To explore the effect of amylose within starch granules on the efficiency of starch hydrolysis by acid, we chose the warm water extraction method to treat red adzuki bean starch to obtain different degrees of amylose removal granule models and to prepare samples in combination with acid hydrolysis. The amylose content was reduced after acid hydrolysis, reducing the peak viscosity (2599–1049 cP), while the solubility was significantly increased. In contrast, the short-chain content of the deamylose–acid hydrolysis samples was reduced considerably, exacerbating the trend towards reduced starch orderliness and increased solubility. This work reveals the granular structure of starch from the point of view of deamylose and contributes to a thorough understanding of the mechanisms of acid hydrolysis. It might add to knowledge in starch science research and industrial applications for the acid processing of starch-based foods, particularly with regard to the most important factors controlling the structure and function of starch. Starch is an important renewable resource in nature and is widely used in food, medical, biological and material applications . HoweverStarch consists of two types of dextran: amylose and amylopectin. Amylose is a relatively long and poorly branched polymer, whereas amylopectin is a highly branched polymer. The starch chains are linked by α-1,4) bonds and branched by α-1,6) bonds. Amylopectin side chains can form continuous amorphous and crystalline flakes . The int bonds an bonds. AMoreover, acid hydrolysis has also been proven to produce low-molecular-weight starches with a large reduction in liquid viscosity after paste formation, which has led to more and better applications in the food industry, such as fondant and starch jelly, in the paper industry as a surface sizing agent, to improve printability, and in the textile industry for warp sizing ,9. High-Much effort has been put into further understanding the structure and properties of different starches in terms of amylose content to obtain better industrial properties through acid hydrolysis. However, the presence of amylose has prevented a clear understanding of the degradation characteristics of starches in terms of structure and properties . We hypoRed adzuki beans were bought from Yangling Haoyouduo supermarket (Xianyang China). 8-aminopyrene-1,3,6-trisulfonate sodium salt (APTS) was obtained from Aladdin Reagent Co., Ltd., Shanghai, China. All other chemical reagents were of analytical grade.g, 15 min), and the supernatant and the top cream-colored layer were discarded. The purified starch isolate was dried in a hot air oven at 45 °C for 1 d.The red adzuki bean starch was isolated by using the method of Li et al.  with minv/v; 3000 rpm, 24 h). The residue was air-dried in a fume hood after standing for 1 h to obtain defatted starch. Next, 10 g of the above starch was mixed with distilled water (300 mL) and placed in a water bath at 50 °C for 1 h and 2.5 h. The supernatant was removed , washed twice with distilled water and centrifuged . Finally, the residue was freeze-dried and passed through a 150 µm sieve. The samples were named DA-1 and DA-2.5.Then, 30 g red adzuki bean starch was dispersed in 300 mL methanol . The residue was placed in a blast-drying oven at 40 °C, crushed and passed through a 150 µm sieve. The samples obtained were named H-0.3, H-0.6 and H-0.9.First, 10% starch emulsion was added to HCl solutions at concentrations of 0.3 mol/L, 0.6 mol/L and 0.9 mol/L and reacted for 2 h in a constant-temperature shaker at 30 °C, and then the reaction was terminated with NaOH (0.5 M). The sample was washed with distilled water and the process was repeated thrice  at 5.0 kV and 3000× magnification.v/v). Finally, a drop was taken on a slide for observation using a CLSM  fitted with a 100× plan apo/1.4011 oil UV. First, 2 mg starch was dispersed in a mixture of 4 µL APTS (10 mM) and 4 µL sodium cyanoborohydride (1 M) . The reacted sample was washed with distilled water and then dispersed in 20 µL of a glycerol–water mixture , and the sample morphology was observed using light microscopy under a 400× objective.A small amount of the sample was homogenized with water–glycerol , and the supernatant was diluted 50-fold and passed through a 0.22 μm aqueous membrane in a sample bottle. Sample preparation: starch (40 mg) was dispersed in 2 mL acetate buffer  and pasted in a boiling water bath for 10 min. After cooling to 25 °C, 5 µL pullulanase was added and reacted . The enzyme was deactivated in a boiling water bath for 20 min, cooled and centrifuged . The condition parameters were as follows: Cu-Kα radiation; 2θ: 4° to 60°; scanning rate: 6°/min. −1, scanning numbers: 16, resolution was 4 cm−1.Fourier transform infrared (FT-IR) red adzuki bean starch spectra were collected with a Vertex 70 spectrometer; scanning range: 4000 to 400 cmThe amylose content of the red adzuki bean starch was determined by referring to an iodine-binding procedure of Gao et al. . First, An RVA-Tec Master  is the device used for pasting property measurement. First, 3.0 g starch and 25 mL deionized water were mixed, which was calculated to a wet basis of 14%. Red adzuki bean starch was equilibrated at 50 °C for 1 min, and then was heated at a rate of 12 °C/min to 95 °C, held for 2.5 min, then cooled to 50 °C at the same speed and held for 2 min. 1) was dispersed in 25 mL distilled water, and the prepared 2% starch solution was placed in a centrifuge tube. The tubes were centrifuged after shaking for 0.5 h in a water bath at different temperatures . The supernatant was then poured into an aluminum box and dried, and the box (m2) and the centrifuge tube (m3) were weighed. The solubility and swelling power were determined as follows:About 0.5 g starch (mp < 0.05) was applied for multiple comparisons. Principal component analysis (PCA) and Pearson’s correlation analysis were analyzed using Origin 2023.All statistical calculations were performed in SPSS version 21.0. Analysis was executed by using ANOVA, and Duncan’s test  decreased further, i.e., hydrochloric acid modification after DA exacerbated the decreasing amylose content. The amylose content decreased significantly with increasing HCl concentration or warm water deamylose treatment time. In addition, the amylose content of red adzuki bean starch decreased with increasing hydrochloric acid addition at the same deamylose time, and the amylose content of the samples decreased gradually with increasing deamylose treatment time at the same hydrochloric acid concentration. This might be because hydrochloric acid mainly affected the amorphous region of the red adzuki bean starch, which was acidolyzed into small short-chain molecules that penetrated the starch granules . It was also possible that the treatment condition of warm water destroyed the amorphous zone of the starch granules, making it easier for the hydrochloric acid to enter the granules and reduce the amylose content. There was no significant difference in the amylose content of samples DA2.5-H0.6 and DA2.5-H0.9. After the water bath deamylose treatment, HCl may have entered the red adzuki bean starch granules more easily and caused damage to the amorphous zone. The high concentration of hydrochloric acid further affected the crystalline zone and the branched starch was cleaved, resulting in rearrangements such as amylopectin–amylose and amylose–amylose [The peak, trough, and final viscosities decreased gradually with the increase in the treatment time. In contrast, the attenuation value, regeneration value, gelatinization temperature and pasting time all increased gradually. This indicates that the maximum swelling power of the red adzuki bean starch granules decreased after the water bath deamylose modification, a result reflected in After HCl treatment, the peak viscosity, final viscosity and trough viscosity of red adzuki starch decreased and were negatively correlated with the concentration of hydrochloric acid added. At the same time, the changes in pasting time and gelatinization temperature were not significant. This indicated that acid digestion destroyed the structure of the amorphous region of the starch and rearranged it to form a more stable crystalline structure , resultiFrom the solubility results in Under low ambient temperature conditions, the swelling power of the hydrochloric-acid-modified red adzuki bean starch granules increased and then decreased without significant difference. This might be related to the rupture of the amorphous region of the red adzuki bean starch granules after the low concentration of hydrochloric acid treatment and the simple rearrangement between molecules. The rearranged starch molecules were unstable, and hydrogen bonding between adjacent starch polymers was disrupted , resultiIn addition, acid digestion destroyed the crystalline zone, reducing swelling power . The sweThe correlation between multiscale structures and physicochemical properties among native starch, DA starch, HCl-modified starch and dual-modified starch can be verified using PCA scoring plots A and loaAs shown in In addition, Chen et al.  also comStarch is divided into crystalline and amorphous regions. To our knowledge, the deamylose treatment causes the amylose with a smaller molecular weight to penetrate the starch surface. With the entry of water molecules, the glycosidic bonds between the starch molecules were broken . At the −1/1022 cm−1 and RC were first increased and then reduced. It was confirmed that the small molecular weight of amylose was mainly enriched on the surface of the starch granules, and the short-time deamylose treatment made the starch structure more compact, while the extended time caused damage to the granule structure. After acid digestion, the starch granules showed wrinkles and a significant decrease in PV. In addition, the A chain proportion and the AC decreased, indicating that hydrochloric acid mainly affected the structure of the amorphous region of the starch granules. DA-HCl treatment revealed a more significant decrease in AC, RC and A chain content and a substantial increase in solubility. This study shows that the amylose content severely affects the multiscale structure and physicochemical properties of starch, which also provides a basis for developing degradable materials and food additives.In this research, one facility was proposed for modifying red adzuki bean starch through warm water deamylose treatment, and its feasibility for improving starch properties was verified by exploring its multiscale structure. The effect of hydrochloric acid digestion on the deamylose was further investigated. The surface of red adzuki bean starch granules was deformed and depressed after deamylose; the AC and A chain content were significantly reduced, while the ratio of 1047 cm


