Arabidopsis thaliana has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form π-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a VMax similar to that of the wild type enzyme but with a 7.5-fold lower KM for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.The β-amylase family in  Starch is the principle storage form of reduced carbon and energy in plants and it accumulates in plastids for short-term or long-term requirements. Starch granules are composed primarily of amylopectin, which contains linear chains of α-1,4-linked glucose with regularly spaced α-1,6 branches . AmyloseArabidopsis thaliana, four encode plastid localized, catalytically active enzymes    BAM was crystallized as a tetramer, but the active form was monomeric  . The soydrolysis . A sweetonomeric . Laederaonomeric .in vivo by physiological concentrations of K+, which are typically greater than 120 mM in the stroma , a BamHI site was introduced into the WT sequence in frame between the acidic region and the catalytic domain. A BamHI site already existed in the vector between the encoded N-terminal His-tag and the start of the BAM2 coding region. The primers used for PCR were B2_NDel, 5′-TGATGAAGAAATTGTGCAGGATCCAGAGCGTGATTTTGCTGGC-3′ and the complementary sequence. The protocol for the PCR was the same as for mutagenesis described above. The resulting DNA was then digested with both DpnI for 1.5 h at 37°C, and then BamHI for 4 h at 37°C. After digestion, the DNA was ligated and then transformed into competent DH5α E. coli cells. All of the nucleotide changes were confirmed by sequencing at Eurofins Genomics. Each plasmid was then transformed into BL21+ E. coli cells, and proteins were expressed and purified as described by Construction of the WT BAM2 expression vector in pETDuet-1 was previously described . Amino a−1 were injected into a 4.6- × 300-mm size-exclusion column with a particle size of 5 μm and a pore size of 300 Å at a flow rate of 0.5 mL min−1 in filter-sterilized (0.2 mm) 10 mM MOPS, pH 7, 250 mM KCl. Absorbance data were collected on an Agilent G1315B Diode Array detector at 280 and 212 nm, and light scattering was measured on a miniDAWN-TREOS (Wyatt Technologies). Data were analyzed using ASTRA (version 6.1.5.22). The predicted molecular mass and extinction coefficient of each monomer were determined using the Protein Identification and Analysis Tools on the ExPASy Server (Size-Exclusion Chromatography Multi-Angle Light Scattering was performed as described previously . Proteiny Server .−1 porcine gelatin. Amylase assays were conducted in 0.5 mL containing 50 mM MES (pH 6) and various amounts of soluble starch (Acros Organics). Unless noted, assays also included 100 mM KCl. Reactions were stopped after 20 min by immersion in a boiling water bath for 3 min; then, reducing sugars were measured by the Somogyi-Nelson assay  lacking its predicted 55 amino acid N-terminal chloroplast transit peptide  we used Figure 1A). Interface A involves a relatively long loop between α-helix 7 and β-sheet 8 identified as L’7 . Moreover, the active sites do not contain amino acids associated with either Interface. The N-terminal and C-terminal regions of each subunit are not integral to the TIM barrel but associate with each other and extend ∼90° from the primary dimer axis .The resulting model has dihedral symmetry and forms an apparent “dimer of dimers” with the primary dimer consisting of two BAM subunits interacting back-to-back (Interface A) to bring together the SBSs. Then two of these dimers pair together to form the tetramer (Interface B) in a bottom-to-bottom manner  with a diameter of 12 Å might be expected to sit on top of these two extensions within what we now refer to as the “starch-binding groove.”Each primary dimer creates a deep groove between the subunits, lined on each side by conserved surface residues previously identified as the SBS . The groFigure 2A). Trp456 is also conserved in BAM5 but it is in a poorly conserved region, and Asp490 is not conserved in BAM5 . After sequencing each plasmid to confirm the changes, proteins were expressed in E. coli and purified using their N-terminal 6-His tags to near homogeneity as previously described , which is ∼3.6 times larger than the predicted mass and consistent with BAM2 forming a tetramer. The W456A mutant had a calculated mass of 135.8 ± 17.7 kDa, and the D490R mutant had a calculated mass of 59.5 ± 6.9 kDa indicating that they were a dimer (2.4:1) and monomer (1.04:1), respectively . When assayed with 80 mg/mL soluble starch, both of the mutants with changes in Interface A had less than 5% of the WT activity .In order to test the validity of the tetrameric homology model of BAM2, several conserved residues located in the interfaces were mutated. To disrupt Interface A, Trp456 was changed to Ala to eliminate its involvement in hydrophobic interactions, and Asp490 was changed to Arg to reverse the charge . SEC-MALS analysis indicated that the F238A mutant had a mass of 143.8 ± 2.9 kDa which is consistent with it being a dimer (2.51:1) . Unlike the mutations that disrupted Interface A, however, the F238A mutant had activity that was similar to that of the WT protein .To disrupt Interface B Phe238 was changed to Ala, which, like the mutated Interface A residues, is also in a highly conserved region of BAM2 . A sequence alignment of 24 BAM2 orthologs from flowering plants, a seedless vascular plant, a non-vascular plant and a charophyte alga revealed that all of these sequences had a similar region that contained a high frequency of acidic residues, although they did not align well in the traditional sense . We refer to this region as the “acidic region.”Unlike all other characterized BAM proteins, BAM2 requires Kns of K+ . In lookFigures 4A,B). Despite the fact that this region appears to be located near both interfaces in our model , SEC-MALS analysis indicated that the mutant protein had a mass of 221.7 ± 16.3 kDa and thus was tetrameric (3.9:1) . Activity assays showed that the NDel mutant had higher catalytic activity than the WT. Interestingly, the activity of the mutant was largely unaffected by the concentration of KCl .To test the importance of the acidic region and its potential role in the salt requirement of BAM2 activity, we generated a mutant lacking the first 30 amino acids following the predicted chloroplast transit peptide  that we hypothesize might be involved in aromatic-sugar stacking (π-interactions) . The functional requirement of key residues in the SBS support its role as an allosteric binding site.The presence of a large, conserved region on the surface of BAM2 ∼20 Å from the active site led us to suspect that it might be a SBS that is involved in allosteric regulation of the protein. Two Gly-to-Met mutations in this region were previously made to attempt to interfere with binding at this site. Both of the mutant proteins were found to be tetramers with very little activity compared with the WT protein in agreement with this hypothesis . Examinaactions) . To testFigures 6A–C). This residue is a Ser in all of the BAM2 orthologs that were examined, whereas the corresponding residue in all of the other active BAMs is a Gly residue. To determine if this Ser was involved in allosteric regulation of BAM2 we mutated it to a Gly and measured the mutant’s kinetic properties . Remarkably, this mutation had no effect on VMax  but the mutant had a KM that was 7.5-fold lower than the WT protein , and with a Hill coefficient of 1.06 ± 0.07 the allosteric regulation of the protein was completely disrupted . Thus the S464G mutation suggests a direct structural connection between the SBS and the active site further supporting our tetrameric model of BAM2.We were also interested in how allosteric binding was communicated to the active site. It is unlikely that the mechanism of allosteric activation of BAM2 by the binding of starch to the SBS involves a radical change in the active site as all of the active-site residues identified to interact with the substrate in the soybean BAM5 crystal structure  are iden+ for activity, is a tetramer with sigmoidal kinetics, and possesses a putative SBS, presumably for starch . The active sites of each monomer are positioned facing out so that it is unlikely that tetramerization would block access of the substrate to these sites. In addition to the TIM barrels that make up most of the monomeric structure, the N- and C-termini of each subunit appear to interact and are positioned at the base of the starch-binding groove extending away from each dimer in such a way that they may facilitate starch binding to the groove . However, this latter aspect of the model is more difficult to validate due to the apparent flexibility of both termini.Using the sweet potato tetramer structure as a template, we positioned four BAM2 monomeric homology models in roughly the same orientation as those in the sweet potato structure, as a dimer of dimers, and noticed that the subunit interfaces contained residues that are highly conserved among BAM2 orthologs. Moreover, the subunit interface in the dimer formed a deep groove with each side of the groove comprised of the large, putative SBS. The groove is large enough to accommodate a starch chain . Interestingly, the two proteins with mutations in Interface A, necessary for forming the starch-binding groove, were nearly inactive, whereas the protein with a mutation in Interface B formed an active dimer . Taken together, these results are consistent with the interpretation that BAM2 activity requires a dimer that contains the starch-binding groove, and that starch binding to the amino acids in the SBS does not stimulate formation of the dimer and the starch-binding groove. Why BAM2 forms a tetramer when a dimer is functional is a further question. These results also suggest that the tetramer interfaces in the model are a reasonably close approximation of the real BAM2 structure.In order to test the validity of the tetrameric homology model of BAM2, we selected several conserved residues in the interfaces and changed them to residues that we expected would disrupt each interface. Once the proteins were purified we tested their activity and used SEC-MALS to determine their association state. Each mutant failed to form a tetramer and was instead either a dimer or a monomer, supporting the orientation of the BAM2 monomers in the model . Because these residues are expected to be negatively charged at physiological pH, their orientation relative to the TIM domain might be affected by the presence of cations. We were curious what effect removal of the acidic region would have on BAM2 activity and sensitivity to KCl, so we generated a mutant lacking the acidic region (NDel). Despite being located near Interface A and being part of the starch-binding groove , we were surprised to find that the NDel mutant was still a tetramer with considerable activity . Importantly, NDel activity was nearly insensitive to KCl, and the reaction rate was higher than that of the WT protein at saturating KCl . Because the effect of KCl on WT activity produces a sigmoidal curve  it seems likely that the interplay between the acidic region and K+, and the effect of starch binding to the groove in increasing amylase activity are somehow related. Further, the presence of this acidic region in BAM2 orthologs from all land plants and even in a charophyte alga, Klebsormidium nitens , suggests that it is involved in a highly conserved BAM2 function.The requirement of K cations , the int cations . ExaminaFigure 5C). This type of aromatic-sugar stacking (π-interactions) is relatively common in sugar-binding proteins . Given the distance of this amino acid side chain from the active site, we do not believe that the activity defect is catalytic but is due to an inability to bind to starch efficiently to the starch-binding groove.Close examination of the starch-binding groove revealed a centrally located Trp449 residue orientated such that the side chain ring might bond with a glucose residue of starch  and is proximal to the helix containing Trp449, which we had identified as important for starch binding to the starch-binding groove . The side chain of Ser464 faces away from the active site and toward the starch-binding groove , and is conceivably involved in an allosteric interaction network. In all other catalytically active BAMs, this residue is a Gly. Changing Ser464 to Gly resulted in a protein that had a similar VMax to the WT BAM2 , but it displayed hyperbolic kinetics (Hill coefficient = 1) with a KM for the substrate that was 7.5-fold lower than the WT protein . The change in the Hill coefficient suggests that the S464G mutation disrupted the allosteric network between the starch-binding groove and the active site. Interestingly, the mutation resulted in an enzyme that was more efficient that the WT protein rather than less as might be expected. Previous studies on allosteric mutations within pyruvate kinase and phosphofructokinase indicate mutations to amino acids near allosteric sites can shift the equilibrium toward the T (inactive) or R (active) state  state . Structuin vivo, but it is possible that BAM2 activity is also negligible at low levels of starch, which are known to occur at the end of the night. If BAM2 is present at this time of the photoperiod, it may be advantageous to prevent it from acting on small, growing molecules of starch. Moreover, this activity might be regulated by a yet unidentified metabolic, signaling, or post-translational modification pathway, which controls the T-R equilibrium of the protein. A possible function for the SBS is involvement in the separation of double helical strands of amylopectin, not unlike the role of an SBS on the surface of β-agarase that helps to separate double-helical chains of agarose  play an important role in starch degradation in plants. Flowering plants contain several active BAMs although their specific roles are not well understood. T-DNA mutants of Arabidopsis have been used to show that BAM3 plays a prominent role in leaf mesophyll cells at night, and that BAM1 plays a prominent role in guard cells across the night-day transition . In contJM, LP, JB, and AS designed and conducted the experiments. CB carried out the modeling. JM, CB, and AS wrote the manuscript.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.


