Oryza sativa L.) are key characteristics affecting variety adoption and market value. Starch viscosity profiles tested by a rapid visco analyzer (RVA) offer a direct measure of ECQs and represent the changes in viscosity associated with starch gelatinization. RVA profiles of rice are controlled by a complex genetic system and are also affected by the environment. Although Waxy (Wx) is the major gene controlling amylose content (AC) and ECQs, there are still other unknown genetic factors that affect ECQs.The eating and cooking qualities (ECQs) of rice  and 136 QTLs were identified in more than one year. Among them, 6 QTLs were stalely detected in four years and 26 QTLs were detected in at least three years including 13 pleiotropic loci, controlling 2 to 6 RVA properties simultaneously. These stable QTL hotspots were co-located with several known starch synthesis-related genes (SSRGs). Sequence alignments showed that nucleotide and amino acid sequences of most SSRGs were different between the two parents. Finally, we detected stable QTLs associated with multiple starch viscosity traits near Wx itself, supporting the notion that additional QTLs near Wx control multiple characteristic values of starch viscosity.Quantitative trait loci (QTLs) for starch paste viscosity in rice were analyzed using chromosome segment substitution lines (CSSLs) developed from the two cultivars 9311 and Nipponbare, which have same Wx, multiple QTLs associated with the RVA profile of rice were identified, several of which were stably detected over four years. The complexity of the genetic basis of rice starch viscosity traits might be due to their pleiotropic effects and the multiple QTL hot spots. Minor QTLs controlling starch viscosity traits were identified by using the chromosome segment substitution strategy. Allele polymorphism might be the reason that QTLs controlling RVA profile characteristics were detected in some known SSRG regions.By eliminating the contribution from the major locus The online version contains supplementary material available at 10.1186/s12864-021-07913-7. Oryza sativa L.) is one of the most important cereal crops worldwide, with about half of the world’s population consuming it as a main source of dietary calories [Rice  on chromosome 6. Wx encodes the granule-bound starch synthase (GBSS) that is mainly responsible for longer amylose chains [ALK) on chromosome 6 encoding starch synthase II a (SSII-3), is the major factor responsible for gelatinization temperature (GT) and the distribution of amylopectin chain length [Starch RVA profiles of rice grains are controlled by a complex genetic system that involves multiple quantitative trait loci (QTLs), several with major effects on the trait, and many starch synthesis-related genes (SSRGs) . Linkagee chains –9. Many e chains . The alkn length –13. Othen length . These Sn length –17.http://www.gramene.org/, http://www.ricedata.cn/). Certain varieties carrying the same Wx allele exhibit different ECQs, indicating that other minor QTLs affect ECQs. Identifying these minor QTLs will be critical to precisely modulate rice grain quality in the future. However, because the effects of minor QTLs might be covered by major QTLs such as Wx, fine mapping and the cloning of the underlying locus can be challenging. Populations derived from two parents with similar AC or with the same Wx allele have been developed to eliminate the effects of Wx and detect minor QTLs for starch RVA and ECQs of rice [The heredity of starch RVA profile characters is complex and could easily be effected by environmental factors, especially the temperature during the grain filling stage , 19. Thi of rice , 26, 27.indica variety 9311 (recipient) and the japonica variety Nipponbare (donor) [Wx-b allele [Wx locus.The genetic basis of complex traits in rice and the contribution of single-nucleotide polymorphisms have been explored by genome-wide association studies, allowing the generation of testable hypotheses relating allele variation and starch RVA traits . Allelic (donor) . Since bb allele , this CSWx locus for parameters of starch RVA profiles using CSSLs. Our results establish the foundation for fine mapping and subsequent cloning of genes responsible for these QTLs, which will help to improve ECQs in rice by molecular breeding.In this study, we aimed to detect stable QTLs in the absence of variation at the indica rice 9311, almost all parameters were much smaller than those in Nipponbare, with the exception of setback viscosity (SB) and PeT.The two parents 9311 and Nipponbare showed similar apparent amylose content (AAC), 15.5 % ± 0.2 and 14.6 % ± 0.2 %, respectively, in Nanjing from 2017 to 2018. The AAC of CSSLs ranged from 13.1 to 17.2 %, with an average AAC of 14.92 % in 2017. The starch RVA profiles of 9311 and Nipponbare differed significantly over four years of the study. Most RVA parameters varied greatly over four years, with the exception of peak time (PeT) and pasting temperature (PaT), which were less affected by environment . Over four years, the relationship between SB and TV or FV changed , BD, and FV, but negatively correlated with SB and PaT. TV was positively correlated with FV and PeT. FV was positively correlated with PeT and PaT but negatively correlated with SB. BD was negatively correlated with all other RVA parameters. SB was positively correlated with PeT and PaT. These correlations were highly significant P < 0.0. Over foEvery year, 9311 and Nipponbare bloomed on July 20th and August 25th, respectively. The heading time of CSSLs ranged from August 15th to the 25th. Although the maximum temperature of July and August was more than 40 °C in 2017, CSSLs and parents showed normal seed sets across the four years. The mean daily maximum temperature during the recorded periods ranged from 28.7 to 34.3 °C over the four years of this study. The daily average temperatures during observational periods in 2013, 2016, and 2017 were about 28.0 °C, higher than the average temperature of 25.1 °C measured in 2014  on chromosome 10, overlapped with the mapping interval described for certain starch RVA profile QTLs related to FV, SB, PeT, TV, PaT, and PV [Several reports have described QTLs affecting rice starch RVA profiles –23, 34. , and PV , 34.Wx, SSIII-1, SSIV-2, and SBE3 , ADPsma , SSII-1, and ISA were near our starch RVA QTLs. In the case of ADPlar, ADPiso (Adenosine diphosphate glucose pyrophosphorylase large subunit 2), GBSSII, SSII-2, SSIII-2, SSIV-1, and PUL, the nearby or overlapping QTLs were identified in only one or two years, suggesting that the underlying QTLs are easily influenced by the environment. No QTLs associated with starch RVA traits was detected in regions that overlap with the genomic location of SSI, SSII-3, SBE1, or SBE4. The region between markers RM6748 and RM5473 on chromosome 4 contained a QTL for SB and overlapped with the genomic location of SSIII-2, which is related to SB. SSIII-2 is a critical gene controlling Pat and PeT [We noticed that several of our stable QTL hotspots co-located with some known SSRGs, such as E3 Table . BesidesSSII-1 and SSII-3 were previously reported to have major effects on PT and minor effects on gel consistency (GC), AC, PV, CPV, BD, and SB [SSII-1 is associated with BD. A QTL hotspot near SBE3 on chromosome 2 was also reported, in which four major QTLs associated with HPV, consistency viscosity, viscosity at 95 °C, and BD over two years [SBE3 and are associated with FV, TV, SB, and PaT.The QTL hotspots near , and SB . Here, wwo years . We alsoSSI and SSII-3 were identical in sequence in 9311 and Nipponbare, possibly explaining why we failed to detect a QTL overlapping with either of these intervals in this study. Likewise, we detected no QTL near SBE1 and SBE4, whose sequence identity between the two parents was 99.71 and 99.87 %, respectively.The stable QTL hotspots were co-located with several known SSRGs. To explore the potential sequence variation between the two parental cultivars at these SSRGs, we aligned the nucleotide and predicted protein sequences from 9311 and Nipponbare, which revealed limited allelic diversity between the two parents, with some exceptions Table . For exaADPlar and SSIV-2, whose genomic coordinates did not overlap with any of our QTL intervals. However, our QTLs related to RVA profile parameters overlapped with or in close proximity to 12 SSRGs. Although the gene sequences of Wx are identical between 9311 and Nipponbare, we identified QTLs associated with multiple RVA traits in the Wx region. In addition, the nucleotide sequences and encoded protein sequences for ADPsma, SSII-1, SSII-2, SSIII-1, SSIII-2, SSIV-1, SBE3, ISA, and PUL were different between the two parents, suggesting that sequence polymorphisms at these loci contribute to the observed QTLs controlling RVA profile characteristics.The two parents also shared the same allele at Recent studies have confirmed that changes in the environment largely affect rice starch PV, TV, and FV, while AC and other starch RVA parameters are mainly influenced by genotypes , 11. In We observed that PV is positively correlated with TV, BD, and FV but negatively correlated with SB and PaT. Similar correlations were previously reported , 3, 34. Wx make it difficult to identify more minor QTLs for starch RVA profiles. Only a few genes and QTLs related to starch RVA profiles have been cloned, such as qAC2, qGC6, ALK, Chalk5, and Dul (Dull) [Wx allele, Wx-b, to generate CSSLs as the mapping population [SSII-3, the major gene controlling GT, showed no differences in its genomic sequence and only a 2-amino acid difference in its protein sequence between 9311 and Nipponbare. We therefore hypothesize that 9311 and Nipponbare carry the same allele at SSII-3. New QTLs that exert minor effects on starch RVA profiles and GT could be identified without the influence of major Wx and SSII-3. Zhang developed a CSSL population consisting of 38 lines with the same parents selected here. Following the same mapping strategy, 10 stable QTLs for RVA properties and two minor stable QTLs for GT were identified over two years and in two environments [The confounding effects of the environment and the major effect gene pulation . SSII-3,ronments , 23.SSII-3 and Wx.The QTLs for the RVA properties they mapped also displayed pleiotropy but didn’t map to the same or similar chromosomal regions with our QTLs, which might not be surprising in considering that the two sets of CSSL lines harbored different chromosome segments from Nipponbare. Liu et al. (2011) also performed an extensive QTL mapping analysis for 16 rice quality traits across eight environments using a set of CSSLs developed from two rice varieties with similar AC, and detected 10 stable RVA profiles cross four environments . HoweverqSB6 and qBD11, which had not been previously reported, may be useful for research on marker-assisted selection of ECQs.In our research, although 310 QTLs were detected for seven paste viscosity properties for rice starch RVA profile traits, only 136 QTLs were mapped repeatedly. These results indicated that the RVA profile characteristics