While many starch biosynthesis enzymes have been characterized, the mechanisms of certain genes (including Sugary enhancer1) are yet undefined, and very little is understood about the regulation of starch biosynthesis. As a model, we utilize commercially important sweet corn mutations, sh2 and su1, to genetically perturb starch production in the endosperm. To characterize the transcriptomic response to starch mutations and identify potential regulators of this pathway, differential expression and coexpression network analysis was performed on near-isogenic lines (NILs)  in six genetic backgrounds. Lines were grown in field conditions and kernels were sampled in consecutive developmental stages (blister stage at 14 days after pollination (DAP), milk stage at 21 DAP, and dent stage at 28 DAP). Kernels were dissected to separate embryo and pericarp from the endosperm tissue and 3′ RNA-seq libraries were prepared. Mutation of the Su1 gene led to minimal changes in the endosperm transcriptome. Responses to loss of sh2 function include increased expression of sugar (SWEET) transporters and of genes for ABA signaling. Key regulators of starch biosynthesis and grain filling were identified. Notably, this includes Class II trehalose 6-phosphate synthases, Hexokinase1, and Apetala2 transcription factor-like (AP2/ERF) transcription factors. Additionally, our results provide insight into the mechanism of Sugary enhancer1, suggesting a potential role in regulating GA signaling via GRAS transcription factor Scarecrow-like1.In maize, starch mutants have facilitated characterization of key genes involved in endosperm starch biosynthesis such as  Zea mays) is one of the most important cereal crops in terms of economic and nutritional value and, together with rice and wheat, provides over 60% of calories consumed by people throughout the world (Maize (he world . The majhe world . The endlarge subunit of AGPase Shrunken2 (Sh2) and isoamylase type DBE Sugary1 (Su1). The AGPases catalyze the first committed step of starch biosynthesis and require both large and small subunits for enzymatic function. The sh2 mutation was caused by a complex structural variation -linked glucose units, and amylopectin, a highly branched glucan polymer of α-(1→6)-linked glucose units connecting linear chains. Starch begins accumulating in the endosperm of maize kernels approximately 10 days after pollination (DAP) and continues through approximately 30 DAP, when starch production declines and kernels begin to enter the dehydration phase (reviewed in ariation  and prodlopectin .Sugary enhancer1 (se1) gene is commercially relevant in sweet corn production, but its role in starch and carbohydrate metabolism is still undefined. Although its exact mechanism is unclear, the Sugary enhancer1 mutant allele is caused by deletion of Zm00001eb115450  in six genetic backgrounds. Kernels were harvested in three consecutive developmental stages to study the responses of gene networks over time. Transcriptomes were compared to identify both conserved and background-specific differentially expressed genes (DEGs) in response to disruptions in the starch biosynthesis pathway. We show here that responses to the sh2 mutation include increased expression of sugar (SWEET) transporters and an increase in genes involved in ABA signaling. We identify Class II trehalose 6-phosphate synthases (TPS synthetases), Hexokinase 1, and Apetala2 transcription factor-like (AP2/ERF) transcription factors as potential regulators impacting the transcriptional response to sh2. Additionally, we propose a potential role of ZmSE1 in regulating the hormone balance of GA and ABA to ultimately regulate starch breakdown and sugar metabolism.Mutations throughout the starch biosynthetic pathway provide genetic perturbations that allow us to study the transcriptional response to the changing endosperm sugar and starch levels to identify coexpression networks controlling these responses. Sugars can act as signaling molecules as well as carbohydrate sources, with regulatory control over various processes including establishment of the basal endosperm transfer layer (BETL), inflorescence branching, and activation of specific transcription factors  to create near isogenic lines. Plants were grown in 2018 in Belle Glade, Florida at the University of Florida Everglades Research and Education Center in Belle Glade, FL, United States. Soil type at the research center is Histosol. Plots of 12 plants per NIL were grown in a completely randomized design with five replicates. Plants were spaced at 18 cm; rows were 76 cm apart with 0.91 m alleys between ranges. All plants were self-pollinated, and two ears were harvested from each plot at blister stage, milk stage, and dent stage  and immediately frozen. The kernels were removed from both frozen ears, bulked per plot, and stored at −80°C for RNA extraction and quantification of starch and sugars.Eighteen sweet corn lines that included three near-isogenic lines (NILs) in six genetic backgrounds were obtained from Dr. William Tracy at the University of Wisconsin-Madison. Each inbred line, which were all originally D-fructose and D-glucose assay procedure from Megazyme (K-SUFRG). The assay was scaled to 96 well plate format. Briefly ears, harvested in the field, were frozen immediately in liquid nitrogen. Fifteen kernels were then taken from the equatorial region midway between the tip and base, weighed and lyophilized. The dried kernels were pulverized in a geno-grinder and 10–20 mg were transferred to each of three 1.5 mL tubes. One mL of cold water (4°C) was added to each tube and vortexed. Samples were then incubated at 80°C for 15 min. Samples were centrifuged at maximum speed for 10 min and transferred to clean 1.5 ml tubes. Appropriate dilutions adjusted sugar concentrations to within the linear range of the standard curve (0–5 μg). Typical dilutions were 1:2 for glucose and fructose, and 1:10 for sucrose. Enzymes, ATP and NADP+ were added according to the Megazyme assay procedure but were scaled to accommodate a 96 well format. Standard curves for glucose, fructose and sucrose were used to quantify sugars in each sample. The amount of sucrose was calculated by subtracting the amount of glucose obtained in each sample and subtracting it from the amount of sucrose plus glucose in the sample. Starch was quantified as described by Sugars were determined using the sucrose, For RNA sequencing, two kernels were randomly selected from each pooled-kernel sample from each of the five biological replicates and frozen in liquid nitrogen. In all the treatments, endosperm was dissected from each frozen kernel by removing the embryo, pericarp, and pedicel. Total RNA was isolated from the dissected endosperms using a CTAB based extraction as previously described . SamplesP-value threshold of 0.05. DEGs are defined as having an absolute value log2 fold change > 2 unless otherwise specified.Single end reads were demultiplexed, trimmed and quality filtered using Trimmomatic v0.32 . The reaGene coexpression modules were identified using the R package Weighted Correlation Network Analysis (WGCNA) . Expressp-values ≤0.05 were considered over-represented. For pathway enrichment analysis, KOBAS3.0 software was used to test the statistical enrichment of DEGs in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways  terms for each gene were obtained from the B73v5eb annotation. The R package topGO v.2.38.1 was used to conduct GO enrichment analysis . GO termpathways . Pathwaysh2 mutation led to a 255.9 mg g–1  increase in all quantified sugars on average  . Some backgrounds, most notably Ia5125, exhibit a stronger increase in sugars with the sh2 mutation (mean sugar levels increase by 365.6 mg g–1)    . Even ta mg g–1) . The sug kernels . Sugar l< 0.001) . By 21 D< 0.001) .sh2, su1, Wt) was represented by combining results across backgrounds. By using different genetic backgrounds as replicates, we aim to identify conserved responses to these mutations in the maize endosperm. Using an adjusted p-value threshold of 0.05 and a log2 fold change cutoff of two, 667 DEGs were identified between sh2 and Wt endosperm and 77 DEGs were identified between su1 and Wt endosperm in at least one stage of development  is found at blister stage  and milk stage (12 DEGs), followed by a more dramatic increase to 60 DEGs at dent stage  were tested for KEGG pathway enrichment using KOBAS 3.0 , near the completion of starch synthesis . An F-box containing protein (Zm00001eb067000) is significantly downregulated in su1 endosperm with a log2 fold-change of −7.1. Additionally, a number of auxin related genes including ZmIAA24 are downregulated in su1 endosperm (log2 fold-change = −2.8). Interestingly, the aforementioned alcohol dehydrogenase and ZmEREB4 are also upregulated in sh2 endosperm  and Brittle2 (log2 fold change = 1.34) are upregulated in the sh2 mutant (2 fold change = 2.3) in sh2 endosperm (Granule-bound starch synthase2 (ZmGBSS2), Pullulanase (ZmZPU1), and multiple β-amylases show significantly decreased expression in sh2 