The interaction between the motor control and the morphological design of the human leg is critical for generating efficient and robust locomotion. In this paper, we focus on exploring the effects of the serial and parallel elasticity on hopping with a two-segmented robotic leg called electric-pneumatic actuation (EPA)-Hopper. EPA-Hopper uses a hybrid actuation system that combines electric motors and pneumatic artificial muscles (PAM). It provides direct access to adjust the physical compliance of the actuation system by tuning PAM pressures. We evaluate the role of the serial and parallel PAMs with different levels of compliance with respect to four criteria: efficiency, performance, stability, and robustness of hopping against perturbations. The results show that the serial PAM has a more pronounced impact than the parallel PAM on these criteria. Increasing the stiffness of the serial PAM decreases the leg stiffness of the unloading phase during hopping. The stiffer the leg, the more efficient and the less robust the movement. These findings can help us further understand the human hopping mechanism and support the design and control of legged robots and assistive devices. Stability, performance, robustness, and efficiency are four essential metrics to evaluate controlled machines, such as legged locomotor systems. Since legged biological systems such as humans are still far beyond robots in providing an optimal trade-off between these metrics, understanding the biomechanics of legged locomotion can help us develop robust and efficient high-performance robots . Interestingly, the principles of human locomotion can be explained and reproduced by simple template models   and the locomotion behaviors . Two PAMs are in parallel with the knee joint for extending  and flexing  the knee joint , respectFor verification of the results, an extension of the EPA-Hopper comprising a third segment  was utilized shown in . This roFmtc can be described as a function of the muscle activation signal A, muscle length lm, muscle velocity vm, MTC length lmtc, and MTC velocity vmtc.It has been shown that the bio-inspired neuromuscular reflex control with positive force feedback (PFF) can produce human-like stable and robust hopping behavior both in simulation  and the PFF reflex are defined by the following equationsThe relation between the muscle activation S(t) is the muscle stimulation signal, S0 is the constant stimulation bias, δt is the time delay in the feedback pathways, and GF is the force feedback gain. A detailed description of the virtual MTC model and the PFF can be found in our previous study . Each PAM pressure was tuned to a predefined constant value before the hopping experiments. The same control parameters of the reflex-based controller were used in all experiments. Based on previous studies , which is defined as the ratio between the robot's energy consumption (E) during a hop cycle and the robot's stable hopping height (h). The PAMs do not consume energy during hopping because all valves are closed. Therefore, we only consider the electric energy consumption of the hip and knee motors because the energy consumption of the other parts  is constant with respect to different hopping behaviors. The energy consumption E is calculated asHopping efficiency can be quantified by the normalized energy consumption of the robot , which is computed as followsHopping performance is quantified by the hopping height (hmax and htd denote the maximum hip height (apex) during the flight phase and the hip height at the touch-down moment, respectively. The touch-down moment is defined as the timing when the vertical ground reaction force is larger than 5N. The mean hopping height of the 15 hops before the perturbed hop is considered the stable hopping height.where h) to quantify hopping stability, i.e.,A small perturbation can be used to verify the stability of a cyclic motion. The robot hopping motion can generate a stable limit cycle if the robot states return to the same equilibrium after the perturbation. In theory, if the resulting limit cycle after the perturbation is different from the original one, the original equilibrium is not stable. However, with uncertainties in the hardware setup, a slight deviation from the original cycle is inevitable. Thus, to verify stability in the robot experiment, we define a small range around the cycle. To simplify this procedure, we reduce the system order using the Poincaré return map. We utilize the apex height to define the Poincaré section and the difference between hopping height before and after perturbation , the robot responses can be divided into stable and unstable solutions.where N) required to return to the 5% neighborhood of the stable hopping height after convergence. This measure is used to evaluate the robustness of hopping.In control theory, robustness is evaluated by identifying the maximum range of perturbations or uncertainties that cannot destabilize the stable response of the system  . Interestingly, the normalized energy consumption of the highest hopping height which occurs at PAM pressure settings of  is relatively low (3179J/m), as shown in The mean normalized hopping energy consumption of the 15 hops before perturbation is shown in Pse = 0.5MPa, Pfle = 0.5MPa, Pext = 0.4MPa). The lowest hopping height is 7.1cm . On average  is higher than the other two pressure settings . The average hopping height does not change consistently with respect to different pressures in the flexor and extensor PAM  is lower than 10% of the original hopping height in most of the PAM pressure settings (25 out of 27), as shown in EPA-Hopper can regain stable hopping settings after the 5 cm ground dropping perturbation for all PAM pressure combinations. The hopping height difference ΔPse = 0.4MPa, Δh = 0.36 ± 0.24cm; Pse = 0.5MPa, Δh = 0.13 ± 0.08cm; Pse = 0.6MPa, Δh = 0.29 ± 0.19cm, Medium pressure in the serial PAM results in both the lowest average hopping height difference and the most stable hopping  (N) to recover from perturbation is used to evaluate the hopping robustness. Pse = 0.5MPa, average N = 1.00; Pse = 0.6MPa, average N = 1.78; Pse = 0.4MPa, average N = 2.33)  with increasing extensor PAM pressure  . It requpressure . This trshown in . The higThe spring-like leg behavior can be observed in human hopping experiments  than the hops before the perturbation.The results show that the average leg loading stiffness does not change too much with respect to different serial PAM pressures . On the h and the number of recovery-hops N of the low stiffness group are 32.3% and 50.1% lower than the high stiffness group  and performed perturbed hopping experiments with different serial PAM pressures . In order to minimize the gastrocnemius PAM's potential influences on the hopping behavior and ensure a reasonable ankle joint motion, the gastrocnemius PAM pressure was set to 0.1MPa. To prevent ankle hyper-extension in the flight phase, we added a tibialis spring stiffness and its rest length was selected based on a previous study  on the hopping behavior with four evaluation metrics: efficiency, performance, stability, and robustness. We found that, compared to the parallel PAMs , the serial PAM has a more pronounced impact on the hopping behavior. Interestingly, the results show that increasing serial PAM stiffness results in lower leg stiffness during the unloading phase. The leg stiffness results also indicate a trade-off between energy efficiency and robustness. These findings can help us further understand the role of elastic components in dynamic locomotion and develop more efficient and robust robots and assistive devices.With a neuromuscular controller using PFF  on movement behavior. Then, the relation between the physical compliance at the knee joint and the leg stiffness is analyzed. Finally, the influence of leg stiffness on hopping behavior is elaborated.Among the three PAMs added in parallel and series to the knee joint, the serial PAM demonstrated the most consistent influence on different movement criteria. In addition, the results also support our hypothesis that there is no unique set of PAM pressures that can optimize all metrics simultaneously. In the following, we discuss the role of different PAMs in producing different hopping behaviors.Our results show that increasing the serial PAM pressure reduces energy efficiency see . It has Medium pressure in the serial PAM provides the most stable, robust, and highest performance . The impDepending on the serial PAM stiffness, the extensor and flexor PAMs can further influence efficiency, performance, and robustness. In general, if the parallel passive element is not tuned precisely, it could partly oppose the actuation and increase energy consumption. Hence, in addition to the stiffness of the parallel PAM, the parallel PAM's rest length, which determines the timing of its contribution, is also essential. This might be the reason that we did not find a clear trend in changing the parallel PAMs pressure on energy efficiency. Nevertheless, we found a pronounced influence of parallel PAM extensor on the robustness. Our results confirm that increasing extensor stiffness could improve hopping robustness. Improved damping of the impacts resulting from perturbation with increased extensor PAM stiffness could reduce the perturbation recovery time. This finding supports previous theoretical . In other words, the overall knee joint stiffness can increase while the serial PAM stiffness decreases. As the leg stiffness is proportional to the knee joint stiffness in the robot, reducing the serial PAM pressure could increase leg stiffness as observed in our experimental results . In the Before discussing the different leg stiffness in loading and unloading phases, we want to interpret the robot hopping behaviors from the conceptual modeling perspective  with more complex robotic systems.