are largely affected by the environment, consistent with previous reports , 19. Thehttp://qtaro.abr.affrc.go.jp/, [Based on our mapping, the clustering of multiple QTLs controlling RVA traits indicate that pleiotropic effects and QTL hotspots are key factors affecting starch RVA traits in rice. Similar observations of pleiotropy were reported previously . QT.go.jp/, . Starch Wx, which should be taken into consideration in rice quality breeding. The effect of SSRG allele combinations on starch quality and ECQs should be investigated more in the future. We failed to identify QTLs associated with RVA profiles near some SSRGs, such as SSII-3 and SSI, likely due to the lack of allele polymorphism between the two parents. The 5ʹ untranslated region (UTR), genomic and protein sequences of Wx are identical between 9311 and Nipponbare, although we noted several RVA traits associated with the Wx genomic region. Xu also reported several SNPs close to Wx that were significantly associated with RVA parameters in subpopulations with the same Wx allele [Overlap between the intervals of our mapped QTLs and known SSRGs was common in this research Table . Most knx allele . These lThe highly consistent genotypes between SSRGs and QTLs reported here confirmed the accuracy of our mapping. The stable or newly developed QTLs located in the region that no known SSRGs in are meaningful to identify new genes controlling starch RVA profiles in the future, which will facilitate further research into the genetic mechanism regulating RVA profiles and ECQ of rice.Wx could be eliminated by generating CSSLs developed from 9311 and Nipponbare with the same Wx-b allele, which allowed the identification of new QTLs associated with RVA profile traits. We analyzed the genetic basis of variation in RVA profile over four years, and identified 136 repeated QTLs. Among them, 6 stable QTLs were detected every year, which are therefore likely to be very important for the RVA characteristics. In addition, we uncovered 13 intervals detected for 3 of the four years that showed pleiotropy with respect to controlling two to six starch RVA profile properties simultaneously. Finally, we highlighted four intervals, such as the interval between markers RM469 and RM587 on chromosome 6, that are associated with more than four RVA parameters for a given year. Pleiotropic effects and QTL hotspots appear to be key factors affecting starch RVA traits in rice. There was high consistency between allelic diversity at known SSRGs and the QTLs reported here. Future research will explore these stable QTLs and hotspots in more detail.We mapped QTLs associated with starch viscosity profile, one of the most important factors contributing to ECQs in rice. The effect of major locus indica variety ‘9311’ (recipient) and the japonica variety ‘Nipponbare’ (donor). 9311 was obtained from its breeder, Yangzhou Institute of Agricultural Sciences, Jiangsu Academy of Agricultural Sciences. Nipponbare was obtained from Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm.An advanced backcross population was developed by our lab using the 4F2 lines. Backcrossing and simple sequence repeat marker selection were performed as described in detail by Zhu and Zhao [The backcrossed population consisted of 119 BCand Zhao , 31. EacThe CSSLs and two parents were planted in fields at the Jiangsu Academy of Agricultural Sciences  in Nanjing in 2013, 2014, 2016, and 2017. Each year, all seeds were planted within two blocks on May 15th and transplanted on June 20th. Each line was planted in three rows with a row-to-row distance of 30 cm and plant-to-plant distance of 13.3 cm. The seeds of five plants were sampled from each CSSL line and dried naturally. Milled rice was grinded into powder and then passed through a 100-mesh sieve. After drying at 4 °C in an oven, each powder sample was balanced for 2 days at room temperature and kept at 4 °C for 3 months. Then, paste viscosity was measured as described below. For each line, five samples were used to determine the starch RVA profile.The climate parameters at the field site  were measured using a data logger . The sensor was placed at a height of 170 cm; air temperature and RH were collected every 10 min. The climate parameters were collected from the beginning of flowering to full maturity each year.Starch paste viscosity was measured with a Rapid Visco Analyser  according to the American Association of Cereal Chemists Standard Method (AACC 61 − 02) with TCW software 3  . 3 g ricStarch paste viscosity characteristics are characterized by five parameters : PV, TV, FV, PeT, and PaT. BD and SB were calculated as: BD = PV – TV, and SB = FV – PV . CorrelaThe AAC was determined using the iodine staining method described in the European Standard EN ISO 6647-2-2015. The absorbance of the solution was measured at 620 nm against the blank solution using a continuous flow analyzer . The AAC was calculated using a standard curve made from four rice samples with known AAC.Genotype data for 250 polymorphic loci, including 211 simple sequence repeat and 39 sequence tag site markers, were used for QTL detection. Molecular linkages were established using composite interval mapping with version 3.3 of QTL IciMapping software .P < 0.05. If more than three CSSLs showed differences, then the QTL was estimated as being located within the chromosomal region shared by those CSSLs [QTL were detected according to the method described by Eshed and Zamir . The sigse CSSLs . QTL nomse CSSLs .http://www.gramene.org/). The genomic sequences for 9311 were obtained from the Rice Genome Project (Beijing Genomics Institute) [https://www.ncbi.nlm.nih.gov/). Sequence alignment was performed with DNAMAN (version 6).The genomic and predicted protein sequences of known SSRGs that co-locate with starch RVA QTLs were downloaded from online resources, as 9311 and Nipponbare genomes are sequenced. Nipponbare sequences were downloaded from Gramene (stitute) . The preAdditional file 1: Table S1. Correlation analysis of seven RVA parameters in 2013 and 2014. Table S2. Correlation analysis of seven RVA parameters in 2016 and 2017. Table S3. Correlation analysis of starch RVA parameters.Additional file 2: Table S4. Genotype and phynotype of CSSLs.Additional file 3. Sequence Alignments of SSRGs between 9311 and Nipponbare.


Vibrio cholerae O139 could reemerge, and proactive development of an effective O139 vaccine would be prudent. To define immunoreactive and potentially immunogenic carbohydrate targets of Vibrio cholerae O139, we assessed immunoreactivities of various O-specific polysaccharide (OSP)-related saccharides with plasma from humans hospitalized with cholera caused by O139, comparing responses to those induced in recipients of a commercial oral whole-cell killed bivalent (O1 and O139) cholera vaccine (WC-O1/O139). We also assessed conjugate vaccines containing selected subsets of these saccharides for their ability to induce protective immunity using a mouse model of cholera. We found that patients with wild-type O139 cholera develop IgM, IgA, and IgG immune responses against O139 OSP and many of its fragments, but we were able to detect only a moderate IgM response to purified O139 OSP-core, and none to its fragments, in immunologically naive recipients of WC-O1/O139. We found that immunoreactivity of O139-specific polysaccharides with antibodies elicited by wild-type infection markedly increase when saccharides contain colitose and phosphate residues, that a synthetic terminal tetrasaccharide fragment of OSP is more immunoreactive and protectively immunogenic than complete OSP, that native OSP-core is a better protective immunogen than the synthetic OSP lacking core, and that functional vibriocidal activity of antibodies predicts in vivo protection in our model but depends on capsule thickness. Our results suggest that O139 OSP-specific responses are not prominent following vaccination with a currently available oral cholera vaccine in immunologically naive humans and that vaccines targeting V. cholerae O139 should be based on native OSP-core or terminal tetrasaccharide.Cholera caused by IMPORTANCE Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae serogroup O1 or O139. Protection against cholera is serogroup specific, and serogroup specificity is defined by O-specific polysaccharide (OSP). Little is known about immunity to O139 OSP. In this study, we used synthetic fragments of the O139 OSP to define immune responses to OSP in humans recovering from cholera caused by V. cholerae O139, compared these responses to those induced by the available O139 vaccine, and evaluated O139 fragments in next-generation conjugate vaccines. We found that the terminal tetrasaccharide of O139 is a primary immune target but that the currently available bivalent cholera vaccine poorly induces an anti-O139 OSP response in immunologically naive individuals. Vibrio cholerae serogroup O1 or O139 organisms  that contains killed V. cholerae O1 and killed O139 organisms , and fragments and derivatives of the latter, using bovine serum albumin (BSA) or a recombinant protein (rTTHc) as the carrier. rTTHc is a nontoxic 52-kDa recombinant tetanus toxoid heavy chain fragment , analyz, and 3a ). We fouP < 0.05 ) on day P = 0.08 ). Since P < 0.001; IgG P < 0.001). In comparison, we found no increase in immunoreactivity to any saccharide preparation other than native OSP-core IgM responses in vaccine recipients of WC-O1/O139  of sera-O1/O139 .P < 0.05  ). We werV. cholerae O139 emerged as a cause of cholera in the early 1990s, rapidly spreading through 11 Asian counties before all but disappearing as a clinically important cause of cholera by 2005 -α-linked (4-N-3-deoxy-l-glycero-tetronyl)-perosamine, with or without a methyl group at O-2 on the terminal perosamine, distinguishing the Ogawa and Inaba serotypes, respectively. In comparison, the OSP of V. cholerae O139 is a single hexasaccharide whose structure was identified more than two decades ago , d-galacturonic acid , N-acetyl-d-glucosamine (GlcNAc), d-galactose  and two colitose (Col) residues. It contains two negatively charged groups, including a carboxyl group of d-galacturonic acid and a cyclic phosphate bound to O-4 and O-6 of the d-galactose  and not to any synthetic or related preparations of O139 saccharides, and we detected no IgA or IgG responses to any O139 antigen in recipients of WC-O1/O139. Interestingly, baseline immunoreactivities (as judged by immunoreactivity of prevaccination day 0 samples) to a number of the polysaccharides analyzed in our profiling were higher in the samples from Haitian vaccinees than from Bangladeshi patients. This may reflect previous exposure to various polysaccharide or related antigens in these different populations. Cholera caused by V. cholerae O139  and induced protection in the mouse model, while vaccination with the latter did not. Importantly, vaccination with native OSP-core conjugate was also more immunogenic than synthetic OSP hexasaccharide and provided better protection against challenge than did vaccination with the synthetic OSP lacking core. This could suggest that core-specific antibodies themselves might contribute to protection against O139 cholera. However, the fact that the cores of V. cholerae O139 and O1 are identical, but that there is no evident cross protection of O1 versus O139 cholera, suggests that even if core-specific antibodies do contribute some degree of protection, they are not clinically highly significant.Based on our immunoprofiling of O139 OSP-core and various fragments , we furtrae O139 , 46–49.  