sister. He presented at around 1 year of age with reluctance to stand and walk unsupported. Subsequently, he showed difficulty walking long distances, getting up from the floor, and frequent falls. Speech acquisition was delayed. Gradual improvement in motor activities was then noted and, at the age of 3.5 years, he was able to walk half a mile, although with frequent falls, and he acquired the ability to walk upstairs. Gowers’ time from lying was 6.4 sec. At the age of 6 years, he showed further improvement, showing less falls and a Gower's time of 3.6 sec. Weakness was proximal more than distal, affecting lower limbs (MRC power grade 3–4/5) more than upper (MRC power grade 4/5), with the exception of the peroneal muscles that were MRC power grade 3+/5. He presents mild end of range tightness of Achilles tendons but no other joint contractures. Deep tendon reflexes were present and symmetric; Babinski was negative. A degree of poor coordination, especially of the left hand, was noticeable from the age of 3 years. At the age of 6 years, he showed a fine intentional tremor bilaterally at finger‐nose test, dysdiachokinesia and difficulties with rapid hands repeated movements but no nystagmus. He shows no cognitive involvement but presents problems related to speech articulation. He has no cardiac or respiratory involvement, and forced vital capacity was 91% of predicted values. Ophthalmological examination was normal. An EMG/NCS showed changes compatible with a myopathic process. Brain MRI at the age of 3.5 years showed hypoplasia of cerebellar vermis and hemispheres . These findings remained static at a follow up of 2 years. Serum CK was 4,520 IU/L and serum lactate and transferrin were normal. Right thigh muscle biopsy at 2 years showed dystrophic changes with nonrimmed vacuoles. Oxidative histochemistry  did not show COX‐ or SDH‐deficient activities. Ultrastructural examination showed striking vacuolar degeneration of mitochondria .MSTO1, a gene regulating mitochondrial distribution and morphology, not previously associated with human diseases  (GenBank accession no. NM_018116.3): c.1033C>T; p.R345C and c.1128C>A; p.F376L  in ExAC database. Sanger sequencing in the probands and their parents confirmed the presence of the  c.1033C>T and the  c.1128C>A variants in both patients (A1 and A2), as expected for an autosomal‐recessive nonconsanguineous inheritance , two novel missense mutations were identified in  and A2, MSTO1 variants on transcript and protein, we studied available specimens from the patients. For A1 and A2, we used skin fibroblasts: quantitative PCR analysis of the retrotranscribed MSTO1 cDNA showed partial reduction of transcript amount  in both cell lines, but no aberrant species were observed. Notably, the total amount of MSTO1 protein detected by western blot (WB) analysis was drastically reduced in patients’ fibroblasts compared with controls . Then, we tested the antibody on total lysate, but no immunoreactive signal was observed at the expected molecular weight even in control samples , suggesting that the antibody does not work on muscle homogenate.In order to evaluate the effect of the identified MSTO1 variants by assessing the mitochondrial network in patients’ fibroblasts. Fibroblasts grown in either standard glucose or galactose medium were stained with a mitochondrial dye (Mitotracker) and examined by fluorescence microscopy. Patients’ cells showed a clear fragmentation of the mitochondrial network compared with controls. Altered mitochondria were also more abundant in immortalized fibroblasts from patient A1 than from controls  are also involved in peroxisomal morphology, we did check this aspect by immunofluorescence using an antiperoxisomal antibody (PMP70): in patients’ cells, we observed a normal morphology and distribution of peroxisomes that had only a slightly, not significantly, reduced mean area compared with control cells .Given the predicted role of MSTO1 in regulating mitochondrial morphology ; nevertheless, by Picogreen staining, we did not observe any evident alteration  in mitochondrial nucleoid distribution . Respiratory capacity, assessed by SeaHorse micro‐oxygraphy, as well as citrate synthase activity, were normal in patients’ fibroblasts (not shown).g supernatant, corresponding to the postmitochondrial fraction, but not in the mitochondria‐containing 13,000g pellet. Ultracentrifugation of the postmitochondrial supernatant at 100,000g indicated that MSTO1 is a soluble protein present in the 100,000g (cytosolic) supernatant but absent in the corresponding  pellet . Notably, the distribution of MSTO1 upon differential centrifugation is similar to that of DNM1L . Since the antibody against MSTO1 was not suitable for immunofluorescence studies, we expressed a HA‐tagged MSTO1 protein (MSTO1‐HA) in HeLa cells. The HA signal was distributed throughout the cytoplasm with only