Introduction: Meshed split-thickness skin grafting represents a rapid and effective technique for surgical wound closure. Factors such as ongoing inflammation, microbial colonization, and a poorly vascularized wound bed increase the rate of skin autograft failure up to 33%. Because of the inherent angiogenic, anti-inflammatory, antimicrobial, and antifibrotic properties of human placental membranes, the complementary use of human placental membranes may promote graft survival and improve success rate for complete ulcer resolution. Methods: In this case series, a viable cryopreserved placental membrane was used as a meshed split-thickness skin grafting overlay in 6 high-risk patients with various comorbidities and recalcitrant nonhealing lower-extremity wounds. Results: The mean size of grafted wounds was 130.3 cm2. The average graft take-rate by postoperative days 10 to 14 was 92.5%, with complete epithelialization of all skin graft interstices observed between days 10 and 21. Transplanted autograft tissues did not lyse or dissolve, and sites remained free of infection and maceration throughout postoperative follow-up. Complete wound closures remained intact at the 12-month follow-up visit. Discussion: Thus far, our clinical experience has warranted the complementary use of viable cryopreserved placental membrane and meshed split-thickness skin grafting to reduce the need for repeat surgical interventions or prolonged local wound care due to graft loss or failure in high-risk patients. The authors describe the use of vCPM as an overlay in conjunction with standard surgical protocols for mSTSG and report clinical outcomes related to autograft survival and complete ulcer resolution at long-term follow-up.This aseptically processed vCPM is a commercially available product used in the management of acute and chronic wounds. vCPM is fully tested, stored, and distributed according to the US Food and Drug Administration and the American Association of Tissue Banks requirements and has a 3-year shelf life at −80°C.The purpose of this investigator-initiated study was to report the clinical outcomes of a 1-year evaluation for vCPM as an adjunct to standard surgical treatment protocols. Institutional approval was granted for the retrospective collection and presentation of material and data. De-identified data sets, consistent with the Health Insurance Portability and Accountability Act of 1996 (HIPAA), were collected from electronic health records. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2008.In this study, patients (n = 6) with recipient sites considered to be at high risk for autograft failure were identified and managed using the vCPM-mSTSG technique. Informed written consent was obtained from all individual participants included in this study. Recipient sites were characterized to be at high risk based on the following criteria: (1) presence of complex comorbid disease states known to impact lower-extremity wound healing, that is, diabetes mellitus, peripheral arterial disease, venous insufficiency, congestive heart failure with lower-extremity edema, obesity, or autoimmune disorder; in addition to, (2) long-term (>1 year) failure to demonstrate measureable reductions in wound size using good wound care (GWC) practices; and (3) failure to achieve delayed primary closure through surgical interventions, that is, previous failure of autologous tissue transfers. For the purposes of this evaluation, a postoperative complication was defined as total autograft failure or surgical site infection.Staphylococcus aureus [MRSA], Pseudomonas aeruginosa, Proteus mirabilis). If warranted, a prescribed course of topical antimicrobials or systemic antibiotics was administered preoperatively.Preoperative patient and wound assessment followed standard clinical practice guidelines to medically manage comorbidities and address underlying issues such as infection, pressure, edema, ischemia, and exudate. After a minimum of 2 weeks of GWC that included regular sharp debridement and local cleansing, wound swabs were taken for evaluation of wound pathogens prior to surgical intervention. Qualitative or semiquantitative swab cultures with Gram stain were considered sufficient to identify and manage pathogens of primary concern  and selective use of a weck blade, the area was recleansed, redraped, and readied for the autografting portion of the procedure . At thisOnce the mSTSG had been secured to the recipient wound bed using skin staples, the vCPM was carefully detached from the plastic applicator  and appl2). Areas with good mSTSG adherence to the recipient wound bed with or without interstitial granulation were considered viable. The areas of necrotic, nonadhered, and lysed portions of grafted skin were considered as graft-loss. These areas were measured, and the percentage of graft loss was calculated relative to the total surface area of the wound.NPWT was discontinued at the first postoperative visit on days 7 to 10. Primary nonadherent dressings and surgical staples were also removed for surgical site assessment. During the second postoperative visit on days 10 to 14, the take-rate of mSTSG was quantified in percentages based on the total surface area of wound , followed by a lightly compressive 3-inch self-adherent wrap . Patients were instructed to continue elevation of the affected limb and to limit fully weight-bearing ambulation. This cleansing and dressing change protocol was repeated on a weekly basis until complete epithelialization of meshed interstices and graft maturation was achieved.2. The average graft take-rate by postoperative days 10 to 14 was 92.5%, with complete epithelialization of all skin graft interstices observed between days 10 and 21 (a and 5b). Wounds were free of infection, edema, or maceration throughout postoperative follow-up. All successfully grafted sites matured, and wounds remained closed at the 3-, 6-, 9-, and 12-month follow-up visits. There were no postoperative complications attributed to the use of vCPM in conjunction with standard surgical protocols for mSTSG noted amongst study subjects.Patient demographics and clinical outcomes using the mSTSG-vCPM overlay technique are summarized in 0 and 21 a and 5ba and 5b). These wounds required either continued periods of prolonged local GWC or repeat surgical autografting procedures—a challenging pursuit in patients with limited and poor tissue quality at remaining donor sites.Prior to the adjunct of vCPM, all wounds—including those considered to be at high risk—were managed with the perioperative protocol using mSTSG alone in conjunction with 7 to 14 days of NPWT. Even with the initial NPWT, transferred skin was notably necrotic with poor adherence, contracted edges, or lysed portions, which led to partial or complete sloughing off of the graft and near total mSTSG failure during the immediate postoperative time frame a and 5b2 following excisional debridement of the subcutaneous tissue and muscle (a). Following fixation of the mSTSG, vCPM, NPWT, and a posterior splint, the patient was discharged home and evaluated at the prescribed postoperative intervals. At the 3-month follow-up visit, wound closure remained intact (b).A 79-year old man .21,The use of fresh amnion as a protective meshed-skin autograft dressing has been previously described. In 1984, Lin et al21Pseudomonas aeruginosa or Staphylococcus aureus serves as a predictive factor for skin graft failure and is associated with increasing reoperation rates by 4.2 times.23,Pseudomonas aeruginosa and Staphylococcus aureus (P < .001).Our 1-year evaluation period of the vCPM-mSTSG technique demonstrated positive outcomes. During postoperative evaluation, mSTSG tissues covered with a vCPM overlay did not lyse or dissolve and sites remained free of infection and maceration. Autologous graft loss due to infection is one of the primary causes for postoperative “melting” and destruction of the tissues transplanted into chronic wounds, even when the positive cultures occurred up to 12 weeks before surgery.In our study, high-risk patients experienced improved graft take-rates at postoperative days 7 to 10, complete epithelialization of graft interstices by day 21, and no wound reoccurrence during the first year of follow-up. Although vCPM may increase supply-related expenditures for the surgery, the authors considered the cost justifiable in patients at a high risk for skin graft loss, as repeat surgical interventions were not required to achieve definitive closure in this group. Our initial clinical experience has warranted the ongoing use of vCPM in the high-risk patient population, as it reduces long-term costs associated with repeated surgical interventions or prolonged local wound care secondary to skin graft loss or failure. The retrospective nature, lack of control arm, small sample size, and short-term presence of NPWT—a variable known to aid with graft take—are limitations of the study. The authors take caution while interpreting these results, and it is recognized that further studies are required to support the initial findings.