animals , 48. In  animals –52 to atlerae O1 , 53–55. Shigella dysenteriae type 1 and their binding to a cavity-type monoclonal antibody (A plausible explanation for higher immunogenicity and protective capacity of the conjugate made from tetrasaccharide (conjugate 13) than of the one made from the complete synthetic OSP lacking the core (conjugate 4) could be that the absence of the downstream GalA-(1→4)-QuiNAc sequence in conjugate 13 allows the tetrasaccharide to be presented to immune cells in a more immunogenically relevant manner, e.g., allow it to attain a more favorable conformation, better fitting the subsite in the antibody binding area. Similar results have previously been reported in an analysis of immune responses to oligosaccharides related to the OSP of antibody . NuclearV. cholerae O139 but were able to show these when using a thinly encapsulated strain. The vibriocidal assay rests on binding of complement to the Fc fragment of bacterial surface-bound antibody that leads to generation of a terminal complement-based pore-forming membrane-attack complex (MAC) that inserts into the bacterial lipid membrane, resulting in lysis. The presence of a thick capsule could be assumed to lessen the likelihood that the MAC would be able to form or insert into the bacterial lipid membrane. The ability to assess vibriocidal responses against V. cholerae O139 is well known to be more variable than the ability to assess vibriocidal responses against V. cholerae O1  is the primary antigenic determinant. Our results also define two distinct epitopic regions as putative targets for antibody recognition: the two colitose residues and the cyclic 4,6-O-phosphate. If either of these structural elements is absent, the immunoreactivity of human plasma from patients who recover from O139 infection is significantly reduced. Our results also demonstrate that core oligosaccharide can contribute to immunogenicity, but its presence is not essential to induction of immune responses to O139 OSP. Our data show that relatively crude preparations of native OSP-core are as immunogenic and immunoreactive as the highly purified material and suggest that the crude native O139 OSP-core . Plasma samples separated from blood were also collected as part of a research protocol approved by Zanmi Lasante Institutional Review Board (ZL0006303), from individuals who received the WHO-prequalified and -endorsed bivalent (WC-O1/O139) oral killed cholera vaccine, Shanchol , in Haiti. This study was also approved by the Ethical Review and Research Review Committees of the ICDDR,B and the MassGeneral Brigham (MGB) Institutional Review Board, Boston, MA.Our use of animals met all institutional and governmental requirements, guidelines, and policies. The Massachusetts General Hospital Subcommittee on Research Animal Care (SRAC) approved this work. This work adheres to the USDA Animal Welfare Act, PHS Policy on Humane Care and Use of Laboratory Animals, and the  Animals . Plasma V. cholerae O139 from strain CIRS245 as previously described (V. cholerae O139 strain CIRS245 (containing a thicker capsule) and strain CIRS134B (containing a thinner capsule) (We prepared native LPS and OSP-core of escribed . We usedcapsule)  in vibriV. cholerae O139 OSP-core fragment as previously described (V. cholerae O139 OSP (hexasaccharide), as well as a series of derivatives and fragments of the OSP from disaccharide to hexasaccharide (conjugates 4 to 18 ) for growth control wells was between 0.2 and 0.3. At the conclusion of the assay, the vibriocidal titer was considered the serum dilution that generated a 50% reduction in optical density compared to that in the wells containing no serum  were removed from dams 2 h prior to inoculation. The mice were orally inoculated with 109 CFU of V. cholerae O139 strain CIRS134B  or CIRS245  mixed with a 1:1 dilution of pooled convalescent-phase day 56 serum from mice previously immunized with conjugate vaccines. Neonates were maintained at 30°C after the oral challenge and monitored every 3 h up to 36 h, following which the survivors were euthanized.We assessed the ability of serum from vaccinated mice to protect against challenge with wild-type virulent escribed , 53. In P value of <0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 8 .The data from different groups were compared using Mann-Whitney U tests. In addition, intragroup comparisons between the baseline (day 0) and other time points were made with Wilcoxon signed-rank tests. Survival curves generated from the neonatal challenge study were compared and analyzed with Kaplan-Meier and log rank analysis. The analyses were two tailed, and the cutoff 10.1128/mSphere.00114-21.1TEXT S1V. cholerae O139 OSP and its derivatives/fragments to carrier proteins using squaric acid chemistry. Download Text S1, DOCX file, 1.4 MB.General protocol for conjugating Copyright © 2021 Kamruzzaman et al.2021Kamruzzaman et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Journal of Materials Science: Materials in Medicine (2015) 26:59Correction to: 10.1007/s10856-014-5322-1The Editor-in-Chief has retracted this article  because Author Xing Wei agrees to this retraction but not to the wording of the retraction notice. The editor was not able to obtain a current email address for any of the other authors.


TM camera, in comparison with empty scaffolds. The biocompatibility of the composite material was ascertained with an MG-63 cell viability assay. The microtomography proves the homogeneous distribution of pores throughout the whole sample, whereas the presence of the biocompatible copolymer among the ceramic filaments, referred to as a polymer-infiltrated ceramic network (PICN), results in a safety “damper”, preventing crack propagation and securing the desired material flexibility, as observed by an infrared camera in real time. The study represents a challenge for future dental implant applications, demonstrating that it is possible to combine the fast robocasting of ceramic paste and covalent bonding of polymer adhesive for hybrid material stabilization.The aim of this work was to prepare and characterize polymer–ceramic composite material for dental applications, which must resist fracture and wear under extreme forces. It must also be compatible with the hostile environment of the oral cavity. The most common restorative and biocompatible copolymer, 2,2-bis(p-(2′-2-hydroxy-3′-methacryloxypropoxy)phenyl)propane and triethyleneglycol dimethacrylate, was combined with 3D-printed yttria-stabilized tetragonal zirconia scaffolds with a 50% infill. The proper scaffold deposition and morphology of samples with 50% zirconia infill were studied by means of X-ray computed microtomography and scanning electron microscopy. Samples that were infiltrated with copolymer were observed under compression stress, and the structure’s failure was recorded using an Infrared Vic 2D Per-Ingel MgAl2O and zirca (ZrO2) ,9,10,11.2) was first reported as being used in dental implantation surgery in 1975 when Cranin and co-workers employed zirconia and alumina (Al2O3) to coat Vitallium alloy [Zirconia  with 50% infill of solid yttria-stabilized tetragonal zirconia (3Y-TZP) filaments and 50% of macropores filled with methacrylate polymer, in a cubic geometry . The wor−3, after the sintering process. Pluronic® F-127 hydrogel (25% w/v); γ-MPS (3-(trimethoxysilyl)propyl methacrylate); Bis-GMA (bisphenol A glycerolate dimethacrylate); TEGDMA (triethylene glycol dimethacrylate) and BPO  were all supplied by Sigma-Aldrich  and were used to prepare the polymer-modified pieces.Zirconia powder was provided by SEPR Saint-Gobain ZirPro under the commercial name CY3Z-R, . It consists of 3 mol% yttria-stabilized tetragonal zirconia polycrystal (hereafter denoted as 3Y-TZP), with a particle size of about 300 nm. The ceramic density is 6.05 g·cm® F-127 hydrogel (30 wt. %) using a D-VM 16 vacuum mixer from Harnisch-Rieth, . Aiming to prevent drying, immediately after mixing, an extrusion syringe was filled with the paste, then the paste was extruded by a 3D printer using a 3D Dima Elite dispenser , with a cylindrical nozzle of 800 µm and equipped with DimaSoft CAD/CAM software. Extrusion was followed by a 5-day drying period before sintering. After a two-step sintering process (at 700 °C for 1 h and at 1450 °C for 2 h), zirconia cubes with dimensions of 1.2 × 1.2 × 0.8 cm3 and with 50% infill of zirconia filaments were obtained. Afterward, the 3D-printed scaffolds were submerged in a previously stirred solution of γ-MPS (24 mmol of liquid silane in 100 mL of 3:1 ethanol:water volume ratio) for 1 h, then they were removed and left to partially dry before the acrylate polymerization. Then, the silane functionalized pieces were immersed in a viscous solution with 39.5 wt. % of Bis-GMA, 59.5 wt. % of TEGDMA monomers and 1 wt. % of BPO initiator (previously stirred for 1 h at room temperature); and left to penetrate the scaffold for an additional 1 h. The hybrid system was then removed from the solution and placed in an aluminum foil-covered glass petri dish to complete the curing process at 115 °C for 10 h in a vacuum oven. The hybrid material thus produced received the name PICN, as previously indicated.The detailed procedure for scaffold fabrication was as recently reported elsewhere . In brieB is the Bragg angle for tetragonal and monoclinic diffraction peaks.The crystalline structure of 3Y-TZP was investigated by means of X-ray diffraction (XRD). The equipment used was a Bruker D8-Discover diffractometer  with a vertical goniometer (Bragg-Brentano configuration θ-2θ), XYZ-motorized stage mounted on a sample holder, and provided with a PSD Lynx-Eye detector. The data were collected in the 2θ range from 10° to 80° with an angular step of 0.02° at 1 s per step and a fixed incidence angle of 1°. Cu Kα radiation was obtained from a Cu X-ray tube operated at 40 kV and 40 mA. The average crystal size was estimated using the Debye-Scherrer equation:The topography of zirconia filaments and grain size were investigated using scanning electron microscopy (SEM). The study was carried out using a focused-ion-beam Zeiss Neon 40 scanning electron microscope  and equipped with an energy-dispersive X-ray (EDX) spectroscopy system. Secondary electrons detector (SE) was employed to study the topography of the surfaces and an immersion lens detector (InLens) was preferred for high lateral resolution. The scanning was carried out at 5 kV. The samples were mounted on a double-sided adhesive carbon disc and sputter-coated with a thin layer of carbon to prevent sample charging problems.The structure, pore distribution, and overall architecture