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.GZ, MS, and AS were responsible for the conceptualization of this work. GZ, OM, and MM conducted the experiments. GZ analyzed the data. GZ, MS, and OM drafted the manuscript. All authors approved the final version of the manuscript.This work was supported by the German Research Foundation (DFG) grants number AH307/2-1, AH307/4-1, and SE1042/29-1.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


S. cerevisiae is a member of the XMAP215/Dis1/CKAP5/ch-TOG family of MAPs and has multiple functions in controlling microtubules, including microtubule polymerization, microtubule depolymerization, linking chromosomes to the kinetochore, and assembly of γ-TuSCs at the SPB. Whereas phosphorylation has been shown to be critical for Stu2 localization at the kinetochore, other regulatory mechanisms that control Stu2 function are still poorly understood. Here, we show that a novel form of Stu2 regulation occurs through the acetylation of three lysine residues at K252, K469, and K870, which are located in three distinct domains of Stu2. Alteration of acetylation through acetyl-mimetic and acetyl-blocking mutations did not impact the essential function of Stu2. Instead, these mutations lead to a decrease in chromosome stability, as well as changes in resistance to the microtubule depolymerization drug, benomyl. In agreement with our in silico modeling, several acetylation-mimetic mutants displayed increased interactions with γ-tubulin. Taken together, these data suggest that Stu2 acetylation can govern multiple Stu2 functions, including chromosome stability and interactions at the SPB.Stu2 in  Microtubules are proteinaceous polymers that play several important roles in cell division and segregation of the genetic material to each daughter cell. The functions of microtubules are critically dependent upon their dynamic properties in which tubulin subunits are added or removed from the microtubule end, allowing microtubules to grow or shorten in length. These dynamic properties are controlled by several types of microtubule associated proteins. In this study using bakers yeast, we describe our discovery of a previously unappreciated way to regulate the microtubule associated protein Stu2 by a modification called acetylation. When we created mutations in the Stu2 protein that can’t be properly acetylated, the cell lost some of its chromosomes. Some of these mutations actually caused the microtubules to be resistant to drugs that normally disassemble the microtubule polymer. As similar versions of the Stu2 protein are found in diverse organisms that range from yeast and fungus, to plants, insects, mammals and humans, our work could provide unique insights into how microtubules malfunction in some human diseases. With further studies, this may provide a new understanding of chromosome loss in birth defects and/or cancer. Stu2 is a member of the XMAP215/Dis1/CKAP5/ch-TOG family of MAPs that has multiple microtubule-dependent functions. Stu2 is well known for its role as a microtubule polymerase . Stu2 prWhile Stu2 is perhaps best known for contributing to microtubule stability and growth ,3,4,6,17Stu2 has multiple functions at the kinetochore. It is important for attachment of kinetochore microtubules (K-fibers) to the outer plaque of the kinetochore, where it also aids in the selection of correctly oriented kinetochore attachments . Stu2 acS. pombe CDK mediated phosphorylation was found in Alp14 .  with β-t.  with βWhile evaluating the interactions of K469 with β- and γ-tubulins, we observed that the side chain of K469 was not solved in the crystal structure. To gain insight into this region, we used PyMOL to reconstruct the K469 side chain and screened for optimal rotamer confirmations. As shown in The surface map of β-tubulin with TOG2 in this region is well-characterized, based on crystallography data  Fig 9E)Fig 9E). If Stu2 interacts with both β- and γ-tubulin through this motif, then our model predicts that acetylation at K469 will affect each of the interactions differently because the charge of the EFPD and RYPK loops are different. In this model for γ-tubulin, Stu2 interaction should be promoted by the acetylation of K469 (imitated by Q). This would be due to reduced ionic repulsion between the acetyl-lysine of Stu2 and the positively charged residues within the RYPK loops of γ-tubulin. However for β-tubulin, the non-acetylated form of lysine (mimicked by R) should be preferred over an acetylated lysine.To test this prediction, we assayed the interaction of Stu2 and the tubulins with a co-immunoprecipitation assay. We precipitated wild-type Stu2, as well as each of the K-to-R and K-to-Q mutants, and then assayed for γ-tubulin in the precipitates. Whereas the single K-to-R mutant K469R (as well as the K252R and K870R single mutants) consistently showed little or no reduction in the levels of γ-tubulin co-immunoprecipitated , the 3KRIn these blots, it is evident that an additional higher-molecular weight band of γ-tubulin that runs at 62.5kDa co-precipitates with Stu2. As γ-tubulin has been reported previously to be ubiquitinated in mammalian cell lines HEC293T cells –43, we sThe model also predicts that the positive charge on K469 is important for Stu2 interactions with β-tubulin. To test this prediction, we assayed the Stu2 precipitates for the presence of β-tubulin, using its α-tubulin partner as a surrogate. As seen in This work represents the first functional characterization of acetylation for any Stu2/XMAP215 family member. Our report details how acetylation regulates multiple functions of the Stu2 protein. Using a collection of acetyl-inhibitory (K-to-R) and acetyl-mimetic (K-to-Q) mutants, we show that acetylation of three lysines within Stu2 at K252, K469, and K870 is important for microtubule stability, chromosome segregation, and affinity for its γ-tubulin binding partner. Collectively, these findings suggest that multiple activities of the Stu2 protein are coordinated by acetylation, including those at the kinetochore and SPB, and these are summarized in Several of the mutants display improved Stu2 function . This suggests that these effects are not simply the result of compromised protein function, but rather they represent modulation of Stu2 activity based on regulation of critical ionic charges.Our analysis of these lysines suggest that several functions of Stu2p can be separated based on their acetylation status. This was particularly true for acetylation at K469 and K870. The influential nature of K870 acetylation was especially evident in the benomyl sensitivity assays. At this residue, the Q and R states produced distinctly different phenotypes. Regardless of its acetylation status, the K870 mutation suppresses the benomyl phenotype produced by corresponding double mutation in the TOG domain (the 2K). For example, the non-acetylatable K870R produces robust benomyl resistance, whereas the 2KR mutant does not. When the K870R is added to the 2KR to produce the triple (3KR) mutants, the strain is again benomyl resistant. A similar pattern is seen for the acetyl-mimetic K870Q . Thus, tMoreover, the pseudo-acetylation of the three lysines all trend toward increasing interactions with γ-tubulin. Notably, the pseudo-acetylation mutants K469Q and 3KQ have the largest increases . AlthougOur data also shows that Stu2 acetylation regulates chromosome transmission fidelity. Acetylation at K252 and K469 (found in TOG1 and TOG2 domains respectively) were both important for chromosome segregation, since mutants unable to undergo acetylation at these sites displayed decreased chromosome transmission fidelity .In addition to the Ndc80 complex interacting through the c-terminal MAP domain, Stu2 