Every beginning is under the auspices of positive emotions, being a good reason for introspection and for sharing one’s vision for the future. Both journals “Oftalmologia” (from 1994 to 2015) and “Romanian Journal of Ophthalmology” (from 2015) brought an invaluable scientific contribution to ophthalmologists, not only in Romania, but throughout more than 25 countries from all over the world. With small, but confident steps, and with the increased and tireless efforts of the previous Editor-in-Chief and the Editorial Board, Romanian Journal of Ophthalmology turned into an increasingly visible journal, becoming a landmark for Eastern Europe and Romanian doctors and scientists. At present, the journal is indexed by PubMed and has a large scientific contribution especially from foreign authors.I accepted to take over the position of Editor-in-Chief for Romanian Journal of Ophthalmology from Mihail Zemba, MD, PhD. I am currently an Associate Professor in the Ophthalmology Clinic of the Emergency University Hospital of Bucharest. As an ophthalmologist I had the chance to learn from amazing professors and doctors and to share their values, professionalism, and vision for the future. Having the opportunity of continuing the work of my predecessors, makes me aware of the fact that such a rich and meaningful heritage implies a difficult task and at the same time, a great responsibility towards the readers of the journal and towards the future.We are presently living a time of continuous change, so we must keep up with it. My colleagues from the editorial team and I are committed to continuing the legacy of our predecessors. Our plans include the following: to increase the quality selection of reviewers and supportive editing of articles, to further improve the quality of the published papers, to keep the journal indexed by PubMed by increasing the contributions of articles from foreign authors and thus, increasing the journal’s visibility and the number of cited articles, to continue and, hopefully, succeed in the efforts of obtaining the journal first impact factor, turning the journal into a pillar of science for the ophthalmologists and scientists throughout Eastern Europe. My colleagues from the editorial team and I are embarking in this new journey with the hope of enhancing the journal’s value, visibility, and perspectives and of making it an asset in which medical and scientific worlds can merge, and from which we can all gain as professionals.Editor-in-Chief Ruxandra Pîrvulescu, MD, PhD,Associate Professor, Ophthalmology Department,“Carol Davila” University of Medicine,Emergency University Hospital, Bucharest, Romania


Regulating ankle mechanics is essential for controlled interactions with the environment and rejecting unexpected disturbances. Ankle mechanics can be quantified by impedance, the dynamic relationship between an imposed displacement and the torque generated in response. Ankle impedance in the sagittal plane depends strongly on the triceps surae and Achilles tendon, but their relative contributions remain unknown. It is commonly assumed that ankle impedance is controlled by changing muscle activation and, thereby, muscle impedance, but this ignores that tendon impedance also changes with activation-induced loading. Thus, we sought to determine the relative contributions from the triceps surae and Achilles tendon during conditions relevant to postural control. We used a novel technique that combines B-mode ultrasound imaging with joint-level perturbations to quantify ankle, muscle and tendon impedance simultaneously across activation levels from 0% to 30% of maximum voluntary contraction. We found that muscle and tendon stiffness, the static component of impedance, increased with voluntary plantarflexion contractions, but that muscle stiffness exceeded tendon stiffness at very low loads (21±7 N). Above these loads, corresponding to 1.3% of maximal strength for an average participant in our study, ankle stiffness was determined predominately by Achilles tendon stiffness. At approximately 20% MVC for an average participant, ankle stiffness was 4 times more sensitive to changes in tendon stiffness than to changes in muscle stiffness. We provide the first empirical evidence demonstrating that the nervous system, through changes in muscle activations, leverages the non-linear properties of the Achilles tendon to increase ankle stiffness during postural conditions. Summary: During postural conditions, the stiffness of the ankle in humans is determined primarily by the stiffness of the Achilles tendon, not that of the triceps surae muscles. Determining how the triceps surae and Achilles tendon mechanics contribute to ankle mechanics across a broad range of physiological conditions would provide fundamental insight into the mechanisms underlying humans' ability to navigate their physical world. Such knowledge could also aid in developing targeted interventions when musculotendon mechanics are altered due to neuromuscular pathologies, or in developing biomimetic assistive devices  displacement, which serves as a proxy for changes in tendon length , suggesting that it is less stiff than the muscle . During in vivo in humans. As such, it is unknown under which conditions it is reasonable to assume that muscles and tendons behave as simple springs with only a stiffness component and when they exhibit more complex mechanical properties. We, therefore, quantified these regimes in this study.The objective of this study was to determine how the triceps surae and Achilles tendon contribute to the impedance of the ankle during conditions relevant to postural control. We used an innovative technique that combines joint-level perturbations with B-mode ultrasound to quantify ankle, muscle and tendon impedance . Given tSeventeen healthy young adults  participated in this experiment. All participants were right-leg dominant and had no history of neuromuscular or musculoskeletal injuries to their right leg. All participants provided written informed consent prior to participation. The Northwestern University Institutional Review Board approved the study, and all methods were carried out according to the approved protocols (STU00009204 and STU00213839).Participants were seated in an adjustable chair , with their trunk and torso stabilized with safety straps . The parSingle differential bipolar surface electrodes  measured muscle activity from the medial and lateral gastrocnemius and soleus (ankle plantarflexors) and the tibialis anterior (ankle dorsiflexor). Standard skin preparation methods were performed prior to electrode placement , and eleA B-mode ultrasound system using a linear transducer  recorded images of the medial gastrocnemius MTJ. A custom-made probe holder and elastic adhesive wrap  secured the probe to the leg. We positioned the ultrasound probe to center the MTJ on the image. At the start of ultrasound data collection, a trigger signal was used to synchronize the ultrasound data collection with all other measurements. Ultrasound images were acquired with a mean frame rate of 124 Hz. All ultrasound data were saved for processing offline.At the start of each experiment, we collected three 10 s isometric MVC trials in both the plantarflexion and dorsiflexion directions. These data were used to scale the visual feedback provided to the participants.Our primary objective was to determine how muscle, tendon and ankle impedance vary across various levels of plantarflexion torque. This was accomplished by instructing participants to produce different levels of isometric plantarflexion torque while the rotary motor applied small rotational perturbations in the sagittal plane. We used pseudo-random binary sequence (PRBS) perturbations with an amplitude of 0.175 radians, a maximum velocity of 1.75 radians per second, and a switching time of 153 ms. We tested seven isometric plantarflexion torque levels from 0% to 30% MVC in 5% increments. Participants were provided with real-time visual feedback of their normalized plantarflexion torque. Tibialis anterior EMG was also provided to prevent co-contraction. Rectified EMG and torque signals were low-pass filtered at 1 Hz to remove high-frequency components from the applied perturbations (2nd order Butterworth). Participants completed three trials at each level of plantarflexion torque in a randomized fashion. Each trial lasted 65 s. Rest breaks were provided as needed between trials to prevent fatigue.The measured ankle torque included the gravitational and inertial contributions from the apparatus connecting the foot to the motor. A single trial was collected with only the cast attached to the rotary motor enabling us to remove these contributions from the net torque measured in each trial.All data were processed and analyzed using custom-written software in MATLAB. The same individual manually digitized the MTJ within each frame of the ultrasound videos . All ultWe computed ankle, muscle and tendon impedance as described previously . BrieflyA single approximation of the Achilles tendon moment arm (51.4 mm) was used for all analyses. This was estimated as the mean across participants from KA represents the modeled ankle stiffness, torque (τ) was the input to the model, and β, KA1 and KA0 are the optimized parameters. A similar model has been used to characterize load-dependent changes in the stiffness of a MTU (Ankle and tendon stiffness – the low-frequency component of impedance – varied non-linearly with plantarflexion torque (or musculotendon force). Therefore, the ankle and tendon stiffness experimental data were fitted with non-linear models to synthesize our results. The model used to characterize torque-dependent changes in ankle stiffness was:of a MTU .KT represents the modeled tendon stiffness, musculotendon force (F) was the input to the model, and KTmax, a and b are the optimized parameters. This model was chosen because exponential models have been used previously to characterize the non-linear toe region of the tendon stress–strain curve  is determined by the serial connection of the muscle and tendon and can be described as a function of these stiffnesses :We evaluated the sensitivity of ankle stiffness to changes in muscle and tendon stiffness at different levels of force. We first consider that ankle stiffness  for each participant from the respective mixed-effects model. We tested the null hypothesis that the muscle and tendon contribute equally to ankle stiffness. We used a bootstrapping procedure to determine the range of musculotendon forces when muscle and tendon stiffness were not significantly different from each other to a level of P>0.05. The bootstrapping involved randomly resampling the data from each participant with replacement to create a new dataset for the entire pool of participants. This process was repeated 200 times. Each synthesized dataset was analyzed as described above to create a distribution of estimates for which muscle and tendon stiffnesses were the same. Our null hypothesis – that muscle and tendon stiffness contribute equally to the stiffness of the ankle – was accepted within the 95% confidence intervals of this distribution and rejected elsewhere. All metrics reported are means±95% confidence intervals unless otherwise noted.We sought to determine how the triceps surae and Achilles tendon contribute to the impedance of the ankle over a range of activation levels. Non-linear mixed-effects models were used to characterize the ankle stiffness–torque relationship and the tendon stiffness–force relationship (Eqns 1 and 2). A linear mixed-effects model was used to describe the muscle stiffness–musculotendon force relationship. For all models, subject was treated as a random factor, and plantarflexion torque or musculotendon force was a continuous factor. A restricted maximum likelihood method was used to estimate all models . The modAt all levels of activation, the magnitudes of the frequency response functions for muscle and tendon impedance were nearly constant from 1 to 6.5 Hz , indicatR2=0.94; tendon: R2=0.91; R2=0.94±0.01; tendon: R2=0.94±0.01; Muscle and tendon stiffness increased with increases in musculotendon force . Fig. 4AWe found that muscle stiffness increased at a greater rate with increases in force than did tendon stiffness. Data for a representative participant shown in R2=0.99), and the full population of tested partcipants (R2=0.98±0.007). Our values of ankle stiffness are consistent with previous reports using a similar experimental protocol   . Previouurbation . Therefourbation , it is amportant . We alsomportant  and connmportant . These cin vivo studies conducted during active conditions have only estimated its stiffness above 30% MVC to satisfy the methodological assumption that tendon stiffness is constant. Our approach is not constrained by this assumption, allowing measurements to be made at lower forces corresponding to activation levels that occur during everyday activities such as standing and walking  . This asLastly, our measurement technique assumes that we are estimating the net impedance of the triceps surae and Achilles tendon, despite only making ultrasound measurements at the medial gastrocnemius MTJ. However, we have previously validated this assumption and found that our estimates during active contractions were similar when imaging the medial gastrocnemius, lateral gastrocnemius and soleus MTJs .While the data presented were obtained during isometric conditions, our findings may explain an underlying physiological mechanism of previous estimates of ankle impedance during walking. We observed that the Achilles tendon is less stiff than the triceps surae at almost all loads, but these results may not apply to other MTUs. For the Achilles tendon, the compliance of the tendon is essential for the storage and return of elastic energy, increasing the economy of locomotion . Howeverin vivo sonomicrometry, studies in running guinea fowl (in vivo ultrasound imaging (Our results from human participants provide a mechanical explanation for a commonly observed phenomenon in both animal and human movement. Many animals have distal muscles connected to long compliant tendons, similar to the human ankle . Throughnea fowl  and hoppnea fowl  have dem imaging . All of Finally, our results have implications for targeted rehabilitation. Changes in Achilles tendon stiffness that occur as a result of injury , or heal