partial colocalization with a mitochondrial marker, whereas no signal was detected in the nucleus , thus confirming the results of the immunoblot experiments. The same pattern was observed also in COS7 cells transfected with MSTO1‐HA .MSTO1 was previously suggested to be a mitochondrial protein, localized in the outer membrane  in order to easily identify cells expressing the exogenous proteins . After transient overexpression of wild‐type MSTO1 in mutant and control fibroblasts, we observed that the mitochondrial network was unaffected at 24 hr, but at 48 hr, most of the cells with green signal collapsed, detached from the plate or floating in the medium; only a few cells were still adherent but showed fragmentation and perinuclear aggregation of mitochondria, both in patients and control cells . At 72 hr, no cells with green signal were found. Contrariwise, the single transfection of mtGFP had no major effect on the viability of fibroblasts and on the mitochondrial network even after >96 hr (not shown). These findings suggest that the overexpression of MSTO1 is deleterious, as already reported by Kimura and Okano (2007), who observed reduced vitality and perinuclear clusters of mitochondria after overexpression of MSTO1‐GFP in COS7 cells.Next, we tested whether the expression of wild‐type MSTO1 could recover the fragmented mitochondrial network observed in mutant fibroblasts. To avoid artifacts caused by fixation, we analyzed the effect of MSTO1 can be a new genetic cause for rare human disease, impairing the mitochondrial dynamic maintenance circuit. The fragmented mitochondrial network observed in mutant cells and vacuolar degeneration of mitochondria are in agreement with the proposed role of MSTO1 in mitodynamics. In fact, RNAi‐driven MSTO1 depletion leads to mitochondrial fragmentation, whereas overexpression of human EGFP‐tagged MSTO1 causes alterations in mitochondrial morphology and aggregation of mitochondria in the perinuclear region (Kimura & Okano, S. cerevisiae, where expression of DML1, the bona fide yeast ortholog of MSTO1, leads to mitochondrial dispersion and abnormalities in cell morphology (Gurvitz, Hartig, Ruis, Hamilton, & de Couet, The WES analysis in our families with myopathy and ataxia revealed that mutations of D. melanogaster were associated with irregular chromosome segregation during mitosis (Miklos et al., MSTO1 mutant flies showed severe morphological abnormalities and early death, the phenotype presented by our patients seems milder, suggesting a hypomorphic effect of the identified MSTO1 variants. Another possibility is that the function of the protein may diverge between arthropoda and humans.Given its homology with eukaryotic tubulin and prokaryotic FtsZ (Gurvitz et al., MSTO1‐mutant subjects have indeed a myopathy associated with cerebellar ataxia, underlined by hypotrophy of the cerebellar vermis; the sisters A1 and A2 also present a multisystem condition with developmental delay, particularly motor impairment with ataxia and dysmorphisms.Most of the patients with mutations in genes related to mitodynamics presented a neurological phenotype; our OPA1 (Mayorov et al., DNM1L mutations (Nasca et al., Our biochemical analyses were not indicative of a mitochondrial disorder: patients showed normal lactate levels, and hardly any defect of MRC complex activities or cell respiration, as frequently seen in other defects of mitodynamics, such as MSTO1 lead to impairment of mitochondrial dynamics, and are associated, in our affected patients, with a disease characterized by neurological, muscular, and sometimes skeletal involvement. Notably, a family with an autosomal‐dominant mutation in MSTO1 was recently identified (Gal et al., MSTO1‐related disease.Our study confirms the proposed role for MSTO1 in regulating mitochondrial morphology, favoring fusion versus fission events, despite its mainly cytoplasmic localization. We showed that compound heterozygous recessive mutations in MSTO1 cause mitochondrial dynamics impairment, leading to myopathy and ataxiaONLINE SUPPORTING INFORMATION Recessive mutations in Click here for additional data file.


Eremomastax speciosa on “unhealed gastric ulcers” associated with gastric acid hypersecretion. This work investigated the healing and antisecretory effects of the aqueous extract of “Unhealed gastric ulcers” were induced using indomethacin following the establishment of acetic-acid-induced chronic gastric ulcers. The extract  was administered concomitantly with indomethacin . The effects of the extract on both basal and histamine-stimulated gastric acid secretion were determined. Mucus secretion and oxidative stress parameters were measured, and histological assessment of ulcer healing was carried out.p < 0.05) increase of mucus secretion and concentrations of antioxidant enzymes compared with the controls. The extract at the highest dose showed normalization of the mucosa, without glandular destruction and with the disappearance of fibrosis and lymphocyte infiltration. The extract significantly promoted the healing process in rats subjected to “unhealed gastric ulcers” . Treatment with the extract significantly reduced the basal (25.95–49.51% reduction rates) and histamine-stimulated (24.25–47.41%) acid secretions. The healing effect of the extract was associated with a significant ( The abilities of the extract to increase mucus secretion, to reinforce antioxidant status, and to inhibit acid secretion would be some of the mechanisms by which this extract would accelerate the healing process in “unhealed gastric ulcers.” Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) are among the most commonly used drugs in the world. In the United States, approximately 70 million prescriptions are written each year, while in Europe these medications represent more than 7.7% of all prescriptions , 2. The To counteract the deleterious actions of NSAIDs on the gastrointestinal tract, potentially less toxic NSAIDs (COX-2 inhibitors) have been introduced. In spite of the use of these COX-2 inhibitors, the treatment of gastric ulcers associated with NSAIDs consumption is performed using proton pump inhibitors (PPIs) . Several Eremomastax speciosa is not cited in the available literature for its use against gastric complications in folklore medicine, personal information obtained from local traditional healers suggested the possible antiulcer potential of the plant. Thus, in a preliminary study, the water extract of E. speciosa significantly inhibited the formation of gastric lesions induced by HCl/ethanol and pylorus ligation [ E speciosa were evaluated on “unhealed gastric ulcers” associated with gastric acid hypersecretion stimulated by histamine, in addition to the duration of its antisecretory effect. The effects were compared with that of sucralfate, a nonantisecretory mucosal protective agent which has been experimentally shown to speed up gastric ulcer healing.However, treatment using these PPIs presents many adverse effects. For example, the adverse effects attributed to omeprazole, which is the most prescribed PPI, include headaches, diarrhea, abdominal pain, nausea, sleep deprivation , pneumonligation . Subsequligation . Other wligation . Amang eligation  have sholigation . On the  E. speciosa were collected in April 2015, at 6 a.m., in Yaoundé, Center Region of Cameroon. Botanical identification was done at the National Herbarium, Yaoundé, by Paul Mezili, by comparison with existing herbarium specimen number HNC/136984.The fresh leaves ofMale albino Wistar rats aged 12-13 weeks, weighing between 150 and 200 g, were used for the experiments. The animals were raised in the Animal House of the Animal Physiology Laboratory, Faculty of Science, University of Yaoundé I. They were kept at room temperature under natural day/night cycles. They were fed with a standard laboratory diet  and given tap water ad libitum. Prior authorization for the use of laboratory animals in this study was obtained from the Cameroon National Ethics Committee (registration number: FWA-IRB00001954), which permits, among other procedures, the use of ether anesthesia for animal research. Otherwise, the use, handling, and care of animals were done in adherence to the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Purposes (ETS-123), with particular attention to Part III, articles 7, 8, and 9 .The leaves were chopped and quickly dried under the shade to prevent them from getting moldy and then ground using a mechanical grinder to obtain a fine powder. 400 g of the powder was extracted by infusion in 4 liters of boiled water for 15 minutes. After filtration through Whatman filter paper No. 3, the filtrate was evaporated using a Raven ventilation oven . The brownish solid obtained  was stored at 4°C. The extract was dissolved in distilled water which was used as the vehicle.Induction of “Unhealed Gastric Ulcers” and Basal Acid Secretion. The method described by Pillai and Santhakumari [hakumari  was usedhakumari . BrieflyFive days after the acetic acid injection, indomethacin suspended in saline solution was given once daily to all the remaining rats  for 2 weeks in order to establish “unhealed gastric ulcers.” The extract (200 and 400 mg/kg), sucralfate (50 mg/kg), or vehicle (1 ml/200 g b.w.) alone was given per os (p.o.) once daily 0.5 hours after indomethacin treatment for 2 weeks. On day 13 of treatment , the animals were deprived of food but allowed free access to water. 