Small-for-size graft (SFSG) syndrome after living donor liver transplantation (LDLT) is the dysfunction of a small graft, characterized by coagulopathy, cholestasis, ascites, and encephalopathy. It is a serious complication of LDLT and usually triggered by excessive portal flow transmitted to the allograft in the postperfusion setting, resulting in sinusoidal congestion and hemorrhage. Portal overflow injures the liver directly through nutrient excess, endothelial activation, and sinusoidal shear stress, and indirectly through arterial vasoconstriction. These conditions may be attenuated with portal flow modulation. Attempts have been made to control excessive portal flow to the SFSG, including simultaneous splenectomy, splenic artery ligation, hemi-portocaval shunt, and pharmacological manipulation, with positive outcomes. Currently, a donor liver is considered a SFSG when the graft-to-recipient weight ratio is less than 0.8 or the ratio of the graft volume to the standard liver volume is less than 40%. A strategy for transplanting SFSG safely into recipients and avoiding extensive surgery in the living donor could effectively address the donor shortage. We review the literature and assess our current knowledge of and strategies for portal flow modulation in LDLT. Due to severe organ shortages and the increasing gap between supply and demand, living donor liver transplantation (LDLT) has become an accepted alternative to expand the donor pool -4. VariaCurrently, a donor liver is considered a SFSG when the graft-to-recipient weight ratio (GRWR) is less than 0.8% or the ratio of the graft volume (GV) to the standard liver volume (SLV) is less than 40% , 20. FacOne Korean group no longer measures portal pressures routinely. Rather, they consider establishing excellent venous outflow, preventing acute rejection, and having young donors to be key mechanisms for avoiding SFSG syndrome in grafts with a GRWR as small as 0.7%. That group noticed that successful SFSGs were from young donors with young recipients in whom perfect venous outflow was ensured . They maIt is important to consider donor safety and avoid subjecting healthy living donors to excessive surgery –28. A stGenerally, splenectomy is performed to reduce the bleeding tendency resulting from thrombocytopenia, or as part of surgical procedures such as devascularization of the upper stomach and esophageal transection to control variceal hemorrhage. It is also performed for hematologic disease such as idiopathic thrombocytopenic purpura, splenic tumors such as malignant lymphoma, trauma, splenic artery aneurysm, and liver or renal transplantation from an ABO-incompatible donor . In roden = 50) [Simultaneous splenectomy during LDLT improves graft outcomes by reducing the portal pressure and flow and increasing the vascular compliance of the graft –38. We pn = 50) . SFSG syn = 50) . Therefon = 50) . They fon = 50) . Survivan = 50) . In mostn = 50) .In Japan, the benefits of simultaneous splenectomy in LDLT are well documented, whereas the negative effects, including potential adverse events such as portal venous thrombosis (PVT), infectious complications, pancreatic fistula, and postoperative bleeding, are not discussed in detail . Ito et 3 /mm3), and spleen weight was correlated with splenic vein diameter and white cell count [PVT is a severe complication of LDLT that can result in increased morbidity and mortality . The incll count .The relationship between splenectomy and an increased incidence of PVT after LDLT is not clear. Kurata et al. reported a very high incidence of PVT (33.3%) after LDLT with splenectomy, but no incidence after LDLT without splenectomy . They coSplenic artery ligation (SAL) was initially applied to prevent thrombocytopenia in LDLT . Troisi Compensatory portosystemic shunts develop in about 40% of patients with cirrhosis and the frequency increases with severity , 63. LarTo prevent graft failure resulting from portal hyperperfusion, diversion of the superior mesenteric flow with a mesocaval shunt was first described for LDLT using a SFSG . Animal Pharmacologic manipulation is another method of portal flow modulation. This method is reversible, unlike surgical procedures such as splenectomy or SAL. Propranolol and somatostatin decrease both portal flow and portal pressure reliably and reproducibly in cirrhotic patients ; therefoIn conclusion, portal flow modulation with a prudent combination of shunt control such as ligation or hemi-portocaval shunt, a portal decompression procedure such as simultaneous splenectomy or SAL, and pharmacologic manipulation, is crucial when performing LDLT with an SFSG.Tomoharu Yoshizumi and Masaki Mori have no conflicts of interest to declare.