endosperm. Starch phosphorylase ZmPHO1 is slightly upregulated in sh2 endosperm (log2 fold change = 1.1)  synthetase 11 (ZmTRPS11) is downregulated in sh2 endosperm while T6P phosphatase2 (ZmTRPP2) is upregulated  . Notablyegulated , 4B. In ndosperm . Sugar endosperm .sh2 endosperm results in a significant enrichment of DEGs in the glycolysis and gluconeogenesis pathway and in the pentose phosphate pathway (ZmADH2 (log2 fold change = 2.1), aldehyde dehydrogenases ZmALDH3 and ZmALDH13 , a pyruvate kinase , a pyruvate-phosphate dikinase , and a moderate upregulation (average log2 fold change = 1.2) of the three annotated maize pyruvate decarboxylases  (2 fold change −2.1) (sh2 endosperm. Eleven of these genes are moderately but significantly upregulated (average log2 fold change = 1.34)   . Interesge −2.1) . The pen = 1.34) . These i = −1.4) . Taken tsh2 endosperm. Notably, eight Apetala2 transcription factor-like (AP2/ERF) domain transcription factors are differentially expressed in the sh2 endosperm  in the sh2 endosperm while two  are downregulated  but significant upregulation in sh2 endosperm. These targets include ZmPYK3, pyruvate dehydrogenases , acyl carrier proteins , ZmTHL1, and ZmGLPDH4  . However, a homolog of AtAAP1 (Zm00001eb384590) and nitrate-reductase ZmNNR2 are upregulated   (Zeins compose the majority of storage proteins in the maize endosperm and are encoded by a large gene family (reviewed in nd −5.6) . Most (8ctively) . The GABctively) . Two gluctively) .sh2 endosperm, DEGs were compared across genetic backgrounds. Backgrounds were classified as “high-sugar-accumulating” or “low-sugar accumulating” based on whether the increase in sugars of sh2 kernels was significantly above or below the average change (+255.9 mg g–1)  and either slightly upregulated  or not significantly DE in high-sugar-accumulating genotypes (ZmBHLH157) is specifically upregulated in low-sugar-accumulating backgrounds (ZmMKKK55) and the upregulation of a glutathione S-transferase (Zm00001eb397280) (To gain insight into the background effect of sugar accumulation in the  mg g–1) . Most noenotypes . A helixkgrounds . Other eb397280) .ZmTRPS10 has the highest betweenness centrality, suggesting a central role in regulating this group of genes  is a key regulator of this network with the highest closeness centrality and betweenness centrality values. The hub gene, or gene with the highest number of connections to other genes in the module, is the GRAS transcription factor Scarecrow-like 1 . Module Lightsteelblue1 appears to be involved in the regulation of genes at the protein level with enriched GO Terms related to protein phosphatase regulatory activity and an overrepresentation of genes in the KEGG RNA transport pathway  (ZmARABIDILLO1 (Zm00001eb023660)  . Lightstb023660) . Module b023660) . Module b023660) , S8. Thes module .se1 kernels sampled at 11, 15, and 19 DAP  between se1 and Wt kernels over the three timepoints. Six DEGs were identified between Wt and se1 kernels at 11 DAP, 12 DEGs at 15 DAP, and 112 DEGs at 19 DAP (se1 kernels and sh2 endosperm. A homolog of AtSNF4 (Zm00001eb139880) is upregulated in sh2 and downregulated in se1 kernels, while the GRAS transcription factor ZmSCL1 is upregulated in se1 kernels and downregulated in sh2 endosperm. In addition to ZmSCL1, other gibberellin-related genes are DE in se1 kernels. A gibberellin degradation enzyme, GA4 2β-hydroxylase (Zm00001eb412670), is the most upregulated gene in se1 kernels (log2 fold change = 7.07). B-glucosidase 6, a gene involved in gibberellin-ester hydrolysis, is also upregulated in se1 kernels (log2 fold change = 2.7). Additionally, starch degradation genes known to be activated by GA  (se1 kernels. ZmRAFS3 is downregulated (log2 fold change = −8.9) at the 19 DAP timepoint in se1 kernels while another raffinose synthase (Zm00001eb005800) is upregulated at this timepoint (log2 fold change = 2.7). Additionally, two β-galactosidases  are upregulated in se1 kernels at 19 DAP   . It is actively) .Shrunken2 encodes the large subunit of the endosperm AGPase, responsible for synthesis of ADP-glucose used for starch production in the kernel. The mutant, sh2, is a complete knock out of this gene, and as such creates a drastic kernel phenotype where kernels have ∼25% wildtype starch content and a shriveled (shrunken) pericarp  (sh2 has on the kernel development and endosperm composition. Starch biosynthesis and sugar accumulation begins at ∼10 DAP and continues until ∼30 DAP. We isolated mRNA from wildtype and mutant (sh2 and su1) endosperm at multiple developmental stages spanning starch biosynthesis and sugar accumulation . Developmental timepoint had a strong impact on the overall transcriptome profile  , S5. Thi profile . As suchegulated . Betweent 21 DAP . In wildy 22 DAP . During  kernels . The dift 28 DAP .sh2 mutation resulted in a strong increase in sugars compared to both wildtype and su1 kernels in all genetic backgrounds at 21 and 28 DAP  are upregulated in the sh2 endosperm (sh2 endosperm (Starch phosphorylase 1 is upregulated in sh2 endosperm (Starch phosphorylase1 (ZmPHO1) is the plastidial starch phosphorylase and is typically active in the endosperm amyloplasts . Members of the SWEET  protein family transport sucrose and/or hexoses in the direction of the concentration gradient . Membersndosperm  include ndosperm  has a clsh2 endosperm, most notably those containing AP2/ERF domains  in the same module (ZmNAC130 is a key regulator of grain filling and could bind the promoter motif of 50 kD gamma zeins (one of these being a hub gene in the coexpression network identified here)  . Furthersh2 mutation affects both the sucrose- and hexose-based sensing systems. Differential impacts of sucrose and hexose “states”  is upregulated in sh2 endosperm and a class-II TPS (ZmTRPS11) is downregulated (ZmTRPS10)  as a QTL for overall starch content and, using mutant analysis, validated this role plus a contribution to seed yield. Taken together, these results indicate that Class-II TPS’s, and specifically ZmTRPS10, are central players in the transcriptomic response to the sh2 perturbation of maize endosperm.Input into the TPS-TPP system may differ here despite the elevated levels of sucrose in affected . In the affected . This paaffected . Here, aegulated . Class-Iegulated . InteresSugary Enhancer1 (SE1) as a key regulator of the maize endosperm transcriptome. The SE1 gene is a long-known effector of starch and sugar metabolism in maize kernels (Scarecrow-like1 (SCL1). Also present are multiple genes related to protein-level regulation including a 26S proteasome, a ubiquitin conjugating enzyme, and an F-box protein ARABIDILLO. Notably, the 26S proteasome-ubiquitin pathway has a role in gibberellin signaling , decreased starch  and genes related to grain filling and C:N balance in the endosperm, such as zein-encoding genes. Through a comparison of genetic backgrounds between NILs we also identified putative modifiers of the sh2 response, including maize BHLH157 and MKKK55, that can potentially be used in sweet corn breeding or for targeted starch production. Coexpression analysis has also provided insight into the role of Class II TPS’s and the ZmSE1 gene in the maize endosperm. We propose that the previously identified starch content QTL TRPS10  of starch biosynthetic mutants L TRPS10  functionPRJNA790930.The datasets generated for this study can be found in the NCBI Sequence Read Archive under BioProject MR and CF designed the experiments and wrote the manuscript. CF isolated and prepared RNA for sequencing, performed transcriptomic analyses, and performed statistical analysis of kernel carbohydrate data. SB extracted and quantified kernel carbohydrates. WT, KK, LH, KL, and GM contributed to interpretation, discussion, and manuscript review. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The conjugate of nido-carborane with cholesterol, containing a charge-compensated group in the linker, can be used as a precursor for the preparation of liposomes for BNCT (Boron Neutron Capture Therapy). The solid-state molecular structure of a nido-carborane derivative with the 9-Me2N(CH2)2Me2N-nido-7,8-C2B9H11 terminal dimethylamino group was determined by single-crystal X-ray diffraction.9-HC≡CCH Nido-Carborane or 7,8-dicarba-nido-undecaborate anion and its derivatives have highly polarizable spherical aromaticity as a result of their σ-delocalized electron density  cycloaddition reaction of alkynes to azides is widely used, leading to the formation of 1,2,3 triazoles; this is known as the “click”-reaction” ,35,36. Ee6 , as welle6 ,27,33,39 dianion  and nidoarborane . In thisnido-carborane derivative, with two ammonium centers in its spacer Br (1) Br 1 , azido-3β-cholesterol 2 + calcd 642.6016, found: 642.6028.9-3β-Chol-O2Me2N-nido-7,8-C2B9H11]Br in the copper(I)-catalyzed azide-alkyne cycloaddition reaction with azido-3β-cholesterol, and revealed that during this process, in the presence of a strong base, acetylene-allen rearrangement occurred, resulting in the elimination of the propargyl fragment in carbonyl acetylene and the formation of 9-Me2N(CH2)2Me2N-nido-7,8-C2B9H11. The solid-state molecular structure of this previously described compound was determined by means of single-crystal X-ray diffraction studies. Comparing the structure of 9-Me2N(CH2)2Me2N-nido-7,8-C2B9H11 with the previously described analogs shows that the flexibility of side substituent does not significantly affect the crystal packing. Based on synthesized zwitter-ionic conjugate, the preparation of the boronated liposomes was planned in order to deliver boron clusters into a cancer cell for BNCT experiments.In this work, the “click” reaction of 9-HC≡CCH