The full-digital protocol (SCAN-PLAN-MAKE-DONE®) consisted of 8 phases: (1) intraoral scan of the implant position with scanbody; (2) computer-assisted design (CAD) of the individual abutment  and temporary crown; (3) milling of the individual zirconia abutment and of the temporary polymethyl-methacrylate (PMMA) crown, with extraoral cementation of the zirconia abutment on the relative titanium bonding base, to generate an individual hybrid abutment; (4) clinical application of the individual hybrid abutment and cementation of the temporary PMMA crown; (5) two months later, intraoral scan of the individual hybrid abutment in position; (6) CAD of the final crown with margin line design on the previously saved “supplementary abutment design”, superimposed on the second scan of the abutment in position; (7) milling of the final crown in monolithic translucent zirconia, sintering, and characterization; and (8) clinical application of the final crown. All patients were followed for a period of 1 year. The primary outcomes of this study were the marginal adaptation of the final crown , the quality of occlusal and interproximal contact points at delivery, and the aesthetic integration; the secondary outcomes were the 1-year survival and success of the implant-supported restoration. An implant-supported restoration was considered successful in the absence of any biological or prosthetic complication, during the entire follow-up period. Results: In total, 25 patients  who had been restored with 40 implant-supported monolithic translucent zirconia crowns were included in this study. At delivery, the marginal adaptation was perfect for all crowns. However, there were occlusal issues (2/40 crowns: 5%), interproximal issues (1/40 crowns: 2.5%), and aesthetic issues (1/40 crowns: 2.5%). The overall incidence of issues at delivery was therefore 10% (4/40 crowns). At 1 year, one implant failed; thus the survival of the restorations was 97.5% (39/40 crowns in function). Among the surviving implant-supported restorations, three experienced complications . The success of restorations amounted to 92.4%. Conclusions: The restoration of single Morse-taper connection implants with monolithic translucent zirconia crowns cemented on customized hybrid abutments via the novel SCAN-PLAN-MAKE-DONE® full-digital protocol seems to represent a reliable treatment option. However, further studies on a larger number of patients and dealing with different prosthetic restorations  are needed to confirm the validity of this protocol.Purpose: To present a novel, full-digital protocol for the design and fabrication of implant-supported monolithic translucent zirconia crowns cemented on customized hybrid abutments. Methods: The present retrospective clinical study was based on data from patients who had been treated with single Morse-taper connection implants (Exacone The intraoral scanner (IOS) represents an important innovation in the world of dentistry, able to transform conventional prosthetic ,2, surgiIn fact, IOS allows one to capture impressions of the dental arches of patients without having to use conventional impression materials  with relative trays, but using only a light source  that is projected onto the teeth to be scanned ,2,5. TheThe advantages of using IOS are numerous: the superior comfort of the patient  ,6,7,8,9;To date, as reported in the literature, IOS can be successfully used in the prosthetic field for the capture of accurate impressions for the modeling and manufacturing of single crowns ,14,15 orTheoretically, in implant prosthodontics, IOS should find its ideal application. In fact, with dental implants there is no need to capture subgingival margins with light: the impression must exclusively detect the spatial position of the fixtures, through the acquisition of the scanbodies  . In factThanks to the optical impression, it is possible to model and then mill the components necessary for prosthetic restoration on implants, whether screwed or cemented ,23. In pThis workflow, dedicated exclusively to cemented prosthesis, is particularly elegant, and allows managing and individualizing different clinical situations, maximizing aesthetics through the customization of the abutment ,23,24,25Despite these undoubted advantages, within this workflow there are two critical moments that can cause errors, and therefore clinical problems. The first moment is that of the replacement of the mesh of the scanbody with the corresponding library file. If, in fact, the coupling managed through the CAD software is not perfect, and presents for example a small  rotation, a position error can arise, so there will not be an exact correspondence between the position of the hybrid abutment in the virtual CAD scene and the actual position of the assembly in the mouth . A seconUp to now, these two problems have represented for many colleagues a limit to the use of hybrid customized abutments in the cemented digital prosthesis. In fact, a second scan of the actual position of the zirconia abutments in the mouth, at the end of the provisional period, is not viable; the margins of the preparation of the abutments are not visible, since they are subgingival after tissue maturation. As a consequence, it can be impossible for the technician to precisely draw the margin line of the final restoration ,22.The most experienced dental technicians tried to solve this problem by scanning the physical piece/assembly of the individual hybrid abutment outside the mouth, with a desktop scanner, before sending it to the dentist for application. The .STL file of that scan can be saved, and used at the time of transition from a temporary to the final restoration, after receiving a second scan from the dentist (that of the zirconia abutments in the patient’s mouth). In fact, the dental technician can import the .STL file of the individualized hybrid abutment (with visible margins) in the final CAD scene derived from the second patient’s scan (where margins are not visible), replace it and then design the final restoration.Although this approach can solve problems; however, it forces the technician to model on a mesh, derived by an extraoral scan ; this can lead to errors in the identification of the margins of the restoration and to potential misfits between the final prosthesis and the customized hybrid abutment. In addition, it requires an additional step, i.e., scanning the hybrid abutment outside the mouth with a desktop scanner.®) for the design and fabrication of fixed implant-supported monolithic translucent zirconia crowns cemented on customized hybrid abutments, without any additional desktop scan, but only with a second intraoral scan of the actual position of the abutments in the mouth.The aim of this article is to present a novel, patented, full-digital protocol (SCAN-PLAN-MAKE-DONE®) devised and registered by our research group, for the prosthetic rehabilitation of patients enrolled in this study, consisted of the following clinical and laboratory phases, as summarized in SCAN1: intraoral scan of the implant position with scanbody;PLAN 1: design of the individual abutment from library files (bonding bases) and design of the temporary PMMA restoration; the files of the individual abutment  thus modeled are saved (as .STL files) in a specific folder, labeled as “supplementary abutment design”, ready to be recalled in the following phases:MAKE1: milling of the individual zirconia abutment and of the temporary PMMA crown, and subsequent extraoral cementation of the individual zirconia abutment on the relative titanium bonding base, to generate an individual hybrid abutment;DONE1: clinical application of the individual hybrid abutment and cementation of the PMMA temporary crown above it;SCAN2: intraoral scan of the individual hybrid abutment in the mouth, in position, after removing the temporary crown;PLAN2: in the definitive CAD scene, the mesh of the abutment in the correct position in the mouth is replaced by the .STL file of the individual abutment  that was previously stored in a specific folder; the dental technician proceeds to model the final crown;MAKE2: milling of the final crown in monolithic translucent zirconia, sintering and characterization;DONE2: clinical application of the final crown in monolithic translucent zirconia.The innovative full-digital protocol  in the posterior areas (premolars and molars) of both arches (maxilla and/or mandible) in the period between June 2016 and June 2017, and which had been prosthetically rehabilitated in the same dental center through a full-digital protocol, without the use of physical models. This full-digital protocol consisted of the design and manufacture of monolithic translucent zirconia crowns, cemented on customized hybrid abutments. To be included in this retrospective study, the digital patient records/folders had to present all the clinical and radiological data necessary for enrollment: the intraoral scans captured for the design and manufacture of the individual hybrid abutment and for temporary and final restorations; the temporary and definitive CAD scenes; and the documentation of all the steps of prosthetic rehabilitation. Finally, to be enrolled, patients had to read and sign a document of adhesion to this retrospective study, on the nature of which they were informed in detail. On the contrary, all the patients who were rehabilitated through analogical prosthetic procedures  were excluded from the present study, thus envisaging the development of plaster models, as well as all patients rehabilitated through mixed analog-digital protocols, and all the patients in which 3D-printed models were used. Finally, all patients restored through a full-digital protocol who nonetheless did not give consent for the inclusion in the present study were excluded. The present study was carried out in full compliance with the principles set out in the Helsinki Declaration on human experimentation of 1975 (and revision of 2008) and obtained the approval of the Local Ethics Committee at Sechenov University, Moscow, Russia.The present retrospective clinical study was based solely on data from patients who had previously undergone implant therapy with the insertion of one Morse taper connection implant . The impression was captured by selecting, in the acquisition software, the module for capturing dental implants. The impression consisted of the capture of the antagonist arch, of the master model just after the healing abutment removal, and of the occlusal bite/registration; then continued with the placement of the scanbody, the capture of it, the check of the quality of the file, and the subsequent saving , the mesh of the implant scanbody was replaced with the corresponding library file, with the selection of the different bonding bases (bases in titanium) for modeling. In particular, the library of implants used in this study  envisaged the choice between a straight titanium base 4 mm in height  or, alternatively, a 6-mm-high titanium base  available in either a straight or an angled version (with angle of 15°). For maximum prosthetic versatility, in the case of the need to correct implant angles, the angled abutments library provided the possibility of rotating horizontally the titanium bases 30° in 30° (12 positions instead of 6). Having chosen the bonding bases on which to design, the individual zirconia abutment was modeled, with the prosthetic superstructure of the PMMA provisional crown , sintered , and cemented extraorally on the selected titanium base, according to the CAD project. The