Objective: Descemet membrane endothelial keratoplasty (DMEK) is the gold standard procedure for treatment of primary corneal endothelial disorders. Graft detachment is a frequent complication of DMEK, which often requires re-operation or re-bubbling. However, several cases of spontaneous corneal clearance despite graft detachment after DMEK have been reported. The underlying mechanisms of this phenomenon are poorly understood. We report three cases of corneal clearance after graft detachment in patients with Fuchs endothelial dystrophy and provide a review of the literature. Methods: An 81-year-old and a 69-year-old phakic patient as well as a 56-year-old pseudophakic patient with Fuchs endothelial dystrophy underwent Triple-DMEK and DMEK, respectively. All three patients presented postoperatively with blurred vision due to an almost complete detachment of the graft, as shown by slit-lamp photography and anterior segment optical coherence tomography (OCT). Results: Without additional intervention, gradual corneal clearance and presence of endothelial cells on the posterior recipient’s stroma were observed in all patients three months postoperatively. Increase in endothelial cell density, decrease in central corneal thickness (CCT), recovery of corneal transparency, and improvement of visual acuity were documented in all patients.Conclusions: Our findings support the theory of corneal clearance after Descemet membrane endothelial transfer (DMET) (“free-floating” donor Descemet graft in the recipient anterior chamber after descemetorhexis). Further understanding on endothelial homeostasis might lead to innovative approaches in handling endothelial disorders.  Descemet membrane endothelial keratoplasty (DMEK) is a standardized technique, which is utilized for treatment of primary or/and secondary corneal endothelial disorders  such as DMEK is described as a superior technique over other lamellar techniques like Descemet’s stripping automated endothelial keratoplasty (DSAEK) for treatment of corneal endothelial disorders , due to A successful outcome of DMEK has been strongly associated with the adherence of the donor graft to the posterior surface of the corneal stroma , 5]. Th. Th5]. TRemarkably, successful outcomes after DMEK, despite graft detachment, have also been reported. In this report, we present three cases of positive outcome, following late graft detachment, left without re-intervention and we attempt a comprehensive discussion of this condition by providing a review of the literature. An 81-year-old phakic female, a 69-years-old phakic male, and a 56-year-old pseudophakic female presented in our clinic with progressive deterioration of visual acuity, epiphora and photophobia of both eyes. ® Scheimpflug camera.In all patients, preoperative slit-lamp examination revealed corneal edema with extensive cornea guttata in both eyes, caused by Fuchs endothelial dystrophy. A best-corrected visual acuity (BCVA) of 4/20, 6/20 and 2/20 was measured in the worst eye of each patient, respectively. In all patients, reliable measurements of endothelial cell density by corneal specular microscopy could not be obtained due to the advanced stage of endothelial dysfunction. Central corneal thickness (CCT) was 687 µm, 606 µm, and 832 µm, respectively, as measured by Oculus PentacamThe first two patients underwent Triple-DMEK (phacoemulsification and intraocular lens implantation combined with DMEK) while the third one underwent single DMEK as described by Melles and colleagues . In all The first and the third patient were discharged on the fifth postoperative day without presentation of any complications and with attached graft as documented by anterior segment OCT. In the second patient, re-bubbling was necessary due to incomplete attachment of the graft. During the re-bubbling procedure, the graft was accidentally partially folded. This patient was discharged after absorption of the air bubble. Approximately one week following discharge, all three patients were referred to our clinic again with photophobia and increased blurry vision. Slit-lamp examination demonstrated diffuse corneal edema and corneal decompensation, highly likely as a result of graft detachment. Anterior segment OCT Figure 1  confirmeWe followed a “wait & watch” approach in all three patients and close follow-up visits were undertaken. Remarkably, gradual corneal clearance was observed within three months in all patients. Upon slit lamp examination three months postoperatively, the corneal stroma was more transparent in all three patients, despite the fact that it was covered neither from donor tissue nor from the patient’s Descemet’s membrane. Central corneal thickness demonstrated a significant