A waveguide model for a pre-compressed cylindrical natural rubber vibration isolator is developed within a wide frequency range—20 to 2000 Hz—and for a wide pre-compression domain—from vanishing to the maximum in service, that is 20%. The problems of simultaneously modeling the pre-compression and frequency dependence are solved by applying a transformation of the pre-compressed isolator into a globally equivalent linearized, homogeneous, and isotropic form, thereby reducing the original, mathematically arduous, and complex problem into a vastly simpler assignment while using a straightforward waveguide approach to satisfy the boundary conditions by mode-matching. A fractional standard linear solid is applied as the visco-elastic natural rubber model while using a Mittag–Leffler function as the stress relaxation function. The dynamic stiffness is found to depend strongly on the frequency and pre-compression. The former is resulting in resonance phenomena such as peaks and troughs, while the latter exhibits a low-frequency magnitude stiffness increase in addition to peak and trough shifts with increased pre-compressions. Good agreement with nonlinear finite element results is obtained for the considered frequency and pre-compression range in contrast to the results of standard waveguide approaches. Natural rubber is an interesting polymeric material. It exhibits frequency- and pre-strain-dependent mechanical properties, including its shear storage and loss modula, and an immense dissimilarity between the transversal and longitudinal wave velocities . The latSimplified models such as the classical long rod example are extensively applied to vibration isolators ,3,4,5, dHowever, the above approaches do not take into account the influence of pre-compression, which is from a practical point of view, an inescapable parameter since most vibration isolators carry the static load of the source or isolated system they are attached to. These effects involve geometrical and material non-linearities, rendering the modeling task substantially more complex. Indeed, the original, free outer surface of the isolator is transformed into a deformed shape, clearly non-identical to the original surface, by the application of the static load—a geometrical non-linearity. The geometrical non-linearity results in a nonlinearity in the axial stiffness due to geometrical changes. In addition, the response to a superimposed dynamic excitation displays a seemingly anisotropic and non-homogeneous feature in the transformed state—a material non-linearity. The material non-linearity results in a nonlinearity in the axial stiffness due to nonlinearity in the stress–strain relation for the material itself. A way to account for the pre-compression dependence is by adopting a nonlinear approach based on single integral viscoelastic constitutive theory. The mechanical response of two natural rubber compounds were examined ,10 under100Hz .Although good results were obtained using the aforementioned models, the intrinsic non-linearity of the theory of pre-strained isolators, involving a series of complex mathematical difficulties, renders its application arduous. Furthermore, the use of non-linear finite element methods makes the results difficult to interpret as no closed-form solutions are obtained.The present paper proposes a waveguide approach for the dynamic stiffness modeling of natural rubber vibration isolators accounting for both the pre-compression and frequency dependencies. This original, mathematically cumbersome task is simplified by transforming the complex shape of the pre-compressed natural rubber isolator into a globally equivalent, homogeneous, and isotropic form, combined with a straightforward waveguide approach to satisfy the boundary conditions by mode matching. A fractional standard linear solid is applied while using a Mittag–Leffler function as the stress relaxation function . The proH and diameter The studied natural rubber isolator in t is time.Consider the vibration isolator, a simple body consisting of continuously distributed natural rubber material occupying a reference configuration Accounting for the nearly incompressibility of natural rubber, an approximate globally equivalent configuration is derived from the pre-compressed natural rubber isolator configuration The non-linear relation between the quasi-static axial force nt of u0  readsu˜=grIn passing, alternative and equivalent forms of the equilibrium shear modulus and the non-dimensional relaxation density are  modulus  is a fra Koeller  for moreThe displacement field is provided by the Helmholtz theorem , readings.Using the traction-free boundary condition along the radial surface The dispersion relation solution  is calcuThe potential fields solutions to Helmholtz equations (Equations  and 11)11)) can The boundary conditions at the cylinder ends are satisfied by mode matching the above fields.The boundary conditions for the pre-compressed, transformed natural rubber isolator undergoing a superimposed infinitesimal motion relation . Using , , a, aΔφ:S→Srelation , the reqand readu˜·er=0aer=0andu˜·ez=−Δuon ∂d1S;u˜·er=0aer=0andu˜·ez=0,y, using , 18), , , , ,,40), 4444, 45),,45), 4747, and  is to ap order j , the connditions , 45),   W=J0Ξ1−Jditions (∑n=1NDn+e)=0,and∑n=1NDn+eThe relations  to 53)  53) and ibration .N while satisfying the traction free boundary condition at the radial surface exactly and the displacement and traction boundary conditions at the cylinder ends in mean. They globally represent the dynamic stiffness for a pre-compressed natural rubber vibration isolator as given by a waveguide model, thereby reducing an original, mathematically arduous, and complex non-linear problem into a vastly simpler assignment.Finally, the transfer and driving point stiffness readpression  with r=Rhey readk˜tω;u0=∑0)/2andk˜dpω;u0=Lahey Fortran 90®  with all calculations performed in double precision and the results presented using Matlab® . Two test objects are studied with a diameter of The formulas above are implemented in The natural rubber material applied in this numerical investigation is supposed to be identical to that of Kari et al. —namely, To obtain accurate stiffness results, the number of modes is required to be large; R at the point The expression  slices tEquation ) into  edge boundary condition on The displacement and stress fields at the cylinder ends and Clearly, the displacement boundary conditions are well satisfied for Likewise, the normalized axial and shear stress Clearly, the stress boundary conditions are well satisfied for relation . Howeverquations  and 64)1<r/R≤r0  and tougThe dynamic stiffness solutions are shown to converge for a moderately high number of modes—such as The dynamic stiffness is found to depend strongly on the frequency displaying resonance phenomena such as peaks and troughs;The dynamic stiffness is found to depend strongly on the pre-compression, displaying low-frequency stiffness magnitude stiffness increase in addition to peak and trough shifts with increased pre-compressions;The waveguide model is shown to yield dynamic stiffness results close to those of the non-linear finite element method;The non-linear finite element method was previously shown to give results close to those of the experiments for similar natural rubber vibration isolators ;The applied Mittag–Leffler shear relaxation function was previously shown to give shear modulus results close to those of the experiments for the same natural rubber material as studied in this paper ; andThe free-free cylinder edge boundary condition is shown to result in superior pre-compressed dynamic stiffness results.A waveguide model is developed for the dynamic stiffness of pre-compressed natural rubber vibration isolators by transforming the isolator into a globally equivalent homogeneous and isotropic form, where the boundary conditions are satisfied by mode matching. The conclusions are as follows:An interesting continuation of the work presented is to apply the proposed method to other excitation directions, such as torsional and shear, and to other natural rubber vibration isolators, such as strips and rectangular shaped natural rubber vibration isolators. Another possible extension is to additionally include physically and chemically aging of the natural rubber, for example, by applying shear modulus models that incorporate physical and chemical aging ,53.