0.5 or 12 hours after the final administration (day 14), basal acid secretion was studied by the pylorus ligation technique described by Shay et al. [Induction of “Unhealed Gastric Ulcers” and Histamine-Induced Gastric Hypersecretion. The same protocol described above was performed but with slight modifications. Following the establishment of “unhealed gastric ulcers,” 0.5 or 12 hours after the final administration of vehicle, sucralfate, or extract, pylorus ligation was performed in the rats. Histamine-induced gastric acid hypersecretion was studied by injecting histamine  in all the remaining rats 1 hour after pylorus ligature. Four hours later, the animals were sacrificed and submitted to the same procedures as described above.The mucus covering each stomach was gently scraped using a glass slide and the mucus was weighed carefully using a sensitive digital electronic balance. The same experimenter performed this exercise each time .The gastric contents were collected and centrifuged at 4000 rpm for 10 min, to remove residual debris. The volume of the gastric juice was measured using a graduated test tube. 1 ml of centrifuged gastric juice was used to determine the hydrogen ion concentration by pH-metric titration against NaOH solution (0.1 N) using a digital pH meter. The acid content was expressed as mEq/l .Sections of stomach walls were made perpendicular to the surface of each ulcer crater. Sections of the normal stomach were also made for comparison. Hematoxylin and eosin stains of stomach sections were then prepared following standard histological procedures described by Bayelet-Vincent  and the μmol of H2O2/min/mg of protein. Reduced glutathione (GSH) was measured based on the reaction between 2,2-dithio-5,5-dibenzoic acid and the thiol (SH) groups of glutathione to yield a complex whose absorbance was read at 412 nm [ε = 13600 cm/mol and the results are expressed in nmol/g of tissue. Lipid peroxidation was assessed by measuring the levels of malondialdehyde (MDA) in gastric tissue samples [5 M−1 cm−1 and expressed as μmol of MDA/g of wet stomach tissue. Tissue protein was measured using the Biuret method of protein assay [Oxidative stress parameters were measured in gastric tissue samples. Superoxide dismutase (SOD) activity was measured using a standard method  and exprt 412 nm . The glu samples . Quantifin assay .p < 0.05.Values in tables are given as arithmetic means ± standard error of the mean (SEM). The significance of differences between groups was analyzed by means of Analysis of Variance (ANOVA) followed by Student-Newman-Keuls comparison tests using GraphPad Prism 5.03 software. All the differences were considered significant at 2 (9.07% of glandular area). In the control rats (vehicle control) that were given the vehicle concomitantly with indomethacin during the two weeks following the establishment of ulcers, the ulcer area dropped to 39.20 ± 7.71 mm2 (pylorus ligation) or 42.50 ± 5.29 mm2 (histamine + pylorus ligation) indicating autohealing of 35.95% and 30.56%, respectively. The extract, given concomitantly with indomethacin for 2 weeks, significantly promoted the healing process , with healing rates of 82.35 and 85.17%, respectively , catalase (CAT), and reduced glutathione (GSH) decreased, and malondialdehyde (MDA) increased, in the vehicle control compared with normal rats. In the two experimental methods, extract administration significantly increased concentrations of SOD, CAT, and GSH compared with the vehicle controls. On the contrary, the levels of MDA significantly decreased in extract-treated rats compared with the vehicle controls. However, these levels of oxidative stress parameters did not return to those observed in normal rats.Tables  E. speciosa on gastric ulcers [Previous studies revealed the prophylactic and healing activities of the aqueous extract ofc ulcers –19. In t+ ions through the channels in the mucus layer, towards the internal layers of the gastric membrane. This results in tissue necrosis which triggers the release of arachidonic acid metabolites and attracts leucocytes (polynuclear neutrophils and macrophages) leading to the transformation of superficial injury into deeper mucosal lesions and to inactivation of growth factors essential for mucosal integrity and repair [2) observed in ulcerated rats (Control 1) sacrificed 4 days after acetic acid ulcer induction.The experimental model of acetic-acid-induced chronic ulcers easily and reliably produces round, deep ulcers in the stomach that highly resemble human ulcers in terms of pathology and healing . These ud repair , 33. Thiα, interleukin-1β) and the impairment of growth factor biosynthesis  might contribute to the mechanism underlying “unhealed gastric ulcers.”Previous work  showed t E. speciosa extract (200 and 400 mg/kg) significantly prevented the delayed ulcer healing in response to indomethacin without histamine  and with histamine  stimulation. The healing process of the extract was associated in all the tested models with a significant increase in mucus production compared with the vehicle control. The importance of increased mucus strength and quantity in protecting the regenerating gastric epithelium is