decrease from 687 to 572 µm (17%) in the first patient, a moderate decrease from 606 to 556 µm (8%) in the second one while in the third patient a more significant decrease from 832 to 580 µm (30%) was documented Figure 2 . Those changes in corneal pachymetry were accompanied by an increase of visual acuity from 4/20 to 8/20 in the first patient, from 6/20 to 12/20 in the second and from 2/20 to 4/20 in the third one within the same time period. Although a marked improvement of the CCT was seen in all three cases, BCVA has not been a reliable outcome parameter. The reduced visual acuity despite the more advanced corneal clearance in the second and third patient was attributed to a concomitant retinal disorder and central retinal vein occlusion, respectively. Furthermore, the “free-floating” Descemet graft was positioned within the visual axis, causing blurry vision. However, despite the detached donor tissue, enlarged and irregularly shaped endothelial cells were found by noncontact corneal specular microscopy on the recipient’s posterior corneal stroma in all patients Figure 3 .The above described positive findings were maintained over one-year follow-up, despite the still detached grafts. However, re-DMEK was performed after 14 months in the first patient, with successful graft attachment and a final visual acuity of 20/20. The second and third patient refused further interventions.In this case report, we present three cases of spontaneous corneal clearance, improvement of BCVA and corneal re-endothelialization after almost complete graft detachment following DMEK, in three patients with Fuchs endothelial dystrophy. A decrease in central corneal thickness together with a presence of endothelial cells on the recipient’s posterior stroma was observed three months after the diagnosis of graft detachment without any additional operative intervention.Similar to our observations, corneal transparency and improvement of BCVA along with presence of functional endothelial cells on the posterior stroma, despite graft detachment, have been previously reported. Of note, such positive outcomes were observed independently of the time of diagnosis and the extent of graft detachment. Dirisamer et al. described the involved patterns of corneal re-endothelialization following complicated DMEK, categorizing them in 4 groups depending on the extent of detachment and the positive or negative outcome . CornealEndothelial cell regeneration has also been discussed as a possible mechanism of re-endothelialization, found after a solely mini central stripping of Descemet’s membrane in patients with focal corneal edema without keratoplasty as well as in eyes with corneal graft detachment after DSAEK . in vivo confocal laser scanning microscopy. Comparison of the morphology of the endothelial cells between the denuded areas and the opposite or adjacent corneal quadrants revealed that the endothelial cells covering the patient’s denuded corneal stroma resembled those of the donor graft. This observation means that endothelial cells might migrate from the donor graft to the denuded corneal recipient’s stroma. However, this theory fails to explain the different response after DMET of patients with Fuchs endothelial dystrophy and bullous keratopathy.Jacobi et al.  addresseBased on the above theories, we assume that the healthy endothelial cells seen in our patients are the result of either cell migration from the detached donor graft onto the posterior recipient’s stroma, regeneration of the remaining endothelial host’s cells, or a combination of both. The concept of regeneration allows the assumption that the endothelial cells themselves may not be primarily dystrophic but their dysfunction may be the result of specific conditions . PresencIn conclusion, spontaneous recovery of corneal transparency along with improvement of visual acuity and increase in endothelial cell density is possible after graft detachment following DMEK, even without any further intervention. Such positive outcomes can be attributed to the increased endothelial cell repopulation by migration or/and by regeneration. These findings merit further study in regard with the corneal endothelial homeostasis in order to develop innovative approaches in handling endothelial disorders.BCVA: best-corrected visual acuityDMEK: Descemet membrane endothelial keratoplastyDMET: Descemet membrane endothelial transferDSAEK: Descemet’s stripping automated endothelial keratoplastyCCT: central corneal thicknessre-DMEK: repeat Descemet membrane endothelial keratoplastyTriple-DMEK: phacoemulsification and intraocular lens implantation combined with DMEKOCT: optical coherence tomographyThe authors declare that they have no competing interests.