also interacts with Bik1 and Bim1 through this domain . These Met al. (2021) [K. marxisus, zebra fish D. rerio, and the bird G. gallus each possess a region from L869-K877 that is important for association with the Ndc80 complex [S. scitamineum, P. antarctica, and U. maydis) and the protozoan (A. deanei)), the amino acids in the K870 A871 R872 motif are flipped to R870 A871 K872. Lastly, Alp14 found in S. pombe and XMAP215 family proteins from the Trypanosome T. conorhini, the mosquito A. aegypti, and fruit fly D. melanogaster possess a lysine at amino acid 872 in addition to or in the place of the lysine 870 position.Recent novel findings describe how Stu2 functions at the kinetochore as a mechanosensor to promote correct bilateral anchoring of sister-chromatids to opposing spindle pole bodies in yeast undergoing mitosis ,14,18. H. (2021)  showed t complex . Our wor complex ), and ra complex  showed t complex . This inWe speculate that in some of these systems a putative acetylation at a K872 may target that face of the Stu2 alpha helix to impair salt bridges with Ndc80 rather than those of Spc24. Nevertheless, these findings make the prediction that acetylation at K870 will regulate these Ndc80-complex dependent interactions.Our conclusion that acetylation of Stu2 at K469 regulates its interactions with different tubulins is also informed by modeling of these interactions using previously published structures . In examet al., (2016) [To frame these interactions in the context of MT nucleating complexes, we modeled Stu2 interactions with γ-tubulin using PyMOL. For this, we juxtaposed the structure of the β-tubulin/TOG complex derived from x-ray crystallography coordinates found in pdb:4U3J [, (2016) . We did , (2016) . For theFrom this modeling, we predict that Stu2 interactions are more likely to occur with the γ-TuSCs in their closed conformation rather than in their open conformation. This concept is based upon three observations that are apparent in the model. First, in the open γ-TuSC structure, a loop within γ-tubulin (amino acids S307-N312) extends into spaces normally occupied by the TOG2 domain, which would interfere with Stu2 binding. This steric hindrance is apparent in the backbone ribbon structure of Second, we observe in this model that alpha helix 10 of Stu2’s TOG2 domain interferes with the space occupied by multiple gamma ring proteins (GRIPs) of the γ-TuSC. Alpha helix 10 of TOG2 domains overlap with two alpha helices of Spc98  and threThird in this model, a loop of γ-tubulin (comprised of amino acids D411 through S415) moves into position and forms additional binding contacts with TOG2 α-helices 2 and 4 specifically in the closed confirmation  was shown to regulate interactions with CLIP-170, p150 and APC . Additio and APC . Acetyla and APC . Lastly, and APC . TogetheIn summary, this work proposes a novel hypothesis for the regulation of Stu2 association with kinetochores, spindle pole bodies, and microtubule plus ends. As the expression status of the human XMAP215 family member, CKAP5/ch-TOG is associated with a poorer prognosis in cancers , future et al., 2018 [et. al., 2018 [Yeast expressing Stu2-HA (yRM10637) or a vector control (yRM10641) were grown to mid-exponential phase and disrupted using cryo-milling as described in Greenlee l., 2018 . Frozen l., 2018 . ProteinPeptides from the digested samples were dissolved in 0.1% aqueous formic acid, and injected onto a 0.075 x 400 mm nano HPLC column packed with 3-μm Magic AQ C18 particles. Peptides were separated using a 120-min gradient of 3–30% acetonitrile/0.1%formic acid and eluted through a stainless-steel emitter for ionization in a Proxeon ion source. Peptide ions were analyzed by a high/high mass accuracy approach. Parent ions were measured using the Orbitrap sector of a Fusion mass spectrometer (Thermo), followed by data-dependent quadrupole selection of individual precursors and fragmentation in both the ion trap (CID at 35% energy) and in the HCD cell (25% or 35% energy). Lastly, fragmented ions were measured again in the Orbitrap sector at 30,000 resolution.Raw mass spectra files were converted to .mgf files using msconvert from Proteowizard . SearchG600 value of 0.3 and treated with a final concentration of 200 ng/mL TSA in DMSO or DMSO alone for 2 hr at 30°C. Following TSA treatments, cells were harvested, cryo-milled, resuspended in 1x PBS containing 0.1% Tween, 20 mM N-Ethylmaleimide, 20 mM 2-Iodoacetamide, 1:500 Sigma protease inhibitor cocktail #8849, and 1 mM PMSF, and clarified as described in Greenlee et. al., 2018 [For western blotting analysis, the lysine deacetylase inhibitor Trichostatin A (TSA) was used to preserve Stu2 acetylation for subsequent detection. Yeast cells were grown to an ODl., 2018 . ProteinS. cerevisiae (P46675), M. sympodialis (A0A1M8A6U5), D. discoideum (Q1ZXQ8), P. pallidum (D3BU59), A. deanei (S9W6U7), P. marinus (C5LPC0), C. elegans (G5EEM5), T. conorhini (A0A3R7L7I8), E. multilocularis (A0A087W255), A. thaliana (Q94FN2), S. scitamineum (A0A0F7S5S5), O. sativa subsp. Japonica (Q5N749), P. humanus subsp. Corporis (E0VU40), A. darlingi (W5J5U5), A. aegypti (A0A1S4FBN3), D. melanogaster (Q9VEZ3), X. laevis (Q9PT63), M. musculus (A2AGT5), H. sapiens (Q14008), S. pombe , A. gossypii (Q75CQ1), P. antarctica (A0A081CMB9), G. candidum (A0A0J9XCS4), C. albicans (A0A1D8PTZ8), C. glabrata (A0A0W0D3T8), E. nidulans (Q5B1Q9), and U. maydis (A0A0D1CA73). These MT polymerases were grouped as either fungal organisms or broadly as eukaryotes, and aligned using Clustal Omega [To determine the conservation of lysines shown to be acetylated, we compiled a list of twenty-eight XMAP215 family members from twenty-seven different organisms including: al Omega .S. cerevisiae (P02557), C. elegans (P12456), A. thaliana (P12411), D. melanogaster (Q24560), X. tropicalis (Q6GLE7), M. musculus (A2AQ07), and H. sapiens (P07437) and γ-tubulins from S. cerevisiae (P53378), C. elegans (P34475), A. thaliana (P38557), D. melanogaster (P23257), X. tropicalis (P23330), M. musculus (P83887), and H. sapiens (P23258).Conservation analysis of β- and γ-tubulins was carried out using Clustal Omega as previously described using β-tubulins from URA3-marked plasmid expressing wild-type STU2 (pRM10693) and a LEU2-marked plasmids expressing STU2 (yRM12337), vector (yRM12338), K252R (yRM12340), K469R (yRM2341), K870R (yRM12342), 2KR (yRM12343), 3KR (yRM12344), K252Q (yRM12345), K469Q (yRM12346), K870Q (yRM12347), 2KQ (yRM12348), or 3KQ (yRM12349) were grown overnight. OD600 values were determined for each culture and normalized to 0.100 each. Normalized cultures were further diluted to prepare 0.100, 0.055, and 0.010 dilutions in 96 well plates. These were transferred with a multi-pronged device to synthetic defined media lacking histidine, leucine, and uracil to confirm equal loading of cells. Cells were transferred to 5-FOA plates lacking leucine to select for cells that lost the WT-STU2 URA3+ plasmid, while retaining the Stu2 version on the LEU2 plasmid.To evaluate the viability of Stu2 acetyl mutants, we performed a plasmid shuffle assay. Cultures (5 mL) of yeast containing a STU2/stu2-Δ1::HIS3 heterozygous diploid strain (yRM 11105) for subsequent yeast mating assays, we noticed that the Stu2-3KR mutant failed to produce viable spores in the wild-type STU2 background. Of the 36 tetrads dissected from multiple independent transformations, 72 spores were expected to contain the Stu-3KR plasmids in the wild-type STU2 background. However, zero viable spores of this genotype were obtained  for 1 hr. The resulting zygotes were isolated by micromanipulation using an Olympus BX41 dissection microscope. The resulting diploid (yRM11988) was heterozygous for STU2/stu2Δ and homozygous for the ade2-101. The artificial chromosome was generated by the fragmentation of Chromosome III, induced through the integration of NotI linearized pJS2/pRM11972 plasmid. . Successful candidates were identified by comparing them to a previously published haploid cell-line possessing the 125-kb chromosome III fragment (YCTF58/yRM11971 from ) as a poTo proteolytically digest the protoplasts, the liquid overlay was removed and replaced with a solution containing 1 mg/mL proteinase K resuspended in 270 mM EDTA (pH 9.0), 10 mg/mL N-Lauroylsarcosine (pH 9.0), and 10 mM Tris (pH 8.0). Protoplasts with proteolysis buffer were incubated overnight at 50°C in a sealed plastic bag. To store digested cells, the proteolysis liquid overlay was removed and replaced with 500 μLs of pH 9.0 0.5 M EDTA.2O for 1 hr and destained in ddH2O for 1.5 hr. Using electrophoretic mobility to separate the four smallest chromosomes in S. cerevisiae, we identified two strains with Chromosome III fragmentation  were then transferred to four SD plates lacking both uracil and leucine. Approximately 17,000 Cells from the 5-FOA treated cells were plated to three 1 mg/mL 5-FOA plates lacking leucine and grown for 4 days at 30°C. To quantify chromosomal loss, initial cellular densities were back calculated using the uracil leucine deficient plates. Colonies growing on 5-FOA plates were quantified as individual chromosomal loss events. The total number of 5-FOA colonies was then divided by the total number of colonies predicted to determine a quantitative value for chromosomal loss.To quantitatively determine the rates of chromosome transmission fidelity for the strains described above, cells were initially grown over night to ODSUP11 artificial chromosomes and plasmids expressing WT-Stu2 (yRM12253), vector with endogenous Stu2 (yRM12300), K252R (yRM12261), K469R (yRM12301), 2KR (yRM12266), 3KR (yRM12269), K252Q (yRM12262), K469Q (yRM12260), K870Q (yRM12270), 2KQ (yRM12267), and 3KQ (yRM12268) were initially grown overnight to OD600 values between 0.100 and 0.200. Overnight cultures were further diluted to approximately 0.0001 OD600 values. 