provisional crown was instead milled in PMMA using a 4-axis milling machine . Upon delivery of the provisional restoration, the individual hybrid abutment was activated with a percussion hammer and the PMMA crown was cemented using a zinc-oxide eugenol cement . After a careful check of the perfect occlusal adaptation and congruency of the contact points, polishing and characterization were done. Articulating papers  were used to check the occlusion, and gingival floss was used to verify the quality of contact points. The provisional restoration remained in situ for a period of 2 months . This time, however, the impression was made after removing the temporary restoration, in order to capture the actual position of the individual zirconia abutment in the mouth . In the CAD software, the clinician replaced the mesh of the abutment with the previously saved .STL file of the individual hybrid abutment design . This was done in order to allow a correct visualization of the margins of the abutment, otherwise not visible because subgingival, and to allow the operator to design the final restoration on an original CAD drawing (with perfect margins) instead of on a mesh derived from an intraoral scan. In short, the “supplementary abutment design” .STL file saved from the previous CAD scene was imported into the new CAD scene as an “additional scan.” This mode allowed us to replace the scan mesh with the original CAD drawing, with perfectly designed and visible margins. In other words, the original CAD drawing of the individual hybrid abutment was superimposed on the mesh of the abutment itself, and replaced it  that revealed little deviations between the two surfaces , subsequently sintered in an oven , characterized, and ready for cementation. On delivery, the clinician carefully checked the marginal adaptation with magnifying glasses  and a periodontal probe. The goodness of the contact points with adjacent teeth (if present) was carefully checked with dental flosses and the absence of occlusal precontacts was controlled with the aid of articulating papers . The clinician reported in the patients’ electronic records/folders all possible problems and issues encountered at the delivery of the final crowns. Finally, cementation was carried out with the same zinc-oxide eugenol cement  used to cement the temporary restorations  were evaluated at the delivery of the final restorations, whereas the last two (secondary outcomes) were evaluated 1 year after the delivery of the final crowns. Accordingly, the first three (primary) outcomes of the present study were: (1) the marginal adaptation of the final crown, which was checked clinically with the aid of magnifying glasses and radiographically with an endoral periapical radiograph; (2) the quality of occlusal and interproximal contact points; and (3) the aesthetic integration of the final monolithic translucent zirconia crown. Moreover, all patients were included in a recall control program that included at least two professional oral hygiene sessions per year. During these sessions, the clinician could check the status of the restoration and possibly note in the electronic medical record/folder the presence of any problem or complication occurring during the follow-up period. The clinician had therefore the possibility to evaluate the last two (secondary) outcomes of this study: (4) survival and (5) success of the implant-supported restoration.®, Zeiss, Oberkochen, Germany) and phisically probing the margin area with a periodontal probe. This procedure was performed circumferentially all around the crown in order to intercept any possible misfit, gaps, or undercuts. Finally, a radiograph of the implant-supported restoration was taken to radiographically check the perfect seating and marginal adaptation of the final restoration. If the marginal adaptation was satisfactory, the clinician could proceed to cement the final crown; conversely, if the marginal adaptation was unsatisfactory, the crown was not cemented and the work was sent back to the dental technician for re-evaluation. In this case, anyway, the work had to be completely remade.The marginal adaptation of the final restoration was carefully checked by the clinician at the delivery of the monolithic translucent zirconia crown. The marginal adaptation was checked clinically using magnifying glasses , was particularly accurate. In the case of small boosters, the crown was polished directly in the dental clinic and so could be applied. In that case, however, the precontacts were excessive and the shape of the occlusal surface was not satisfactory, the crown was not cemented, and the work was sent back to the dental technician for re-evaluation (or remaking). Similar considerations were valid for the contact points with the adjacent teeth, which, if present, necessarily had to be satisfactory. The control of the contact points with the adjacent teeth was done using dental flosses. If the contact points were not satisfactory, in order to avoid stagnation of food and hygienic problems the crown was not cemented and was sent back to the laboratory for remaking.The occlusion check, using articulating papers  and the location/position of the crowns. Absolute distributions were calculated for qualitative variables  while means, standard deviations, medians, and 95% confidence interval (CI) were estimated for quantitative variables (patient’s age at start of the prosthetic treatment). The survival and success of the zirconia crowns and the incidence of complications were calculated at the restoration level.®, 10 Multitech® straight, and 12 Multitech® angled 15°), which were retained through a Morse taper connection inside fixtures of different diameters  and different lengths .In total, 25 patients  who had been restored with 40 implant-supported monolithic translucent zirconia crowns  were included in this study. Among these patients, only 6 were smokers. The restorations present in this study were supported by zirconia abutments cemented on different titanium bases  the marginal adaptation was optimal as clinically (inspection with magnifying glasses and probing) and radiographically  verified. Occlusal adaptation, on the other hand, presented some problems. In fact, in 5 cases  the occlusion of the definitive restorations was not perfect and had to be retouched by polishing the cusps before cementation; in two other cases (2/40: 5%) the crowns had to be sent back to the dental technician, remodeled, and milled again, as the occlusal precontacts were such that it was not possible to polish and apply them. Interproximal adaptation was instead optimal in all cases, except for a crown (1/40: 2.5%) which had rather weak and loose contact points and was therefore sent back to the dental technician for remodeling and milling. Finally, from an aesthetic point of view, the integration of monolithic translucent zirconia restorations was quite satisfactory, with only two cases of non-perfect chromatic integration; in one of these (1/40: 2.5%); however, the crown was sent back to the dental technician for further characterization. In the other case, in fact, the patient was still satisfied with the color and asked for the final restoration to be cemented without further chromatic changes, or delay. In summary, therefore, only four monolithic translucent zirconia crowns (4/40: 10%) were sent back to the dental technician ; 3 of these had to be completely redone (therefore redesigned and milled) and 1 was differently characterized. In any case, the dental technician corrected the errors and the imperfections and the new restorations, reviewed and corrected, could be subsequently cemented. The problems encountered at the delivery of the final crowns are summarized in ®); this required a new extraoral cementation of the individual abutment on the titanium base. This procedure was performed after careful cleaning and sandblasting of the base (to increase the adhesion surface). Finally, one zirconia crown decemented from the hybrid abutment. In this case, recementing it sufficed. Globally, these three complications did not involve particularly long or difficult interventions by the clinician, and could be resolved quickly. In any case, the success rate of implant-supported restorations was 92.4% (36/39 restorations were in function and did not present complications throughout the study period). The failures and complications found during the follow-up period and at the end of the present study—subdivided according to patient characteristics, type of bonding base, and type of implant—are reported in During the follow-up period there were no dropouts, because all patients regularly presented themselves at the two annual check-ups for the professional oral hygiene session. At the end of 1 year, only one crown (in the maxillary molar area) was lost (because the patient lost the implant). This implant failure, which occurred at 2 months from the positioning of the final crown, occurred in the absence of infection; the patient was a smoker and the implant supporting the restoration was extra-short (6.5 mm). No other failures occurred; hence, the survival of the restorations at 1 year was 97.5% (39/40 crowns in operation). Similarly, in the group of patients selected for this study, there were no biological complications . However, complications of a prosthetic nature were more frequent, affecting 3 crowns (3/39 functioning: 7.6%). In a single patient there was a loss of connection between the hybrid abutment and the Implant. This abutment was repositioned and reactivated through axial percussion and did not present any more problems at the end of the study. In another patient, the upper portion of the hybrid abutment (part in zirconia) decemented from the bonding base  ,23,36, tIf, for instance, the transfer of the implant position from actual to virtual  is positioned correctly, the error that occurs during acquisition with intraoral scanner is rather limited in the case of single crowns, as shown in several scientific articles ,37,38,39In summary, when working with individual hybrid abutments, the sum of all these errors can result in a discrepancy between the planned CAD position of the abutment and the actual position of the abutment in the mouth, with potential problems in the transition from the provisional to the final restoration. This may be true for any implant system.A possible strategy to overcome these errors has been found by the most experienced dental technicians, who scan the hybrid abutment outside the mouth with a powerful desktop scanner, immediately after extraoral cementation and before sending it to the dentist for application; then they save this mesh in a specific folder and retrieve it when designing the final restoration. In fact, they ask the clinician to perform a second scan of the abutments in the mouth, after the provisional period has ended. Although in this second scan the prosthetic margins of the abutments are not visible because subgingival, advanced CAD tools allow the dental technicians to accurately replace the same abutments with the meshes previously captured via desktop scanner (in which the prosthetic margins are visible). At this point the dental technician can model, in a second CAD, the definitive restorations, starting from the correct position of the abutments and with clearly visible margins .Although this technique is deserving, and allows one to solve the problems mentioned above, it itself has some critical issues: it forces the technician to perform additional desktop scans (one per each