Interestingly, pineapple stem starch provided the highest slowly digestible starch (SDS) (48.84%) and resistant starch (RS) (15.77%) contents compared to other starches. The oil-in-water (O/W) emulsion stabilized with gelatinized pineapple stem starch exhibited higher emulsion stability than that stabilized with gelatinized cassava starch. Pineapple stem starch could therefore be used as a promising source of nutritional SDS and RS, and as an emulsion stabilizer for food applications.In this study, the physicochemical, rheological, in vitro starch digestibility, and emulsifying properties of starch extracted from pineapple stem agricultural waste were investigated in comparison with commercial cassava, corn, and rice starches. Pineapple stem starch had the highest amylose content (30.82%), which contributed to the highest pasting temperature (90.22 °C) and the lowest paste viscosity. It had the highest gelatinization temperatures, gelatinization enthalpy, and retrogradation. Pineapple stem starch gel had the lowest freeze–thaw stability, as evidenced by the highest syneresis value of 53.39% after five freeze–thaw cycles. Steady flow tests showed that pineapple stem starch gel (6%,  Nowadays, there is a growing global interest in exploring new starch resources and developing bio-based or environmentally friendly chemical products. Consequently, a green alternative material such as starch derived from agricultural waste would be valuable for both food and non-food products due to its low cost and availability from renewable resources ,2,3. StaPineapple is one of Thailand’s most commercially important fruit crops, producing more than 1.67 million tons annually [According to previous studies, the pineapple stem was used as agricultural waste to obtain the enzyme bromelain, which can be used in the food and pharmaceutical industries ,9. RecenAnanas comosus, Smooth Cayenne variety) were collected as agricultural waste after harvest at a pineapple farm  (Pineapple stems (hailand) A. All fr2S2O5) at a weight ratio of 1:3 in a mill. The pineapple stem slurry was then stirred at room temperature for 18 h. The slurry was then filtered with a filter cloth to remove coarse fibrous material and then filtered through 100, 140, and 270- mesh screens, respectively, to remove fine fibrous material. Starch in the filtrate fraction was collected by centrifugation at 3000× g for 10 min. The liquid fraction was decanted, and the yellow layer on the surface of the sediment was scraped off manually. The starch sediment was washed several times with distilled water until the supernatant was clear. The starch cake was dried overnight in an oven at 45 °C. Finally, the dried pineapple stem starch was ground and sieved through a 100-mesh sieve  ethanol. The precipitated starch was dissolved in an acetate/salt solution and subsequently used for amylose determination. The lectin concanavalin A (Con A) was added to the starch suspension to precipitate amylopectin, which was removed by centrifugation. Glucose obtained from the enzymatic hydrolysis of amylose in the supernatant was analyzed using a GOPOD reagent.The amylose content of pineapple stem starch and commercial starches  was analyzed using an amylose/amylopectin test kit . Starch samples were defatted by heating them in dimethyl sulfoxide (DMSO). Lipids were removed by precipitating the dispersed starch in 95%  was used to express the viscosity.Pineapple stem starch and commercial starches were analyzed for pasting characteristics using a Rapid Visco-Analyzer (RVA)  based on a standard 1 profile. The 28 g starch suspension was prepared by mixing the starch sample with distilled water to obtain a final starch concentration of 6% The pineapple stem and commercial starch pastes obtained from the RVA measurement were placed on a glass plate and cooled to 25 °C. All starch pastes were photographed with a digital camera  against a black background to qualitatively evaluate their appearance and clarity.Prior to microscopic observation, the starch pastes from the RVA were stained with 0.1% iodine solution, transferred to a slide, and covered with a coverslip. The morphology of the pastes was observed with a normal light microscope  at 40× magnification.g for 10 min by the initial gel weight and multiplying it by 100.Five grams of starch paste from the RVA were placed in a 15-mL centrifuge tube, frozen at −18 °C for 21 h, and then thawed in a water bath at 30 °C for 3 h, which was repeated for five cycles. Percent syneresis was determined for each freeze–thaw cycle by dividing the weight of water separated after centrifugation of the sample tube at 2200× Hret is the retrogradation enthalpy and ∆Hgel is the gelatinization enthalpy.The starch samples were analyzed for their thermal properties using a differential scanning calorimeter (DSC)  as described by Wongsagonsup et al. . An alumG′), loss modulus (G″), and loss tangent, defined as the ratio of G″ to G′ (tan δ), were then plotted as a function of frequency.The freshly prepared starch gels from the RVA were analysed for their rheological properties at 25 °C using a rheometer  equipped with a cone and plate sensor . For the dynamic viscoelastic measurements, a linear viscoelastic range was determined by varying a strain of 0.01–100% at a fixed frequency of 10 rad/s. A dynamic frequency sweep test was then performed in the linear viscoelastic range with a constant strain of 0.2% in a frequency range of 0.1–10 rad/s. The storage modulus , σ0 is the yield stress (Pa), γ̇ is the shear rate (s−1), K is the consistency coefficient (Pa.sn), and n is the flow behavior index (dimensionless).For steady flow measurements, samples were sheared continuously from 0.1 sStarch digestibility was determined in vitro according to the method of Englyst et al.  with sliw/w) starch in distilled water was boiled in a water bath at 100 °C for 15 min. Emulsions were prepared by mixing 10% (w/w) soybean oil and 90% (w/w) aqueous phase of the cooked starch suspension after cooling to room temperature in a disperser  at 10,000 rpm for the first 1 min and then at 14,000 rpm for an additional 4 min. Sodium azide was used as a preservative at a concentration of 0.02% (w/w). The freshly prepared emulsions (10 mL) were filled into glass vials and stored at room temperature for 15 days. The stability of the emulsion was evaluated by the creaming index (CI), defined as follows:HS is the height of the serum layer and HE is the total height of the emulsions.The emulsion was prepared according to the method of Sriprablom et al. . The stap ≤ 0.05).All measurements were performed in three replicates. Results were expressed as means ± standard deviations. Statistical analyses were carried out using the SPSS version 26.0 program for Windows . A significant difference between means was analyzed using one-way ANOVA and Turkey’s multiple range test at 5% significance level  (p ≤ 0.05) increased to 51.52% after three freeze–thaw cycles, and then changed little (53.21–53.39%) in cycles 4–5. While the syneresis of cassava starch gel was not observed after the first cycle and was 23.16% after five freeze–thaw cycles, which was the significantly lowest syneresis. Syneresis occurs in freeze–thaw treatments due to starch crystallization (retrogradation). Amylose retrogrades much faster than amylopectin due to its predominantly linear structure [The term “freeze–thaw stability” refers to the ability of starch to withstand the unfavorable physical changes that occur during freezing and thawing. Syneresis is the process by which unbound water is readily released from the polymeric network during thawing . It is a ≤ 0.05) . Repeatetructure . TherefoHgel value indicates the loss of molecular order inside the granule and reflects the total crystallinity of the starch granules both in terms of the quality and quantity of the starch crystals [The DSC thermograms of the gelatinized and retrograded starches are shown in crystals . It can crystals . Amylosecrystals . TherefoTo, Tp, and Tc) and melting enthalpy (ΔHret) of the retrograded starch gels were lower than the gelatinization temperatures and ΔHgel of the native starch counterparts. The crystallites and ordered structure of the amorphous region of retrograded starch are not as perfect as those of native starch [To, Tp, Tc, and ΔHret values, resulting in the highest degree of retrogradation (46.07%) compared to retrograded cassava, corn, and rice starches. Retrogradation enthalpy was reported to be positively correlated with amylose content [In the case of retrograded starch, the transition temperatures , and tan δ as a function of frequency of starch gels is shown in σ0) for the range of shear rates used in this study (σ0), the consistency coefficient (K), the flow behavior index (n), and the coefficient of determination (R2) of the upward flow curves. All starch gels exhibited a clockwise hysteresis loop area, indicating structural breakup caused by the shear field and a change in an existing structure, which then continues to exhibit a shear thinning characteristic during subsequent shear sweeps [n values of less than 1, indicating their pseudoplastic shear thinning behavior. In this study, the pineapple stem starch gel exhibited the lowest K value and hysteresis loop area, as well as the highest n value, indicating the lowest viscous properties, the highest shear resistance and structural recovery, and the lowest degree of shear thinning. The cassava starch gel had the highest K value and the lowest σ0 and n values, indicating the greatest viscous properties, the least structure formation of the gel, and the highest degree of shear thinning. Similar observations were reported by Wongsagonsup et al. [K value and hysteresis loop area than native cassava starch gel with complete gelatinization, and no swollen granules remained. From these results, it can be concluded that the steady flow properties, i.e., the K value, of all starch gels, correspond to the results of the pasting properties  and resistant starch (RS). The emulsifying property of pineapple stem starch was better than that of cassava starch. Because of these properties, pineapple stem starch could be used as a good source of nutritional SDS and RS for food applications. Pineapple stem starch with a high content of SDS and RS can be used in the formulation of a wide range of low-moisture foods such as pasta, bread, cookies, cakes, and breakfast cereals to enhance health benefits.