25 or 50 cells of each strain were plated onto SC deficient for leucine with either 4 μg/mL (20%) or 8 μg/mL (40%) of normal adenine concentrations. Each set of conditions was repeated three times to generate error bars. Yeast were then grown at 30°C for 5 days and stored for 1 day at 4°C. Because sectors slowly become more apparent at low temperatures, all visible red sectors were counted within 8 hr of each other. For each plate, the total number of sectors relative to colonies was determined as qualitative values.To determine relative rates of chromosome transmission fidelity between mutants and WT-Stu2, yeast strains containing 600 value of 0.7, harvested, cryo-milled, and resuspended in either PEM buffer  containing 20 mM N-Ethylmaleimide, 20 mM 2-Iodoacetamide, 1:500 dilution of protease inhibitor cocktail , and 1 mM PMSF or 1x PBS (pH 7.4) containing 0.1% Tween, 20 mM N-Ethylmaleimide, 20 mM 2-Iodoacetamide, 1:500 dilution of protease inhibitor cocktail , and 1 mM PMSF. Resuspended extracts were then clarified at 27,000 x g. To detect the interactions of α-tubulin, Stu2-HA was immunoprecipitated from 67 mg of yeast extracts resuspended in PEM buffer using 20ul with anti-HA magnetic beads . To detect interactions with γ-tubulin, Stu2-HA was immunoprecipitated from 90 mg of yeast extracts resuspended in 1x PBS, as described above. To detect co-precipitated tubulins, pull-downs were subsequently western blotted and probed with 1:100 mouse anti α-tubulin (4A1 developed by Dr. Margaret Fuller (Stanford University) as a gift from Dr. Jeff Moore ) and 1:1000 rabbit anti γ-tubulin . Stu2 was detected using mouse anti-HA . Secondary antibodies for these blots were goat anti-rabbit conjugated with HRP from Jackson Immuno Research Labs (cat #111-035-144) (1:5000) and goat anti-mouse-HRP (1:5000) .Yeast were grown to an ODPyMOL was used to visualize atomic coordinate data from the following pdb files: TOG/GTP β-tubulin complex from 4U3J, GDP β-tubulin polymerized in MTs from 5W3F, and GDP hydrolyzed γ-tubulin from 3CB2. We used the ColorByRMSD Python Module  to alignThe structural prediction of Stu2 was generated with I-TASSER –72 usingS1 FigStu2-3K mutants with mutation of lysines 252, 469, and 870 in HA epitope- tagged strains were analyzed for acetylation by western blot. (A) Cells expressing Stu2-HA (yRM12358), untagged Stu2 (yRM12359), Stu2-3KR-HA (yRM12364), or Stu2-3KQ-HA (yRM12369) were immunoprecipitated and western blotted with anti-HA and anti-AcK to evaluate Stu2 acetylation. Band densitometry was performed to quantify reductions in Stu2 acetylation. (B) The ratio of Stu2 present in WCEs relative to the Pgk1 loading control was evaluated to determine differences in Stu2 steady state abundance from logarithmically growing cell cultures. (C) The ratio of anti-AcK to Stu2-HA signal from immunoprecipitations was determined to evaluate reductions in acetylation for the Stu2-3K mutants compared to wild type. Ratios of Stu2-HA to Pgk1 protein from WCEs and AcK signal to Stu2-HA signal from pulldowns displayed as mean ± SEM from four biological replicates. Student’s t-tests performed on densitometry values prior to normalization to WT controls; * p < 0.1, ** p < 0.05, *** p < 0.01 (raw quantitative data are available in (TIF)Click here for additional data file.S2 FigLEU2 plasmids with STU2 (yRM12358), K252R (yRM12360), K469R (12361), K870R (12362), 2KR (yRM12363), 3KR (yRM12364), K252Q (yRM12365), K469Q (yRM12366), K870Q (yRM12367), 2KQ (yRM12368), or 3KQ (yRM12369) were transferred to YPD containing DMSO or DMSO + 20 μg/mL benomyl and grown at 30°C.Haploid yeast containing (TIF)Click here for additional data file.S3 FigSUP11 present on the 125-kb artificial chromosome (pRM11972/pJS2). (B) Colonies with STU2 plasmids (pRM2119) form red sectors less frequently than cells containing K252R plasmids (pRM11481). Yeast in this assay contained plasmids with STU2 (yRM12253), K252R (yRM12261), K469R (yRM12301), 2KR (yRM12266), 3KR (yRM12269), K252Q (yRM12262), K469Q (yRM12260), K870Q (yRM12270), 2KQ (yRM12267), and 3KQ (yRM12268) in a stu2Δ::HIS3 background, except for the WT-genomic control which had an intact STU2 at its genetic locus and an empty vector instead of the STU2 on a plasmid (yRM12300). (C) The total number of sectors counted was divided by the total number of colonies present to determine chromosomal loss rates of mutants relative to WT controls. Qualitative chromosomal loss data displayed as mean ± SEM of triplicate plates; * p < 0.05, ** p < 0.01, *** p < 0.005  Chromosome transmission fidelity was qualitatively assessed using a color-sectoring colony assay in which the red phenotype is suppressed by (TIF)Click here for additional data file.S4 FigThe side chain of K469 was not solved in crystal structures between TOG2 and tubulin heterodimer in pdb:4U3J (A). To gain insight into how K469 may interact with β-tubulin, we modeled the side chain of K469 using PyMOL. The TOG2 K469 side chain is reconstructed in panel B. We modeled side chains for the (C) K-to-R acetylation prevention mutation, the (D) K-to-Q acetylation mimetic mutation, and (E) acetylated K469. Lastly, K469 rotamer confirmations were examined in this model to predict this residue’s interactions with (F) E158 and (G) D161 of β-tubulin.(TIF)Click here for additional data file.S5 FigA previous acetylomic screen identified K711 as an acetylated lysine in the yeast XMAP215 family member, Stu2 . To unde(TIF)Click here for additional data file.S6 FigFragmentation of Chromosome III was generated as described in , which c(TIF)Click here for additional data file.S1 TableAdditional mass spectra were identified for each of the acetylated residues in Stu2. Listed are all of the confidence scores associated with each putative AcK specta. AcK 252 had a total of 32 spectra with the following confidence scores: 9 with 100%, 5 with 99%, 4 with 97%, 8 with 96%, 2 with 95%, 1 with 94%, 1 with 83%, 1 with 82%, 1 with 32%. AcK 469 had a total of 10 spectra with the following confidence scores: 1 with 97%, 3 with 96%, 1 with 95%, 1 with 94%, 1 with 93%, 1 with 91%, and 1 with 70%. AcK 870 had a total of 18 spectra with the following confidence scores: 3 with 100%, 4 with 99%, 3 with 98%, 3 with 97%, 1 with 96%, 1 with 86%, 1 with 85%, 1 with 82%, and 1 with 80%. All spectra fell well within a 30 ppm mass accuracy with the vast majority of parent ion masses deviating less than 3 ppm.(TIF)Click here for additional data file.S2 TableSTU2/stu2-Δ1::HIS3 heterozygous diploid (yRM11105) was transformed with wild-type STU2-HA (pRM2119), empty vector (pRM2200), untagged wild-type STU2 (pRM6507), K252R (pRM11481), K469R (pRM11249), K870R (pRM12016), 2KR (pRM11966), 3KR (pRM11976), K252Q (pRM11482), K469Q (pRM11254), K870Q (pRM12015), 2KQ (pRM11969), and 3KQ (pRM11974). Each strain was then sporulated and tetrads dissected. STU2 deletion was marked with HIS3 (stu2-Δ1::HIS3). Yeast centromeric plasmids expressing STU2 or stu2 mutants were marked with a LEU2 gene. In this analysis, the total tetrads for each dissected strain are reported. The expected viable spores reflect the 2:2 nature of sister chromatid distribution as well as the inability of our vector control to rescue stu2 deletion. Observed spores represents the total number of spores that grew. The four categories of possible genotypes are reported in columns 5–8. Notably, the K252,469,870R (3KR) mutant fails to produce viable spores in the presence of an endogenous copy of STU2. In the absence of endogenous STU2 (stu2-Δ1::HIS3), spores containing the 3KR mutant grow.A (TIF)Click here for additional data file.S1 Supplementary fileThis Excel spread sheet contain six tabs, one each for: 1. The microscopy analysis of (XLSX)Click here for additional data file.