customized hybrid abutment) and, above all, forces the technician to model on a mesh and not on an implant library file (as it should be). This is certainly the main problem, since a mesh is always a geometric approximation of reality, with inherent geometric-dimensional limits . The fac®) for rehabilitation on implants with monolithic translucent zirconia crowns, cemented on customized hybrid abutments, which overcomes this problem and which moreover is perfectly suited to the implant system used here. The implant system used in this study  is in fact characterized by a locking-taper screwless implant-abutment connection [® 4 mm, and Multitech® 6 mm straight and 15* angled) have an apical index (an hexagon that is useful only for repositioning the abutment) [®, this hexagon is removable, due to the fact that the implant library has 12 positions (and not 6). This gives excellent versatility in CAD, but it can lead to difficulties in positioning that can be associated with possible errors—in scanning, in overlap in CAD, and, not least, in precision—during extraoral cementation of zirconia over titanium. It should also be noted that, in Morse taper screwless implants, the abutment is usually not removable once placed in the mouth (according to the one abutment—one time concept) [The best possible approach is certainly to model the final restoration on library files, in order to have a clear, easy-to-read margin line, to facilitate the dental technician in the CAD design, the machines that produce in the CAM phase, and ultimately to improve the marginal closure of the restorations on the abutments. This is why we have studied and present here a new full-digital protocol  . Howeverconcept) . This isconcept) ; yet it ®. At this point the library file, with clear and visible margins, is superimposed on the scanning mesh, and replaces it. A model is therefore obtained in which it is possible to clearly visualize subgingival margins, and the technician can easily model the final restoration on a library file. The whole procedure is based on the powerful superimposition algorithm of the Exocad® software, mathematically verifiable with powerful, additional reverse engineering software.Our approach basically consists in saving as a .STL file the modeling of the hybrid abutment made in the first CAD, and in re-importing this file (a library file with the preparation margins of the abutment clear and visible) in the second CAD, derived from the second intraoral scan , so as to replace a scanning mesh (but in the correct position) with a library file. This can be done simply through CAD operators such as the import of “additional scan” into ExocadThe present retrospective study  included 25 patients  who had been restored with 40 implant-supported monolithic translucent zirconia crowns .At delivery, the marginal adaptation was perfect for all zirconia crowns (40/40: 100%). This seems to validate the present CAD superimposition protocol. However, there were occlusal issues (2/40 crowns: 5%), interproximal issues (1/40 crowns: 2.5%), and aesthetic issues (1/40 crowns: 2.5%). The occlusal and interproximal issues may be mainly related to mistakes during the CAD process of the final crowns, rather than being determined by dimensional variations of zirconia during sintering. Since these issues represent major problems—problems that force the clinician to send the restoration back to the dental technician for re-making, with (high) costs—appropriate solutions should be found and the possibility of testing the occlusal and interproximal adaptation of the definitive design via a try-in crown milled in low-cost material (polyurethane) should be considered. This polyurethane try-in is tested directly in the mouth and can also be useful for verifying marginal adaptation. Once the marginal adaptation, occlusion, and contact points have been validated, this polyurethane try-in crown is eliminated and the corresponding design is used to mill the final zirconia restoration. In this study, moreover, one monolithic zirconia crown failed to aesthetically integrate in the mouth of patient. With monolithic, non-stratified crown milled from zirconia discs and then painted/characterized manually, aesthetic integration is still a issue; it must be pointed out that translucent zirconia is aesthetically better than a non-translucent one; and in any case this issue will probably be solved with the advent of ceramic 3D-printing technology. Overall, therefore, the incidence of issues at delivery was 10% (4/40 crowns). This can be considered rather high, and efforts should be made to reduce it. The failures and complications encountered in the follow-up period were, however, rather low. At 1 year, one implant failed; therefore, the survival of the restorations was 97.5% (39/40 crowns in operation). Among the surviving implant-supported restorations, three experienced complications . These complications were considered minor in nature because they did not require long interventions by the clinician. In accord with the definition of success established for this study , the rate of complications encountered in this study was rather low (7.6%) and the success rate of restorations was 92.4%. These results are in accordance with the previous literature, which reports excellent survival and success rates for monolithic implant-supported zirconia crowns in the short term ,48.Naturally, the present study has its limits. First is the limited number of patients enrolled, besides the fact that they were restored with single crowns and not with more complex restorations like partial fixed prostheses. To be able to draw more definitive conclusions on the validity of this method, studies on a larger number of patients are necessary, and with short- and long-span fixed partial prostheses. For more complex applications, it might perhaps be appropriate for the abutments in the first CAD phase to be drawn with geometric shapes impressed, so as to facilitate superimposition during the second CAD phase. Finally, the present study is retrospective in nature and it is well known that only by prospective clinical trials and randomized clinical trials we can draw more definitive conclusions about the validity of new protocols.® full-digital protocol seems to represent a reliable treatment option, with an excellent marginal adaptation and a rather low incidence of failures  and complications  one year after delivery. Still, some issues linger, regarding occlusal, interproximal, and aesthetic integration of the monolithic restorations at delivery. Further studies on a larger number of patients and dealing with different prosthetic restorations  are needed to confirm the validity of this protocol.The restoration of single Morse-taper connection implants with monolithic translucent zirconia crowns cemented on customized hybrid abutments via the novel SCAN-PLAN-MAKE-DONE


The details of the histological analysis of the explants with different initial stiffness and porous structures at various time points are presented. The images and data presented support the conclusion about the coupled effects of stiffness softening and the hierarchical porous structure modulating tissue ingrowth, vascularization and macrophage polarization in the article .This DiB article contains data related to the research article entitled “Cellular responses to thermoresponsive stiffness memory elastomer nanohybrid scaffolds by 3D-TIPS” . Thermoresponsive poly (urea-urethane) nanohybrid elastomer (PUU-POSS) scaffolds were implanted in rats for up to 3 months. The porous structure and tensile mechanical properties of the scaffolds are listed and compared before and after  Specifications tableValue of the data•in vitro and in vivo tests. The data magnify more insights about the changes of structures at multi-scales and mechanical properties of the scaffolds under biophysical and biological conditions.Data presented in this article provide direct comparison of the stiffness softening and hierarchical structure of the 3D-TIPS scaffolds before and after •in vivo.The histological images of the scaffolds with different initial stiffness and porous structure by immunohistochemistry elucidate for the first time how the stiffness softening and digitally printed hierarchical porous structure regulate tissue ingrowth, vascularization and macrophage polarization towards an M2 phenotype at the early (week 4) and late (week 12) stages 1in vitro over day 0–28 and how they relax towards their intrinsic elasticity. The dimensions of the 3D printed preforms and the scaffolds as produced are shown in in vivo implantation at various time points, in terms of tensile mechanical properties and XRD characterization respectively. 1.11.21.31.422.1A 3D-TIPS technique, based on reverse 3D printing and phase separation of the polymer solution, as described in 2.2An Instron 5655 was applied to test static tensile mechanical properties of the scaffolds, before and after incubation over 28 days at body temperature, as described in 2.3As detailed in


This makes it difficult to differentiate the epidermis from the dermis, thus increasing the risk of incorrect placement. This is associated with morbidities such as the need for repeated procedures under general anesthesia, additional donor site morbidity, prolonged dressing, increased cost, and unhappy parents.For the survival of the skin graft, the dermal side needs to be placed against the wound bedAt our center, the KK Women’s and Children’s Hospital in Singapore, we describe a simple and novel technique to identify the dermal side of the harvested graft to ensure safe placement during split-thickness skin grafting. To aid the identification of epidermis and dermis, a letter of the alphabet is drawn on the epidermis before harvesting. The markings are made using a sterile skin marker or methylene blue dye that will fade postoperatively. As shown in the picture, the letter “p” is marked out on the epidermis. The skin graft is then harvested using a powered dermatome with an adjustable guard set to a variable thickness in accordance with the age of the child and the donor site selected. On the freed graft, the side with the letter “p” suggests the epidermal side Figure , whereas5 The skin graft (meshed or unmeshed) is then laid out on a piece of tulle gras. Placement on the tulle gras may cause the skin graft to be shiny on both sides, along with the paraffin oil used for harvesting the skin graft, increasing the difficulty of differentiating between dermal or epidermal side. With the epidermis side in direct contact with the tulle gras, the reversed alphabet will show up clearly through the dermis, which is facing the operator, and is ready for a safe application.When indicated, meshing or fenestration is then performed on the skin graft without changing its orientation.The advantages of RAG are:Safe—minimizes the risk of misplacement of graft.Simple—easily reproducible for multiple strips of skin graft or for technically challenging cases, especially in younger children. This uncomplicated step can be carried out by all operators regardless of the level of experience. This can also reduce confusion in a surgical team with multiple operators.Decreases operating time by decreasing time taken to determine the correct side of skin graft.In summary, with good preoperative optimization and postoperative care, RAG is a novel adjunct that involves the use of alphabets to facilitate accurate on-table judgment of skin graft surface orientation, a crucial step in the success of skin grafting.