The Journal of Clinical Hypertension, 10: 348–354. The above article, published online on May 02, 2008 on Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal's Editor‐in‐Chief, Dr. Ji‐Guang Wang, and Wiley Periodicals LLC. Following publication, concerns were raised by a third party regarding the statistical analysis presented in the article. The Journal conducted an independent statistical review of the article and concluded that the results were misleading due to issues regarding the sensitivity and specificity of the medical adherence scale used. The authors responded to the Journal's request to address the findings of the independent statistical review, but were unable to adequately address the concerns. As a result, the Journal no longer has confidence in the reported conclusions and is issuing this retraction. The Journal of Clinical Hypertension, 10: 348–354. The above article, published online on May 02, 2008 on Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal's Editor‐in‐Chief, Dr. Ji‐Guang Wang, and Wiley Periodicals LLC. Following publication, concerns were raised by a third party regarding the statistical analysis presented in the article. The Journal conducted an independent statistical review of the article and concluded that the results were misleading due to issues regarding the sensitivity and specificity of the medical adherence scale used. The authors responded to the Journal's request to address the findings of the independent statistical review, but were unable to adequately address the concerns. As a result, the Journal no longer has confidence in the reported conclusions and is issuing this retraction.


This was only made possible by the high quality of submissions from authors who chose the journal as a vehicle to publish their results, the team of subject editors, numerous reviewers, and the efficient editing and publishing of the journal. This issue is a great occasion to look back and evaluate the performance of MycoKeys.Since its inception in 2011 , MycoKeyFungi, however, the majority of papers deal with Ascomycota or Basidiomycota, including lichenized fungi.MycoKeys started with only 13 submissions in 2011, whereas the number of submitted manuscripts has been above 130 annually for the past six years (Fig. MycoKeys has attracted researchers from various parts of the world to publish their results Fig. . AltogetThe top 10 most cited MycoKeys papers up until 31 October 2023 include papers addressing a wide array of issues, including: potential bias in the use of high throughput molecular identification methods ; qualityMycoBank. Since its launch, 1108 new species, 71 new genera and four new families have been described in MycoKeys. In addition, 248 new combinations of taxa have been proposed in the journal.All nomenclatural changes in the journal are indexed in When the journal received its first Journal Impact Factor in 2015, it was at 1.846 and has subsequently increased to the current 3.3, demonstrating the quality of the peer review of submitted manuscripts, stringent quality control and management of manuscripts. The current CiteScore – a journal-level citation metric by Scopus – of MycoKeys is 5.8. Although the journal is currently in the Q2 Mycology quartile of the Web of Science, it is in the Q1 quartile in all three Scopus categories: Agricultural and Biological Sciences; Ecology, Evolution, Behavior and Systematics; and Plant Science.X and Facebook, where updates about the most recent publications and news from the journal are currently shared to approximately 1,500 and 2,200 followers, respectively. As a result of regular press campaigns, over the years, studies published in MycoKeys have been publicized in major news media outlets, such as The Washington Post, CNN, Newsweek and Spiegel.MycoKeys is also active in popularizing research on social media via its own channels on In its short history, MycoKeys has already played a vital role in contributing to the understanding of the evolution, diversity and taxonomy of fungi. Exciting new methods provide further insights and allow us to address questions we could not dream of a few decades ago.


TM, 37% phosphoric acid, 9.5% hydrofluoric acid, Al2O3 sandblasting, low-speed dental stone, and high-speed dental stone) on the fracture resistance, microstructure, and surface roughness of monolithic and multilayered zirconia. The as-received and sandblasted zirconia was used as a control. One-way ANOVA and t-test were performed. As-received monolithic zirconia was stronger (856 ± 94 MPa) than multilayered zirconia (348.4 ± 63 MPa). Only phosphoric acid (865 ± 141 MPa) and low-speed dental stone (959 ± 116 MPa) significantly increased the flexural strength of sandblasted monolithic zirconia (854 ± 99 MPa), but all tested decontamination methods except phosphoric acid (307 ± 57 MPa) and Al2O3 (322 ± 69 MPa) significantly increased the flexural strength of sandblasted multilayered zirconia (325 ± 74 MPa). Different decontamination methods did not significantly affect the flexural modulus, but introduced irregularities in the crystal as well as deep surface flaws in both types of zirconia. The surface of sandblasted monolithic zirconia is more resistant to change than multilayered zirconia. Among different decontamination methods, a low-speed dental stone could be beneficial as it significantly increased the surface roughness and fracture resistance of both types of zirconia. This study aimed to evaluate the effect of seven different decontamination methods (water, ZirClean Due to its superior mechanical properties, zirconia has become an excellent alternative to metal–ceramic restorations, but due to its high opacity it was used as a framework that is typically veneered with a glass–ceramic material . It has Zirconia exists in three crystalline forms which are monoclinic, tetragonal, and cubic ,8. ZircoIn the literature, there is no clear decontamination method before the final cementation of zirconia restorations . In a ch2O3, Zhermack, Polesine (RO), Italy), low-speed dental stone , and high-speed dental stone  on the fracture resistance, microstructure, and surface roughness of sandblasted monolithic and multilayered zirconia. This study aimed to investigate the effect of different decontamination methods including water, ZirCleanTM , phosphoric acid , hydrofluoric acid , sandblasting  and multilayered zirconia disks  were used. Their shrinkage factors (SF) were 1.266 and 1.233, respectively. n = 10). Since 9 groups from each material were tested, the total number of samples was therefore 180 . Based on previous research ,16, samp3) samples were used . Samples were prepared from zirconia disks using imes-icore CORiTEC 150i pro milling machine  after considering the SF of each material. Samples were milled and sintered at 1400 °C for 3 min using a furnace  to achieve their maximum strength. Ten samples from each material were randomly selected to be tested immediately as the as-received group (G0). The rest of samples were air-blasted on one surface with 50 µm Al2O3 at 2.5 bars for 15 s, 10 cm distance, and perpendicular to the surface. Then, another 10 air-blasted samples from each material were randomly selected to be tested immediately as sandblasted/uncontaminated group (G1). Both G0 and G1 were used as controls. The rest of the samples were kept in saliva, obtained from one healthy donor who refrained from eating and drinking two hours before collection, for 1 min at room temperature in polypropylene containers, after obtaining ethical approval from the Research Ethics Committee at the University of Sharjah (#REC-21-10-4-S). Then, samples were randomly allocated to G2–G8 groups according to different decontamination methods as given in A total of 180 rectangular (25 × 5 × 2.5 mmn = 10 per group). The load was applied at 1 mm/1 min until failure, using the universal testing machine . The dimension of each sample was measured before testing using a caliper. The flexural strength and modulus were calculated using the machine software . The broken samples were used for microstructure and surface roughness measurements. The flexural strength and modulus of the samples were tested using the 3-point bending test  Thermoscientific Apreo C .n = 5 per group) using Atomic Force Microscope  under a non-contact mode at a scanning speed of 1 s/line and scanning points of 256/line. The scanned area was 10 µm. Surface roughness was measured for all tested groups , ANOVA tests were utilized to compare different treatment surfaces while independent p < 0.05) when compared to the sandblasted/uncontaminated control group—p < 0.05) except the phosphoric acid and Al2O3 sandblasted group that showed no significant difference from the sandblasted/uncontaminated control group—TM or phosphoric acid, the grains become more irregular and smaller than those seen in G0–G2—2O3 produced sod-like spherical crystals on the surface—TM, an increased surface heterogeneity and irregularity in grain size and morphology were observed—2O3, the surface showed the presence of small spherical grains—Regarding the multilayered zirconia, after sandblasting the as-received group, the grain showed more irregularities in both size and morphology—AFM images of monolithic and multilayered zirconia subjected to different decontamination methods are shown in 2O3 significantly increased the surface roughness. Different decontamination methods did not change the surface roughness when compared with G1 control except hydrofluoric acid and low-speed bur that showed a significant increase in surface roughness (p < 0.05)—p < 0.05), except the high-speed bur group that showed no significant difference from the sandblasted/uncontaminated control. The increase in surface roughness however with G2 group is not expected and warrant further investigation —Regarding monolithic zirconia, sandblasting the as-received group with 50 µm AlZirconia has great affinity to phosphate ions, found in saliva and blood, that encounter the restoration surface during try-in . These pTM [TM is potassium hydroxide (KOH) that has a pH > 13. These highly alkaline cleaners work by interrupting or neutralizing the existing ionic bonds formed between salivary phosphate ions and the zirconia surface [4ZrO4), and this could explain the significant increase in irregularity and surface roughness, particularly with multilayered zirconia, after decontamination with ZirCleanTM when compared with the sandblasted uncontaminated control. As reported in previous studies, complete decontamination of the zirconia surface cannot be achieved with water rinsing, saliva needs to be attacked