Verification using corn flour and corn starch showed a significant increase in RS contents from 15.84 to 27.78% and from 15.27 to 32.53%, respectively, and a significant decrease in starch digestibility from 67.02 to 35.74% and from 76.15 to 28.09%, respectively. Treated sample also showed the pasting profile that was stable under heating and stirring.Autoclaving-cooling is a common starch modification method to increase the resistant starch (RS) content. The effect of this method varies depending on the type of crop and treatment condition used. The objectives of this study were to verify the autoclaving-cooling treatment based on a meta-analysis result and to evaluate the physicochemical properties of modified starches. The meta-analysis study used 10 articles from a total of 1,293 that were retrieved using the PRISMA approach. Meta-analysis showed that the optimal treatments of autoclaving-cooling process that increase the RS content significantly, was in starch samples from the cereal group  (SMD: 19.60; 95% CI: 9.56–29.64;  It is r® 330.A modification process can increase the amount of RS content in a material, and autoclaving-cooling is one of the methods that is widely used. This modification does not use chemicals, so it does not produce by-products in the final product. However, this modification process requires equipment such as an autoclave that can provide high temperature and pressure. It involves heating a starch sample suspended in water in an autoclave, then cooling it to produce retrograded amylose . The appSeveral studies stated that the more cycles used, the higher the RS content increase , 9–11. HThe findings of studies did not clearly describe the effect of autoclaving-cooling treatment on RS contents. Therefore, a meta-analysis study needs to be carried out to evaluate the effect of the treatment used. Meta-analysis is a quantitative scientific synthesis of various research results that have been used in many scientific fields. Meta-analysis aids in the practice of evidence-based research and the resolution of conflicting study findings. The meta-analysis extracts one or more study outcomes in terms of effect sizes. The purpose of effect sizes is to put the results of a large number of research on the same scale by employing a number of metrics such as oddity and risk ratios, standardized mean differences, transformed correlation coefficients and logarithmic response ratios , 18. TheMaterials for meta-analysis study were articles obtained from several reputable online journal databases and published from 2000 to 2020. Corn flour and corn starch for verification are obtained from commercial products in Indonesia, under the brands “Mugo Tepung Jagung” and “Maizena 328,” respectively.The studies were retrieved using PRISMA  statement guidelines . SeveralBased on inclusion and exclusion criteria, studies were selected through screening and eligibility stages. Research articles from international journals indexed by Scopus (Q1–Q3) and web of science published from 2000 to 2020 were used as inclusion criteria. Articles also have to include sufficient data, namely source of starch, the value of RS from native (as control) and from autoclaving-cooling process (as experiment), standard deviation or standard error, and number of replications. Exclusion criteria included studies from books or patents, studies with raw materials other than starch, and studies with other treatments or multiple modifications.Data were extracted and inputted into a worksheet developed by Afandi et al.  using thVerification of the autoclaving-cooling treatments was carried out based on the results of the meta-analysis. Sample (fineness 100 mesh) was weighed as much as 60 g, then suspended with distilled water in a ratio . The sample was put into an autoclave for 30 min at a temperature of 121°C. The sample then continued was cooled in a refrigerator at 4°C for 24 h. The autoclaving-cooling was repeated once again, so that the total cycle is two times. The samples obtained were then dried in an oven at 50°C for 24 h. After that, the sample was mashed and shieved (100 mesh) for further characterization. Native and modified samples were analyzed for moisture, ash, protein and fat content according to AOAC , starch I2 value  using SPSS 26 software.The meta-analysis data were analyzed by the effect size value using Hedges’d (standardized mean difference/SMD), with 95% CI  pooled through a random-effects model , 23 to aI2 value , while pI2 value . The chaA total of 1,293 studies were initially retrieved and screened by the search strategy. Then, 135 full articles were evaluated, with 125 studies being excluded, leaving 10 studies for meta-analysis . These sIn autoclaving-cooling process, heating above the gelatinization temperature resulted in the dissociation of hydrogen bonds from the double helix structure of amylopectin, melting of crystallites and the release of the amylose fraction from the granules , 8, 33. p = 0.149) included zero. It is revealed that single modification by autoclaving-cooling process statistically had no significant effect on increasing the RS content. This insignificant outcome may be due to the numerous variations used in the autoclaving-cooling process.The forest plot of individual studies was shown in I2) among studies is 89.70% and classified as high heterogeneity (greater than 75%) (n ≥ 2.The heterogeneity (han 75%) . The stahan 75%) . To evalp < 0.001) but did not significantly affect the legume group . The amounts of RS are closely linked to the levels of amylopectin and amylose contained in a substance. In general, the higher amylose/amylopectin ratio, the more RS is present. This is because the chain structure of amylose is small and easy to orient and regenerate, while amylopectin has a dendritic structure and is difficult to orient , while the ratio 1:3.5  and 1:5  had no significant effect. The higher water content during gelatinization increases starch gelatinization and retrogradation enthalpy. This value is associated with the amount of single and double helical structures , while autoclaving-cooling 1 cycle , 3 cycles  and 4 cycles  had no significant effect.Based on the number of autoclaving-cooling cycles, the studies were sub grouped into 4 groups  and presented in the forest plot SMD, 95% CI . The anaMany studies applied autoclaving-cooling starch modification with varying numbers of cycles. The addition of cycles is expected to increase the formation of short chain amylose fraction and increase the amount of retrograded amylose, resulting in more RS . Setiartp < 0.001) has a significant effect on increasing the RS content compared to heating time 60 min . Meanwhile, heating time 15 min  has no significant effect on decreasing the RS content. The application of heat to a certain temperature and time is required to confirm that the starch is gelatinized fully thus it will undergo retrogradation later.Analysis for subgroups based on heating time of autoclaving was shown in p < 0.001) compared to autoclaving at 110°C  and 120°C   . Aside f< 0.001) . The temp < 0.05 being statistically significant for publication bias  ratio 1: 4 and autoclaving for 30 min at 121°C. The samples used were in the form of starch and flour from corn. Corn flour is also used as a sample so that it can be used as a comparison for use in application, because corn flour is easier to obtain than corn starch which requires an extraction stage. The chemical properties of native and treated samples are presented in In corn flour, all parameters showed significant changes from native and treated samples, while in cornstarch, the ash content, protein content, and amylose content did not change significantly. The moisture of each sample is at a low value, which is below 12%. Low moisture is needed especially for samples in the form of flour, to increase the shelf life of the product. The ash content in the corn flour decreased from 0.6 to 0.14%, while in cornstarch, the ash content value did not change. The decrease in ash content in the sample after treatment indicates that the autoclaving-cooling process in this experiment resulted in a cleaner sample . AutoclaCharacterization of the samples was also carried out through analysis of amylose content, RS content and starch digestibility. In A significant increase in the levels of RS was seen in corn flour and cornstarch after treatment . This reThe value of RS content in treated cornstarch (32.53%) was higher than in the corn flour (27.78%). This could be caused by the amylose content of the cornstarch (39.75%) which was higher than in the corn starch (32.47%). The higher the amylose content in a material, the higher the RS content produced –60. HighThe digestibility of starch in the treated corn flour decreased by 46.67% (from 67.02 to 35.74%), while the maize starch sample decreased by 63.11% (from 76.15 to 28.09%). The decrease in starch digestibility in the corn flour was also in line with the smaller increase in RS content when compared to the cornstarch. In autoclaving-cooling process, there is a rearrangement of starch molecules, both between amylose-amylose and amylose-amylopectin, so that it can strengthen starch bonds which make starch more difficult to digest , 61. Apa–1, which is a fingerprint of the conformation and hydration of starch. Wave numbers 1,045–1,047 cm–1 and 1,020–122 cm–1 are bands for the crystalline region and amorphous region in starch granules. The ratio 1,045 cm–1/1,022 cm–1 indicates the arrangement of the crystalline regions, while the ratio 1,022 cm–1/995 cm–1 indicates the arrangement of the amorphous regions  are shown in The final viscosity value in each sample also decreased. The final viscosity indicates the ability of the starch paste to form a gel after the heating or cooling process and the resistance of the paste to the stirring process. The gelatinization temperature of the treated sample decreased in the corn flour. The decrease in gelatinization temperature also occurred in arrowroot tuber samples that were treated with autoclaving-cooling . The gelGelatinization and retrogradation occur during the autoclaving-cooling and result in RS type 3. The retrogradation rate in cereal starch is higher than those of tuber due to higher precipitation rate of the cereal starch. Thus, the RS content increases significantly in cereal starch. Based on analysis, autoclaving-cooling on corn flour and cornstarch caused the decrease of amylose content and starch digestibility and the increase of RS content. Morphological changes were also observed after autoclaving-cooling. There was crystalline formation due to retrogradation.Meta-analysis study found that RS content significantly increased after autoclaving-cooling treatment using a sample:water ratio of 1:4, two cycles of autoclaving-cooling, and 30 min of autoclaving at 121°C. This RS increase was significant in cereal  with corn as the type of sample that has the highest effect size value. Verification using corn flour and cornstarch showed a significant increase in RS contents and a significant decrease in starch digestibility. Treated sample also showed the pasting profile that was stable under heating and stirring. This study can provide the optimal treatment of autoclaving-cooling process to achieve a significant increase in RS content for research treatment or industrial purpose.