For En/DMT ≥ 18 µm cut-off (at any pre-rejection visit), the Cox proportional hazard ratio was 6.89 , and it increased to 9.91  with a ≥ 19 µm cut-off. In high-risk corneal transplants, the increase in En/DMT allowed predicting rejection prior to the clinical diagnosis.To determine whether measurements of Endothelium/Descemet complex thickness (En/DMT) are of predictive value for corneal graft rejection after high-risk corneal transplantation, we conducted this prospective, single-center, observational case series including sixty eyes (60 patients) at high risk for corneal graft rejection (GR) because of previous immunologic graft failure or having at least two quadrants of stromal vascularization. Patients underwent corneal transplant. At 1st, 3rd, 6th, 9th, and 12th postoperative month, HD-OCT imaging of the cornea was performed, and the corneal status was determined clinically at each visit by a masked cornea specialist. Custom-built segmentation tomography algorithm was used to measure the central En/DMT. Relationships between baseline factors and En/DMT were explored. Time dependent covariate Cox survival regression was used to assess the effect of post-operative En/DMT changes during follow up. A longitudinal repeated measures model was used to assess the relationship between En/DMT and graft status. Outcome measures included graft rejection, central Endothelium/Descemet’s complex thickness, and central corneal thickness (CCT). In patients with GR (35%), the central En/DMT increased significantly 5.3 months  prior to the clinical diagnosis of GR, while it remained stable in patients without GR. During the 1-year follow up, the rejected grafts have higher mean pre-rejection En/DMTs ( Despite the use of immunosuppressants, graft failure rate secondary to a rejection varies from 5% in low-risk penetrating corneal transplant (PK) after five years to 35% in high-risk PK at three years1. These failures inflict a heavy burden on the patients’ productivity and the health care system1. Therefore, early diagnosis of graft rejection and aggressive therapy with corticosteroids are crucial to long-term graft survival for high-risk corneal transplant recipients.Corneal graft rejection is the most common cause of graft failure in the intermediate and late postoperative period. Up to 30% of the yearly 60,000 corneal transplant patients experience at least one episode of rejection3. Increased central corneal thickness may offer an early warning of rejection; however, central corneal thickness alone is not a reliable index of graft immunological status5. Using specular microscopy, a significant decline in the endothelial cell density in severe graft rejection was reported, while mild rejection episodes were not associated with a loss in cell density surpassing the expected6. Using contact confocal microscopy, central immune cell densities have been reported to increase prior to graft rejection via manual counting7. Likewise, aqueous humor cytokine levels have been reported to increase during rejection of PK8. In addition, retrospective analysis of specular microscopy and Scheimpflug imaging has been shown to improve detection of allograft rejection following DMEK9. Using anterior segment OCT (AS-OCT), endothelial/Descemet membrane complex thickness (En/DMT) has been reported to accurately assess the immunological status of the corneal graft as compared to both endothelial cell density, and central corneal thickness11.Early diagnosis of graft rejection is necessary to initiate the treatment before irreversible endothelial cell damage to improve graft survivals15. Using this tool, the three-dimensional thickness of the endothelial/Descemet membrane complex has shown excellent sensitivity and specificity in diagnosing corneal graft rejection that strongly correlated with the rejection severity13. Hereafter, we hypothesized that En/DMT could potentially reveal early changes that herald corneal graft rejection. In this study, we report on the use of segmental tomography algorithm, and examine the relationship between the central three-dimensional (3D) En/DMT and graft rejection in this population at high risk for rejection.Recently, we developed and validated a segmental tomography algorithm to generate 3D thickness maps of different corneal layers using AS-OCT corneal scans to enhance the diagnosis of different corneal diseasesThis study was approved by the University of Miami Institutional Review Board. All participants provided written informed consent before enrollment. The study design followed the tenets of the Declaration of Helsinki for biomedical research.2. Corneas eligible for transplantation were from donors aged 10 to 70 years with a preoperative endothelial cell density (ECD) of at least 2500 cells/mm2 determined by the eye bank. Surgical technique and perioperative care were provided according to each surgeon’s customary routine. Every patient was required to complete regularly scheduled postoperative ophthalmic examinations at 1 week, 2 weeks, and 1, 3, 6, 9, and 12 months after surgery. Patients were instructed to contact the surgeon immediately if they had a dropped vision, redness, photophobia, or pain. At each visit, the slit-lamp examination was performed on each eye by a cornea specialist, masked to the OCT data, to assign the examined cornea into either a healthy, rejection or failed category. Corneal graft rejections were considered as the primary endpoint in this study. Endothelial graft rejection was diagnosed by detecting new keratic precipitates (KPs) or a Khodadoust line in the presence of anterior chamber cells and new persistent corneal graft edema in a graft that was previously clear2. Failed graft was considered if there was an irreversible loss of central graft clarity for a minimum of 3 consecutive months after endothelial rejection. Exclusion criteria included epithelial or stromal rejections and microbial infection after corneal transplantation.Sixty eyes of 60 patients were prospectively and consecutively recruited from 2016 to August 2019 at Bascom Palmer Eye Institute, University of Miami. For inclusion in the study, eyes were required to have a high-risk feature for graft rejection as described elsewhere16. To confirm optimal centration, each candidate was asked to fix at a target to visualize a saturation artifact in all images. The fixation target was customized in decentered grafts and post-PK eyes with high irregular astigmatism to maintain the geometric centration of the scan upon the graft. A custom-built segmental tomography algorithm was used to automatically generate the 3D thickness of the En/DM in the central 2 mm region of the corneal transplant. The automatic segmentation was checked by 2 experts, masked to the clinical diagnosis, with manual editing of segmented images if needed. We have reported excellent reliability of our image processing techniques for the total corneal thickness and En/DMT measurements in healthy corneas, Fuchs’ endothelial cell dystrophy, and corneal graft rejection16. In Brief, Random Sample Consensus (RANSAC)17 method with a polynomial model is used to estimate the borders of corneal layers. Then, reconstruction of the corneal layers is done via bi-cubic interpolation18. Lastly, 3D ray-tracing algorithm is used to generate the mean 3D thickness values of the central 2-mm region of the corneal transplant20.Anterior segment HD-OCT  with 6 mm radial cuts centered on the corneal vertex was performed at 1st, 3rd, 6th, 9th, and 12th month after surgery. The characteristics of this device were described in our previous work21.Central 3D-En/DM complex and central corneal thickness were the diagnostic indices generated from the segmented HD-OCT images. We previously reported the 2 interfaces of the En/DM as the two most posterior hyperreflective bands of the cornea on HD-OCT imagesStatistical analyses were performed using SPSS software version 22.0  to calculate descriptive statistics for all eyes. The obtained CCT measurements were verified to have a normal distribution by assessment of histograms and a Shapiro–Wilk’s test of normality, while En/DMT was not. Therefore, mean ± standard deviations (SD) were used to characterize the distribution of the corneal thickness values, while the median values were used to characterize En/DMT.Multivariate analyses were used to explore the relationships between baseline  factors and En/DMT. Longitudinal repeated measures models were used to evaluate En/DMT change throughout the follow-up. The final multivariable model was generated through a stepwise selection of covariates at a significance level of 0.05.We used time-dependent covariate Cox survival regression to assess the effect of post-operative En/DMT changes on the risk of graft rejection. For this purpose, a variety of cut points for En/DMT thickness, measured at the 1, 3, and 6 months follow-up visits were specified prior to data analysis. These were included in a series of time-dependent proportional hazards models to estimate the increased risk of graft rejection with attainment of each En/DMT thickness cut point, postoperatively. The time-dependent Cox analyses estimate the increase in risk associated with each En/DMT cutpoint, regardless of follow up time. They are expressed as ratios of rejection risk in eyes with En/DMT thicker than the cutpoint to eyes with En/DMT thinner than the cutpoint. These risk ratios apply whenever the En/DMT thickness is observed to be greater than the cutpoint and are not specific to any particular post-surgical follow up time.The mean (± SD) age of the 60 patients included in the analysis was 59 ± 3 years, and 35 (58%) were males. At the beginning of the study, these participants underwent corneal transplantation  for the following indications: previous graft failure (35%), therapeutic (23%), pseudophakic bullous keratopathy (20%), herpetic eye disease (12%), vascularized leucoma-adherent after repaired corneal rupture (8%), and Stevens-Johnson Syndrome (2%). Table p = 0.01), as compared to the healthy grafts while the mean pre-rejection CCTs showed no significant changes (p = 0.7). It is shown that En/DMT was predictive of graft rejection with larger En/DMT values among the 21(35%) cases whose graft subsequently rejected compared with 39 non-rejection cases.All HD-OCT images were segmented successfully, and no manual editing was required by both observers. Regarding the dynamic changes in En/DMT and central corneal thickness (CCT), Fig. p = 0.002). Increasing En/DMT cut-off to 19 µm, the risk increases to 9.91 . This data-driven analysis determined that once En/DMT thickened to 19 µm or greater the risk of graft rejection increased by a factor of almost 10. Of the 21 grafts that were rejected, 17 (81%) had a postoperative progressively increasing En/DMT ≥ 19 µm prior to rejection, while of 39 grafts remaining clear only 7 (18%) had a postoperative stable En/DMT ≥ 19 µm as compared to their respective baseline measurements. For cases where En/DMT thickened to 19 µm, graft rejection did not necessarily follow immediately, the range of intervals was 2 to 11 months (mean = 5.3). The time dependent Cox analysis produces estimates for increased risk that are not specific to follow up time; therefore, the observation of an En/DMT thicker than 18um or 19um is concerning whenever it occurs during follow up.Time-dependent covariate Cox survival regression was used to assess the effect of post-operative En/DMT changes during follow-up. We explored the increased risk of graft rejection after En/DMT thickening with a variety of cut-points Table . Using tQualitatively, Fig. p = 0.026), which did not affect the significance of En/DMT (still p < 0.001). In this model, the risk of rejection increases by a factor of 10.9 times  after En/DMT reaches a thickness of 19 µm. The effect of age was that risk of rejection decreases by a factor of 0.19 , about 20% with a 10-year increase in age. The only other variable which came close to statistical significance was transplant type (p = 0.079) but it did not enter the model because p > 0.05.Running the time-dependent covariate analysis with the En/DMT cut point set to 19 µm and allowing forward stepwise inclusion of all the continuous variables as well as the indication for and type of the corneal transplant was done. Of these, the only variable that entered the model was age . This thickening could be explained by a resolved subtle rejection episode that might have occurred in-between the follow-up visits and was not evident during the clinical examination. In our series, the risk of rejection decreases about 20% with a 10-year increase in age, in agreement with Vabres et al.29 and Alexandre et al.30 who reported that the rejection rate decreases with aging. Future studies are required to allow better utilization of En/DMT as a way of evaluating prophylaxis or treatment measures for graft rejection. For example, if low-grade rejection or inflammation exists as a cause of graft failure, more frequent steroid treatment might be evaluated based on En/DMT measurements.In the current study, we reported the predictive role of the central 3D thickness of En/DM in corneal graft rejection. We have included high-risk corneal transplants due to the higher rates of rejection. PK, DSAEK and DSAEK under PK were included in our study. We reported a 35% one-year rate of rejection in our cohort, within the range of those reported in other series of patients who underwent high-risk corneal transplantThere are a few limitations in our study. First, our results stemmed from a limited number of patients, and a larger study is required to replicate our results with longer follow-up time. Second, owing to the high-risk nature of the recipient beds, our study was limited to the central En/DMT due to the poor signal to noise ratio in the periphery of the OCT images that hinder the ability to delineate the corneal En/DM consistently in all the frames. Hypothetically, an ongoing rejection in the periphery could be missed. Despite this limitation, central En/DMT was found to increase at least 2 months prior to clinically detected rejection. Third, our cohort was restricted to eyes at high-risk of graft rejection. Eyes at low to mid risk of graft rejection following corneal transplantation should be included in future studies with longer follow-up duration. Lastly, the influence of possible confounding factors on the results of our series such as differences in surgical techniques could not be assessed.In conclusion, in our series, a major advantage for En/DMT compared to CT is that a one-time measurement could predict graft rejection in high-risk corneal transplants during the first year postoperatively. The progressive thickening and the appearance of nodular excrescences of the En/DM compared to the previous visit should raise a red flag of an upcoming or existing rejection. Also, as an objective parameter of graft immunological status, we believe that En/DMT would be appropriately included in any future studies examining graft survival after a high-risk corneal transplantation.This study was approved by the University of Miami Institutional Review Board.