chemically by a strong alkaline such as ZirCleanTM ,20. The  surface . KOH reaTM or phosphoric acid did not adversely affect the flexural strength and modulus of both types of zirconia. Nevertheless, a significant increase in the flexural strength of multilayered zirconia was observed with ZirCleanTM, and this could be related to the formation of orthocarbonate potassium (K4ZrO4), but further investigation is needed to support this finding. On the other hand, with phosphoric acid, the increased flexural strength was observed with monolithic zirconia. Decontamination with 37% phosphoric acid increased the surface roughness of both types of zirconia; the significant increase was observed with multilayered zirconia compared to the sandblasted/uncontaminated control. This increase in surface roughness could be correlated to the great chemical affinity of zirconia to phosphate ions found in phosphoric acid as is the case with those in salivary proteins. The formation of chemical bonds on the zirconia surface could be responsible for increased surface heterogeneity and roughness . HoweverDecontamination with 9.5% hydrofluoric acid significantly increased the surface roughness of both types of zirconia. Hydrofluoric acid interacts with salivary organic contaminants and dissolves them and produces nanoporosities that increase the surface irregularity and thus the roughness of both types of zirconia when compared to the sandblasted/uncontaminated control ,22,23. R2O3 significantly increased the surface roughness of the multilayered zirconia that contained a higher amount of yttria (9.3 ± 0.3 mol%) to stabilize the cubic crystal content at room temperature. Increased amounts of yttria and cubic crystals were attributed to the good esthetics but low strength of the material. Therefore, sandblasting with Al2O3 particles produces greater effect on the surface roughness of multilayered compared to monolithic zirconia. Highly translucent zirconia showed more pronounced topographic changes and increased surface irregularity compared to conventional zirconia [Decontamination with 50 µm Alzirconia . Changeszirconia . The decontamination with low-speed bur significantly increased surface roughness and flexural strength of both types of zirconia. On the contrary, high-speed bur decreased surface roughness of both types of zirconia but increased the flexural strength of multilayered zirconia. The change in roughness could be correlated to the diamond bur grit size, with coarse grinding showing higher surface roughness than fine grinding . In thisDue to the importance of surface roughness of the fitting of the zirconia crowns in enhancing the bond strength with the luting cement , the effFlexural strength and modulus are important properties for the restoration to resist the occlusal load . Higher Based on the findings of this study, it can be recommended that a low-speed bur could be used for decontamination of a contaminated zirconia surface, as it significantly increases the surface roughness and flexural strength. A limitation of this in vitro study is that findings are based on laboratory experimental conditions that may not answer complex clinical questions. Within the limitations of this in vitro study, it was concluded that multilayered zirconia is weaker, and its surface is less resistant to change than monolithic zirconia. In clinical settings following saliva contamination during the try-in stage, decontamination with low-speed bur can be beneficial as it significantly increases the surface roughness as well as the flexural strength of both types of zirconia. The effect of different decontamination methods on the contact angle, shear bond strength, and fatigue resistance will be considered in our future work. For this purpose, zirconia crowns will be used.


The first case of Vaccine-Induced Thrombotic Thrombocytopenia (VITT) was reported in the letter-to-editor submission in the journal of Indian Journal of Hematology and Blood Transfusion which was published online on 29th Sep 2021. Whereas, an article published in your journal on 04th Mar 2022 has been titled as first report of VITT from India which is a very conflicting statistic. The former article under reference has been diagnosed by a confirmatory functional assay as per the recommended guidelines and is thus genuinely the first case reported in this country. Dear Editor,As we all know that COVID-19 has created a grave situation ever since the inception of pandemic in Nov 2019. To curb the situation, the mankind worked tirelessly and produced vaccines with the vision of mass vaccination. However, an extremely rare vaccine associated adverse event was observed in a sparse subpopulation of individuals especially those who received adenoviral vector-based vaccines. This complication was termed as Vaccine-induced Immune Thrombotic Thrombocytopenia (VITT).“COVID-19 Vaccine-Induced Thrombosis and Thrombocytopenia: First Confirmed Case from India” [In this context, I would like to bring to your notice that the first confirmed case from India was reported by Mishra et al. in his article titled “Vaccine-Induced Thrombotic Thrombocytopenia (VITT): First Report from India” by John et al. was published in your esteemed journal [The article titled  journal . This suYou are requested to delve into this matter and make necessary refinements so that the article in reference number 1 gets the due credit and the article printed in your journal looks error-free.


Saccharomyces cerevisiae strain and this research was also to investigate the optimum conditions for the pretreatment of biomass and technical procedures for the production of bioethanol. It successfully resulted in high yields and productivity of all the experiments from the laboratory scale and the pilot plant. Ethanol yields from pretreated starch are comparable with those in commercial industries that use molasses and hydrolyzed starch as raw materials.A processing methodology of raw starch extraction from avocado seeds (ASs) and a sequential hydrolysis and fermentation bioprocess in just a few steps was successfully obtained for the bioethanol production by a single yeast pilot-scale bioethanol production, studies of starch extraction and dilute sulfuric acid-based pretreatment was carefully conducted. The amount of starch extracted from dry and fresh avocado seed was 16.85 g ± 0.34 g and 29.79 ± 3.18 g of dry starch, representing a yield of ∼17% and 30%, respectively. After a dilute sulfuric acid pretreatment of starch, the released reducing sugars (RRS) were obtained and the hydrolysate slurries containing glucose (109.79 ± 1.14 g/L), xylose (0.99 ± 0.06 g/L), and arabinose (0.38 ± 0.01 g/L). The efficiency of total sugar conversion was 73.40%, with a productivity of 9.26 g/L/h. The ethanol fermentation in a 125 mL flask fermenter showed that Saccharomyces cerevisiae  produced the maximum ethanol concentration, pmax at 49.05 g/L (6.22% v/v) with a yield coefficient, Yp/s of 0.44 gEthanol/gGlucose, a productivity or production rate, rp at 2.01 g/L/h and an efficiency, Ef of 85.37%. The pilot scale experiments of the ethanol fermentation using the 40-L fermenter were also successfully achieved with essentially good results. The values of pmax,Yp/s, rp, and Ef of the 40-L scale were at 50.94 g/L (6.46% v/v), 0.45 gEthanol/gGlucose, 2.11 g/L/h, and 88.74%, respectively. Because of using raw starch, major by-products, i.e., acetic acid in the two scales were very low, in ranges of 0.88–2.45 g/L, and lactic acid was not produced, which are less than those values in the industries.Before the Saccharomyces cerevisiae strain is practicable and feasible for realistic and effective scale-up strategies of bioethanol production from the starch of avocado seeds.The sequential hydrolysis and fermentation process of two scales for ethanol production using the combination of hydrolysis by utilizing dilute sulfuric acid-based pretreatment and fermentation by a single yeast  Starch is a polymer of glucose and mainly consists of amylose and amylopectin. It is the second most important and abundant source of carbon and energy in a large variety of higher plants . Starch Bioethanol is mainly obtained from corn starch and sugarcane . HoweverA few studies have demonstrated that using avocado seed wastes can be saccharified and fermented with bacterial strains in a laboratory, pilot scale, and semi-industrial levels process to produce bioproducts with successful results , 27–30. Saccharomyces cerevisiae, Scheffersomyces (Pichia) stipites, Kluyveromyces marxianus, Pachysolen tannophilus, and Candida shehatae can assimilate and ferment sugars derived from hydrolysis of starch [Alternatively, the direct hydrolysis of raw starch to glucose with the dilute acid pretreatment (DAP) could significantly simplify processing and reduces the cost of producing starch-based products e.g., bioethanol and other bioproducts , 23. Thif starch –45. Therf starch –48. Therf starch –54. Howef starch , 56. Forf starch . So, thef starch .Saccharomyces cerevisiae without additional nutrient supply into hydrolysate, less time-consuming and lower operating cost at the laboratory scale and the 40-L pilot plant.Therefore, finding not only cheap and renewable feedstocks but also the high availability of this starchy material in Mexico, such as avocado residues, represents a field of opportunity in the industrial fermentation process of agroindustrial residues for microbial bioproducts. So, more research and development in pilot scale and semi-industrial levels for starch hydrolysis by diluted acid and subsequent sugar fermentation to bioethanol in just a few steps for bioprocessing of renewable lignocellulose biomass using natural yeast strains are required in order to render the process even more cost-effective. The objective of this research was to obtain a processing methodology for raw starch extraction from avocado seeds for sequential hydrolysis and ethanol fermentation. The second objective of this study was also to obtain a starch hydrolysate by a dilute acid-based pretreatment method for raw starch conversion to ethanol by fermentation using the yeast The Hass-type avocado seed was provided by the SIMPLOT company . The chemical characterization of avocado seeds was performed according to the procedures described by the National Renewable Energy Laboratory (NREL). The analysis included the determination of moisture content , extractwhere: FMS: Final mass starch (g); MASs: Mass of avocado seeds used in the extraction (g).2SO4. The reaction of hydrolysis was carried out in an Erlenmeyer flask of 250 ml at 87 °C for 12 h at 100 rpm with a magnetic stirrer in a water bath using a cryothermostat . The experiment was carried out in triplicate.In this study, a mixture of 100 g containing 15% w/w of starch powder of avocado seed and the balance with diluted acid 2% (w/w) HAfter the pretreatment procedure, starch hydrolysates were used as nutritious media to obtain bioethanol. Also, 1 mL aliquots of hydrolysate were centrifuged at 13,500 rpm  and the supernatants were analyzed for glucose, xylose, and arabinose content using high-performance liquid chromatography (HPLC). Released Reducing sugars (RRS) were characterized by the 3,5-dinitro salicylic acid method (DNS) before and after mild thermal-acid hydrolysis pretreatment.Saccharomyces cerevisiae (DistilaMax® DS strain) was hydrated in YPD medium at 35 °C for 30 min and directly inoculated into the starch hydrolysate with an initial inoculum concentration of 5 g/L. Fermentation conditions were: 30 °C, 50 rpm, and pH 5.0 adjusted with the addition of 14.6% NH4OH (v/v). All experiments were carried out in triplicate. The scale-up of dilute acid-based hydrolysis of starch from avocado seeds for ethanol fermentation was performed from the 125 mL shake-flask cultures until the pilot scale . The pilot plant contains a 40-L fermenter, which has a jacket that allows temperature control with steam, as well as a variable revolution impeller stirrer that can stir up to 125 rpm.For fermentation on the laboratory scale, the 125 mL shake-flask fermenters were utilized and 100 mL of the raw hydrolysate was used as a cultivation medium. 