of zirconia 3D-printed scaffolds (50% infill) were analyzed with an X-ray computed microtomography (micro-CT) Skyscan 1272 by Bruker . The measurement was performed at a source voltage of 100 keV and a current of 100 µA, with an isotropic pixel size of 4.6 µm. The analysis and the scan stacking were performed with Nrecon and CTAn software from Bruker.The Vickers hardness of the empty 3Y-TZP scaffold and the copolymer-infiltrated composite were obtained using a DuraScan 10 G5 unit by Emco-Test . Abraded 3D-printed samples were used, with and without infiltrated copolymer. The hardness imprint was made on the filament area of each sample using an applied load of 7 kN; thus, the data measured are reported as HV10.TM camera and analyzed with Vic 2D software from Correlated Solutions, Inc. . Vic-2D software uses optimized algorithms to provide full-field displacement and strain data for mechanical testing on planar samples. Definite in-plane movement can be determined for every point within the measurement surface, as well as by using the Lagrangian strain tensor. For this analysis, the samples were abraded to attain straight sides that were exposed to the camera recording. Compression tests were conducted using an Instron 8511 universal testing machine, under a displacement rate of 0.1 mm/s. As a result, the high-stress areas and crack propagation were recorded in real time until the sample cracked or until a maximum applied force of 7 kN was reached.The failure mode of the 50% infill 3D scaffold, 100% infill 3D scaffold, and polymer infiltrated 3D scaffold under pressure was observed with an Infrared Vic 2D−1 were chosen for spectra acquisition in the wavenumber range from 4000 to 600 cm−1. The spectra are reported as a percentage of transmittance versus wavenumber.The chemical composition of the adhesive was investigated by infrared spectroscopy. The infrared spectra of Bis-GMA/TEGDMA copolymer and pure monomers were measured with an FTIR 4700 Jasco spectrophotometer . The equipment is coupled to an attenuated total reflection (ATR) accessory (Specac model MKII Golden Gate Heated Single Reflection Diamond ATR). For this, 32 scan accumulations and a resolution of 8 cm3 penicillin, 50 mg/mL streptomycin, and L-glutamine 2 mM at 37 °C, in a 10% humidified atmosphere with CO2 and air in the proportion of 5% and 95%, respectively. Culture media were changed every two days, as standard protocol. For the sub-culture, cell monolayers were rinsed with PBS solution  and detached by incubating them with 2 mL TrypLETM  for 2–5 min at 37 °C. The incubation was stopped by re-suspending in 5 mL of fresh medium. For cell counting, a Neubauer camera and a dye  were employed.MG-63 cells  were cultured in Dulbecco’s modified Eagle’s medium (DMEM). It contains 4500 mg/L of glucose, 110 mg/L of sodium pyruvate, and 2 mM of L-glutamine, complemented with 10% fetal bovine serum (FBS); 50 U/cm4 cells/well to rate cell adhesion, and 5 × 104 cells/well for a cell proliferation assay, were seeded in each well. The plates were incubated for 24 h to check the cellular adhesion and, further, for 7 days to determine the cell proliferation. In parallel, the control was performed in each without any scaffold.3D scaffolds were placed in 12-well tissue culture plates and sterilized by exposure to UV light for 15 min for each side of the cube. A culture medium (4 mL) was added to each well to cover the scaffolds. Then, 100 µL containing 2 × 10The percentage of cells adhered (after 24 h) and proliferated (after 7 days) was determined through an MTT -2,5-diphenyltetrazolium bromide) assay . The proThe fabrication of dense and geometrically complex ceramic structures through 3D-printing technology by using polymer-derived ceramic precursors is relatively easy with 100% infill of ceramic deposition. However, the preparation of 3D-printed highly porous structures is rather difficult, due to the sample deformation. Considering that the objective of this study was the development of a hybrid material intended for dental applications, the most important properties to be evaluated for the new PICN material are the mechanical resistance and osseointegration via a cell viability test. The first property will be affected by the piece’s geometry, the proper infiltration of polymer adhesive to create the PICN structure, and the crystal structure of the zirconia filaments after the robocasting and sintering processes. In this regard, the geometry was fixed to have the maximum percentage of macropores as possible without the loss of the CAD/CAM 3D cubic design, characterized by a layer-by-layer zigzag filament deposition and the number of layers being 10  and monoclinic  zirconia.It is well known that the monoclinic and cubic phases of zirconia are less mechanically stable than the tetragonal one, and that phase changes are usually observed after aging, hydration, or temperature increase ,34,35. TThe crystalline size of the tetragonal lattice was found to be 35 nm and 51 nm, before and after robocasting, respectively . From thThe computed tomography technique (commonly referred to as micro-CT) allowed 3D- and 2D-investigations of the marginal and internal gaps produced by the computer-aided manufacturing of zirconia scaffolds, within the range of a few micrometers . The cer® hydrogel under vacuum, which helped to avoid bubbles, and, thanks to the employment of an adequate printer nozzle (800 µm in diameter), which avoided particle agglomerations and the formation of defects during the printing process. In The successful deposition of zirconia filaments was only possible thanks to the mixing of 3Y-TZP powder with PluronicOnce the material was printed and sintered at high temperatures, the SEM images at low magnification revealed the good stability of the zirconia filaments c, corrob−1 belongs to the Bis-GMA units. This compound also produces O–H bending vibrations but the latter couple with other vibrations and produce complex bands in the fingerprint region, coinciding with that of ester and ether groups (1000–1200 cm−1). The spectra of monomers also show a sharp absorption band, corresponding to the C=C stretching wavenumber at 1635 cm−1, which has been reduced in the copolymer spectrum, evidencing successful radical copolymerization. The presence of aromatic rings from the Bis-GMA monomer also manifests at the region between 900 and 800 cm−1. Moreover, the extensive presence of methylene groups can be confirmed by the absorption band at 1452 cm−1, corresponding to scissoring frequencies [The PICN pieces, with the zirconia scaffold voids filled by the copolymer adhesive, were polished and the infiltrated polymer was analyzed by ATR-FTIR spectroscopy, comparing the main chemical bonds of cured copolymer with the pristine monomers ; the O–Hquencies ,37. The The authors of this study have previously demonstrated that 3D-printed zirconia with 50% zirconia infill and PICN  exhibit different mechanical resistance to compression forces . In thiset. al. [The new material is intended to be used in the dentistry field, combining biocompatible ceramic and polymer adhesive. The dental implants are subjected to direct compression and abrasion forces with teeth. Therefore, it is important to evaluate its hardness and mechanical response under compression forces. Vickers hardness tests were conducted on the 50% infill scaffold and the polymer-infiltrated one. The Vickers hardness of the hollow scaffolds (50% infill) was 1414 ± 176 HV10 whereas, for the PICN scaffolds, it was lower (1078 ± 49 HV10). The lower hardness of the PICN sample can be explained by its higher flexibility and resistance to breaking under pressure, due to the filling of the zirconia macropores with Bis-GMA/TEGDMA copolymer, which is a softer material. In summary, the filled macropores transform a rigid and brittle ceramic into a more compliant and soft material. Lin et. al.  have repet. al. . The harThe breaking of the first filament and crack propagation starts at 0.55 kN in the case of zirconia scaffolds with macropores . FailureAs can be seen in Escherichia coli and Streptococcus salivarius bacteria are not able to proliferate on the 3D-printed PICN scaffolds with Bis-GMA/TEGDMA acrylate adhesive [p < 0.05) than those measured for cell adhesion in the zirconium scaffolds with a coating of silane and copolymer (Bis-GMA/TEGDMA), i.e., 68 ± 12%. Although, this percentage is high, a significant part of the cells seeded in the well-drained area toward the plate surface, suggesting that a block with a larger surface area on the upper face could improve the percentage of adhesion and retention of the cells in the 3D scaffold. The proliferation results indicate that the three types of 3D scaffolds prepared were compatible with cell growth (p < 0.05). In this same direction, cell proliferation is noticeable in the 3D scaffolds coated with silane and copolymer, where the percentage of viability was 87 ± 5%, which was not different from the control. Qualitative support of the above findings and statement is given in In a previous study by the authors, it was shown that adhesive . This isl growth b. The peSeveral works have demonstrated the viability of zirconia material towards human-osteoblast cells ,39,40. CIn a very recent study, reported by ours , a catecTherefore, the insertion of acrylate copolymers on zirconia has demonstrable advantages for bioactivity, since they have the ability to induce the cell adhesion that is essential for subsequent bone proliferation. The PICN samples studied also display a good correlation regarding structural integrity and biocompatibility. Future work needs to be performed in order to focus on its application in clinical practice. In this sense, mechanical properties, together with cell viability and reduced bacteria proliferation as published elsewhere , should Here, the successful obtaining of a stable and mechanically resistant PICN 3D-printed scaffold with a biocompatible copolymer acrylate inside the structure macropores was described. The superior cohesion of 3Y-TZP particles in the ceramic layer-by-layer filaments, after sintering, together with the very stable zigzag ceramic configuration, strategically designed to create the 50% infill cubic structures, led to the obtaining of promising hybrid material for dental applications.For the first time, it was possible to demonstrate that the copolymer infiltrated among the ceramic filaments, created by CAD/CAM design, acts as a mechanical stabilizer and adhesion promoter at the same time.The compression-testing, combined with the in situ observation of cracking phenomena with an infrared camera, led to the conclusion that PICN samples have a larger capability to resist higher deformation than 50% infill ceramic scaffolds. Moreover, the presence of the polymer also aids in decreasing the ceramic hardness, both properties being desirable for the use of zirconia material in the dentistry field.The results of MG-63 cell adhesion and proliferation of PICN, compared to 3D-printed zirconia without copolymer adhesive, are also a positive point for future biomedical applications.