Back in June 2009 the first issue of “Facts, Views and Vision in Obgyn” (FV&V) was published as an international journal with a new concept in the world of scientific journals.With the paramount goal of reaching our public and in the scope of a globalized world, we opted to offer an open access journal, rendering the content of each and every of our published issues free and available without any charge to the user or his/her institution, plus allowing the reader to download, copy, distribute, print, search, or link full texts and content of the articles in this journal without asking for prior permission from the publisher or the author.From the very beginning it was clear that the scope in our papers would be different from that in most scientific journals. Indeed, we were convinced with the promise of offering a unique forum for socio-cultural, ethical, philosophical, health-economical and political debates related to our work as gynaecologists and reproductive health care providers. Considering the facts: original studies, pilot-studies, reviews and PhD summaries became the backbone of our journal. We witnessed the publication of many viewpoints and opinion papers critically commenting the facts provided in the literature and last but-not-least we also published many strong opinions of international key opinion leaders, shedding a visionary dimension to what currently raises interest in the broad medical field of human reproduction. With surprise, we were fascinated to observe that many papers grasped lots of interest in the global perspective of our specialty, thus, reaching our paramount aim.Thanks to our artistic reviewer, Koen Vanmechelen, FV&V realised a different but very attractive look compared to other scientific journals. Indeed, the confrontation of art and science turned out to be something unique, a cross-breeding at its best.As a major achievement in the history of our Journal, in 2014, we succeeded to become PubMed-cited. Nowadays the Journal is covered by major indexing services such as PMC , Pubmed, Web of Science, Google Scholar, BMJ Clinical Evidence and Europe PMC.During the past 10 years, from 582 submissions, 310 papers have been published. As such, our journal extended an acceptance rate of 53.2 % with more than 20 % of our publications (66/310) being “Opinion and Viewpoint” articles.After 10 years, we can only be positively surprised about the number of citations we have achieved, a good parameter of the impact of our papers on the actual scientific literature of obstetrics, gynaecology andreproductive health .From 2019 onwards, our Journal will be owned by the European Society for Gynaecological Endoscopy (ESGE) in cooperation with the Walking Egg non-profit organization. Hence, it will become the official Journal of the ESGE, with a majority of papers related to the surgical aspects of endoscopic imaging and allied techniques. Another part of the Journal will be creserved to continue publishing selected papers as we did before.Additionally, from its very beginning our journal has embraced all ObGyn disciplines and placed a major interest in images, art contributions and visionary ideas. Therefore, we believe that FV&V has provided and will continue to provide the perfect scientific environment to initiate the use of video’s, a novelty in our scientific community.With much satisfaction, we are still associated on our artistic reviewer, Koen Vanmechelen, who is responsible for the very attractive and unique look of our journal. Koen is one of most influential contemporary artists today; finding his inspiration in the wide crossroad of art, science, politics, philosophy and ethics.Opinion and viewpoint papers will continue to be confronted with his artistic interpretation to further stimulate and encourage discussions in a truly holistic context, thus, adding a unique dimension to our journal.As the Editor-in-Chief, I look back on 10 exciting years and therefore I would like to thank the editorial board, all the authors and referees who contributed to the journal since 2009. Also, allow me to express my special gratitude to the ‘Universa Press’ team for their highly dedicated work.It has been an honour and a real pleasure to be part of this exciting project. A bright future for our journal is awaiting us.Editor-in-ChiefWillem Ombelet


To assess the advantages offered by the air pump assisted PDEK technique that utilizes pressurized Anterior Chamber (AC) air infusion.Pressurized air infusion was provided through an anterior chamber maintainer connected to the fluid air exchange system of a posterior vitrectomy machine during surgery.Pressurized air infusion within the AC helped perform Descemetorhexis, prevented bleeding during Peripheral Iridectomy (PI) and synechiolysis, prevented oozing of blood from peripheral corneal neovascularization into the AC and thus helped maintain a non-fibrinous AC environment. In addition, it helped in precise graft manipulation, centration, edge unfolding and unwrinkling after it was floated against the stroma as well as faster graft adhesion. It also prevented AC depth fluctuations during intra-cameral maneuvers and prevented intra-operative as well as post-operative graft detachment.This technique makes several steps of surgery easier and improves graft adhesion. This facilitates graft handling within the Anterior Chamber (AC) and makes the procedure comparable in ease to DSAEK , 10 whil2Institutional review board approval was obtained and procedure confirmed to declaration of Helsinki. Informed consent was obtained from all patients. Data was analyzed using Microsoft Excel .2.1The corneo-scleral graft was placed endothelium side up on a flat surface. A 30 gauge needle mounted on a 5 ml syringe half filled with air was bent bevel-up and inserted into the scleral part of the rim close to the limbus. It was then moved forward superficially such that the bevel of the needle lay completely within the peripheral part of the donor cornea. Air was then injected quickly in order to create a type 1 big bubble. If required, the bubble was further expanded carefully using either air, storage medium or 0.1% Trypan blue  after moving the needle into the bubble space. A 15 degree blade was used to enter the bubble at the base and Trypan blue was injected into the space to stain the graft. The graft was then cut all around its base with a curved Vannas scissor held flat to obtain a PDEK graft. The graft was