well known [In this work,ll known . Indeed,ll known . E. speciosa extract against HCl/ethanol-induced gastric ulcers was suppressed when the rats were pretreated with indomethacin, and it was interpreted that the extract was acting through the intermediary of endogenous PGE. This interpretation was supported by the significant increase of mucus production in extract-treated animals compared with the vehicle control. Endogenous PGs play an essential protective role in the stomach by stimulating the synthesis and secretion of mucus and bicarbonate, increasing mucosal blood flow, and promoting epithelial proliferation [2. Thus, the stimulation of mucus secretion mediated by PGE could partly explain the mechanism by which the extract prevents the delayed healing caused by indomethacin in acetic-acid-induced ulcersIn previous work, Amang et al.  demonstrferation , 40. Okaferation  showed tferation  reported2•−) into less noxious hydrogen peroxide (H2O2), which is further degraded by catalase or glutathione peroxidase. Catalase enzyme accelerates the degradation of H2O2 into water and oxygen [2O2 metabolism depends on the activity of glutathione peroxidase (GPx) and cooperating glutathione reductase. The reduction of H2O2 into water by GPx is accompanied by the conversion of glutathione from the reduced form into the oxidized form [ E. speciosa. These phytochemical compounds have well-known antioxidant activity and have been associated with gastric mucosal protection [The pathogenesis of “unhealed gastric ulcers” also involves the generation of reactive oxygen species (ROS)  due to td oxygen . The seczed form –51. The zed form  reportedotection –57. E. speciosa has antisecretory effects which involve a mechanism common to both cholinergic and histaminergic pathways. Consequently, the observed significant drop of basal and stimulated (histamine) acid secretion by the extract in the present work suggests the implication of its antisecretory effects in the healing process.Gastric acid secretion is significantly involved in the delayed healing of gastric ulcers, and the control of gastric acid secretion represents a cornerstone for the promotion of ulcer healing . There i E. speciosa extract on the indomethacin-induced delay of chronic ulcer healing may occur by the same mechanism.The extract, given repeatedly for 2 weeks, significantly inhibited both basal and histamine-stimulated gastric secretion in rats with “unhealed gastric ulcers” up to 12 hours after the last administration. Wang et al.  reported E. speciosa accelerated the healing of “unhealed gastric ulcers” following the combined action of several mechanisms which involve the increases of mucus production and antioxidant levels on the one hand and the decrease of gastric acid secretion on the other hand. All these factors taken together can enhance angiogenesis, cellular proliferation, cellular migration, and granulation tissue maturation. The results of the present study suggest that E. speciosa extract can be a useful resource for the production of a standardized antiulcer product, especially given that toxicological studies have demonstrated the innocuousness of this plant extract [Histological sections of rat stomachs illustrate the damage caused by acetic acid and indomethacin as well as the healing process induced by the extract. In general, following ulcerative injury, ulcer healing is initiated by the formation of the “healing zone.” At this stage, inflammatory infiltration occurs close to the necrotic tissue and ulcer crater. In response to growth factors, the ulcer margin is formed by cells adjacent to the margin and granulation tissue develops at the ulcer base. During healing, the granulation tissue undergoes continuous remodeling, whereby the inflammatory cells that appeared in the early phase of healing are replaced by fibroblasts and microvessels in the late healing phase . The maj extract .Study Limitations. This study did not provide information on the identification of the active components of E. speciosa extract. Thus, a detailed discussion of the exact mechanism of antisecretion and healing action of the extract could not be given.Second, data for sucralfate antisecretory effects against histamine-induced hypersecretion are not provided for detailed discussion of results in Tables  E. speciosa extract to increase mucus production, to reinforce the antioxidant status, and to inhibit acid secretion by a mechanism involving both cholinergic and histaminic pathways constitute possible ways by which the extract accelerates the healing of “unhealed gastric ulcers.” This healing process might be reinforced by the long-lasting antisecretory effect of the extract. Thus, the use of this plant extract could be recommended for the management of chronic gastric ulcers for patients who are concomitantly taking NSAIDs to treat other ailments.The abilities of