Exercise is a critical factor that impacts arterial stiffness. In this narrative review, we noted multiple findings that could not be reconciled with one another. Some studies indicated that arterial stiffness increases after a regimen of resistance training. However, such studies were limited by a lack of specification of the resistance training protocols, as well as varying results reported from different areas of the body, undermining the internal validity of the studies. Another factor explored in this review was how the order of performing exercises can affect arterial stiffness. Low-intensity resistance training before high-intensity resistance training resulted in increased arterial stiffness, whereas vice versa showed no change in arterial stiffness. Other studies indicated that resistance exercise results in reduced arterial stiffness. Intensity is a variable in studies that produces inconsistent results of arterial stiffness, with some studies suggesting high-intensity resistance training increases arterial stiffness and low-intensity resistance training decreases arterial stiffness, while other studies pointing to a significant decrease in arterial stiffness, regardless of the intensity of resistance training. Demographic factors such as gender, age, and diet play an important role in explaining these differences. In terms of future implications, there is potential clinical significance as increased arterial stiffness serves as a prognostic marker in diagnosing coronary heart disease. It is widely known that cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity in developing countries. In the United States alone, CVDs have led to the death of 600,000 individuals, which is approximately 25% of the total deaths . SeveralExercise affects the cardiovascular system in a multitude of ways. Dynamic exercise results in an increase in heart rate, an increase in stroke volume, an increase in cardiac output, and a reduction in cholesterol . There aA loss of arterial stiffness is a major independent cause of common chronic cardiovascular diseases and increased morbidity and mortality. Arterial stiffness indicates the rigidity of the blood vessels around the heart. Low stiffness implies that there is low pressure exerted on the walls of the arteries or blood vessels during systole . If the Arterial stiffness is associated with changes in the mechanical properties of the arterial wall . These aSeveral risks are associated with increased arterial stiffness. Increased arterial stiffness serves as a prognostic marker in diagnosing coronary heart disease . MultiplAlthough it is commonly understood that resistance training increases arterial stiffness, there remains a gap in knowledge regarding the overall effects of resistance training on arterial stiffness. Thus, this review aims to analyze 23 randomized controlled trials to determine the relationship between full-body resistance training and arterial stiffness. Our efforts to understand the impact of resistance training on one of the major causes of CVDs can pave the way for future medical research and advancements.Stiffness associated with large arteries is a proven risk factor for CVD . PatientSignificant increases in arterial stiffnessWithin the review, out of the 23 randomized controlled trials analyzed, seven showed significant increases in arterial stiffness after a regimen of resistance training -22. HoweSignificant decreases in arterial stiffnessIn our analysis, six out of the 23 papers reported a decrease in arterial stiffness after resistance training . OkamotoNo significant changes in arterial stiffnessIn our review, 10 papers out of the 23 showed no significant changes to arterial stiffness as a result of resistance training -38. ThreThe conclusions drawn from this study challenge the current understanding of the effect of resistance training on arterial stiffness. The analysis of the 23 randomized controlled trials found that the majority of the studies claim that resistance training has no significant impact on arterial stiffness. However, it is important to acknowledge that multiple conclusions were drawn from this meta-analysis. The type of resistance training performed had an effect on the central pulse wave velocity values. Those that recorded arterial stiffness values found major differences while training distinct parts of the body, leading to concerns regarding the reliability of the study. Some studies indicated that hypertension may have either led to an increase in arterial stiffness or resulted in no change. Moreover, the order in which the resistance training is performed can also impact arterial stiffness. Although a few studies reported that resistance training did not have an effect on the arterial stiffness of obese individuals, some claimed that diet composition can affect central and peripheral arterial stiffness. A hypocaloric diet was found to reduce arterial stiffness, and a high-fat diet with resistance training decreased peripheral arterial stiffness and increased it when there was no training involved. The current data will allow future researchers to identify various factors  that need to be considered when determining the effect of resistance training on arterial stiffness. Additionally, future investigations should also include a larger sample size that takes racial and ethnic variation of the participants into account before drawing any conclusions. Considering that a loss of arterial stiffness is a major factor in CVDs, this data can be beneficial in developing new and advanced treatment plans that involve resistance training.


The resurgence of heroin use and the misuse of pharmaceutical opioids are some of the reasons for a worldwide increase in opioid dependence. Opioid Medication Therapies (OMT) have amply demonstrated their efficacy. From a medical point of view, the main objectives of OMT concern medical and social outcomes, centred on risk reduction and the cessation of opioid use. But patient points of view can differ and few studies have explored opioid-dependent patient viewpoints on their OMT. This variable seems important to consider in a patient-centred approach. The aim of our study was to explore points of view of people who use drugs (PWUD) treated with OMT, in a large multicentre sample.A cross-sectional multicentre study explored the points of view of PWUD with Opioid Use Disorder following OMT. Data regarding the patients’ points of view were collected using a self-administered questionnaire developed by the scientific committee of the study. A descriptive analysis and an exploratory factor analysis were performed to explore the structure of items exploring patient viewpoints.263 opioid dependent PWUD were included, a majority were men consuming heroin prior to being prescribed OMT. 68% were on methadone, 32% were on buprenorphine. Most PWUD identified a positive impact on their lives, with 92.8% agreeing or strongly agreeing that OMT had changed a lot of things in their lives. The exploratory factor analysis identified three factors: (F1) items related to points of views concerning the objectives and efficacy of OMT; (F2) items related to the legitimacy of OMT as a treatment compared to a drug, (F3) items related to experiences and relationships with OMT.Patient viewpoints on efficacy were correlated with the pharmacological benefits of OMT and with the associated psychosocial measures. The implications of OMT in relationships, such as the feeling of being judged, concerned a majority. Points of view were ambivalent concerning the role of OMT as a treatment or as a drug. Involving patient points of view in therapeutic strategies decisions could help enhance positive views among PWUD on OMT and help PWUD towards their recovery.Trial registration: OPAL study was registered: (NCT01847729). The resurgence of heroin use in some countries such as the United States, and the misuse of pharmaceutical opioids, such as fentanyl, are some of the reasons for the current “opioid crisis” –3. The nOUD is associated with rapidly-appearing, strong withdrawal symptoms and craving, both responsible for continuing opioid use and unsuccessful attempts to stop consumption, despite PWUD s' awareness of the damage . OUD treBoth OMT drugs have proved to be efficacious in terms of public health, access to care and risk reduction, particularly in France where buprenorphine is widely available on prescription by any physician , 30, 31.OMT prescription has different objectives: (1) to avoid opioid withdrawal symptoms, (2) to block the effects of illicit opioids, (3) to reduce opioid craving, to stop or reduce the use of illicit opioids and to prevent relapse, (4) to stop drug injection to reduce risks of infection, (5) to promote and facilitate patient commitment to recovery-oriented activities including psychosocial interventions , 37, 38.In literature, barriers to OMT are explored, but in majority regarding access to care for PWUD, and care provider training, or care organization , 46. TheThe aim of our study was to explore the points of view of PWUD treated with OMT for at least six months, in a large multicentre sample.OPAL NCT01847729) was an observational, cross-sectional, multicentre study involving 10 centres located in the Western region of France. It