stored again in storage medium till further use.2.21 and 2).A 1 mm paracentesis incision was created tangentially such that the Anterior Chamber Maintainer (ACM) could be placed directed towards the angle rather than to the centre of the AC. The fluid air exchange system in a posterior vitrectomy system was used to provide pressurized air infusion into the AC through the ACM. Because of continuous leakage of air, especially when any instruments were passed through the incisions, air pressure on the gauge of the machine was adjusted to obtain slightly higher than physiological IOP within the anterior chamber as checked by finger pressure. A partial entry, either clear corneal or scleral tunnel was created using a 2.8 mm bevel up keratome. Under air, an inverse Descemetorhexis was performed using a reverse Sinskey hook. In cases with extensive and long standing bullous keratopathy with thickened DM and largely non-functional endothelium, the entire host DM was stripped. In eyes with early bullous keratopathy, the area of DM stripped was planned to be 1 mm larger than the PDEK graft size. Gross edge irregularities and DM tags were smoothened out by localized Descemet's stripping with the reverse Sinskey hook or a microforceps. Under air, a small inferior Peripheral Iridectomy (PI) was made using the vitrector at 400 cuts per second and vacuum of 200 mmHg. Air was replaced with BSS once any iris bleeding stopped and a bubble test was performed to assess stability of the air bubble in the AC. In cases with iris defects or where the air bubble was unstable and tended to migrate posterior to the iris, the ACM was connected to balanced salt solution and a pupilloplasty was performed to attain a stable iris IOL diaphragm as described by Jacob et al in 2014 . The ACM32 and at 6 months was 1916±309 cells/mm2 .12 patients were included in the study. Ten patients had pseudophakic bullous keratopathy, one patient had ICE syndrome, while one had viral endothelitis. All patients underwent underwent air pump assisted PDEK, 4 patients also required additional synechiolysis and three patients required pupilloplasty to reform the iris-IOL diaphgram. Continuous pressurized air infusion helped perform intra-operative steps and tamponaded any intra-ocular bleeding during PI and synechiolysis. Two patients had partial graft detachment. One was small and peripheral and reattached spontaneously while the other needed rebubbling on the slitlamp. All corneas were clear post-operatively. The patients were followed up for 6 months. Spectacle corrected distance visual acuity improved from 0.13±0.13 to 0.62±0.31 post-operatively. Pre-operative donor specular microscopy was 3033±74 cells/mm4Graft handling, folds and wrinkles in the floated graft, edge folds and decentred graft are major intra-operative challenges for both PDEK and DMEK as is graft detachment post-operatively. The use of continuous pressurized air infusion makes PDEK easier by helping perform various steps in a reproducible manner. It thus makes PDEK an easier surgery to adopt and more repeatable.Descemetorhexis initiated under an air bubble -16 or co16 This is especially important as young donor grafts can scroll tighter and increased manipulation may be required if attempting to unfold the graft completely before floatation, sometimes still resulting in unsuccessful edge-to-edge unscrolling. Increasing graft manipulation can result in increased cell loss. Air pump assisted PDEK is therefore helpful in allowing earlier flotation of graft followed by handling of graft edge folds, wrinkles and creases with a reverse Sinskey hook after floatation. Care should however be taken to avoid repeated, rough or large areas of graft touch with the reverse Sinskey hook which can increase endothelial cell loss. In our cases, we found graft edge unfolding easy to perform under pressurized air infusion once some graft adhesion had initiated and it was generally possible with single touch points, only at the extreme edges of the graft. Care should however be taken to ensure graft centration before graft adhesion initiates as this may become difficult later. Though similar manoeuvres may be performed in DMEK with very low infusion pressures, in our experience, DMEK grafts had a higher rate of graft wrinkling and graft edge tearing because of the thinner and more fragile nature of the graft. Hence this technique, if used, should be performed only with great caution in DMEK.The air-pump assisted PDEK technique is more forgiving with respect to graft edge folds and decentration as the sturdy nature of the graft allows these to be adjusted once the graft is floated up against the stroma. The graft can therefore be floated earlier without having to ensure perfect centration or an edge-to-edge unfolded graft unlike in DMEK. A third hand effect is obtained that prevents AC depth fluctuations and the graft is prevented from detaching by the air pressure during various manoeuvres. Graft centration and unfolding of edge folds therefore becomes easier.Pressurized air infusion also allows adequate operating space within the AC that allows easy insertion of instruments without causing damage to the graft endothelium. The set infusion pressure on the machine does not equate to the intra-cameral pressure achieved because of continuous leakage through the incisions. The infusion pressure on the machine is therefore set to achieve a slightly higher than physiological intra-cameral pressure estimated clinically by gently indenting the cornea. This slightly higher air pressure within the AC also allows earlier and better graft adherenceOne of the disadvantages of this study was the absence of comparison with a control group where pressurized air infusion was not used. Future, randomized, comparative studies assessing long term endothelial cell loss between these two groups is therefore desirable. Further studies are also required on the duration of the pressure rise and the quantum of pressure rise required in different patient scenarios and correlating this with endothelial counts.To conclude, air pump assisted PDEK increases the ease, rapidity and success of surgery while decreasing complications and acting as a third hand within the AC to stabilize the graft for certain important manoeuvres. Addition of this newer technique will help the learning surgeon to perform the surgery with greater ease and confidence while also helping the established surgeon.