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.DF: content, methodology, and schema research. RS: writing – original draft and processing data. DI: review manuscript. FA, AJ, and MA: processing data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Small and Medium Enterprises (SMEs) contribute significantly to the European GDP and play a pivotal role in the ecological transition from a linear to a circular economy (CE). According to transition management theory, which emphasizes the active role of firms as accelerators of global transition processes, and based on qualitative content analysis of the literature, we found key pillars of CE  that SMEs should manage in an integrated way to increase the speed of the transition towards circularity. The result of this study is a conceptual framework that explains the development of the identified pillars in the context of the transition towards CE. This study addresses a gap in the literature concerning SMEs’ transition towards circularity, emphasizing the importance of a dynamic vision and the integrated management of a variety of key dimensions. The study also provides pragmatic advice to facilitate self-assessments by SMEs with respect to their path of transition and to maximize the effectiveness of policy-makers’ interventions to support SMEs. Finally, the study has societal implications: promoting the transition of SMEs towards CE can accelerate the global green transition due to the proximity of SMEs to the local environment and work force. The health emergency associated with COVID-19 and the current situation of material price volatility, resulting from high demand and limited supply due to resource scarcity, highlight the vulnerability of the traditional model of development with regard to the protection of the environment, the economy and people’s wealth, thus emphasizing the importance of resilience in generating economic opportunities while returning environmental and social benefits. The shift from a linear to a circular economy (CE) can be a legitimate answer to these many issues.CE is a more sustainable way of managing natural resources that incorporates a regenerative system  to facilitate a process of transition that involves business, societal values, norms and behaviours . Research suggests that firm size is an important factor in determining the extent and quality of sustainability practices; that is, smaller firms engage in fewer environmental practices  must embrace the values of circularity due to the need to engage with the companies of suppliers and customers, even if those companies are smaller in size. However, some characteristic elements of SMEs, such as the notion of the owner-manager and informal processes of relations and communication, facilitate the use of informal sustainability practices, causing the transition to formalized and structured policies to be difficult in this context  theory as its theoretical background  to another (such as a circular economy). TM theory was introduced in the context of the debate concerning sustainability to address problems associated with complex challenges to societal sustainability  approach  approach  database to identify contributions related to our research. WoS was the first broad-scope international bibliographic database to emerge, and over time, it has become the most influential bibliographic data source used for journals selection and research evaluation . A total of 86 contributions were excluded because they were inconsistent with the research question. A total of 110 contributions were submitted to such abstract analysis. Regarding Objective I, we followed the suggestions of Scipioni et al. . We traced these barriers back to the firm’s general relations with stakeholders. Responsible production and consumption typical of CE is promoted by the ability to create and maintain virtuous interactions with upstream and downstream actors throughout the value chain, thus enabling the creation of a market in which all players feel involved concretely in the achievement of collective objectives according to a win‒win logic. Technological issues refer to the ability to change the firm’s usual mode of operation. Innovation serves as a foundation for and accelerator of sustainable development as a result of disruptive technologies that enable changes in terms of time and cost that would have been inconceivable decades ago. Regulatory barriers refer to policy-makers’ actions with respect to the implementation of obstructing laws and regulations. These barriers are not viewed as critical in the research by Kirchherr et al. , even ifAnalysis of the literature allowed us to collect a series of factors that can as barriers to or potential enablers of the application of CE by SMEs. We excluded factors that were not subject to the direct control of firms , and we created codes that accounted for all remaining factors cited by the authors and standardized synonymous factors . The resulting coding frame is depicted in Fig. Step 3.2 Coding scheme for objective II: The following step in the research, which is related to the second objective of the research, pertained to the search for information regarding the different attitudes displayed by SMEs regarding CE in accordance with previous studies concerning the process of transition towards CE, as codified in Fig. Step 4. Validation of results via empirical testing: Finally, this framework is defined as the result of our research analysis, and we aim to validate this framework by conducting empirical research concerning a small enterprise (11 employees) operating in the bioenergy industry in the region of Lombardy .The bioeconomy has received attention from policy-makers who, motivated by the orientation of the EU, encourage investments to reduce the use of fossil fuels with the aim of mitigating climate change and reducing dependence on scarce natural resources. The bioeconomy can have a positive impact on the transition from a linear economy to a circular economy as long as the CE includes the concept of a circular bioeconomy ; this approach allows the loop to be closed in accordance with the principles of CE.We aimed to explain the conceptual framework to the entrepreneur during a face-to-face interview using an open interview method , while the other direction was related to circular management thinking and organizational eco-capabilities. Investments in innovation with respect to business models with the aim of finding alternative solutions to issues associated with linear production must also be approached in the context of a culture focused on long-term development as well as by reference to adequate performance indicators to enable the assessment of the economic and socioenvironmental impact of such investments, awareness of financing sources , which contributes to the formalization of environmental management processes. At this stage, organizations do not have special expertise or skills  air pollution. His relations with suppliers of waste products that could support a biogas plant (such as cocoa skins or pomace olive) were based on opportunistic negotiations. The absence of collaborative networks made negotiations more expensive for the entrepreneur and less effective for relevant suppliers, thus mitigating the potential benefits of this approach for the whole supply chain.These results address our research question concerning the ways in which SMEs can manage their transition towards CE in accordance with an integrated and strategic approach. We uncovered three pillars  that have already received attention in the business administration literature. Previous studies focused on CE in the context SMEs, however, have failed to employ an integrated approach or to emphasize the TM approach that characterizes the transformation from a linear economy to a circular economy  with regard to the pillars of CE prevents the value chain from taking full advantage of CE and acts as a barrier to value cocreation. SMEs are not always aware of the importance of the transition towards circularity, which requires the various key aspects on which such a successful transition is based to be considered jointly. Simultaneously, stakeholders are not always conscious of their role in the value chain or of the possibility of obtaining benefits from such engagement.Meso- and macrolevel politics are limited in terms of their beneficial effects if they prioritize the founding pillars of CE separately, i.e., without knowledge of the different ‘speeds of transition’ exhibited by different SMEs (whether they are considered as single units or as industries). Policy-makers must, therefore, act on the various pillars supporting SMEs by implementing well-rounded interventions.Ecological transition at the global level is possible by developing awareness of a just transition at the local level that can be tailored to local characteristics. This fact implies that SMEs, which are the engine of growth in the EU, should be guided and accompanied in their paths towards more sustainable business models. The case used as an example emphasizes two general aspects of SMEs that are critical to this process:The results of our research have the following implications. First, the key pillars emphasized in the literature as being fundamental to SMEs’ transition to CE  are difficult-to-implement concepts because they are rather broad with respect to the ways in which the approaches taken by the firms can differ: the grounded theory approach that we used . SMEs should be equipped with managerial tools suitable for their complexity, and the framework that we suggest, which is based on a qualitative circular assessment, points in this direction. The use of quantitative assessment tools for circularity should be preceded by an awareness of the ways of improving the circularity of the business in all its various aspects.Third, TM theory recommends catalysing the efforts of the different actors involved in transitions with respect to the same factors of change. According to this recommendation, this framework could also be used at the industry level  to assess sectors’ maturity with regard to these three key dimensions. The availability of a synthetic view of the ‘state of the art’ concerning the ways in which SMEs within a certain sector approach CE in terms of the aspects that are being handled or overlooked can help policy-makers implement more focused interventions. This catalysing role could also be played by trade associations, which may have greater proximity to specific territories and local realities. In other words, an initial assessment of the approach that the SMEs with a territory adopt towards CE can activate different ways of facilitating the development of circular networks and value chains .Increasing the commitment of SMEs to CE offers benefits for the global green transition due to the proximity of SMEs to the local environment and work force, thus broadening the societal implications of our research. SMEs can contribute to fighting climate change. Furthermore, shifting jobs towards CE requires the translation of global targets into local strategies involving industries, supply chains, and territories and requiring collaboration across different enterprises and workers . The shift to full maturity in circular thinking requires an active effort on the part of firms to adopt a holistic and integrated circular approach.In this context, our study does not aim to provide specific circular tools but rather to raise awareness of the need to change the global approach to business to enable SMEs to prepare for the inevitable changes that they must face in the near future and thus guarantee a new model of economic development.The contributions of this study are twofold. As a key contribution to theory, this study addresses the gap in the literature concerning the application of a circular approach in SMEs and emphasizes the importance of a dynamic vision and integrated management of various key dimensions. We conceptualize the transition associated with CE from a firm-level perspective by proposing a theoretical framework that highlights the different attitudes that SMEs adopt with respect to the founding pillars of CE depending on their knowledge and awareness of circular business.As a key contribution to practice, we suggest the use of this theoretical framework by both firms and policy-makers. In fact, SMEs can exploit the long-term benefits of CE when they attain a greater level of awareness concerning their approach to the principles of a circular economy and make an active effort to change the firm’s vision. In other words, SMEs must manage the transition and not merely undergo it. SMEs, despite their limitations, have great potential to participate in global change and must be aware of the key levers of CE that can allow them to exploit the opportunities emerging during the postpandemic period.Awareness of the fact that a global societal transition towards sustainability is a complex process of coevolution that involves the participation of actors at different levels also highlights a debate concerning the effectiveness of the financial, technological or knowledge interventions of governments and institutions with respect to supporting the adoption of CE by SMEs. All these interventions bear fruit over the long term when they are planned in a way that encourages SMEs to voluntarily embrace the relevant global, societal and cultural changes. Finally, all stakeholders should be made aware of their contributions to the transition of modern economies towards more sustainable ways of producing, consuming and financing.This study has the following limitations. We suggest a theoretical framework that is based on the extant academic literature, founded on qualitative content analysis and tested by reference to a single example case. Using qualitative content analysis allows the researcher to introduce relatively elementary codes, introducing simplifications that can blur a complex picture (Kracauer, Additional contributions from scholars are also necessary to increase the awareness of SMEs concerning the path towards a circular vision of their future, which is not limited to single or market-pressured projects, as well as to increase the awareness of policy-makers concerning the fact that the effectiveness of their interventions and future key priorities depend on the adoption of a culture of sustainability and a global approach to circularity among SMEs.