combined a clinical evaluation 729 was a. OPAL waDSM-IV) [The study included PWUD 18 years or older, receiving OMT (methadone or buprenorphine) for at least 6 months for opioid dependence (according to the DSM-IV) . The 6-mDSM-IV) . PWUD atThe clinical evaluation consisted of a hetero-assessment (structured interview conducted by one of the investigators) and a self-assessment (self-administered questionnaires completed by the patient). Different clinical data were collected, including impulsivity and ADHD evaluations. The results have been published previously . Only vaWe collected data on gender, age, educational level, marital and parental status, housing, social support, professional status, and financial situation.Data concerning the main substance used was collected , the main route of administration , and negative consequences related to opioid dependence .OMT characteristics were collected .PWUD were asked about the current frequency of any co-addictions . Current co-addictions were considered in case of nicotine dependence (Fagerström nicotine dependence test ), and/orA questionnaire was developed by the scientific committee of the study to explore the patients' subjective points of view .A descriptive statistical analysis of the sociodemographic and clinical characteristics was conducted for the entire sample. Continuous variables were described by means and standard deviations if Gaussian and by quantiles otherwise. Categorical variables were presented by numbers and percentages.An exploratory factor analysis was then performed to explore the structure of the items reflecting the patients’ points of view concerning OMT. A principal component analysis with varimax rotation was performed to explore the structure of variables related to patients’ opinions on OMT. Factor loadings were considered as meaningful if they were above a 0.3 threshold in absolute value. The assumption was that there would be two components, one related to the perception of OMT as a medication as opposed to a drug, and one related to the perception of OMT as a means of social control as opposed to a means of controlling addiction.263 PWUD (opiate-dependent) were included in the study. Descriptive data of the sample is presented in Table OMT Success defined as an Opioid abstinence without other substance use or gambling**getting worse Unsuccessful OMT defined as either Persistence of opioid use without other substance use or gambling getting worse , or Persistence of opioid use with other substance use or gambling getting worse  or Opioid abstinence but other substance use or gambling getting worse .Successful or unsuccessful OMT defined in a previous article : *OMT SuA descriptive analysis of patient viewpoints on their OMT is presented in Table to points of view concerning the objectives and efficacy of OMT.The first factor (F1) included items related legitimacy of OMT as a treatment as opposed to a drug.The second factor (F2) included items related to the items related to experiences and relationships with OMT use.The third factor (F3) included Finally, four items did not load on any of the three components.A Principal Component Analysis led us to prefer a three-component model rather than the expected two-component model. The three components explained 33.62% of the total variance. The factor loading estimates for the three-component solution after varimax rotation are presented in Table The main objective of this study was to collect points of view on their treatment from PWUD on OMT prescribed for an OUD. Points of view were collected from 263 opioid-dependent patients.The main result of the study was that most PWUD had positive points of view on the efficacy of their OMT. First, a great majority identified a positive impact on their lives, as 92.8% agreed or strongly agreed that OMT had changed a lot of things in their lives. Only 11.2% agreed or strongly agreed that OMT had induced health disorders. OMT occasionally causes severe side effects,  that can deteriorate patients’ quality of life , which mIndeed, OMT is a pharmacological treatment, but it is not just that . Most ofRegarding relationships with health professionals, we also showed that 51.4% of the sample agreed or strongly agreed that they had already felt judged by health professionals for their OMT. In addition, 61.8% of the sample agreed with the feeling that health professionals did not seem sufficiently informed on OMT. These results could seem surprising, as OMT has now shown considerable benefit to public health, and has been authorized in France for more than 20 years. But it could also reflect the fact that addiction is still not considered as a chronic disease, and opiate use disorders are seen as a social problem rather than a medical problem .It could also reflect a lack of training of health professionals on this topic. Many physicians feel ill-prepared when prescribing OMT  or lack In the PCA, the items concerning feelings of being judged by health professionals, and feelings that health professionals were not sufficiently informed about OMT, were correlated with the item about feelings of being judged by relatives. These items constitute a "relationship factor" in our study, and could reflect the patients’ personal experiences of their treatment, and the fear of being judged. The question of identity is central to the management of OUD. Relating to an "addict identity" is exciting, it is associated with a free spirit and is not bound by conventional expectations, which contrasts with PWUD who relate to a treatment identity. OMT can be perceived as a loss of autonomy  by the PRegarding pharmaceutical diversion of OMT, it is considered no more considered in literature as a "problem" but as an alternative perspective in the PWUD trajectories , 69. DivThe views and medical training on OUD treatments need to change, and more particularly concerning heroin dependence . OUD treWe also showed the importance of support from physicians prescribing OMT, which was associated with the "efficacy factor". This identified factor showed that PWUD had integrated the short-term objective of alleviation of withdrawal symptoms, which is an important step in treatment, as failure in this management is associated with poorer outcomes . But PWUIn our study, as shown in the preliminary study , the pat. showed that in a methadone maintenance treatment program, there was low concordance between the patients’ perceptions and the clinical staff’s perceptions of improvement [This "treatment factor" and the patients’ points of view correspond to an axis previously defined in the literature concerning the role of OMT perceived by PWUD and also by physicians either as a medical treatment or as a legal drug , 42, 54.rovement . One hyprovement , 78. In rovement , MTD androvement .Another variable that could influence patients’ perceptions of OMT is the way OMT is prescribed. Despite the public health impact, patterns of OMT prescription can lead to poor quality treatment. Inadequate doses of OMT can contribute to poorer outcomes, and to treatment dissatisfaction among PWUD . GuideliAnother hypothesis is that this situation is the result of differences between 2 theoretical points of view on OMT. Dole and Nyswander suggested a "medical model" of OUD treatment, in which methadone was a medication for treating an acquired disease , 64. In This study collected a large dataset concerning PWUD with OUD and OMT. To the best of our knowledge, this is the first study to explore this topic in such a broad manner, as this topic is rare in the literature, particularly with such a large sample. The PWUD included filled in the self-report questionnaire on their points of view on OMT, which could reflect patients’ interest in this topic. Despite a single-nationality sample, which could limit the representativeness and extrapolation of the results, our recruitment was multicentre with a variety of centres with different practices . This provided heterogeneity in the population. Heterogeneity of the sample was intended in order to improve its representativeness of real-life OUD PWUD in treatment in specialized centres. Missing data was marginal, and the number of PWUD enabled statistical analyses with sufficient power. This study was based on a global evaluation of the PWUD and an exhaustive assessment of substance and behavioural addictive disorders.Our study has certain limitations. First, its design was cross-sectional with recruitment in specialized centres. The sample combined many severity factors, and was not representative of the PWUD under OMT in France, and the sample did not match national data, where the majority of opiate-dependent PWUD receive buprenorphine via pharmacy-based delivery, prescribed for two-thirds by family physicians , 84, 85.The results of this study underline how patient expectations and points of view on efficacy are coherent with and correlated to the pharmacological benefits of OMT, and to the combined psychosocial measures. Patient implication in treatment decisions, a patient-centred approach to individualized goals, with an adaptation of pharmacological prescription and delivery modes could certainly help enhance positive points of views among PWUD on OMT and help PWUD towards their recovery.