Biological networks are complex (non-linear), redundant (cyclic) and compartmentalized at the subcellular level. Rational manipulation of plant metabolism may have failed due to inherent difficulties of a comprehensive understanding of regulatory loops. We first need to identify key factors controlling the regulatory loops of primary metabolism. The paradigms of plant networks are revised in order to highlight the differences between metabolic and transcriptional networks. Comparison between animal and plant transcription factors (TFs) reveal some important differences. Plant transcriptional networks function at a lower hierarchy compared to animal regulatory networks. Plant genomes contain more TFs than animal genomes, but plant proteins are smaller and have less domains as animal proteins which are often multifunctional. We briefly summarize mutant analysis and co-expression results pinpointing some TFs regulating starch enzymes in plants. Detailed information is provided about biochemical reactions, TFs and cis regulatory motifs involved in sucrose-starch metabolism, in both source and sink tissues. Examples about coordinated responses to hormones and environmental cues in different tissues and species are listed. Further advancements require combined data from single-cell transcriptomic and metabolomic approaches. Cell fractionation and subcellular inspection may provide valuable insights. We propose that shuffling of promoter elements might be a promising strategy to improve in the near future starch content, crop yield or food quality. Plant cells are autotrophic organisms fully exposed to many environmental signals. While plants must cope with a wide range of conditions , animals enjoy more stable environments since they are able to escape from danger and to migrate searching for food. Plants are totipotent while animal cells are non-totipotent due to regulatory restrictions by cytosolic and nuclear factors. Photosynthesis in plants leads to sucrose and starch providing food for heterotrophic organisms. This review summarizes what we know about transcriptional regulation of starch metabolism in flowering plants. Most genes of starch synthesis and degradation have been widely studied due to their importance for plant physiology and growth . The expThe review started with an electronic literature survey that was expanded iteratively. Scientific articles were searched in PubMed, ISI Web of Science, Google Scholar and other databases such as EndNote and Mendeley. The first search terms included following key words: starch metabolism, TFs, regulation and plants. The abbreviated names of genes and the enzyme commission (EC) numbers of key reactions of starch metabolism were also included in the literature survey. The search also included the names of the first and senior authors of publications in high impact journals during the last 20 years about starch metabolism. The pathway of sucrose to starch conversion has been intensively investigated mainly in Arabidopsis and in potato  (ADP-glucose pyrophosphorylase is the first committed step in the starch synthesis pathway . The plaagpL1-3) –3.Some starch biosynthetic enzymes assemble in high molecular weight complexes . One conSeveral starch synthase isoforms use ADPglc to add its glucose moiety to amylose and amylopectin molecules in the ordered and crystalline structure of the starch granule . DiffereStarch synthases are divided into four subfamilies of soluble SSs  and one subfamily of granule-bound starch synthases (GBSS) . Starch Starch synthesis in leaves has been said to be largely similar to that in storage organs . Table 2Metabolic and transcriptional regulation are commonly thought to be equivalent in both plant and animal systems. According to Tom Ap Rees and Mark Stitt, central metabolism of pea is like the subway map of London , 1980. CThere are some important differences between metabolic and transcriptional networks that must be taken into account when trying to explore them by correlation analysis. Plant cells may produce a larger number of chemically distinct metabolites  than the number of enzymes encoded by their DNA . In metabolic networks, connections  are theoretically reversible, bidirectional and may have certain stoichiometry . MetabolAccording to the classifications of gene ontology (GO) ∼4–8% of the genes are involved in DNA transcription and regulation, whereas 10–20% of the genes are involved in metabolism . In planAcross species there is a negative correlation between protein size and protein number in eukaryotic genomes . Plant pDrosophila and 1.7 times that of Caenorhabditis elegans and Saccharomyces   and 2. GBAM5 is regulated by two TFs, WRKY DNA-binding domain 75  and NAC domain-containing protein 96   promoter and ZmDOF2 blocks this transactivation and represses PEPC expression  Fig. 2)Fig. 2). 5g46590)  granules . In riceisoforms  Microbial volatiles promote the accumulation of starch in leaves via a photoreceptor-mediated control . The traTranscripts of many starch genes are regulated by both an endogenous clock and by the diurnal cycle   and alsoAlso, some SS isoforms are affected by photoperiods . Even thThe duration of the photoperiod has two major consequences for plant growth and metabolism. Firstly, a longer night requires alterations in the timing of growth and the diurnal allocation of carbon , 2014. SThe coordinated regulation of gene expression in sink and source sink tissues is orchestrated by light, sugars and energy status . In addiAGPS1a, AGPL1, SUT1, SuSy2, GBSSI, SSI, SBEI, PUL1 and ISA1genes AGPS1a, A and ABA . An ethy and ABA . OverallISA1 promoter and exhibited a similar expression pattern as ISA1 ISA1 prom as ISA1  o pGBSSI . OsSERF1nd GBSSI  Ibbfruct2)  . In cass ataf1 knotrans regulation for adapting isogene expression to increase fitness under a changing environment (AtSUS2 expression such as the W-box (ttgact) and SEF3 (aaccca) motifs ; mutation of this cis-element induced APS1 expression in roots, indicating that this cis-element could mediate transcriptional repression  motifs . An bZIPand SBE1 . A cis-apression . A shiftpression . Some bZpression . In riced OsISA2  Fig. 3)trans reg1 and Wx  and pSSI .2 inside photosynthetic leaves, or whenever enough oxygen (energy), sucrose and hormones are supplied to storage organs. However, microscopy reveals that not all cells make starch, thus we wonder why some differentiated cells are full of it while others completely lack it. With the advantage of new transcriptomic technologies, it will be possible to build regulatory networks that can help to elucidate the TFs behind the expression patterns of starch metabolic genes. But we must solve the old problem as when studying metabolism, that whole organs and cell mixtures are homogenized and analyzed in bulk. Subcellular analysis of metabolism is needed to pinpoint key regulation sites. For example, detailed subcellular inspection using fluorescent microscopy allowed to distinguish the metabolic source of blue glow in banana leaves, fruit skin and pulp (Identification of all TFs and cis-elements would enable a future strategy of rational metabolic design in order to turn on starch synthesis in tissues that lack starch . Increasand pulp . When epThis review highlighted the importance of distinguishing different types of biological networks, namely metabolic interconversion networks and transcriptional regulatory networks . Compari


The cross-linked rice starches were hydrolyzed by α-amylase and native, hydrolyzed, and hydrolyzed cross-linked rice starches were comparatively studied. It was found that hydrolyzed cross-linked rice starches showed a lower the degree of amylase hydrolysis compared with hydrolyzed rice starch. The higher the degree of cross-linking, the higher the capacity to resist enzyme hydrolysis. Hydrolyzed cross-linked rice starches further increased the adsorptive capacities of starches for liquids and decreased the trend of retrogradation, and it also strengthened the capacity to resist shear compared to native and hydrolyzed rice starches.Native rice starch lacks the versatility necessary to function adequately under rigorous industrial processing, so modified starches are needed to meet the functional properties required in food products. This work investigated the impact of enzymatic hydrolysis and cross-linking composite modification on the properties of rice starches. Rice starch was cross-linked with epichlorohydrin (EPI) with different concentrations (0.5%, 0.7%, 0.9%  Rice is one of the most important cereals and commercially more than two thousand varieties of rice are grown throughout the World. Native rice starches have many properties that differ from those produced from other plant sources. These inherent characteristics can be exploited to produce various types of starch-based food products, but native rice starch lacks the versatility necessary to function adequately under rigorous industrial processing. Thus, a variety of ways have been developed to modify starch to provide the desired stability and tolerance toward a wide range of processing techniques. The native starch can readily be modified by physical, chemical or enzymatic processes to afford products with improved starch functionality and consequently these products find diverse applications . Starch was treated by enzyme  at a temperature below the pasting temperature of starch. Enzymatically hydrolyzed starch is a natural susbtance which has strong adsorbent capability and retarded retrogradation. However, hydrolyzed starch showed lower capacity to resist shear compared with native starch. Cross-linked starches constitute a major class of modified starches. Cross-linking of starch by reaction with bi- or polyfunctional reagents is widely used in numerous industrial applications such as preparation of wet-rub-resistant starch paper coatings, permanent textile sizes and water-resistant adhesives . Cross-lThe degree of cross-linking of the cross-linked rice starch was determined from the RVA viscosity values and the results are presented in et al. reported that EPI cross-linking was not homogeneous and was concentrated in the non-crystalline domain of starch granules [et al. found that cross-linking of starch chains occurred mainly in amylopectin and some amylose molecules were cross-linked to amylopectin [The hydrolysis of native and cross-linked rice starches with different degrees of cross-linking is shown in granules . Jane etlopectin . The croThe adsorptive capacities of native, hydrolyzed, and hydrolyzed cross-linked rice starches for water and oil are presented in et al. proposed the concept of “centripetal” hydrolysis (surface-to-core directed) and “centrifugal” hydrolysis (involving regions and layers) of native granules by α-amylase [It is well known that α-amylase can drill and widen the pinholes in starch granules. Helbert -amylase . The stu-amylase ,12 foundet al. [The RVA viscosity characteristics of native and modified starches are shown in et al.  indicateHgel values primarily reflect the loss of double helical order rather than loss of crystalline register. The decrease in transition temperatures and ΔHgel values of the hydrolyzed starch after amylase treatment suggested that some of the double helices present in the crystalline and non-crystalline regions of the granule may have disrupted under the conditions prevailing during amylase treatment. The decrease in ΔHgel values indicated that hydrolyzed starch required less thermal energy for gelatinization compared to the native starches. Heat of gelatinization reflects the energy required for disrupting the starch granule structure and since cross-linking reinforces the structure of starch granules, more heat will be required for gelatinization.The transition temperatures and the enthalpy values of gelatinization and retrogradation of native, hydrolyzed, and hydrolyzed cross-linked rice starches are presented in The molecular interaction (hydrogen bonding between starch chains) after cooling of the gelatinized starch paste has been called retrogradation . The entet al., cross-linked starch had the most stable structure and the strong ability to resistant to shear, the structure of hydrolyzed starch was weakened and it lost the ability to be resistant to shear [Previous results indicated that rice starches were pseudoplastic and shear thinning liquids. As was shown by the flow curves in to shear . Strengtw/v) raw starch in a buffer solution  and releasing 1 mg glucose in 1 h at 40 °C. All chemicals and reagents used in this study were of analytical grade. Native unmodified rice starch was purchased from Puer Yongji Biological Technology Co. Ltd. . The contents of moisture, ash, and protein in this rice starch are 11.2%, 0.15%, and 0.50%, respectively, while the amylose content is 19.5% . EPI and α-amylase  were purchased from NongHe Co. Ltd. . Here, IU/mg refers to 1 mg enzyme solution acting on 2.5%  was added directly to the slurry with stirring at 25 °C