0.5 g of lyophilized yeast 2SO4(w/w). The mild pretreatment condition was: 87 °C and 30 rpm for 12 h. After hydrolysis, the pilot-scale ethanol fermentation was carefully conducted directly into the starch hydrolysate in the 40-L fermenter with an initial inoculum concentration of 5 g/L Saccharomyces cerevisiae strain. Fermentation conditions were: 30 °C, 30 rpm, and pH 5.0 adjusted with the addition of 14.6% NH4OH (v/v). The pilot plant experiments were carried out in duplicate.In this pretreatment scale, the fermenter contained 40 Kg of the fermentation mixture containing 15% w/w of starch powder of avocado seed and diluted acid 2% HEthanol production kinetics were performed to determine the parameters of hydrolyzed slurries from the starch of avocado seeds were utilized as culture media. 1 mL aliquots were taken over the course of 24 h. For each sample, the supernatant was recovered and the concentrations of sugars, ethanol, and acetic acid were determined by HPLC. The experiments were carried out in triplicate substrate and product analysis.The Hass-type avocado seed provided by the SIMPLOT Company located in Morelia, Michoacan, Mexico, was characterized by its potential for use as feedstock for bioethanol production. The results of Hass seeds characterization on a dry basis (%w/w) presented in Table As shown in Table 2SO4 at 87 °C for 12 h. The analysis of the kinetics of glucose and RRS production was carried out for 12 h in the 250 mL flask and fermentation tank of the pilot plant  HoC on a laboratory scale (125 mL Erlenmeyer flasks) using 100 mL of the starch hydrolysate without additional nutrient supply into hydrolysate were conducted under optimized fermentation conditions . Results presented in Fig. Yp/s of 0.44 gEthanol/gGlucose, a productivity or production rate, rp at 2.01 g/L/h and an efficiency, Ef of 85.37%. The glucose degradation increased with fermentation time, and it was almost completely utilized after 20 h of fermentation. The exponential phase for sugar consumption was between 4 and 16 h. As expected, xylose and arabinose were not consumed during the fermentation and the acetic acid concentration was very low, reaching the value of 0.88 g/L. The results showed that the raw hydrolysate of starch from avocado seeds provided sufficient sugars to be fermented to ethanol without requiring additional supply nutrients and without producing inhibitory compounds such as furans.Studies on ethanol production at 30 pmax,Yp/s, rp, and efficiency of the 40-L scale were at 50.94 g/L (6.46% v/v), 0.45gEthanol/gGlucose 2.11 g/L/h and 88.74%, respectively. Because of using raw starch, major by-products, i.e., acetic acid in the two scales were very low, in ranges of 0.88–2.45 g/L, and lactic acid was not produced, which is less than those values in the industries. The exponential phase for sugar consumption was also between 4 and 16 h. As expected, xylose and arabinose were also not consumed during the fermentation. As in laboratory fermentation assays, the results in the pilot-scale batch fermentation also showed that the raw hydrolysate of starch from avocado seeds provided sufficient sugars to be fermented to ethanol without requiring additional supply nutrients and without producing inhibitory compounds such as furfural and 5-hydroxymethyl furfural, after hydrolysis of starch from avocado seeds. As result, these hydrolysates can be further used for large-scale hydrolysis using other microorganisms for the production of commercial bioproducts . As shown in Fig. 0 L. As sSaccharomyces cerevisiae strain, which in turn makes the bioprocess of production of ethanol cost-effective. Furthermore, the laboratory process was technically quite easily feasible to scale up to a pilot scale with promising results. From the results, it could be concluded that the starch of avocado seeds is an attractive raw material for the production of bioethanol and can also be used for large-scale hydrolysis using other microorganisms for the production of other commercial bioproducts.A sequential hydrolysis and fermentation process of two scales for the ethanol production process from the starch of avocado seeds was conducted in this study Fig. . Laborat


Correction to: Journal of Plant Research10.1007/s10265-023-01466-4In the original publication of the article, figure


Neuroblastoma is the most frequent extracranial childhood tumour but effective treatment with current immunotherapies is challenging due to its immunosuppressive microenvironment. Efforts to date have focused on using immunotherapy to increase tumour immunogenicity and enhance anticancer immune responses, including anti-GD2 antibodies; immune checkpoint inhibitors; drugs which enhance macrophage and natural killer T (NKT) cell function; modulation of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway; and engineering neuroblastoma-targeting chimeric-antigen receptor-T cells. Some of these strategies have strong preclinical foundation and are being tested clinically, although none have demonstrated notable success in treating paediatric neuroblastoma to date. Recently, approaches to overcome heterogeneity of neuroblastoma tumours and treatment resistance are being explored. These include rational combination strategies with the aim of achieving synergy, such as dual targeting of GD2 and tumour-associated macrophages or natural killer cells; GD2 and the B7-H3 immune checkpoint; GD2 and enhancer of zeste-2 methyltransferase inhibitors. Such combination strategies provide opportunities to overcome primary resistance to and maximize the benefits of immunotherapy in neuroblastoma. Neuroblastomas are the most common extracranial solid tumour in children, with 90% of cases occurring in children less than 10 years of age.reg) exert highly immunosuppressive effects through functions including secretion of inhibitory cytokines, such as interleukin (IL)-10, which attenuate cytotoxic T-cell responses and inhibit antigen presenting cell maturation.High-risk neuroblastomas are classified as immunologically “cold” due to limited T-cell infiltration, major histocompatibility complex class I (MHC-I) downregulation and low mutational burden leading to an immunosuppressive tumour microenvironment (TME).This review will address the shortcomings and advances in the use of immunotherapy for the treatment of neuroblastoma. The clinical characteristics and risk classifications for neuroblastoma have been extensively discussed elsewhere.MYCN oncogene, which is frequently seen in patients with high-risk neuroblastoma, is a major driver of poor prognosis and is associated with decreased tumour immunogenicity.MYCN amplification is responsible for the suppression of interferon activity as well as the expression of chemokines, and its overexpression enhances tumour cell resistance to immune-mediated cytotoxicity via multiple mechanisms, including MHC-I downregulation and inhibition of NK cell activation.Amplification of the GD2 is a disialoganglioside highly expressed in melanomas and neuroblastomas, implicated in tumour progression by modulating cancer cell proliferation, migration and tumour invasiveness.19The murine IgG3 antibody 3F8 is the first anti-GD2 antibody developed and studied for effectiveness in treating neuroblastoma. An early phase I study of anti-GD2 monotherapy showed anti-tumour responses, ranging from complete clinical remission to mixed tumour regressions, in 7 of the 17 patients with neuroblastoma and melanoma treated. Typical acute side effects of severe pain and urticaria were prevalent throughout this study, thought to be the result of 3F8 interaction with pain sensory fibers, but long-term effects were not observed.25Naxitamab (Hu3F8) is a humanized IgG1 antibody being tested clinically as both a monotherapy and in combinations with immunogenic cytokines . A phaseSeveral anti-GD2 antibodies have been generated and characterized over the past decade, with dinutuximab Ch14.18/SP2/0) being the first to receive marketing approval in 2015 for the treatment of high-risk neuroblastoma./SP2/0 be29Dinutuximab beta (Ch14.18/CHO) is a dinutuximab analog produced by the International Society of Paediatric Oncology European Neuroblastoma Group (SIOPEN) using Chinese hamster ovary (CHO) cells to optimize protein glycosylation patterns and reduce rapid drug clearance by endogenous antibodies which recognize non-human sialic acid epitopes in antibodies derived from SP2/0 cells.Other anti-GD2 antibodies in development have followed a similar approach of humanization to counteract dose-limiting toxicities and HAMA responses. Indeed, phase I clinical testing of humanized 14.18  demonstrated reduced pain and other toxicities associated with ch14.18 treatment, while still inducing an objective response in 18% of patients with refractory neuroblastoma.38Overall, multiple anti-GD2 antibodies have undergone molecular revision and refinement to reduce immune-related toxicities and severe neuropathic pain associated with CDC and HAMA responses. Despite the attractive tissue expression and restricted distribution