A 3‐D biomechanical movement analysis of treadmill running was also conducted. RE was on average 47.8 and 62.3 ml O2 min−1 kg−1 at 14 and 18 km h−1. Maximal difference between the individual athletes was 21% at 18 km h−1. Mechanical work rate was significantly correlated with VO2 measured in L min−1 at both running velocities. However, RE and relative work rate were not significantly correlated. LAch was significantly correlated with RE at 18 km h−1 implying that a short moment arm is advantageous regarding RE. Neither foot volume nor shank volume were significantly correlated to RE. Relative muscle fascicle length of m. soleus was significantly correlated with RE at 18 km h−1. Whole body stiffness and leg stiffness were significantly correlated with LAch indicating that a short moment arm coincided with high stiffness. It is concluded that a short LAch is correlated with RE. Probably, a short LAch allows for storage of a larger amount of elastic energy in the tendon and influences the force–velocity relation toward a lower contraction velocity.Running economy (RE) at a given submaximal running velocity is defined as oxygen consumption per minute per kg body mass. We investigated RE in a group of 12 male elite runners of national class. In addition to RE at 14 and 18 km h Running economy was found to be significantly related to Achilles tendon moment arm, foot length ratio, whole body stiffness and leg stiffness, and muscular fascicle length. RE can also be normalized with respect to distance as VO2 kg−1 km−1. Normalization to body mass allows for comparisons between individuals. RE is a complex measure, which reflects the combined functioning of biomechanical, anatomical, metabolic and cardio‐respiratory factors  at a specific submaximal running velocity is defined as oxygen consumption (VOLAch) about the ankle joint, another is the ratio between the length of the forefoot and LAch  in the genus Homo . Since 1968, African and especially Kenyan runners have dominated the international scene in middle‐ and long‐distance races to a degree that has been termed the greatest geographical concentration of sports excellence in the annals of sports  was found to be longer in Kenyan than Japanese elite runners  and VO1.32) per kg body mass while running at two different submaximal velocities on a motorized treadmill . The speed of 14 km h−1 was chosen as a “safe” velocity with regard to the expected aerobic capacity of the athletes. The speed of 18 km h−1 was chosen to represent a velocity close to the conditions during competition. After a standardized warm up on the treadmill, the subjects ran at two submaximal running speeds 14 and 18 km h−1, 0% grade, for 4 min separated by 1–2 min rest. During the 4‐min stages, in and expired gases were measured continuously by a gas analyzer . Breath‐by‐breath data were processed by the software system JLab (CareFusion). Running economy (RE) was determined as the mean VO2 (ml kg−1 min−1) during the last minute of each 4‐min bout.Running Economy was determined as the rate of oxygen consumption  was measured by the method presented by Scholz et al. . The length of the forefoot was determined as the mean of two consecutive measurements.Lower leg and foot volume were determined by scanning the lower leg and foot with a hand‐held 3‐D surface scanner  . The lower legs were isolated from the 3‐D model by cutting off everything proximally and distally to the two marker pairs, respectively. The feet were isolated by cuts proximal to the distal markers. The volumes of the 3‐D models of the isolated lower legs and feet were calculated using the Artec Studio software. The volumes of two successful scans of each lower leg were calculated, and the volume of the lower leg was determined as the mean of these. The same procedure was applied to calculate the volume of the foot.Body mass and height were measured using standard procedures and, in addition, the following anthropometric variables on the subjects’ right side were determined: total leg length (from the ground to spina iliaca anterior superior), thigh length , shank length , foot length (from the back of the heel to the tip of the longest toe), forefoot length from the lateral malleolus to the fifth metatarsal joint Figure , and toe1.5Lf) was estimated using a B‐mode ultrasound scanner  and transducer . The vastus lateralis (VL), gastrocnemius medialis (GM), and soleus (SOL) muscle of the subject's right leg were scanned. For VL, the transducer was placed at a point midway between trochanter major and the lateral condyle of the femur. For GM and SOL, the transducer was placed at a point approximately 30% proximally between the medial condyle and the medial malleolus of the tibia and midway between the medial and lateral borders of the GM  was determined as the angle between the deep aponeurosis and the fascicles of the specific muscle  was determined by measuring the distance between the deep and superficial aponeurosis of the specific muscle  and relative to the related segment length (cm cm−1).The 1.6fs), stride length (Ls), contact time (tc), swing time (ts), vertical oscillations, and mechanical work. Thirty‐five spherical reflective markers were placed on selected anatomical landmarks  while recorded by a Qualisys system for movement analysis . Eleven high‐speed infrared cameras (300 Hz) recorded a minimum of 15 steps at each velocity. Three‐dimensional coordinates of the markers were exported to the software system AnyBody , which was used to analyze the recordings. Simultaneously, the athletes were recorded on video (120 frames s−1) and these recordings were later used to obtain contact time, stride frequency, swing time, and stride length. Stride length was calculated as velocity divided by stride frequency.Due to injuries (not related to this study), only 10 of the 12 subjects managed to complete a 3‐D biomechanical analysis of treadmill running to determine stride frequency  till Fmax, which is the peak value of the vertical ground reaction force.Stiffness of the whole body was measured during running as previously described . The level of significance was set to 22max of the athletes are listed in Tables 2max was 67.0 ml O2 kg−1 (range: 61.7–78.2), which confirmed that the athletes were all well‐trained elite runners . Running economy (RE) corrected for resting values was (averaged across subjects) 44.1 and 58.7 ml O2 kg−1 min−1 at 14 and 18 km h−1, respectively  indicating a linear relationship between RE and running velocity as shown before  ml O2 kg−1 min−1 at 14 and 18 km h−1, respectively. Uncorrected VO2max was 70.8 (±4.7) O2 kg−1 min−1.Without correction for resting metabolism, RE was on average 47.8 (±2.8) ml O−1 was on average 66.1% (±5.0) and 87.9% (±4.8) with respect to VO2max  were not correlated with RE Table .Pmech) was 3.41 (0.28) and 3.79 (0.54) W kg−1 for 14 and 18 km h−1, respectively  at 14 km h−1  and at 18 km h−1 . Body mass was also significantly correlated with Pmech at 18 km h−1 .The mechanical work intensity  and at 18 km h−1 Rho was 0.84 (p = 0.0045)  as was Pneg .LAch) was on average 3.91 cm and was significantly correlated with RE at 18 km h−1   length the soleus fascicles showed a significant correlation with RE at 18 km h−1   was 4.1 cm on average and varied from 3.2 to 4.9 cm corresponding to a 36% difference between the subject with the shortest and the subject with the longest fascicles. Similar differences were observed for the gastrocnemius (GM)  and the vastus lateralis (VL) . Individual range for the GM corresponded to 34% and for the VL 29%. No significant correlations were found between absolute fascicle length and RE, neither at 14 nor at 18 km h−1   Table   Table , that is−1 kg−1 at 14 km h−1 and 1240 (±240) N m−1 kg−1 at 18 km h−1. Leg stiffness (kleg) was 900 (±220) and 1200 (±230) N m−1 kg−1. None of these stiffnesses were significantly correlated with RE . Whole body stiffness at 14 km h−1  and at 18 km h−1  was significantly correlated with the Achilles tendon moment arm (LAch)  and at 18 km h−1 . The ratio between whole body stiffness and LAch was significantly correlated with RE at 18 km h−1   was significantly correlated with body mass  and to absolute VO2 (L min−1) .Whole body stiffness normalized to body mass was 930 ±227) N m N m−1 kg33.1−1 (13–14 km h−1). Williams and Cavanagh subdivided their subjects into three groups based on RE at 3.57 m s−1 (approximately 13 km h−1) and observed a trend between relative positive power and three “Physiological Efficiency Groups”  and 264 (53.9) Watt corresponding to 3.41 (0.28) and 3.79 (0.54) W kg−1, respectively  was reported between running economy (RE) at 16 km h−1 and the Achilles tendon moment arm (LAch). In the present study, a significant correlation (Rho = 0.66) was found between LAch and RE at 18 km h−1.In the study of Scholz et al.  a signifLAch)  with RE at 14 km h−1 implying that a short moment arm is advantageous regarding RE. The LAch was on average 4.4 cm for males and 3.5 cm for females with r‐values of 0.82 and 0.81 between RE and LAch. Accordingly, males and females had the same RE despite differences in LAch  of collegiate or national level were examined regarding RE and Achilles tendon moment arm (LAch (4.46 cm) than the Japanese runners . LAch of the Kenyans ranged from approximately 3.6–5.1 cm  was found between LAch and a performance index . This indicated a long moment arm to be an advantage, but notably, RE was not measured directly  and, whr‐value of 0.66, and the previous results from the literature with even higher r‐values  has been reported to have had a RE of 170 ml O2 kg−1 km−1  . In fact, the runners in Spurrs et al.  leg thickness than European runners Saltin, , and it Different animal species exhibit often very different anatomy of the legs. Taylor et al.  calculat3.6−1 when fascicle length was normalized to shank length  in 12 long‐distance runners (Giannakou et al., Long fascicles imply long muscle fibers and more sarcomeres in series. As longer muscle fibers can contract at a higher shortening velocity than shorter fibers this would indicate a more beneficial force–velocity relationship of the muscles in question (Abe et al., −1 in the present study (Figure Finally, the most important feature of longer muscle fibers may be an altering of the force–velocity relation so that the muscle can produce more force at the same shortening velocity (Abe et al., y Figure , so addiy Figure .3.7LAch and stiffness (Barnes et al., A significant correlation between muscle–tendon stiffness and RE was found by Barnes et al. . It was,Sano et al. found longer Achilles tendons in Kenyan than Japanese runners but also longer shanks. The cross‐sectional area of the Achilles tendon was also significantly larger than that of the Japanese (Kunimasa et al., LAch will cause a lower shortening velocity than a longer LAch, simply due to geometry. In this way the required muscle force may be produced by fewer motor units and thereby fewer muscle fibers.Another effect of a short Achilles tendon moment arm may be a positive influence on the force–velocity relation of skeletal muscles. At a given angular motion in the ankle joint during plantar flexion, a shorter 4As the first study, we were able to show a significant correlation between biomechanical calculations of mechanical power and absolute oxygen consumption. However, this correlation did not exist when data were normalized to body mass. This is probably partly due to differences in anthropometry not accounted for in biomechanical movement analysis.The Achilles tendon moment arm is considered highly important for RE as a short moment arm theoretically can be beneficial for both storage of elastic energy and for the force–velocity relation of skeletal muscles. The ratio between forefoot and Achilles tendon moment arm is also significantly correlated with RE due to a beneficial gearing of the foot with respect to the external forces. Stiffness of the whole body and the stance leg is indirectly important for RE as stiffness and Achilles tendon moment arm are significantly correlated. High stiffness of the leg muscles is very likely to favor storage and reuse of elastic energy during running.The authors declare no conflict of interest.C. E. Hansen planned and conducted the experiments, participated in the calculations, discussion of results, and in writing the manuscript. E. B. Simonsen planned and conducted the experiments, participated in the calculations, discussion of results, and in writing the manuscript. M. Stensvig participated in the biomechanical data collection and in writing the manuscript. J. Rasmussen participated in the biomechanical calculations, discussion of results, and in writing the manuscript. J. Wienecke participated in data collection and in writing the manuscript. J. Lorentzen participated in collection of data from ultrasonography, data interpretation, and in writing the manuscript. C. Villa participated in collection and interpretation of surface scans and in writing the manuscript.