for 3 h, then the pH was adjusted to 6.0–6.5 with 0.2 mol/L HCl and the cross-linked rice starch was isolated by centrifugation . After washing with distilled water, the sediment was dried at 45 °C for 48 h in a vacuum oven. The cross-linked rice starches produced with three levels of cross-linking were named R1, R2, and R3, respectively.Cross-linking reaction of rice starch was performed by suspending the starch  in distilled water (150 mL) with added NaCl (3 g). The starch suspension was continuously stirred at room temperature. After adjusting to pH 10.0 with 1 mol/L NaOH, EPI with different concentrations  and stirred continually for 24 h at 40 °C according to Yamada [et al. [Samples  of native starch or cross-linked starch and α-amylase (5 g) were suspended in pH 3.4 buffer solution . The viscosities of the starches were recorded with starch suspensions . They underwent a controlled heating and cooling cycle under constant shear where it was held at 50 °C for 1 min. They were then heated from 50 to 95 °C at 5 °C/min and held at 95 °C for 2.7 min, cooled from 95 °C to 50 °C at 5 °C/min and held at 50 °C for 2 min. The initial speed of blending in 10 s was for 960 rpm, after that it was maintained at 160 rpm. Pasting parameters such as pasting temperature, peak viscosity, breakdown (peak viscosity-hot paste viscosity), final viscosity, setback (cold paste viscosity-peak viscosity) were recorded.To) and peak temperature (Tp) were determined from the run heating DSC curves. Enthalpy of gelatinization (ΔHgel) was evaluated based on the area of the main endothermic peak. After the DSC run, gelatinized starch samples were stored at 4 °C for 7 day for retrogradation studies, these stored samples were scanned under the same conditions and the transition temperature and retrogradation enthalpy (ΔHret) was determined from the second run heating. The thermal characteristics of starches were measured using 61 DSC-Pyris Diamond . Starch samples were weighed into aluminum DSC pans, and deionized water was added by micropipette to achieve a water-sample ratio of 2:1. The sample pans were sealed and allowed to stabilize at room temperature for 24 h before heating. Samples were heated at a rate of 10 °C/min from 25 to 100 °C, which used an empty pan as reference. The onset temperature  was suspended in distilled water (100 mL) and heated in boiling water bath for 25 min, followed by cooling to room temperature, the starch paste was obtained. Starch paste was put into the testing platform of dynamic rheometer . Viscometry was performed using a controlled strain rheometer using parallel plate mould (40 mm diameter and 1 mm gap). After trimming off the over-loaded portion of samples around plates, the open side of samples was covered with a thin layer of silicon oil to prevent moisture loss. Shear stress with increasing shear rate (0–500/s) was obtained at 20 °C to characterize flow behavior. The applied measurement regime was as follows: rate of shear was increased from 0 to 500/s logarithmically within 600 s.The data reported in the tables were average of triplicate observations. Data obtained were analyzed by analysis of variance (ANOVA) using SPSS for windows version 13.0. Confidence interval of sample means was reported at the 95% confidence probability. Comparisons of means were made using least significant difference (LSD) and shortest significant ranges (SSR) at 5% significance level.The amylase hydrolyzed rice starch has stronger adsorptive capacity and shows a lower retrogradation tendency compared with native rice starch, whereas hydrolyzed rice starch shows the weakest ability to resist shear in comparison to native rice starch. The cross-linking retarded the caving of pores caused by the extensive penetration of enzyme and kept the integrity of pores by strengthening the structure of starch granules, which resulted in an increase in the adsorptive capacities of starches for liquids. In the meantime hydrolyzed cross-linked rice starch further decreased the tendency of retrogradation and increased the ability to resist shear compared with hydrolyzed rice starch. The higher the degree of cross-linking, the stronger the resistance to shear and retrogradation. However, the capacity to resist enzyme hydrolysis of cross-linked starches increased with the increasing degree of cross-linking. It is concluded that hydrolyzed cross-linked rice starch overcomes the undesirable properties caused by retrogradation and low shear resistance. Considering starch is a common ingredient used widely in many food and non-food applications, and native rice starch lacks the versatility necessary to function adequately under industrial processing conditions, the hydrolyzed cross-linked rice starch could more effectively meet the functional property demands in food and non-food products


The human ankle joint has an influential role in the regulation of the mechanics and energetics of gait. The human ankle can modulate its joint ‘quasi-stiffness’ (ratio of plantarflexion moment to dorsiflexion displacement) in response to various locomotor tasks . However, the direct effect of ankle stiffness on metabolic energy cost during various tasks is not fully understood. The purpose of this study was to determine how net metabolic energy cost was affected by ankle stiffness while walking under different force demands .Individuals simulated an amputation by using an immobilizer boot with a robotic ankle-foot prosthesis emulator. The prosthetic emulator was controlled to follow five ankle stiffness conditions, based on literature values of human ankle quasi-stiffness. Individuals walked with these five ankle stiffness settings, with and without carrying additional load of approximately 30% of body mass .Within the range of stiffness we tested, the highest stiffness minimized metabolic cost for both load conditions, including a ~ 3% decrease in metabolic cost for an increase in stiffness of about 0.0480 Nm/deg/kg during normal (no load) walking. Furthermore, the highest stiffness produced the least amount of prosthetic ankle-foot positive work, with a difference of ~ 0.04 J/kg from the highest to lowest stiffness condition. Ipsilateral hip positive work did not significantly change across the no load condition but was minimized at the highest stiffness for the additional load conditions. For the additional load conditions, the hip work followed a similar trend as the metabolic cost, suggesting that reducing positive hip work can lower metabolic cost.While ankle stiffness affected the metabolic cost for both load conditions, we found no significant interaction effect between stiffness and load. This may suggest that the importance of the human ankle’s ability to change stiffness during different load carrying tasks may not be driven to minimize metabolic cost. A prosthetic design that can modulate ankle stiffness when transitioning from one locomotor task to another could be valuable, but its importance likely involves factors beyond optimizing metabolic cost. The human ankle joint has an important influence on mechanics and energetics of gait. Specifically, the role of the muscles acting at the ankle joint during normal walking is to provide body support, aid in forward propulsion, and to initiate leg swing . The peaOne important feature of the human ankle joint is the regulation of ‘quasi-stiffness,’ which hereby will be referred to as stiffness. The human ankle stiffness is defined as the slope of the moment-angle relationship of the joint, or the ratio of the ankle moment to angular displacement –14. ThisNumerous studies have shown that humans can alter ankle dorsiflexion stiffness in response to changes in mechanical demands of walking –19, mostStudies involving lower-limb ankle-foot prostheses have provided valuable insights on the role of stiffness in regulating metabolic energy during walking. There have been many studies done to determine what the best prosthetic ankle or foot stiffness is for lowering metabolic cost and improving gait for individuals with amputation , 22, 23.The purpose of this study was to determine how net metabolic energy cost was affected by ankle stiffness while walking with different mechanical demands . Walking with an additional load directly increases metabolic cost . As a prParticipants simulated an amputation by wearing the prosthesis with an immobilizer boot attached. An experimental ankle-foot prosthesis emulator  was used to systematically vary resistance to ankle dorsiflexion  Fig. . A similTo control the prosthetic ankle joint stiffness, adjustments were made electronically using MATLAB/Simulink software . In order to create the desired moment-angle relationship, we entered two moment and angle value-pairs into the software to define a linear slope   under the approval of the Institutional Review Board of the University of Nebraska Medical Center. Each participant provided written consent before being screened for inclusion and exclusion criteria.Fourteen healthy young adults   volunteered to participate in the study. Healthy was defined as: free of musculoskeletal or pathological problems including cardiovascular and neurological disorders. Participants did not have any past injuries or surgeries that affected their gait; any current pain in the neck, back, or shoulders; or any current medication that may affect temporal spatial awareness, joint or muscle stiffness and cognitive function. They were able to carry 30% of their body mass as added weight. Since the weight limit of the prosthesis was 113.4 kg, all individuals were under 87.23 kg and had a body mass index under 30 kg/mThis experiment consisted of 10 conditions each visit. The 10 conditions included five different prosthetic stiffness settings and two different load carrying conditions. All 10 conditions were repeated on three different days to account for any learning effects. There were 24 h to 72 h in between each session. This study set up was done in a previous study using this device . ReporteThe five stiffness settings were 0.0928, 0.1044 0.1160, 0.1276 and 0.1392 Nm/deg/kg. Since the goal of this study was to understand the functional importance of human ankle’s ability to modulate stiffness, we selected stiffness values near the typical human ankle during normal walking, as well as during load carriage. Literature values for typical human ankle stiffness  have ranged from ~ 0.089 to ~ 0.1077 Nm/deg/kg , 20, 34., which provided further justifications for the stiffness levels used for this current study. The 30% additional body mass was symmetrically distributed around the participant’s core, in a weighted vest, with 2.5 kg weights. Since the weight was in incremental amounts, the participants wore the weight that was closest to 30% of their body weight. However, the actual weight percentage ranged from 29 to 34% of the participant’s body mass. Conditions were blocked randomized, in which the no additional load conditions were first, followed by the additional load conditions. The five stiffness conditions were randomized within each load carrying condition.The load carrying conditions were an additional 0%  and 30% of the participant’s body mass. 30% additional body mass was chosen because previous studies have shown that metabolic cost increases with added body mass in an almost linear relationship , 36, 37.Participants were asked to fast 3 h before the data collection, as to not affect the metabolic cost estimates. The visit started with obtaining consent and then taking anthropometric measurements using a caliper, including lower limb segment lengths and width. Prior to data collection, participants were asked to put on a tight-fitting spandex suit to limit movement noise from the reflective markers. A six-degree-of-freedom marker set was used to track the motion of the lower extremities and the prosthesis . This maBefore the first trial, a 7-min recording of the metabolic rate was taken as the participant stood quietly on the instrumented treadmill. For each trial, the participant walked for 6 min at a speed of 1.25 m/s. The treadmill started at 0.8 m/s and gradually increased speed up to 1.25 m/s. Once at 1.25 m/s, the 6 min started. During all conditions, the maximum torque and ankle range of motion of the prosthesis were monitored to make sure either were not reaching the hardware limit. Breath-by-breath gas exchange measurements were recorded for indirect calorimetry calculations of metabolic cost . This metabolic system has been shown to provide reliable results for gas exchange measurements . Data frStandard calculations derived by Brockway  were useKinematic and kinetic data were sampled at 250 and 1000 Hz, respectively, and filtered with a 6 and 25 Hz 2nd order low-pass Butterworth filter. This had been done previously with similar data . Six-degTo calculate ankle-foot power, we used a unified deformable (UD) power analysis. The analysis quantified everything distal to the shank as one deformable segment and captures the total power