Sports activities are highly beneficial for improving the human health and reducing the risk of diseases. This cross sectional study aimed to investigate the prevalence of prediabetes and Type-2 diabetes mellitus in cricket players compared to population based non-elite athlete control subjects.2. The cricket players have been playing cricket for 4 (3-4) hours per day; 3.50 (3-4) days per week; for the total period of 24 (12-36) months. American Diabetes Association (ADA) based criteria on Glycated Hemoglobin (HbA1c) was used to investigate the prediabetes and Type-2 diabetes mellitus.The present matched cross sectional study was conducted in the Department of Physiology, College of Medicine, King Saud University, Riyadh, Saudi Arabia during the period October 2019 to February 2020. Initially, 700 volunteer males, (300) cricket players and (400) population based non-elite athlete control subjects were interviewed. After socio-demographic and medical history, (200) nonsmoker cricket players and (300) nonsmoker control subjects were recruited. The age of cricket players was 34 (32-37) years, weight 81 (76-84) kg, height 1.79 (1.74-1.84) meters, and body mass index (BMI) was 25.09 (23.66-26.76) kg/mIn cricket players, the prevalence of prediabetes was 23 (11.5%) and Type-2 diabetes mellitus (T2DM) was 7 (3.5%) compared to population based matched non-elite athlete control subjects the prediabetes was 73 (24.34%) and T2DM was 63 (21.1%) (p=0.001). Among cricket players, there was a 6-folds decrease in T2DM compared to control subjects.The cricket sports activities decrease the prevalence of prediabetes and Type-2 diabetes mellitus (T2DM) among the cricket players compared to population based matched non-elite athlete control subjects. The study findings demonstrate the urgent need for promoting sports activities, more cricket grounds as a physiological preventive strategy against the global growing diabetes epidemic. Diabetes mellitus is a major global challenge, allied with substantial morbidity, mortality and huge economic burden on the healthcare system.3Currently, diabetes mellitus has a high rank on the international health agenda due to being a deathtrap to human health and worldwide economies.6The sports allied physical activities improve the health and capacity of the individual’s and performance. The sports activities including cricket sport has been acknowledged as a potential health promotion strategy to reduce the sedentary behavior. Sport improves endurance capacity and has a positive influence on cardiovascular and metabolic health.The present matched cross sectional study was conducted in the Department of Physiology, College of Medicine, King Saud University, Riyadh, Saudi Arabia during the period Oct. 2019 to Feb. 2020.In this study, various schools, colleges, universities, and small- and large-scale cricket sports grounds were visited and information about cricket players was gathered. Initially 300 volunteer male cricket players, and (400) population based non-elite athlete control subjects were interviewed. After socio-demographic, medical history and examination, a total of 500, 200) nonsmoker cricket players and (300) nonsmoker control subjects were recruited. The age of cricket players was 34 (32-37) years, weight 81 (76-84) kg, height 1.79 (1.74-1.84) meters, and Body mass index (BMI) was 25.09 (23.66-26.76) kg/m.00 nonsmo11Similarly, for the control group, various schools, colleges, universities were visited and initially, 500 population based non-elite athlete control subjects were interviewed. After socio-demographic and medical history examination, 300 control subjects were selected from schools and universities clerical staff, technicians, research assistants. The median age for the non-elite athlete control subjects were 33 32-37) years, weight 76 (72-81) kg, height 1.70 (1.66-1.75) meters, and Body Mass Index was 26.47 (25.35-27.60) (kg/m2). It was warranted that these control subjects were not involved in sports activities such as football, volleyball, badminton, hockey, swimming etc. Moreover, these control subjects were not involved in working exposure to any industries such as cement, coal, cotton, oil, flour, factories as these industries generate pollution, and pollution increases the prevalence of diabetes mellitus.-37 yearsTwo co-investigators interviewed 300 volunteer male cricket players and (400) population based non-elite athlete control subjects were interviewed. A detailed sociodemographic and medical history was obtained. The information about age, gender, height, weight, BMI, duration of playing cricket, demographic characteristics, lifestyle, diet habit, physical activities and other health-related information was collected by the use of a questionnaire. The “socio-demographic characteristics including residential address, living conditions, education level, marital status, monthly income, lifestyle information, and smoking were recorded. Other health-related evidence including family history of diabetes mellitus was also taken”. Both groups were matched for, age, weight, BMI, socioeconomic, and dietary habits. After demographic, medical history and examination, finally a total of 500, (200) nonsmoker cricket players and (300) nonsmoker control subjects were recruited .Participants with a known history of “anemia, blood diseases, blood transfusion, asthma, diabetes mellitus and malignancy were excluded from the study. Subjects who smoke traditional or electronic cigarette, or shisha were also excluded.”11The cricket players and control subjects were allotted an identification number, and a para-medical staff was assigned to measure the HbA1c. The “HbA1c was measured using the Clover A1c system , an automated boronate affinity assay for the determination of the percentage of HbA1c % in the whole body’s blood.15This study was executed in harmony with the “Declaration of Helsinki”, and the protocol was approved by the “Ethics Committee, College of Medicine Research Centre, King Saud University (E-19-4494)” .th-75th quartiles) are reported for non-normally distributed quantitative variables . Levene’s test was applied to check the variability in the anthropometric measures between the study groups. Mann-Whitney U test was applied to compare various parameters among study groups. A two-sample proportion test was also applied to observe the association between qualitative variables. Binary logistic regression was applied to observe the log-odds between HbA1c and study groups controlling for anthropometric measures. An α=0.05 was considered statistically significant. Spearman Rho Correlation was applied to assess the relationship between HbA1c, cricket playing hours, days, and months at 1% level of significance.The data was analyzed using SPSS 26.0  and Microsoft Power BI, 2020. Normality of the data was checked by one-sample Kolmogorov-Smirnov test. Median (25th – 75th quartile) for age was 34 (32-37) years, weight 78 (73.25-83) kg, height 1.73 (1.68-1.80) meters, BMI 26.14 (24.52-27.45) kg/m2 and HbA1c 5.30 (5.10-5.50)%  were cricket players and 300 (60%) were controls. Both the study groups were matched for age, height, and weight . For the0-5.50)% . The medAmong cricket players the prevalence of pre-diabetes 23 (11.5%) was significantly lower than their matched pre-diabetic controls 73 (24.34%) p<0.001, similarly cricket players had a significantly lower T2DM 7 (3.5%) when compared with the matched controls having T2DM 63 (21%) p<0.001. Moreover, the proportion of non-diabetes was again significantly lower in cricket players 170 (85%) than their matched controls 164 (54.66%) p<0.001, Cricket is a highly popular sport played by both gender, all age groups, and have many health benefits. This is the first study to investigate the prevalence of prediabetes and T2DM among cricket players compared to population based non-elite athlete control subjects. In this study we found that the prevalence of T2DM in cricket players was 3.5% compared to their matched control group (21.0%) with age group 32-37 years. These findings suggest that T2DM is almost 6 folds less in cricket players compared to their age, weight, BMI matched control subjects. The findings are interesting from the perspective that the prevalence of T2DM was significantly low in cricket players compared to non-elite athlete control subjects in a region where T2DM is highest across the globe.16The cricket sport is a highly popular game with various recreational activities. The cricket is a high-intensity sport with a positive impact on glucose metabolism.20Lao et al.Meo et al.The potential mechanism involved in playing cricket decreases insulin resistance and diabetes mellitus is highly interesting to understand. The epidemiological literature acknowledges the fact that sports activities decreases the insulin resistance and ultimately leads to decreased risk of T2DM. Cricket creational activities decreases oxidative stress, improves the antioxidant capabilities, glucose intake and decreases T2DM.20The sports activities are beneficial for people to fight against debilitating diseases including cardio-metabolic disorders. it increases the concentration of GLUT-4 in the cell membrane and increases glucose uptake in skeletal muscles.This is the first study to investigate the prevalence of prediabetes and T2DM in cricket players. The study exclusion criteria was well established, cigarette smokers were excluded. Both groups were matched for age, height, weight, BMI, ethnicity, and socio-economic levels to minimize the possible confounding factors. American Diabetes Association diagnosis approach was followed, Glycated Hemoglobin (HbA1c) is a reliable and valid indicator to identify an individual’s long-term mean blood glucose levels criteria was empoyed. This study could therefore be the best reference on the prevalence of prediabetes and T2DM. There are some limitations that we would like to point out, despite trying to recruit a large number of cricket players, we excluded cigarette smokers, age, weight, height and ethnicity matched criteria was employed hence we excluded a large number of participants and finally included 200 cricket players and 300 control subjects. Moreover, due to cultural limitations, we only include the male gender.Cricket allied recreational activities significantly decrease the prevalence of prediabetes and T2DM in cricket players compared to matched control subjects. The decreased prevalence was associated with the duration of cricket activities. The findings have public health implications, and supports the extension of diabetes intervention efforts. Health officials should establish more sports facilities, cricket grounds for the public to provide better sports facilities which will help in minimizing the incidence of prediabetes and T2DM. Public policies to promote sports activities have potential to improve population health and minimize the healthcare expenditure.SAM designed the study, applied research grant, ethics board approval, literature review, data analysis, manuscript writing and overall supervision of the project,AAA, AAA, ASM literature review, data collection.WS data analysis.