Starch is the main storage carbohydrate in plants and an important natural resource for food, feed and industrial raw materials. However, the details regarding the pathway for starch biosynthesis and the diversity of biosynthetic enzymes involved in this process are poorly understood. This study uses a comprehensive phylogenetic analysis of 74 sequenced plant genomes to revisit the evolutionary history of the genes encoding ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE) and starch de-branching enzyme (DBE). Additionally, the protein structures and expression patterns of these four core genes in starch biosynthesis were studied to determine their functional differences. The results showed that AGPase, SS, SBE and DBE have undergone complicated evolutionary processes in plants and that gene/genome duplications are responsible for the observed differences in isoform numbers. A structure analysis of these proteins suggested that the deletion/mutation of amino acids in some active sites resulted in not only structural variation but also sub-functionalization or neo-functionalization. Expression profiling indicated that AGPase-, SS-, SBE- and DBE-encoding genes exhibit spatio-temporally divergent expression patterns related to the composition of functional complexes in starch biosynthesis. This study provides a comprehensive atlas of the starch biosynthetic pathway, and these data should support future studies aimed at increasing understanding of starch biosynthesis and the functional evolutionary divergence of AGPase, SS, SBE, and DBE in plants. Additionally, starch is an important resource for agriculture, human consumption and industry. For example, it is a major contributor to the harvestable starch-storing organs of crop plants, which include cereal seeds , tubers  and storage roots . Moreover, starch provides abundant calories for human diet and animal feed and is an economical, biodegradable and renewable industrial raw material2.Starch is the predominant reserve form of carbohydrate and energy in plants and can be divided into two types, transitory starch and storage starch, based on biological function. In photosynthetic tissues, transient starches accumulate in chloroplasts during the day. During the night, they are then transported and degraded to provide energy and nutritional substances for growth and metabolism. In non-photosynthetic tissues, such as seed endosperm, tubers and storage roots, storage starches are kept for long periods of time in specialized plastids termed amyloplasts, from which they can be remobilized in preparation for germination, sprouting or regrowth3. Linear amylose adjacent parallel side chains are distributed in the semi-crystalline matrix formed by amylopectin, and this organization underlies the semi-crystalline structure of starch1. Thus, the amylose:amylopectin ratio has a major influence on the appearance and structure of starch granules and also affects the quality of crop storage organs, food production and industrial applications.Starch consists of two types of polysaccharides: amylose and amylopectin. Amylose is a linear polymer composed of α-1,4-linked glucan chains and has very few branches connected by α-1,6-glycosidic bonds. The amylopectin molecule is larger than the amylose molecule and contains abundant α-1,6-branches that connect α-1,4-linked glucan chains and make up a structural framework of repeated amorphous and crystalline lamellaein vitro)5. SS can be further divided into granule-bound starch synthase (GBSS), which is responsible for the synthesis of amylose and the extra-long-chain fraction of amylopectin, and soluble starch synthase (SSS), which is mainly responsible for the synthesis of amylopectin7. SBEs belong to the α-amylase family, the branching activity of which is regulated by Q-enzyme, which introduces a branched structure by cleaving the α-1,4-glucan chain in polyglucans and then reattaching the cleaved chain onto an acceptor chain via an a-1,6-glucan linkage, thereby creating a branch in the same or another chain8. DBEs are another glucan-modifying enzyme that occurs in two forms, namely, isoamylase-type DBE (ISA) and pullulanase-type DBE (PUL). The most important functional difference between these forms is that ISA generally acts upon phytoglycogen and amylopectin by hydrolysing the α-1,6-linkages of polyglucans, which play important roles in the modification of excessively branched chains or the removal of improper branches