Going forward, international elites should instead strive for the application of one of the most liberal-minded principles of this current world order: self-determination.


The starch physicochemical properties, fine structure, texture properties and eating quality of two japonica rice were determined. We found that the content of total protein, crude fat and amylose between superior and inferior grains were significantly different. Compared with inferior grains, superior grains had low relative crystallinity, good pasting characteristics and outstanding eating quality. With the increase of nitrogen application rates, the starch volume mean diameter was lower; the average chain length of amylopectin was longer; and the relative crystallinity of starch was higher. The changes above in starch structure resulted in an increase in starch solubility, swelling power and gelatinization enthalpy, and led to a decrease in retrogradation enthalpy, retrogradation percentage and pasting viscosity, consequently contributing to the increase in hardness and stickiness of rice and the deterioration of taste value. These results indicated that topdressing PNF lengthened the amylopectin chain, decreased starch granule size, enhanced crystallization stability and increased gelatinization enthalpy, which were the direct reasons for the deterioration of cooking and eating quality.Nitrogen fertilizer is essential for rice growth and development, and topdressing nitrogen fertilizer at panicle stage has a huge impact on rice grain quality. However, the effect of panicle nitrogen fertilizer (PNF) on starch physicochemical properties and fine structure remain unclear. In this study, four PNF levels (0, 60, 120, 180 kg N ha Oryza sativa L.) is a widely cultivated cereal crop in Asia, and about half of the world’s population depends on rice and its related products for energy and nutritional needs [Rice . The soil type was waterlogged paddy soil with a sticky texture. The pH value of the 0–20 cm soil layer was 7.79, and contained 31.72 g·kg−1), 60 N (60 kg N ha−1), 120 N (120 kg N ha−1) and 180 N (180 kg N ha−1). The source of nitrogen was urea. Half of the nitrogen was applied at the panicle initiation stage, and the other half was applied at the spikelet differentiation stage. Through the investigation of growth process, Nanjing 9108 was applied PNF on 22 July and 3 August, and Nanjing 0212 was one day later than Nanjing 9108, respectively. Variety was the within-plot factor, and the area was 4 m × 4 m. An application of 90 kg N ha−1 was conducted before transplanting, and an application of 60 kg N ha−1 was conducted at the tiller stage. Each treatment was performed in triplicate, and the inter-plot ridges were wrapped with plastic membrane to ensure separate irrigation and drainage. 100 kg ha−1 P2O5  and 150 kg ha−1 K2O  were applied once as basal fertilizers before transplanting. Cultivation measures such as water management and control of pests and weeds were implemented in accordance with corresponding high-yield cultivation requirements.The experiment was arranged in a split-plot design, with the amount of PNF as main plot factor. Four levels of PNF were set, namely 0 N , the amylose content was measured by spectrophotometer with reference to the national standards of the People’s Republic of China (GB/T 17891-2017), and the amylopectin content was from the total starch content subtracted by the amylose content. According to the method of Tao et al. , the tot−5 mol L−1 sodium hydroxide solution, 0.10 g alkaline protease  and two glass balls. The conical flask was well-mixed to disperse the rice flour in the NaOH and protease solution, sealed and then put in a shaker for 24 h at 42 °C to remove protein. The mixture of starch and protease was sieved through a 200-mesh sieve and transferred to a 50 mL centrifuge tube. Deionized water (30 mL) was added, and the centrifuge tube was shaken at 3000 rpm for 10 min. The supernatant was discarded, and the upper layer, yellowish residue mainly composed of unreacted protease, was scraped off. The operation was repeated 3–5 times until the upper layer was free of residue. The sediment was then washed with a mixture of ethanol and chloroform/methanol (1:1 v/v) to remove fat. The starch was dried in an oven at 60 °C for 24 h, and was stirred and dispersed by a glass rod and then be dried for another 24 h. Finally the starch was sieved through a 200-mesh sieve to obtain native starch and stored in a drying container.Brown rice was frozen and then ground with a mixed-type mill , then 10 g brown rice flour was weighed and placed in a 50 mL conical flask with 30 mL of 10The analysis of starch granule size distribution was conducted by a laser particle size analyzer . The starch samples were dispersed in absolute ethanol, and the rotational speed was set to 2500 rpm. The samples were ultrasonically dispersed for 30 s before measurement. The measuring range of the instrument is 0.1–2000 μm. Once the background interference had been eliminated, the diameter of starch samples was determined based on the volume distribution.According to the method described by Wu et al. , starch c) and the area of amorphous region (Aa). The relative crystallinity was determined using the following equation: The XRD analysis of starch and the calculation of relative crystallinity were based on the method of Zhang et al. , and the0 = 9.4T. Starch samples were loaded into a 4 mm diameter PSZ  rotor with KelF end caps, rotated at 5 kHz with an acquisition time of 50 ms and a minimum of 2400 cumulative scans per spectrum. The preparation of amorphous starch followed the method of Tan et al. [According to the method described by Tan et al. , starch n et al. . The spe0) was weighed and placed in a 2 mL centrifuge tube (m1) with 1 mL of ultrapure water added, and then bathed at 90 °C for 1 h with shaking. The sample was cooled to room temperature (25 °C), then centrifuged at 5000 rpm for 10 min. The supernatant was removed by pipette and the total weight of the residue and centrifuge tube (m2) was gauged. Finally, the sample was dried in an oven at 60 °C to constant weight (m3). According to Cao et al. [The determination of starch solubility and swelling power was conducted as described by Cao et al.  with somo et al. , the sol−1. The starch was heated from 20 °C to 100 °C to determine the gelatinization characteristics and from 10 °C to 90 °C to determine retrogradation characteristics. Retrogradation percentage (R) was determined using the following equation:The analysis of starch thermal properties was measured by differential scanning calorimeter  according to the method described by Hu et al. . A 5 mg −1. The temperature in the tank started at 50 °C for 1 min and then increased from 50 °C to 95 °C at 12 °C min−1, held at 95 °C for 2.5 min, decreased to 50 °C and held 2 min. The peak viscosity, trough viscosity, final viscosity, breakdown value (peak viscosity–trough viscosity), setback value  and pasting temperature were analyzed by TCW  program.The pasting properties were determined using a rapid viscosity analyzer . A 3 g sample of milled rice flour with 12% moisture content was weighed and placed in an aluminum RVA can and 25 g of ultrapure water was added. The stirring speed was 960 rpm in the initial 10 s, and then kept at 160 r minThe taste value was determined by the Satake taste meter . The rice was hulled and milled, and the broken rice was removed to obtain head rice. Using the method of Zhou et al. , 25 g heThe texture properties were measured with a physical property analyzer  with the P/36R probe. The cooked rice grains were placed in the center of the sample table, and the program matched with the force sensing element ran to determine the firmness and stickiness properties.p < 0.05.All variable data were the average of three experimental values and are shown in the table as mean ± standard deviation. SPSS Statistics 16.0 was used to conduct analysis of variance. In the analysis, the sources of variation included nitrogen (N), variety (V), position (P), and the interaction . Duncan’s new multiple range method was used to conduct multiple comparisons of variables with statistically significant differences. The data of the same variety in the same column marked with different lowercase letters meant significant difference at Starch was the main content in the endosperm of rice, followed by protein and crude fat. According to The grain filling of superior grains started early with high activity of granule-bound starch synthase (GBSS), and the inferior grains maintained relatively high activities of ADP glucose pyro-phosphorylase (AGPase) and starch branching enzyme (SBE) in the middle and late stages of grain filling, which contributed to the differences in starch accumulation between superior and inferior grains . Topdressing with PNF decreased the content of total starch, amylose, amylopectin and the ratio of amylose/amylopectin, which was consistent with previous studies . CompareIt can be seen from The content of total protein, albumin and gluten of superior grains were 5.93–21.62%, 9.77–21.38% and 3.90–15.24% lower than those of inferior grains, respectively. Topdressing PNF significantly increased the total protein content, and the content of protein component showed an increasing trend, which was significantly higher in 120 N and 180 N treatments compared with 0 N treatment. Starch and protein competed for carbon source for synthesis. Topdressing with PNF promoted nitrogen accumulation in plants, activated protein synthesis-related enzyme, and accelerated protein synthesis [According to In Nanjing 9108, superior grains had a higher level of small and medium starch granules (≤5 μm) than inferior grains, and the content of large starch granules and starch volume mean diameter were less than those of inferior grains . HoweverTopdressing with