Individuals post-stroke experience persisting gait deficits due to altered joint mechanics, known clinically as spasticity, hypertonia, and paresis. In engineering, these concepts are described as stiffness and damping, or collectively as joint mechanical impedance, when considered with limb inertia. Typical clinical assessments of these properties are obtained while the patient is at rest using qualitative measures, and the link between the assessments and functional outcomes and mobility is unclear. In this study we quantify ankle mechanical impedance dynamically during walking in individuals post-stroke and in age-speed matched control subjects, and examine the relationships between mechanical impedance and clinical measures of mobility and impairment. Perturbations were applied to the ankle joint during the stance phase of walking, and least-squares system identification techniques were used to estimate mechanical impedance. Stiffness of the paretic ankle was decreased during mid-stance when compared to the non-paretic side; a change independent of muscle activity. Inter-limb differences in ankle joint damping, but not joint stiffness or passive clinical assessments, strongly predicted walking speed and distance. This work provides the first insights into how stroke alters joint mechanical impedance during walking, as well as how these changes relate to existing outcome measures. Our results inform clinical care, suggesting a focus on correcting stance phase mechanics could potentially improve mobility of chronic stroke survivors. Up to 80% of stroke survivors experience persistent gait deficits even after standard rehabilitation therapies3 that increase their risk for recurrent stroke and vascular death4. Changes in joint kinetics, kinematics and mechanics manifest as reduced gait speed, instability, asymmetry, and exhaustion3. While kinetic and kinematic impairments during walking have been studied extensively, altered joint mechanics (stiffness and damping) are poorly understood. Following stroke, the mechanical properties of muscle are fundamentally altered, causing an increase in passive and active muscle tone, as well as altered reflex facilitation and inhibition5. Clinically, changes to joint stiffness and damping are referred to as spasticity, hypertonia, and paresis6. Spasticity is thought to be a velocity dependent resistance to movement7. Hypertonia and co-activation can cause an increase in joint stiffness, while limb paresis can cause a decrease in joint stiffness8. To assess altered mechanics and their effects on mobility, clinicians often use coarse, qualitative assessments during non-weight bearing or resting conditions . These assessments are used to help guide treatment, and although current physical therapy and pharmacological treatments have been successful in passive conditions10, this success has not translated to functional improvements in locomotion5. Lack of improvement may stem from fundamental differences in how altered joint mechanics present between passive movement and dynamic movement. Furthermore, although stiffness and damping are well defined properties in engineering, their clinical analogs are not as distinct. Spasticity, hypertonia, and paresis are all changes to joint mechanics, but their clinical characterizations are broad and do not map to any specific impedance property (stiffness or damping). Directly characterizing altered joint stiffness and damping during walking may supplement the qualitative assessment metrics used in the clinic, while reconciling the mismatch between passive assessment and desired improvements in dynamic activities.Stroke is the leading cause of adult-onset disability, affecting millions of Americansmechanical impedance, and are fundamental to our ability to regulate interaction with the environment. There is evidence that the mechanical impedance of limbs helps provide stability during unstable tasks and compensates for unexpected environmental dynamics13. Biped walking is inherently mechanically unstable, and is one common task where joint stiffness and damping may play an important role in stability and forward propulsion. In young, healthy adults, ankle joint stiffness and damping vary continuously throughout the stance phase of walking16. The stiffness component of impedance increases during loading response (early stance phase), mid-stance, and early terminal stance phase in preparation for push off14, then decreases to values reported in swing throughout late terminal stance as the heel rises16. Ankle joint damping values in young healthy adults remain constant throughout loading response, mid-stance, and early terminal stance phase of walking, and increased during late terminal stance as the heel rises16. Characterizing ankle joint impedance in young healthy adults during gait has improved our understanding of gait biomechanics and led to the design of novel biomimetic prosthetic devices18. However, knowledge of how joint impedance is altered following neurological injury (e.g. hemiparetic stroke) is limited, and therefore has not yet been incorporated into treatment strategies and standards of care.Joint stiffness and damping  are collectively known as joint 20, but the component of stiffness associated with reflexes was not found to be different between groups21. These increases in ankle stiffness are not consistent throughout the population with some participants showing no difference in passive ankle stiffness from controls without a history of neurological injury23. Characterization of the damping component of impedance has been limited, but Mirbagheri et al. found no significant difference between paretic and non-paretic limbs under static conditions20. Investigations of the relationship between joint impedance and kinetic and kinematic properties of the ankle have also been conducted. Under static conditions, joint impedance been shown to be related to muscle activity26, ankle position29, and ankle torque30. In seated static conditions, stiffness was found to increase proportionally with mean ankle torque30, increase with activation and co-activation of the tibialis anterior and soleus muscles25, and increase with degree of planterflexion or dorsiflexion31. These relationships appear to hold in individuals with chronic stroke under similar static conditions20, however, as seen in studies on unimpaired populations, it is increasingly unlikely that the relationships between stiffness, joint torque, and muscle activation are maintained under dynamic conditions32. Therefore, although these works have provided valuable insight into how joint impedance is altered post-stroke and the relationship to kinetic and kinematic factors, their insights should not be extended to dynamic tasks such as locomotion.Previous research into altered joint impedance following hemiparetic stroke has focused on static conditions, rather than dynamic tasks, such as walking. These studies show that stiffness of the affected ankle in static conditions is significantly increased in stroke survivors34, and decreased during mid-stance and early terminal stance due to reduced muscle activation35. It was also hypothesized that standard clinical measures of mobility would correlate with impedance impairment during walking, but clinical measures of impairment obtained passively would not. This work provides a foundation for a new assessment paradigm where the factors guiding treatment such as orthotic bracing, pharmaceutical management or physical therapy, can be directly measured quantitatively, rather than inferred from coarse, qualitative studies at rest. Furthermore, these results could inform new clinical targets for therapeutic interventions and the development of novel assistive technologies that leverage knowledge of altered joint mechanical impedance during gait. Preliminary results for this work with a subset of participants was presented at the 2019 International Conference on Rehabilitation Robotics36.The purpose of this study was to (1) estimate impedance of the ankle joint during walking in individuals with chronic stroke, (2) characterize the relationship between ankle impedance and muscle activity, and (3) investigate the relationship between impedance impairment and clinical measures of mobility, spasticity, and sensorimotor function. Our primary hypothesis was that joint stiffness of the paretic limb would be increased during loading response (early stance phase), where muscle activity at the ankle is limited and increased passive joint stiffness dominates16. The contributions of stiffness, damping, and inertia to the resultant torque were 30.3% ± 17.7%, 9.72% ± 8.73%, and 26.9% ± 15.8% respectively for the paretic limb. For the non-paretic limb stiffness contributed 34.7% ± 18.1% of resultant torque, damping contributed 7.81% ± 5.58%, and inertia contributed 22.7% ± 15.9%.A second order parametric model described how the perturbation induced displacements corresponded to the resultant torque response needed to estimate mechanical impedance. The second order model characterized ankle mechanical impedance during walking in participants with chronic stroke with Variance Accounted For (VAF) of 75% ± 14% for the paretic limb and 75% ± 13% for the non-paretic limb. The model fit is notably lower than age-range matched older adults (VAF = 97 ± 3%) and previous studiesp < 0.001, F1,55 = 29.6), but not timing point . The interaction between limb and timing point was not significance . For three older adults with no history of stroke, data were collected as age-range and gait speed matched reference for participants with chronic stroke, but were not included in statistical comparisons due low number of participants. Older adults walking at the same speed as chronic stroke participants exhibited a similar pattern of stiffness variation to young healthy adults16, but with a lower peak stiffness prior to heel off.Ankle stiffness and damping values were investigated for both the paretic and non-paretic limbs in the chronic stroke population as well as for healthy age-matched control subjects Fig. . In subjp = 0.70, F3,55 = 0.48), or limb , but the interaction between these variables neared significance . Post-hoc comparisons with Bonferroni corrections found damping varied significantly with limb only during the end of loading response/early mid-stance, 30% stance . Both the paretic and non-paretic limb lack the stereotypical increase in damping in preparation for toe off seen in the young, healthy adult. Ankle damping for older adults without stroke trended toward a larger magnitude when compared to the chronic stroke population across stance phase, which is notably increased compared to previous results for young adults walking at a faster pace16.The estimated damping component of impedance had an inter-subject variability 0.008 ± 0.003 Nms/rad/kg for the paretic limb and 0.009 ± 0.005 Nms/rad/kg for the non-paretic limb. The repeated measures ANOVA for damping estimates found damping estimates did not vary significantly across timing point  or limb . There was no significant interaction between timing point and limb .The average inertial component of impedance of the paretic and non-paretic limbs were 0.046 kg m37  and a negative correlation with the non-paretic limb , but neither significantly correlated. Ankle stiffness of gait-speed-matched older adults without a stroke also did not significantly correlate with CCI , while the young healthy limb demonstrated a significant negative correlation . The damping component of impedance demonstrated positive relationships with CCI, but did not significantly correlate for any limb: paretic  non-paretic , older adults , or young adults . It is noted that although stiffness correlated with CCI of the young healthy adult, this model explains less than 36% of the variance.Muscle electromyography (EMG) were recorded and investigated for trends Fig. . To elucp = 0.0091, R2 = 0.774). Similarly, a significant correlation was found between the damping asymmetry and 10MWT speed for the self-selected speed  but not for the fast speed . Damping asymmetry was not significantly correlated with the MAS score , or the LE-FW motor score . The average absolute difference in stiffness between the paretic and non-paretic limbs did not significantly correlate with any clinical measure (p > 0.58).To investigate the relationship between changes in ankle impedance post-stroke and standard clinical measures used to characterize impairment, a series of linear regressions were performed Fig. . Clinicap = 0.0285, R2 = 0.65), and 85% stance , but not during loading response and mid-stance (p > 0.156). Although the average absolute change in damping across stance correlated with the 10MWT self-selected speed, no individual timing point demonstrated a significant relationship. However, similar to the 6MWT, the relationship neared significance in late terminal stance , but not at the other timing points (p > 0.08).A more detailed analysis of each timing point was performed for the aforementioned significant relationships. For each timing point, the signed difference in damping was regressed with the clinical measure of interest. This analysis showed damping asymmetry significantly correlated with 6MWT distance during powered push off at 70% stance , 6MWT and MAS , 10MWT and LEFM , or 10MWT and MAS .Finally, linear regressions were performed between clinical measures of mobility (6MWT and 10MWT) and clinical measures of impairment (MAS and LEFM) to assess how clinically defined impairment related to locomotor capacity. No significant correlation was found between 6MWT and LEFM ). The distribution of stiffness estimates for included trials with good model fit (> 50% VAF) as well as for excluded trials with poor model fit (< 50% VAF) are included in Supplemental Information Fig. Participants CVA02, CVA03, and CVA12 did not meet inclusion criteria and therefore did not complete the study. Participants CVA05 and CVA08 did not have enough trials with sufficient perturbation and model agreement necessary to reliably estimate impedance for any time point for either the paretic or non-paretic limb, so was not included in analysis. Removing trials due to insufficient perturbation is a common step of these methods34, but decreased during mid-stance associated with reduced peak muscle activity35. As