of GD2 as a target, the ADCC-based mechanism of anti-GD2 induces often intolerable toxicity made worse by combination with activating cytokines such as IL-2. Alternative methods of enhancing anti-GD2 efficacy are required, since of the three clinically approved antibodies available, currently dinutuximab is the only one deemed effective for standard therapy.Immune checkpoint proteins are expressed by a range of immune cell types and perform critical functions in maintaining tolerance and preventing autoimmunity.The fully human IgG4 antibody nivolumab was evaluated in two phase II trials designed to assess the toxicity and pharmacokinetics of anti-PD-1 monotherapy in patients with refractory neuroblastoma . The first study demonstrated that nivolumab is well-tolerated in children with similar pharmacokinetics to those seen in adults, however objective responses were not observed and benefit was limited to stable disease only.Preclinical studies have sought to overcome the challenge of low-sensitivity to anti-PD-1 in neuroblastoma by inducing target cell PD-L1 expression. One provocative study demonstrated that targeting GD2 in LA-N-1 neuroblastoma cells with ch14.18/CHO in co-culture with primary leukocytes induced strong PD-L1 expression in what is usually a PD-L1 negative model.50Ipilimumab is a first-in-class human monoclonal antibody targeting the immune checkpoint CTLA-4. A phase I clinical trial (NCT01445379) assessing pharmacokinetics and immunogenicity in a mixed cohort off refractory solid tumours showed that it is tolerated in children, although immune-related adverse events (irAEs) consistent with those seen in adults were observed. Despite the lack of tumour regressions following single-agent ipilimumab, patients who exhibited irAEs had an increased OS relative to those who did not, suggesting that disruption of immunological tolerance with ipilimumab was beneficial in terms of survival outlook.51Poor clinical responses to single-agent ICB have motivated preclinical investigations into combination strategies. One such study showed that the murine syngeneic NB-SQ neuroblastoma model exhibits an immunosuppressive TME characterized by a high frequency of myeloid-derived suppressor cells and Tregs.The importance of TAMs in TME of neuroblastoma and other cancers is well established.61 62In contrast to GD2, the transmembrane integrin-associated protein CD47 is ubiquitously expressed throughout a range of different tissue types. Being frequently overexpressed in tumours, CD47 enrichment is strongly associated with worse prognosis in both haematological and solid cancers.MYCN model of high-risk neuroblastoma demonstrated significant growth-limiting effects after treating established tumours.+ myeloid infiltration predicts poor neuroblastoma outlook and PD-L1 is strongly associated with CSF-1R expression in human neuroblastomas, this may represent a promising therapeutic avenue.74Colony stimulating factor-1 (CSF-1)/CSF-1R interactions can also be targeted to modulate TAM migration and accumulation in tumour sites.Clinical trials aiming to validate the therapeutic benefit observed with preclinical CSF-1R-PD-1 dual inhibition using both small molecule (NCT02452424) and monoclonal antibody inhibitors (NCT02526017 and NCT02323191) are assessing responses in patients with neuroblastoma.+ and GD2– target neuroblastoma cells in vitro.81NK cells can target tumour cells for cytolytic killing and are known to produce pro-inflammatory cytokines and chemokines which attenuate tumour progression and metastasis.Cell-based therapies involving the adoptive transfer of chimeric-antigen receptor (CAR) T cells has revolutionized the treatment of haematological cancers, inducing striking and durable regressions of B-cell acute lymphoblastic leukaemia.A first-in-human phase I clinical trial (NCT03294954) administered chimeric antigen receptor NKT (CAR-NKT) cells to children with refractory neuroblastoma.The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is an intracellular DNA sensing pathway and integral innate immune defense mechanism to viral infection or cellular transformation. On detection of pathogen-associated molecular patterns, such as cytoplasmic DNA (cDNA), STING signaling drives IFN-I production which can boost anti-tumour effector T-cell function .90 9190 91The effect of STING agonism on tumour-immune interactions has been studied in multiple cancer types, including pancreatic cancer, breast cancer, head and neck cancer, lung cancer and leukaemia.94STING activation and poly-ADP ribose polymerase (PARP) inhibition was investigated in breast cancer gene (BRCA)-deficient breast cancer models; the combination significantly improved anti-tumour responses by driving IFN-I production, T-cell recruitment and DC activation, while inducing immunological memory and reducing adaptive resistance to PARP inhibitors (PARPi).97+ T-cell recruitment and anti-tumour effector responses, while synergising with ICB to inhibit tumour growth significantly more than irradiation alone.Radiotherapy also induces STING activation through the accumulation of DNA damage and triggering of DNA damage responses.MYCN amplificationMYCN amplified neuroblastoma tumours. A study using genetically engineered mouse models of neuroblastoma used the STING agonist DMXAA to evaluate the anti-tumour effect of dual STING activation and MYCN inhibition.MYCN-amplified tumours are associated with reduced IFN-I pathway activity in primary metastatic neuroblastomas and established cellular models, depletion of N-Myc potently augmented IFN pathway activity in response to DMXAA treatment.Around 50% of high-risk neuroblastomas exhibit Targeting neuroblastoma with immunotherapies has been challenging because of its highly immunosuppressive TME, with some promising treatments not translating successfully (see Outstanding questions). The use of checkpoint inhibitors as a treatment for neuroblastoma has not been successful in the clinic and any studies that have been conducted only proved the safety of using these inhibitors in children, suggesting that they might need to be administered in combination with other treatments. The most widely studied and successful immunotherapy is the use of anti-GD2 antibodies and approaches to overcome resistance to GD2 treatments and improve response are currently being explored. Combination of anti-GD2 and anti-CD47 antibodies showed promising results in mouse models and has now been evaluated in a first-in-human clinical trial.Many questions still remain for the use of immunotherapies in neuroblastoma treatment. Combination strategies involving anti-GD2 have been successful in clinical trials, but have not yet received approval for clinical use. Furthermore, the limited impact of ICIs in the clinic and strategies to improve patient responses need further investigation. The possibility of using radiotherapy in combination with STING agonists also needs to be explored. Moreover, possible combination treatments to enhance responses need to be tested and the safety and efficacy of dual CAR-T cells needs to be tested in a clinical setting in paediatric oncology.https://www.clinicaltrials.gov/ct2/show/NCT03363373NCT03363373: https://clinicaltrials.gov/ct2/show/NCT01757626NCT01757626: https://clinicaltrials.gov/ct2/show/NCT00072358NCT00072358: https://clinicaltrials.gov/ct2/show/NCT00492167NCT00492167: https://clinicaltrials.gov/ct2/show/NCT02914405NCT02914405: https://clinicaltrials.gov/ct2/show/NCT01704716NCT01704716: https://clinicaltrials.gov/ct2/show/NCT04751383NCT04751383: https://clinicaltrials.gov/ct2/show/NCT03189706NCT03189706: https://clinicaltrials.gov/ct2/show/NCT04501757NCT04501757: https://clinicaltrials.gov/ct2/show/NCT03033303NCT03033303: https://clinicaltrials.gov/ct2/show/NCT02650648NCT02650648: https://clinicaltrials.gov/ct2/show/NCT01662804NCT01662804: https://clinicaltrials.gov/ct2/show/NCT01419834NCT01419834: https://clinicaltrials.gov/ct2/show/NCT03332667NCT03332667: https://clinicaltrials.gov/ct2/show/NCT01711554NCT01711554: https://clinicaltrials.gov/ct2/show/NCT02573896NCT02573896: https://clinicaltrials.gov/ct2/show/NCT03786783NCT03786783: https://clinicaltrials.gov/ct2/show/NCT01857934NCT01857934: https://clinicaltrials.gov/ct2/show/NCT04385277NCT04385277: https://www.clinicaltrials.gov/ct2/show/NCT00026312NCT00026312: https://clinicaltrials.gov/ct2/show/NCT01445379NCT01445379: https://clinicaltrials.gov/ct2/show/NCT02813135NCT02813135: https://clinicaltrials.gov/ct2/show/NCT02304458NCT02304458: https://clinicaltrials.gov/ct2/show/NCT02914405NCT02914405: https://clinicaltrials.gov/ct2/show/NCT04412408NCT04412408: https://clinicaltrials.gov/ct2/show/NCT04730349NCT04730349: https://clinicaltrials.gov/ct2/show/NCT05302921NCT05302921: https://clinicaltrials.gov/ct2/show/NCT02541604NCT02541604: https://clinicaltrials.gov/ct2/show/NCT00877110NCT00877110: https://clinicaltrials.gov/ct2/show/NCT03209869NCT03209869: https://clinicaltrials.gov/ct2/show/NCT03294954NCT03294954: https://clinicaltrials.gov/ct2/show/NCT03242603NCT03242603:


Background: University of Wisconsin solution (UW) may freeze at temperatures below −0.7 °C, damaging the graft. The present study assessed the effectiveness of the liver graft package protocol, which recommends filling a package with sufficient liquid to prevent grafts from sustaining freezing injury. Methods: We filled ice cubes at two temperatures (−80 and −20 °C) around packages and performed a comparative study with four groups based on the temperature and filling of the second layer with lactated Ringer’s solution (LR) . The bovine liver was used as a graft and preserved for 6 h in the first isolation bag filled with UW. Results: While temperatures dropped below −0.7 °C at some points for 6 h in groups A, B, C, they never dropped to −0.7 °C in group D. The macroscopic findings in groups A, B, C showed freezing of the UW and grafts, but no such results in group D. A pathological study including electron microscopy showed freezing injury in groups A, B, and C but no significant changes in group D. Conclusions: The graft package protocol prevents freezing of the UW and liver grafts. In Japan, the number of liver transplants from brain-dead donors has been on the rise since the revision of the Organ Transplant Law in 2010 ,4. HowevTo maintain a low temperature, ice cubes are usually packed around the procured organs and stored in a portable container, such as a cool box, for transportation. These ice cubes are stored in a freezer or cold storage after being made with an ice machine set at ≤−18 °C to prevent the growth of bacteria by both the Japanese Industrial Standard (JIS)  and the In response, the Japan Society for Transplantation (JST) 7 established a standard protocol for organ packaging . The present study verified whether or not the Japanese standard protocol for organ packaging can prevent freezing injury during organ transportation.A schematic diagram of the experimental liver graft based on the standard protocol for organ packaging presented by the JST is shown .In this study, commercially available beef livers from animals slaughtered by the Nagoya Meat Public Corporation on the day of the experiment were preserved at about 6 °C before the experiment as a liver graft model. Since the weight of the human liver is approximately 1000 g, the bovine livers were trimmed to a weight of 1000 g as a liver graft model and used in the experiment. To prevent tissue disintegration during manipulation that would affect the experimental results, the left lobe of the liver was cut on one or two sides to make one block.® 50 × 50 cm, 3M Corporation, Saint Paul, MN, USA) was packed containing the liver graft model with 1000 mL of UW  and then packed with more two bags as well. After initial wrapping, the second layer was filled with 1000 mL of lactated Ringer’s solution , and the third layer was simply closed without any liquid.The first isolation bag  as a specimen box for organ transportation. Ice cubes with a side length of about 2 cm made by an ice cube machine  were used to fill the cooler box. To bring the ice cubes to temperatures of −20 and −80 °C, ice cubes for filling were placed in a deep freezer  for ≥24 h at −20 and −80 °C, respectively, and to create a low-temperature environment, the cooler box was filled with about 15 L of these ice cubes.Regarding temperature monitoring, a data logger for temperature measurement  was used to monitor the temperature of the UW in the first layer, including the experimental graft liver. The measurement probe terminals were fixed to the surface of the experimental graft liver and the inner wall of the cooler box, and the temperature was measured continuously.After cold storage of the experimental graft liver in a cooler box for 6 h, the macroscopic findings of both the UW and the experimental graft liver were observed. A 1-cm-square piece of liver tissue was then excised and collected as a specimen for a histopathological examination. The liver surface capsule was sectioned and microscopically examined, focusing on the central vein as a guide to tissue structure. For hematoxylin and eosin (HE) staining, all specimens were fixed with a 10% buffered formalin solution, entrusted to KAC Co., Ltd.  embedded in paraffin, and observed at a magnification of 400×.Electron microscopy was outsourced to Hanaichi Electron Microscopy Laboratory . All specimens were prefixed with 2% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C, dehydrated in graded ethanol solutions, briefly washed with propylene oxide, and embedded in epoxy resin. Conventional ultrathin sections were cut and double-stained with uranyl acetate and lead citrate for 5 min. These ultra-thin sections were examined with an electron microscope  at 5000× magnification.Experiment 1To confirm that the cooler box used in the experiment was effective at maintaining a constant temperature, as a model that deviated from the standard packaging, we used a packaging model that did not use LR in the second layer and filled it with −80 °C ice for 24 h while monitoring the temperature of the experimental liver graft and in the box. After 24 h, the macroscopic appearance of the UW and experimental liver graft was observed.Experiment 2We set 2 temperature conditions: at −80 °C assuming a cryogenic environment and at −20 °C assuming ice that could be used in normal clinical practice for the experiments. Comparable experiments were performed at these temperatures while filling the second layer with or without LR. As shown in the figure, the following groups were established: A, at −80 °C and LR−; B, at −80 °C and LR+; C, at −20 °C and LR−; and D, at −20 °C and LR+. Continuous temperature monitoring for six hours from the start of the package and sample collection after cold storage were repeated three times. In the overall experiment, six commercial bovine livers were purchased and trimmed, resulting in 12 packaging models.The temperature in the cooler box was measured by filling it with ice cubes at −80 °C. About 20 min after the start of cold storage, the temperature of the UW with the experimental liver graft dropped below freezing (<−0.7 °C). Two hours after the start of cooling, the temperature converged to −1.2 °C, and the temperature remained almost constant until 24 h after the end of the observation . After 24 h, the cooler box was opened, and a macroscopic observation showed that the UW and experimental liver graft were completely frozen.Given the results of UW temperature monitoring, which showed a plateau at −1.2 °C at 2 h after the start of cooling, and the change in the appearance of the experimental liver graft in Experiment 1, and considering the time required for liver transportation in Japan, Experiment 2 was performed with a 6-h cold storage model for comparison among groups. In groups C and D, which involved filling the box with ice cubes at −20° C, the UW was ≤−0.7 °C during the measurement period in 2 out of 3 experiments in group C (without LR filling). However, in group D , the temperature never dropped to −0.7 °C for 6 h. In addition, in groups A, B, and C, which reached below-freezing temperatures, the macroscopic findings showed freezing of the UW and the experimental graft , In group D, which was filled with LR in the second layer, neither the UW nor the experimental graft showed any freezing macroscopically.In contrast, the normal hepatocyte arrangement was maintained in groups C and D filled with ice cubes at −20 °C under observation at 400× with HE staining, and no significant differences were observed in the tissue structure.Additional observations with an electron microscope were performed to confirm the impact of LR filling in the second layer on structural changes in the hepatocytes. In group C (LR−), the membrane structure of the intracellular organelles was significantly disturbed, and crystallized ice chips appeared in the cells in the areas shown in the figure, indicating freezing injury. In contrast, in group D (LR+), the same observations as in the control before cold storage were obtained concerning the structure of the nucleus and the membranes in the intracellular organelles To perform liver transplantation safely, graft preservation to prevent PNF from organ harvesting and resumption of blood flow in the recipient is one of the essential factors. The first report of organ preservation with normothermic storage was made by Carrel et al.  in the 1However, following a report of a liver transplantation case in which freezing injury caused by excessive low-temperature exposure during transportation resulted in PNF, it became necessary to review the conventional cold preservation packaging method. Potanos et al. , who repThere is no standard protocol for liver packaging in Japan, with approaches varying among regions, facilities, and transplantation teams. However, given our experiences with graft freezing, a standard protocol for liver packaging is needed to prevent such issues going forward. The JST developed a standard protocol for liver packaging based on these experiences, and the present study was conducted to verify whether the protocol was appropriate. The first experiment to verify the cooling status of a cooler box used for normal liver transport showed that the temperature in the cooler box filled with ice cubes at −80 °C, to mimic packaging, reached a plateau at −1.2 °C after about 2 h and remained at this temperature for 24 h. These findings clarified that the commercial-based cooler box used for liver transportation reaches a temperature below the freezing point of the UW when filled with supercooled ice cubes and maintains a sufficiently low-temperature environment for 24 h.Given this finding, we decided to set a cold storage time of 6 h in the next experiment, assuming the typical transport time for such cases in Japan. We also assessed the effectiveness of filling the second layer with LR as temperature interference. As shown in The pathological findings in groups A and B, which involved filling with ice cubes at −80 °C, revealed the disturbance and rupture of the tissue architecture around the central vein, findings that were similar to a report of hepatocellular freeze injury in rats . HoweverTullius et al.  reportedA pathological study including electron microscopic findings in the present study revealed that cell degeneration induced by low temperatures could be prevented by applying the standard protocol for organ packaging, involving filling the second layer with LR in an environment with ice cubes at −20 °C, which is considered possible for liver transportation. However, when ice cubes are used at −80 °C, regarded as an extreme condition and not a clinical situation, graft freezing cannot be prevented, even when the second layer is filled with LR.A limitation and weakness of this experiment is that the temperature measurement was limited to a single point on the liver surface per specimen, and the results were limited to a restricted range. The uniformity of the temperature distribution in the liver during cooling cannot be ruled out. Therefore, as a prospect, more accurate results could be obtained if measurements could be taken at multiple points. Also, as a point to add, how the packing method affects graft function and outcome after transplantation is a very important question. We believe the validity of the standard packing method confirmed in this study must be confirmed in a living transplant model rather than organ units.Depending on the freezer, the temperature of the ice cubes filled in the cooler box used for graft transportation may be −18 °C, as specified by JIS and IEC standards, instead of 0 °C, at which point livers can freeze. The present findings showed that, as long as the second layer is filled with enough liquid as temperature interference, avoiding direct contact between the UW and the ice cubes helps prevent excessive low temperatures and freezing of the UW and grafts. It was found that safe liver packaging is possible in the current method of operation without any additional effort or new resources. Thus, the effectiveness of the standard protocol for liver packaging recommended by the JST was confirmed, and all efforts should be made to comply with this protocol.