Starch from cereal grains, pulse grains, and tubers is a major energy substrate in swine rations constituting up to 55% of the diet. In pigs, starch digestion is initiated by salivary and then pancreatic α-amylase, and has as final step the digestion of disaccharides by the brush-border enzymes in the small intestine that produce monosaccharides (glucose) for absorption. Resistant starch (RS) is the proportion of starch that escapes the enzymatic digestion and absorption in the small intestine. The undigested starch reaches the distal small intestine and hindgut for microbial fermentation, which produces short-chain fatty acids (SCFA) for absorption. SCFA in turn, influence microbial ecology and gut health of pigs. These fermentative metabolites exert their benefits on gut health through promoting growth and proliferation of enterocytes, maintenance of intestinal integrity and thus immunity, and modulation of the microbial community in part by suppressing the growth of pathogenic bacteria while selectively enhancing beneficial microbes. Thus, RS has the potential to confer prebiotic effects and may contribute to the improvement of intestinal health in pigs during the post-weaning period. Despite these benefits to the well-being of pigs, RS has a contradictory effect due to lower energetic efficiency of fermented vs. digested starch absorption products. The varying amount and type of RS interact differently with the digestion process along the gastrointestinal tract affecting its energy efficiency and host physiological responses including feed intake, energy metabolism, and feed efficiency. Results of research indicate that the use of RS as prebiotic may improve gut health and thereby, reduce the incidence of post-weaning diarrhea (PWD) and associated mortality. This review summarizes our current knowledge on the effects of RS on microbial ecology, gut health and growth performance in pigs. In the Starch, the main carbohydrate in cereal and pulse grains, is stored in the amyloplast as discrete granules with distinct morphology in various botanical origins . Starch The term resistant starch (RS) was first used to describe the portion of starch that was cooled in cooked foods but was resistant to digestion by α-amylase . This de+/glucose cotransporter 1 against an electrochemical gradient . In. InBifid digesta . Previou digesta . A meta- digesta  Fig. 1)Bifidobac digesta . Consumphia coli . In pigshia coli . The chahia coli . Finallyuctivity .Table 22, H2, and CH4 [Plant cell wall polysaccharides, oligosaccharides, and resistant starches that are not digested by endogenous enzymes in the small intestine are degraded by microbial polysaccharidases and glycosidases to monomers such as glucose, which are then fermented to produce SCFA  and gases CO and CH4 . Acetate and CH4 , 105. Mi and CH4 .The greatest microbial activity occurs in the cecum and proximal colon of the pigs; however, substantial microbial activity also takes place in the distal section of the small intestine , 108. ThAcross pig studies, diets high in RS or amylose increased intestinal SCFA concentrations. Slowly digestible starch (63% amylose) increased ileal starch and total butyrate content , and SCF, Firmicutes , Bacteroidetes [Escherichia coli, Klebsiella spp., Streptococcus spp., Megasphaera spp., and Acidaminococcus fermentans [Akkermansia muciniphila, a mucin-degrading bacteria, contributed substantially to nitrogen cycling in the small intestine [At weaning, refusal to eat or drastic reduction in feed intake decreased villus height and increased crypt depth, thus reducing brush-border enzyme activities and nutrient digestion , 112. Otroidetes . Proteinrmentans . Akkermantestine . Previountestine , 117. Inntestine .Clostridia [Roseburia spp. and Faecalibacterium prausnitzii to acetate- and propionate-producing bacteria (mainly Bacteroides) [The interaction between diet, intestinal environment and gut bacteria is complex: fermentation of RS leads to increased production of SCFA, which lowers gut pH, thereby affecting microbial composition and in turn, affects both the type and production rate of SCFA  Fig. 1)1). High ostridia , 120. Asostridia . The inceroides) , 123.In pigs, important gastrointestinal development occurs in the first three months after birth , 125. ThPrevotella-rich enterotype that were fed a diet containing high amylose (68% amylose) corn starch had decreased gene expression of inflammatory cytokines in the colonic mucosa [MUC4, MUC5AC, and MUC12) compared with feeding a corn starch diet. Therefore, long-term intake of RS may consequentially improve gut health by increasing mucin secretion and lowering the incidence of bacterial products translocation across the gut barrier [In addition to the potential to alter the diversity and stability of gut microbiota, RS affects the intestinal expression of genes involved in immune regulation and thereby gut health. Among SCFA, especially butyrate produced by RS fermentation serves as the primary energy source for the colonocytes, and regarded as the modulator of intestinal barrier function and immunity , 132. Rec mucosa . Feedingc mucosa . In multc mucosa –137. Butc mucosa , 139. Lo barrier , 140. Us barrier . Pro-inf barrier .in vitro starch digestion was used to accurately predict the kinetics of net portal vein appearance of glucose in pigs by applying the corrections for gastric emptying and intestinal glucose utilization [Apparent total tract digestibility of starch is mostly complete; however, apparent ileal digestibility of starch differs depending on its botanical origin , 143. Felization .Resistant starches affect host glucose and lipid metabolism by altering the profile of metabolites in systemic circulation such as glucose, cholesterols, triglycerides, and SCFA. Pigs fed diets containing a wide range of amylose or RS have a reduction in postprandial glucose and insulin responses, hence, indicating that a specific starch characteristic may not entirely explain the starch effect on postprandial glucose. In portal vein-catheterized pigs, the net portal appearance of glucose, insulin, C-peptide, and glucose-dependent insulinotropic polypeptide was lowered in pigs fed slowly digestible starch . InsulinIn mammalian species, evidence is accumulating that SCFA production directly affects pancreatic secretion of insulin . The SCFAn earlier study in humans identified that dietary inclusion of 5.4% RS (high amylose corn starch) increased fermentation products, SCFA, and fat oxidation, and thereby may decrease fat accumulation in the long term . In pigsin-vitro enzymatically-degradable starch [Previously, RS that is fermented to SCFA was shown to be approximately 17% less efficient than starch that are digested and absorbed as glucose from the small intestine . In the e starch . Using ie starch . The dife starch . The eque starch , 175. Hoe starch , 173. Pae starch –179  rate of passage of digesta or transit time  and (2) ad-libitum access to feed, 30% greater RS intake did not affect feed intake pattern and growth rate of pigs likely because microbial-produced SCFA were absorbed and available as energy source thereby sparing glucose from oxidation [Findings on satiety-related effects of RS or SCFA in pigs are rather limited and inconsistent. Rapidly digestible starch stimulated insulin release that consequently inhibited feed intake and increased adiposity . In contxidation . In growxidation . Separatxidation . Overallin vitro and in vivo studies support the view that RS acts as prebiotic to modulate gut microbiota by changing the intestinal microbial composition and function. In addition, specific bacteria phyla are associated with fermentation of RS and increased SCFA production particularly butyrate. Results of experiments with humans, rats, and pigs demonstrated that benefits of dietary RS on gut health may include increased markers of mucosal barrier function, immune tolerance, and increased abundance of beneficial gut bacteria. This area of research has progressed considerably; however, its benefits during disease challenge remain unclear. Indeed, additional in vivo experiments are required to provide stronger evidence linking prebiotic effects of RS with reduced PWD and mortality. Despite the potential benefits of RS fermentation in the hindgut, utilization of SCFA resulting from RS fermentation reduces energetic efficiency compared with mostly glucose from digested starch and may thus reduce growth. To achieve a balance between increased prebiotic activity and sustained growth performance of pigs, the optimum dietary inclusion of RS, most appropriate source(s) of RS, and duration of supplementation of RS need to be determined. Nevertheless, the current state of knowledge indicates that RS as a prebiotic can enhance gut health of weaned pigs, thereby reducing the incidence of PWD. The evidence linking certain bacterial taxa and its functionality to growth and feed efficiency indicates that manipulation of gut microbiota with dietary inclusion of RS may affect pig growth performance. However, reduced PWD and especially reduced mortality following such diarrhea due to dietary RS as prebiotic is more relevant economically than dietary inclusion of RS to improve feed efficiency. Effects of feeding RS on pig performance may differ depending on the health status of pigs  and gut development, thus, different optimum levels may have to be established for each growth phase and physiological stage .Resistant starch is the proportion of starch that escapes small intestine digestion producing mostly glucose but instead undergoes microbial fermentation in the distal small intestine and colon producing SCFA. Results of