hypothesized, our results showed higher stiffness values at the beginning of stance phase for both the paretic and non-paretic limbs when compared to unimpaired participants. Additionally, paretic ankle stiffness was decreased in comparison to the non-paretic limb across stance phase, but this difference was not associated with differences in muscle activity. The damping component of impedance of the paretic and non-paretic limbs did not increase in preparation for toe-off, and did not significantly differ between limbs. Our secondary hypothesis was that clinical measures of mobility would correlate with impedance impairment during walking, but clinical measures obtained passively would not. The damping component of impedance correlated with mobility (6MWT and 10MWT), but the stiffness component of impedance did not. Furthermore, neither damping or stiffness impairment during walking correlated with clinical measures of spasticity obtained passively (MAS), or sensorimotor function (LE-FM).This purpose of this study was threefold: (1) to determine ankle mechanical impedance in individuals with chronic stroke during the stance phase of walking, (2) investigate how these properties relate to muscle activity, (3) quantify the relationship between mechanical impedance impairment, and clinical measures of mobility, spasticity, and sensorimotor function. We hypothesized that compared to the non-paretic and unimpaired joint, stiffness of the paretic ankle would be increased during loading response (early stance), where muscle activity is limited and passive stiffness dominates22. However, our results show an even greater stiffness increase of the non-paretic limb across stance phase, which was not found in impedance studies conducted in static postures. As hypothesized, paretic limb stiffness did not increase throughout mid stance and was significantly different from the non-paretic limb at the end of mid-stance and throughout terminal stance phase of walking (70% and 85% of stance). Reduced stiffness during mid-stance and early terminal stance phase has important implications for mobility of individuals with chronic stroke; during terminal stance phase, energy is added by the ankle joint. Increased overall stiffness of the non-paretic ankle during walking not present in passive conditions20 may reduce the energy able to be added by the ankle joint during walking.In early loading response and mid-stance the stiffness component of impedance of the paretic limb for participants with chronic stroke were similar in magnitude to results from passive impedance studies20.The damping component of impedance did not vary across stance phase, and was not significantly different between paretic and non-paretic limbs. This aligns with impedance studies in static conditions that showed consistent damping across limbs following chronic stroke238. Increased inertia estimates may indicate that the estimate represents the inertia of the foot and other coupled body segments or the inertia of the platform robot. Misalignment of the ankle and the robot’s centers of rotation could cause the perturbation to also effect displacements of local body segments and contribute to higher, more variable inertia estimates.Inertia estimates were consistent across stance phase and between limbs, but both the paretic and non-paretic ankle inertia estimates were increased relative to ankle–foot inertia reported in literature . Future research should investigate how ankle damping is altered with in a larger cohort of older adults to formally isolate the effect of stroke on ankle damping.The damping component of impedance of the paretic and non-paretic ankles were similar in magnitude to those of the young unimpaired adult prior to heel off, but did not increase throughout late terminal stance in preparation for toe off. Notably, gait-speed matched older adults displayed a similar temporal variation to individual with chronic stroke, but a significantly higher magnitude than both the chronic stroke participants and young healthy adult. This may indicated that overall ankle damping increases with age, and reduced damping in both limbs of the chronic stroke population is an impairment. Alternatively, it is possible that higher damping estimate are a product of the difference in experimental methods. The ankle angle data of age-range-matched older adults was collected using motion capture, while participants with chronic stroke had their ankle data recorded using an electrogoniometer , explaining 77% and 65% of the variance, respectively. However, damping asymmetry was not significantly correlated with 10MWT fast speed. The reduced correlation between ankle damping asymmetry and 10MWT fast speed when compared to the self-selected speed, might be explained by the redistribution of overall power contribution between joints at faster walking speeds. At slower walking speeds for a young unimpaired adult, the ankle contributes slightly more than the hip to positive power generation, while at faster walking speeds this relationship is switched50 as well as a number of studies that have demonstrated that MAS do not relate to hyper reflexivity during walking in spastic gait51. This suggests that MAS may not be measuring impaired impedance of joints in a way that translates to descriptions of impairment during dynamic or functional tasks.Modified Ashworth Scale (MAS) did not correlate with the difference in stiffness or damping between the paretic and non-paretic limb during walking. These results were expected since the MAS is evaluated while the subjects is relaxed and with an open kinematic chain, whereas impedance measurements occurred while muscles are actively contracting during walking, with a closed kinematic chain. Our results align with previous impedance studies in postural conditions, which also did not find a significant correlation between stiffness and MASAsymmetry of either impedance parameter (i.e. stiffness and damping) also did not relate to the Lower Extremity Fugl Meyer (LE-FM) motor score. The motor score evaluates movement, coordination, and reflex action about the hip, knee, and ankle. These results suggest that, while useful in assessment of overall sensorimotor impairment, the LE-FM is not sufficient to characterize changes in mechanical impedance specifically. Therefore, it is unlikely that these changes have been addressed in clinical rehabilitation. However, it is important to note that the difference in stiffness also did not relate to clinical measures of mobility , therefore focusing on correcting impaired ankle joint damping may be more beneficial for improving overall mobility for individuals with chronic stroke.52. Currently, healthcare practitioners treating gait disorders in stroke survivors predominantly attend to the correction of sagittal plane swing phase errors with orthotic management or bracing. This emphasis is understandable given that excessive plantar flexion during mid to late swing phase is easily detected by patient and caregiver, and gives rise to variability in foot clearance which markedly increases risk of trips and falls54. However, the data we present suggest that stroke also changes the less easily detected mechanics of ankle-the foot complex during mid-stance and terminal stance phase of gait. These regions are especially important for energy storage and release55, and impairment to mechanical impedance properties could hinder forward propulsion, reducing gait speed and endurance. Our results show that following stroke, overall ankle stiffness is increased, but temporal variation is lost. The damping component of impedance also lacks the stereotypical increase during terminal stance. Furthermore, asymmetry in ankle joint damping between the paretic and non-paretic limb influence walking distance and speed to a greater extent than a validated composite measure of lower limb sensory and motor function. These results suggest that optimal rehabilitation of hemi-paretic stroke patients may require an expansion of clinical efforts to include the modification of stance phase changes in ankle–foot stiffness and damping.The re-acquisition of the ability to walk community distances at reasonable speed post stroke is of critical importance for stroke survivors given the impact of walking on functional independence and cardiovascular health., who found no improvements in gait speed or distance despite reported improvements in active ankle dorsiflexion, gait quality, and reduced spasticity as determined by MAS in their review of trials using neurotoxin to treat ankle plantar flexor and invertor muscles 56. Similarly, in their meta-analysis, Sun et al. reported neurotoxin-mediated improvements in LE-FM score but no improvement in gait speed57. Finally, it is unclear how pharmacological interventions affect ankle–foot damping, representing a major gap in knowledge given that our data suggests that damping impairment has significant impact on mobility.Most pharmacological interventions used to treat swing phase kinematics also serve to diminish stance phase ankle–foot stiffness. Stiffness during mid-late stance is related to foot supination in mid-late stance, which is mediated primarily by the posterior tibialis muscle. However, during swing phase, posterior tibialis spasticity also leads to plantar flexion and inversion which increases trip risk. Consequently, this muscle is commonly injected with neurotoxin by clinicians, a decision commonly driven by observation of gait and/or clinical assessment with MAS. While the application of neurotoxin would be expected to similarly improve stiffness impairment during loading response (early stance) of walking, the data presented suggest that neurotoxin to the posterior tibialis muscle would be expected to further diminish ankle–foot stiffness in mid-late stance phase; thus improvements in walking speed and distance would be unlikely. These results agree with Lizma et al58. Further research is required to elucidate if FES can address ankle stiffness and damping impairments. In addition to studying the relationship between FES and joint impedance, future lines of research might also focus on a means to reduce hemi-paresis induced muscle spasticity without the contractile paralyzing effects of neurotoxins. Hyaluronidase therapy, which diminishes high impedance-inducing build-up of the compound hyaluronan in paretic muscles, is one such possibility59.Therefore, strategies to diminish swing phase plantar flexion and inversion post-stroke that do not reduce mid stance ankle–foot stiffness as well as strategies that address damping asymmetry are needed. Functional electrical stimulation is one such possibility. One of the few studies to show an increase in walking speed after neurotoxin therapy to the posterior tibilias muscle and plantar flexors combined neurotoxin with functional electrostimulation (FES) of the common peroneal nerveFinally, this research informs clinicians that ankle–foot stiffness and damping during stance phase of gait cannot be accurately gauged at the bedside using the Modified Ashworth Scale. Furthermore, the MAS did not appear to influence walking speed or distance in our participants. Therefore, although clinical strategies aimed at decreasing spasticity as determined by the MAS post stroke may serve to improve positioning or comfort, these strategies may not be expected to impact gait speed or distance. At this point there is no known bedside strategy for measuring or estimating ankle–foot stiffness and damping during the stance phase of gait in the setting of chronic stroke, which represents a further knowledge gap to be addressed in future research.60. Additionally, impedance identification requires a perturbation to be applied to the joint, and therefore current technological limitations prevent the practical application of more sophisticated techniques during walking. While these assumptions proved successful in general for the population, a subset of trials in all individuals with chronic stroke did not reliably exhibit second order ankle dynamics. One possible explanation is that this methodology requires an isolated perturbation of the ankle joint during stance phase while the contralateral limb in in swing. Individuals with chronic stroke exhibit extremely heterogeneous gait patterns, sometimes exhibiting extended double support phase and excessive hip abduction, which would hinder joint isolation in some trials. It is also possible that the assumption of linear quasi-static behavior of the ankle is not maintained in the participants with more inconsistent gait mechanics. Future work should investigate the variability in joint dynamics of individuals post-stroke, and determine potential changes to the experimental methods that could account for this variability.While this study provides first insight into ankle impedance in individuals with chronic stroke during walking, it assumes quasi–static second order dynamics during the analysis window surrounding each timing point . This is a simplification of actual ankle dynamics, which constantly vary throughout stance, however, this model was chosen for initial investigation of ankle impedance based on the previous success of this technique61. The average intra-subject misalignment of rotation axes was 0.8 ± 4.9 cm for the paretic ankle, and 0.3 ± 3.0 cm for the non-paretic ankle. Therefore, this work predicts potential stiffness errors of 4.8 ± 29.4% and 1.8 ± 18% for the paretic and non-paretic ankles respectively.Despite efforts to control the ankle and platform robot’s center of rotation alignment, the location of heel contact on the platform varied across trials. Slight variations in each trial are expected using this methodology, especially when studying a population with gait inconsistencies. Previous studies quantified the sensitivity of stiffness estimates to misalignment, showing a 6% decrease in stiffness per cm misalignment39, which is likely the case for the stroke population as well. Furthermore, statistical comparisons to gait-speed-matched older adults without history of stroke could not be made due to low number of older adult participants. Lower participant numbers stem from the long and arduous data collection process. Due to the lengthy protocol this study only characterized four timing points spanning the stance phase of walking. Future work should