produced by the entire ankle-foot system . This tep < 0.05). The remaining significant variables were included in the predictor equation for the outcome variables. The coefficients for these variables were reported, as well as the R2 value for the equation of the condition averages. This analysis was done for each outcome variable .A linear mixed-effect model was used to determine the factors that affected the outcome variables . Net metabolic power and joint works were normalized to the subject’s biological body mass. The analysis was a linear mixed-model with six-factors . Previous studies involving exoskeletons or prostheses have seen both a linear  and quadIn addition to the primary hypotheses, we performed several additional secondary analyses. A linear mixed-effect model was used to determine the factors that affected ipsilateral knee work, contralateral ankle-foot, knee and hip work, and perception data. A paired t-test was done between the metabolic cost measurements from the second to third day for each condition.The prosthetic emulator systematically changed stiffness consistent with the software input. This was shown by a representative participant’s prosthetic ankle moment-angle relationship for the five stiffness conditions at the normal walking conditions. As the input stiffness in the software increased, the slope of the measured moment-angle curve during walking increased Fig. . The actp ≥ 0.0581) except the highest stiffness for the unloaded walking trial, with significant difference between days 2 and 3 (p = 0.0222).Additionally, we measured the stiffness calculated by the prosthesis software from the prosthesis load cell across all 3 days. , the ankle, knee, and hip had varying joint angles and moments Fig. . OverallThe total power of the ankle-foot, knee, and hip varied across the ten conditions for both the ipsilateral and contralateral side Fig. . Overall2, units: p = 0.0356) and amount of load ) (p < 0.001) were significant predictors of the net metabolic power (Ėmet) (adjusted R2 = 0.8480)  and amount of load (l) (p < 0.001) were significant predictors of the prosthetic ankle-foot positive work (WPRO_ANK_FT) (adjusted R2 = 0.7995)  was related to the interaction of stiffness and load (p < 0.001) and interaction of the square of stiffness and load (p = 0.0023) (adjusted R2 = 0.6622) , the interaction of stiffness and load (p = 0.0157) and the interaction of stiffness squared and load (p = 0.0147) were significant predictors of ipsilateral knee positive work (WIPS_KNEE) (adjusted R2 = 0.8085)  Fig. . Prosthe p = 0.017 were sip < 0.001), as well as the interaction of stiffness squared and load (p < 0.001) were significant predictors of contralateral hip positive work (WCON_HIP) (adjusted R2 = 0.8327)  Fig. . Prosthe27) Fig. 5\documeCON_KNEE) was predicted by load (p < 0.001) and stiffness squared (p = 0.0084) (adjusted R2 = 0.9061)  was predicted by the interaction of prosthetic ankle stiffness and load (p < 0.001) and the interaction of prosthetic ankle stiffness squared and load (p = 0.0326) (adjusted R2 = 0.8622)  Fig. 7\documeFor additional analysis of the net metabolic power and joint power based on the categorical input stiffness, see Additional file We used a robotic prosthetic emulator to simulate various ankle joint stiffnesses above and below a typical human ankle stiffness during different mechanical demands . This experimental approach allowed us to vary the ankle joint stiffness while keeping every other parameter  constant on the device. We tested the hypothesis that the lowest stiffness would minimize metabolic cost for normal walking (no added load), and the stiffness that minimized metabolic cost during load carriage would be greater compared to the no load conditions. However, contrary to our hypothesis, the highest stiffness, out of the range tested, minimized metabolic energy for both walking conditions. We found no significant interaction between stiffness and load on metabolic energy cost.One potential reason why our hypothesis was not supported is that we focused solely on the stiffness of the ankle joint and did not take into consideration other joints within the foot. The joints within the human foot are important and play a role in how the ankle-foot complex behaves during walking –51. A stThe range of stiffness we tested could have influenced why the metabolic energy was minimized at the highest stiffness for both conditions. The range of input stiffness tested was from 0.0928 Nm/deg/kg to 0.1392 Nm/deg/kg, which is around the typical human ankle stiffness for normal walking, as well as walking with additional load , 20, 34.From each participant’s highest stiffness to their lowest input stiffness, metabolic cost decreased by about 0.11 W/kg for the no load condition, or 3.2%, and 0.10 W/kg for the additional load conditions, or 2.57%. This difference would equate to the metabolic cost of transport of 0.01 J/Nm, which is less than the reported minimal detectable difference (0.022 J/Nm) using a portable metabolic system . The stup = 0.246), suggesting that subjects did not perceive later conditions as more difficult than others.We found that the participants’ perception of the difficulty of the condition was predicted by the interaction of load and stiffness  and ipsilateral hip positive work (p < 0.001) were significant predictors of metabolic cost (adjusted R2 = 0.7765). The equation was: At the ipsilateral hip joint, our original hypothesis was that the lowest stiffness would minimize hip positive work, and a higher stiffness would minimize hip work when carrying additional loads. Our statistical model found that hip positive work did not significantly change between stiffness conditions for the no load condition. We found an interaction of both stiffness and load, as well as stiffness squared and load, which indicates that ipsilateral hip positive work varies as a function of both variables. Therefore, our hypothesis was partially supported, since the highest stiffness minimized hip positive work at the 30% load condition. This is important since many individuals with amputation experience an increase in metabolic cost, which can at least partly be attributed to their increase in hip positive work –11. UponWe also did a few secondary analyses to analyze the other joints to see if their behavior was influencing the net metabolic power outcomes. For the additional load conditions, the contralateral ankle-foot had more positive work in the higher stiffness conditions compared to the lower stiffness Fig. , suggestp = 0.0448). However, all other conditions were not significantly different (p ≥ 0.14) . This may suggest that the trends between metabolic cost were similar between the second and third day and no additional changes in metabolic cost happened due to learning.This study had a few limitations. The prescribed prosthetic ankle stiffness was determined by approximating a single linear slope during the dorsiflexion phase based on the moment-angle relationship of human ankles . A studyAnother limitation is the generalizability of these findings. The participant recruitment was limited in that there were two sizes of lift shoes used, and there was a specific weight limit for the prosthesis. Because of these criteria, we had a restricted range of participants based on shoe size and body mass. While this increases the internal validity of the study, the findings of our study are less generalizable. In addition, using a simulator boot with the prosthesis makes these results less generalizable to individuals with amputation. There have been a variety of studies that have used emulator or simulator boots , 33, 52.The purpose of this study was to understand the importance of changing ankle stiffness during different locomotor tasks. While human ankle’s ability to modulate joint stiffness is well documented , 18, 20,Our study revealed that a stiffness higher than the typical human ankle can decrease metabolic energy at both normal walking and walking with additional loads. Additionally, we found no significant interaction between stiffness and load carriage on metabolic cost, which may suggest the modulating ankle stiffness comparable to levels of how the human ankle modulates stiffness is not likely to have a metabolic benefit. Future studies looking at a wider stiffness range or using human-in-the-loop optimization can be done to further solidify if there is an ‘optimal’ stiffness that can be determined when individuals walk under various mechanical demands.Additional file 1: Figure S1. Moment-Angle Relationship from the HuMoTech prosthesis emulator MATLAB/Simulink code. Two moment and angle values-pairs were entered into the software to define a linear slope and created the desired moment-angle relationship (indicated by the circles outlined in black). The first pair of points were always at a plantarflexion moment of 0 Nm and at 0 degrees dorsiflexion. The second pair of points were a condition-specific non-zero dorsiflexion value and plantarflexion moment value. Taking the slope of the best fit line to the moment-angle curve gave an estimate of the ankle dorsiflexion stiffness. The figure above shows a representative moment-angle curve from ‘low’ stiffness (blue) and ‘high’ stiffness (green) conditions.Additional file 2: Figure S2. Input stiffness versus stiffness from inverse dynamics. The stiffness values input into the prosthesis software versus the stiffness values calculated from the inverse dynamics. Each dot represents one participant’s data, and the five stiffness values for each participant are connected with a solid line. The blue lines represent the no load conditions, and the red lines represent the additional load conditions. The black diagonal line shows where the two values would be equal. The black dashed line is a best fit line to all data. This graph shows that while the stiffness that is inputted is not always the exact value found from inverse dynamics, there is still a general trend for increasing stiffness when an increased stiffness is inputted.Additional file 3: Figure S3. Prosthesis stiffness for each condition across all 3 days. The stiffness values calculated in the prosthesis software from the load cell on the prosthesis across the three days of testing. We did a t-test for each stiffness condition between days to determine if the stiffness varied within subjects among the three days of testing. We found that all differences were insignificant (p ≥ 0.0581) except the highest stiffness for the unloaded walking trial, with a significant difference between days 2 and 3 (p = 0.0222).Additional file 4: Figure S4. Metabolic cost for each input stiffness condition. All values are normalized to biological body mass. Blue bars represent the no load conditions, and the red bars represent the additional load conditions. As the colors get darker for both loading conditions, the stiffness values are increasing.Additional file 5: Figure S5. Prosthetic ankle-foot, and ipsilateral knee and hip work per stride for each condition. All values are normalized to biological body mass. Blue bars represent the no additional load, and red bars represent the additional load conditions. As the colors get darker, the stiffness values are increasing.Additional file 6: Figure S6. Contralateral ankle-foot, knee, and hip work per stride for each condition. All values are normalized to biological body mass. Blue bars represent the no additional load, and red bars represent the additional load conditions. As the colors get darker, the stiffness values are increasing.Additional file 7 Figure S7. Ipsilateral ankle, knee, and hip angular impulse for each condition. All values are normalized to biological body mass. Blue bars represent the no additional load, and red bars represent the additional load conditions. As the colors get darker, the stiffness values are increasing.Additional file 8: Figure S8. Contralateral ankle, knee, and hip angular impulse for each condition. All values are normalized to biological body mass. Blue bars represent the no additional load, and red bars represent the additional load conditions. As the colors get darker, the stiffness values are increasing.Additional file 9: Figure S9. Participants’ perception of each conditions’ difficulty, compared to the actual stiffness of the condition. Each dot represents each participant’s individual data, and the solid lines are the predicted equation. The interaction of stiffness and load were significant predictors of the participant’s perception. Perception =  − 1.91 − 0.23 ∙ kl Therefore, for the no load condition, participant’s did not perceive any difference in difficulty, but for the additional load conditions, the conditions seemed more difficult as stiffness increased.Additional file 10: Figure S10. Metabolic Cost for each condition across all 3 days. A paired t-test between the second and third day metabolic cost shows that there was no differences between the metabolic cost values for these days (p < 0.05).