PNF resulted in an increase in the proportion of small and medium starch granules, and a decrease in the proportion of large starch granules and a decrease in the starch volume mean diameter. Compared with 0 N treatment, the small and medium starch granules of high nitrogen (120 N and 180 N) treatments increased by 4.08–12.88% and 6.31–11.39%, while the content of large starch granules and the starch volume mean diameter decreased by 5.72–9.35% and 3.94–4.97%, respectively, all of whose differences reached the significant level. Later nitrogen application postponed the grain filling. Large starch granules mostly appeared in the early filling stage, and decomposed into small and medium starch granules at the later stage of grain filling, which contributed to the decline in the content of large starch granules and volume mean diameter after topdressing PNF .From the analysis of variance, nitrogen and variety had significant effect on A chain, B1 and B2 chain. Position has no significant effect on the B1 chain, B3 chain and average DP . The chaTopdressing PNF decreased the content of A chain and increased the content of B2 chain and B3 chain, increasing the average chain length of amylopectin and reducing (A + B1)/(B2 + B3). Compared with 0 N treatment, the A chain content and (A + B1)/(B2 + B3) of amylopectin in high nitrogen (120 N and 180 N) treatments decreased by 3.54–6.72% and 5.45–9.65%, respectively, while the B3 chain content and average chain length increased by 6.10–12.22% and 1.86–4.53%, respectively, all of which showed significant difference. The same conclusion was drawn by Yang et al. , and theTang et al.  isolatedThe X-ray diffraction pattern of starch from superior and inferior grains of japonica rice under different nitrogen fertilizers showed two strong peaks at 15° and 23°, and unresolved doublet peaks at 17° and 18° (2θ), which were typical A-type starch crystals. It could be concluded that PNF did not change the starch crystal type A,B. AccoThe relative crystallinity of starch from the superior and inferior grains of japonica rice increased as nitrogen application rates increased. Compared with 0 N treatment, the relative crystallinity of inferior grains under treatments of topdressing PNF increased by 1.87–6.08%, and that of superior grains under 180 N increased by 2.47–4.73%, all of which were significantly different. However, Zhu et al. . In this13C solid-state NMR [13C solid-state NMR technique. The native starch spectra was separated into an ordered sub-spectra at the 84 ppm position  treatments decreased by 2.07–8.06% and 15.17–20.82%, respectively, while the double helix content increased by 4.36–18.10%, all of which were significantly different. The crystal structure of starch was reflected by single helix and double helix, and the relative crystallinity of starch was elevated by topdressing PNF, which was consistent with the results of XRD analysis. In addition, the components of single helix and double helix showed opposite trends, which indicated that the formation mechanism of two helix structures differed. According to previous studies, the double helix structure was mainly composed of amylopectin side chains, and the single helix formation was associated with lipids and partly of amylose components ,51, indiAccording to Topdressing PNF improved the solubility and swelling power of starch. Compared with 0 N treatment, the solubility and swelling power of high nitrogen (120 N and 180 N) treatments increased by 10.35–22.55% and 6.06–14.91%, respectively, both reaching significance level . FurtherO), the peak temperature (TP) and the conclusion temperature (TC) of superior grains were higher than those of inferior grains, and some reached a significant level. Nitrogen, variety, position, and N × V had significant effect on starch thermal properties, except for nitrogen on conclusion temperature . The difThe gelatinization enthalpy reflected the energy required to dissociate the helix structure of starch, which was generally considered to be related to the amylose content and relative crystallinity. The retrogradation enthalpy and percentage reflected the recrystallization of gelatinized starch molecules after cooling. Amylose molecules crystallized rapidly during starch retrogradation . Chung eO, TP and TC) under PNF treatments varied with varieties, showed an increasing trend in Nanjing 9108 but a decreasing trend in Nanjing 0212, and the difference became obvious with the increase of nitrogen application. Besides amylose content and relative crystallinity, amylopectin chain length distribution was another important factor affecting the thermal properties of starch. Topdressing PNF significantly increased the gelatinization enthalpy, and significantly decreased the retrogradation enthalpy and percentage . The gelAccording to previous reports, long chains of amylopectin could form ordered crystal clusters, which had better heat resistance . In thisAccording to The breakdown value reflected the heat resistance of starch granules, and the setback value reflected the retrogradation of starch paste, which was related to the rearrangement of the leached amylose . The disWith increasing nitrogen application, the peak viscosity, hot viscosity and final viscosity decreased significantly by 5.09–33.93%, 8.41–43.54% and 7.14–30.74%, respectively . After tThe pasting characteristics comprehensively reflected the swelling, disintegration, and gelation ability of starch granules. Hu et al.  found thNitrogen, variety, position, and two-factor interactions had significant effect on taste value . Figure Topdressing PNF reduced the taste value of superior and inferior grains of japonica rice: the taste values of superior grains of Nanjing 9108 and Nanjing 0212 decreased by 0.72–4.40% and 2.90–11.47%, respectively, and the inferior grains decreased by 2.95–11.47% and 5.35–18.32%, respectively. This indicated that the low taste value varieties and inferior grains were more inclined to be regulated by PNF, which meant that the eating quality of inferior grains could be optimized by selecting varieties with good taste and reducing the application of PNF during cultivation. It was generally believed that the amylose content was closely related to the cooking and eating quality, but we found that the taste value was significantly different with similar amylose content in some treatments. We infer that this was related to the amylopectin chain length distribution and starch thermal characteristics. PNF reduced the proportion of amylopectin A chain and increased the proportion of B2 chain and B3 chain, which increased the double helix content and made starch crystallization more stable, thereby limiting the swelling of starch granules, leading to an increase in the gelatinization enthalpy, and ultimately a deterioration in the eating quality.According to Topdressing PNF increased the hardness and stickiness of superior and inferior grains of japonica rice, and the hardness and stickiness of high nitrogen (120 N and 180 N) treatments increased by 16.27–48.40% and 5.30–19.09% compared with 0 N treatment, reaching a significant level of difference. Proteins, especially prolamins, provided support for rice grains during cooking . Previou−1, so as to meet consumers’ demand for rice palatability. These findings enabled us to better understand the differences in cooking and eating quality of superior and inferior grains and their relationships with starch properties and structure, and to clarify the response of starch physicochemical properties and fine structure to PNF. New ideas were put forward for cultivation measures to optimize rice eating quality and starch functional properties.The results showed that, compared with inferior grains, the superior grains had better pastings properties and high taste value, namely better cooking and eating quality, which was related to the differences in biochemical components and starch crystal structure between superior and inferior grains. Under all nitrogen levels, superior grains had higher amylose content, lower protein and crude fat content than inferior grains, which contributed to the decrease of relative crystallinity and single helix content of starch, making starch granules susceptible to swell during cooking and retrograde after cooling, showing better cooking and eating quality as a whole. PNF deteriorated the cooking and eating quality of superior and inferior grains of japonica rice, which increased hardness, decreased taste value and worsened pasting characteristics. This was related to the fact that PNF increased protein content and decreased starch and crude fat content. However, we also found that there was significant difference in taste value of rice with few differences in starch and crude fat content among some treatments. This meant that the regulation of PNF on physicochemical properties and fine structure of starch was another essential factor in the deterioration of cooking and eating quality. Topdressing PNF reduced the content and volume mean diameter of large starch granules, increased the average chain length of amylopectin and starch relative crystallinity, which limited starch swelling and gelatinization, resulting in increased gelatinization enthalpy and decreased pasting viscosity, thus leading to deterioration of cooking and eating quality, and this effect was more evident under high nitrogen treatments. Moreover, with the increase of PNF, the cooking and eating quality of low taste varieties and inferior grains deteriorated significantly, which suggested that the quality of inferior grains could be optimized by variety selection and reduction of panicle fertilizer in production. We suggest that rice growers improve the eating quality of rice by selecting varieties with good taste and topdressing PNF with 60 kg N ha


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