include data collected from additional participants, a broader range of post-stroke impairments, and other key gait events such as initial contact. Finally, while the methodology used in this study does accurately estimate overall ankle impedance during walking, it does not differentiate between intrinsic and reflex contributions to impedance. However, it is unlikely that our analysis characterizes ankle impedance values associated with reflexes (shown to be increased in some passive studies20) since sustained reflexes are minimized when using transient perturbations60 and the peak torque contribution from reflex activity occurs after a ~ 150 ms delay64, outside the 100 ms window of analysis used in this study.For this initial investigation the chronic stroke population included was limited to nine individuals, all community ambulators, walking at a single gait speed. Even in this small subset of the population, impedance estimates were much more variable across participants than in unimpaired individuals. Therefore, extending the conclusions from this study on how impedance is altered following stroke to individuals with higher levels of impairment should be done with caution; altered ankle impedance post-stroke may differ with impairment level. It has also been shown that ankle impedance varies with gait speed in the young healthy adult populationTwelve individuals with chronic hemiparetic stroke were enrolled in this study, nine of which completed the full protocol . All participants were required to have no history of major ankle injury or Botulinum Neurotoxin (BoNT) treatment for ankle spasticity, as well as being at least 2 years post-stroke. Individuals unable to complete the Six Minute Walk Test (6MWT) and those with a self-selected Ten Meter Walk Test (10MWT) speed less than 0.45 m/s were excluded. Three recruited participants were excluded: CVA02 was unable to complete the 6MWT without stopping, CVA03 had a self-selected walking speed as determined by the 10MWT below the required cut-off, and CVA12 opted not to complete the entire study. Additionally, three age-range matched adults were recruited  controlled by a commercial servodrive  was used to drive the Perturberator to the desired position during perturbations. Finally, a multi-axis force platform  was rigidly attached to the Perturberator Robot to measure ground reaction forces (GRF). A schematic of the Perturberator Robot is included in Supplemental Information Fig. A number of standard clinical measures were conducted to obtain a clinical metric of impairment for each chronic stroke participant, and to ensure that all participants had sufficient speed and endurance to participate. Two clinical measures of mobility were performed: the Six Minute Walk Test (6MWT), and the Ten Meter Walk Test (10MWT). The 6MWT assessed distance walked over six minutes as a submaximal measure of functional capacity in individuals post stroke. Walking speed and functional mobility over short distances was assessed using the 10MWT. Impairment level was assessed in participants meeting endurance and speed requirements using the Modified Ashworth Scale (MAS) and Lower Extremity Fugl Meyer (LE-FM). The MAS measured spasticity and each item was scored from 0 (no impairment) to 4 (severe impairment), while the LE-FM evaluated sensorimotor impairment, and with each item scored from 0 (severe impairment) to 2 (no impairment). Total MAS and LE-FM scores pertaining to the ankle joint were used to analyze the relationship to ankle impedance. The Spinal Cord Assessment Tool for Spastic Reflexes (SCATS) was collected as an additional measure of impairments for participants, but was not included in impedance analysis. All metrics were performed by the same licensed physical therapist.Data for participants with chronic stroke were collected at the Shirley Ryan AbilityLab, while age-range-matched older adults were collected at University of Michigan, therefore minor differences in protocol arise based on locational resources. All participants  walked across the walkway at a pace of 55–60 steps/min. To ensure step frequency consistency between trials, participants were asked to match the frequency of a metronome, and were given time to familiarize themselves with the task prior to data collection. Foot placement on the Perturberator Robot was monitored throughout the experiment, and the starting position of each participant was adjusted to ensure the ankle and Perturberator Robot’s centers of rotation aligned. Ankle angle was measured using electrogoniometers  affixed to each ankle of chronic stroke participants, while ankle angle was measured using motion capture  for older adult participants. GRFs during walking were measured using the force platform embedded in the Perturberator Robot. Electromyography (EMG)  was collected from the tibialus anterior (TA), medial gastrocnemius (MG), semitendinosus (ST), and rectus femoris (RF) muscles of each leg under study (paretic and non-paretic limbs of chronic stroke participants). For older adults, EMG data were collected from the TA, MG, and Soleus muscles of the dominant limb. Each electrode site was cleaned with alcohol to facilitate electrode adherence and conduction of EMG signals. Electrodes were placed on the muscle belly parallel to the muscle fibers. All data were collected using 16-bit data acquisition systems  sampled at 1–2 kHz. As participants walked across the Perturberator Robot, a perturbation was randomly triggered with 50% probability. The Perturberator detected initial contact when the GRF passed the 50 N threshold. During trials that contained a perturbation, a delay timer was set based on walking speed for each time point to ensure the perturbation occurred at the desired location in stance phase. Specifically, ramp-and-hold perturbations occurred at approximately 30%, 50%, 70% or 85% of stance phase for chronic stroke participants, and at 30%, 45%, 65%, 85% of stance for older adults. For chronic stroke participants, 100 perturbation trials were collected for each timing point, and 30 perturbation trials were collected for older adults at each timing point. Perturbations were 2° (0.035 rad) in magnitude; however, it is noted that due to increased ankle stiffness and/or motion of the mid-foot, a 2° perturbation of the robot may not translate to a full 2° perturbation of the ankle joint. Finally, to mitigate slippage and prevent falls throughout the experiment, individuals post-stroke wore a safety harness secured to an overhead gantry system and treaded hospital socks .For each participant, EMG data for all trials were processed prior to dividing data into steps. EMG data were bandpass filtered (50–200 Hz) and full wave rectified. Then a 200 ms moving average filter was applied to the rectified EMG. EMG data for each muscle were divided into steps and normalized to the average peak EMG across non-perturbed steps. Normalized EMG data were used to compare EMG activity between the paretic and non-paretic limbs across participants.To investigate the relationship between the components of impedance and muscle activity, binned EMG at each timing point  were determined by averaging non-normalized EMG for each participant in a 100 ms window beginning with the onset of perturbation . A co-contraction index (CCI) was then determined for muscles spanning the ankle each participant at each timing point  using this averaged EMG data.During stance phase, the TA is the antagonist muscle, and the MG is the agonist muscle.Trials in which the ankle displacement perturbation was less than 0.75° or were not included in analysis to ensure sufficient perturbation in the presence of data variability. Trials were also excluded if second order model explained less than 50% of variance. Data for these trials are included in Supplemental Information Fig. 61. Data were then divided into steps and separated into paretic limb and non-paretic limb. Sagittal plane ankle torque was determined by resolving GRFs to the equivalent force-torque couple at the ankle’s center of rotation:Fx and Fz are the anterior–posterior and vertical GRF respectively, and dx and dz are the corresponding moment arms. Moment arms were determined by transforming center of pressure data to the ankle frame of reference. When the foot is flat on the ground during at the end of loading response (early stance), during mid-stance phase, and during terminal stance prior to heel off, the anterior–posterior distance from the COP at heel contact to the ankle frame was subtracted from COP data. During late terminal stance, as the heel rises, the mid-foot deforms and a more complex transformation is required. A biomechanical model of the foot, was used to transform COP data to the ankle frame while accounting for movement of mid foot segments16.Force plate data, motor encoder data, and ankle goniometer data were low-pass filtered using a bidirectional third-order Butterworth filter with a 20 Hz cutoff frequency. Forces arising from the Perturberator Robot’s intrinsic inertia were removed using previously estimated linear filters16 to provide an estimate of variability. The initial trial in each bootstrap was selected at random, and a probability algorithm selected additional trials such that temporally-similar trials were selected with higher probability until 60% of trials for a specific time point. This method was used to account for slight differences in timing of perturbations at each time point. Offset was removed such that both torque and angle begin with zero. Bootstrapped ankle angle and torque arising naturally during walking  were subtracted from perturbation trials in order to isolate the perturbation response, then trimmed to 100 ms windows beginning at the onset of perturbation.Due to the heterogeneous nature of steps in the chronic stroke population, data were converted to a phase based representation of stance, such that key features of stance phase align across trials. For each limb , perturbation trials were separated from non-perturbation trials, then further segmented into the four perturbation timing points of interest . For older adults, segmented data were bootstrapped in accordance with previously published methods for estimating impedanceangle is the variance accounted for between the perturbed trial’s ankle angle and a non-perturbed trial’s ankle angle excluding the perturbation window, and VAFtorque. is the variance accounted for between the perturbed trial’s ankle torque and a non-perturbed trial’s ankle torque excluding the perturbation window. Data were then trimmed to 100 ms windows beginning at the onset of perturbation Ankle impedance was estimated over the 100 ms window of each trial using least-squares system identification. A second order parametric model mapped perturbation induced displacement to the resultant torque response at the ankle.T and θ are the torque and angle response arising from the perturbation respectively, I is the total inertia of the foot–ankle complex, b is the damping component of ankle impedance, and k is the stiffness component of ankle impedance. Angular velocity and acceleration were determined numerically by differentiating the ankle angle data65.Individuals with chronic stroke exhibited highly variable steps such that consistent trends in the non-perturbed data could not be established. Thus, previous methods were modified to address the heterogeneous nature of post-stroke kinematic and kinetic data. Rather than the bootstrapping procedure used in previous studies, we estimated impedance properties for each perturbed trial. For each perturbation trial, the perturbation was isolated by subtracting the average ankle angle and torque arising naturally during walking from the fifty non-perturbed trials that most resembled that perturbation trial. The 50 non-perturbed trials that maximize kinetic and kinematic agreement (“Fit” as defined by (3)) were selected as non-perturbed trials for a specific perturbation trial.16 were included for reference, but statistical comparisons with the stroke population were not conducted.The primary aim of this study was to characterize the effect of chronic stroke on impedance during the stance phase of walking. Three repeated measures analysis of variances (ANOVA) were performed in which stiffness, damping and inertia were dependent variables. Timing point  and limb (paretic and non-paretic) were treated as fixed factors, and subject was treated as a random factor. The interaction between timing point and limb was examined. A significance level of α = 0.05 was set a priori; Bonferroni corrections were applied for multiple comparisons. Data from gait-speed matched older adults and young adults walking at a faster speedTo investigate the source of changes in impedance post-stroke, the relationship between muscle activity and stiffness was examined for chronic stroke participants (paretic and non-paretic limb), gait-speed matched older adults, and young adults by fitting a linear regression between stiffness (measured at each time point) and co-contraction index (CCI). This analysis was repeated for the damping component of impedance.The secondary aim of this study was to evaluate the relationship between changes in impedance and standard outcome measures used to characterize impairment in the clinic. ΔStiffness scores were calculated for each participant and linearly regressed on each clinical measure . For each limb, stiffness as a function of percent stance phase was determined using an interpolation. A ΔStiffness score was then defined as the average absolute difference in stiffness between the paretic and non-paretic limbs across the stance phase. The same protocol was used to define ΔDamping scores.Impedance impairment post-stroke varied throughout stance phase such that in some portions of stance the paretic limb exhibited increased impedance, while in others stiffness and damping were reduced. The absolute change was selected to capture overall difference from the non-paretic limb. For comparisons that yielded a significant relationship between overall difference in an impedance parameter and a clinical metric, a more detailed analysis was conducted at each timing point separately. In these cases, the signed difference in stiffness between paretic and non-paretic limbs was linearly regressed on clinical measures.Supplementary Information.