of amylopectin formed by branching enzymes to maintain the cluster structure of amylopectin. Moreover, ISA likely provides branched chains for amylose. PUL usually cleaves the α-1,6-linkages of polyglucans in pullulan and, to a lesser degree, amylopectin, and exerts little or no activity towards glycogen9. Recent studies have suggested that the plastidial pathway of starch synthesis exists in all extant higher plants and green algae and that the starch biosynthetic enzymes of higher plants underwent a complex sequence of changes during evolution10. Moreover, the isoform types and functionality of starch biosynthetic enzymes are remarkably similar to those found in green algae10. This similarity indicates that in starch biosynthetic enzyme genes, the functional regions or sites that control starch synthesis are relatively well-conserved because these lineages diverged from a common ancestor.Starch biosynthesis is a complex and highly regulated process that requires coordinated activities among multiple enzymes, including ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE) and starch de-branching enzyme (DBE) Fig. . AGPase,11. Both the GT5 and GT1 domains belong to the GT-B superfamily according to the CAZy database (http://www.cazy.org/), and they possess conserved amino acid residues that can bind the glucosyl donor (ADP-Glc). These enzymes usually merge into the base catalytic region of starch synthases. All SBEs and DBEs belong to glycoside hydrolase family 13 (GH13), an important member of clan GH-H, which is also known as the α-amylase family (Aamy). These enzymes not only share an Aamy domain but also possess conserved carbohydrate-binding module family 48 domains in the N-terminal sequence. Moreover, SBEs have retained a C-terminal β-sheet catalytic domain (Aamy_C), while pullulanase (PUL) has a DUF3372 domain located in the N-terminal sequence, and this domain may play important roles in recognition and/or interaction with certain substrates or be involved in modulating PUL activity and interacting with other starch biosynthetic enzymes in specific environmental conditions13.In plants, AGPase subunits share a common nucleoside triphosphate (NTP) transferase domain, which allows AGPase to transfer nucleotides from one compound to another, providing substrates for starch biosynthesis. SSs share a highly conserved core region located in the C-terminus that generally consists of conserved starch catalytic glucosyl transferase family 5 (GT5) and GT1 domains, which mediate an inverting mode of glucosyl transfer during glucosyl transferase14, potato15, Arabidopsis thaliana16, barley17, wheat19, and rice21. However, far less research has been devoted to the core regulatory network involved in starch metabolism. This paucity of research means that limited information is available to assist breeders and biotechnologists in improving and increasing starch content in a predictable manner. In the present study, we present and discuss the starch synthesis network, which is regulated by multiple starch biosynthetic enzymes, and evolutionary patterns in starch biosynthetic enzymes in 74 plant genomes. By combining data related to the structures, functions and expression patterns of maize starch biosynthetic enzyme genes, we reveal previously undetected information about starch biosynthetic enzymes and the starch synthesis network. It is anticipated that these results will enhance our understanding of the starch synthesis process.Many previous studies have reported enzymes that are directly involved in starch biosynthesis in algaePorphyra umbilicalis genome  domain Fig. . An anal26. Sulfate is an inhibitor of the potato tuber ADP-Glc PPase α subunit homotetramer under certain conditions26. Three areas were found that could interact with the sulfate in maize AGPase subunits. The first sulfate-binding area involved Arg107, Arg119, Asp469, Lys470 and Lys507; the Lys470 was replaced by Met in AGPLS1 and AGPLS2 and by His313 in AGPSS4 To explore the evolutionary relationships of SSs, a phylogenetic tree was constructed using SS-encoding protein sequences collected from 74 plant species Fig. . The phy27  and rice GBSSI ets Fig. . Furtherets Fig. . Althougets Fig. . The disEscherichia coli glycogen synthase (EcGS) and barley SSI protein sequences  and EcGS, we found that the binding sites for ADP and glucose were conserved in maize GBSS isoforms except for residues Lys462, Phe463, Asn464 and Ile490  domain, a central catalytic domain (α-amylase) characterized by a (β/α)ain Fig. . Moreoveain Fig. . Additioain Fig. .Figure 833. The results indicated that some of the binding sites for maltopentaose and glucose were not conserved among SBE isoforms, and the alternative residues were mainly found in SBEIII III Fig. . The catIII Fig. . These pIII Fig. .DBE protein sequences from ancestral lineages of algae, mosses, ferns, monocots and eudicots were used to construct a phylogenetic tree. The analysis showed that DBEs could be clustered into four clades, clade I, clade II, clade III and clade IV, which corresponded to ISAIII, ISAI, ISAII and PUL of maize, respectively  and wheat PUL (74% identity with maize PUL)36. The analysis showed that the binding and catalytic sites of maltotriose and maltopentaose were not conserved in ISAII and that partial residues were changed in ISAIII , Rhodophyceae  and Chloroplastidae 45. In particular, glaucophytes and red algae produce and store starch in the cytoplasm, whereas green algae and all land plants perform starch biosynthesis and then store starches in the plastid compartment46.Initial starch biosynthesis can be traced back to plastid endosymbiosis in photosynthetic eukaryotes. A primary endosymbiotic event occurred in a heterotrophic eukaryotic cell that internalized a cyanobacterial cell, bringing plastids into eukaryotes, thereby rendering them able to perform oxygenic photosynthesis during the continuous evolution of cyanobacterial endosymbionts (cyanobionts)47. Furthermore, the spatio-temporal expression profiles of AGPase gene subunits also indicate that the potential combinations of AGPLS and AGPSS are different in different locations during starch synthesis  was found in the amylose extender (ae1.1) mutant, and SBEIIb deficiencies have been shown to affect SSI and SBEI binding to starch granules53. Moreover, subsequent experiments showed that PHO1 interacts with SBEI or SBEIIb54. The outcome of the amylose extender (ae1.2) mutation experiment also showed that SSI, SSIIa, SBEI and SBEIIb form another multiple enzyme complex, and there were obvious differences in amylose content and granule size between two ae mutations from two near-isogenic maize lines55. Furthermore, a high molecular weight, multiple enzyme complex composed of SSIIa, SSIII, SBEIIa and SBEIIb was found in maize and was shown to further assemble into a 670-kD complex by interacting with pyruvate orthophosphate dikinase (PPDK) and the sucrose synthase isoform SUS-SH133. A lack of SSIIIa function, as in the maize dull mutation, caused a decrease in the activity of SBEIIa and SSII. Researchers were also able to use SSIII to co-precipitate the small subunit of AGPase encoded by bt2, and the large subunit of AGPase encoded by sh2 was recovered from an SSIIIHD affinity column56. In particular, a temporal expression pattern analysis of genes in the 670-kD complex revealed that while the expression levels of SSIIa, SSI and SBEIIb were similar in maize endosperm, they were out of sync in embryos and whole seeds , Vitales (Vitis vinifera Genoscope), Ranunculales (Aquilegia coerulea), Bromeliaceae (Ananas comosus), Zingiberales (Musa acuminata), Amborellales (Amborella trichopoda) and Selaginellales (Selaginella moellendorffii). The phylogeny of these species is provided in Supplementary Table Zea mays. L) hybrid Shandan 609 and the inbred line B73, which were derived from our previously published (BioProject accession number PRJNA299361) and openly published papers 38.In the present study, the sequenced genomes and corresponding proteomes of 74 plant species were used to establish an initial data set., These included twelve species from Fabales, ten from Brassicales, three from Malvales, two citrus species, six from Malpighiales, six aster species, two from Caryophyllales, twelve from Graminales, two from Arecales, three bryophytes, nine chlorophytes, and single genomes and proteomes from Myrtales . All hits obtained using the PFAM (http:// pfam.xfam.org/search), CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), and SMART (http://smart.embl-heidelberg.de/) databases were further verified. Sequences that did not have a detectable domain or a threshold E-value of less than 1e-10 were excluded. Only the longest transcript was retained when two or more transcripts were identified from the alternative splicing of a gene. Finally, the genes verified by the above steps were used in this study.Candidate AGPase, SS, SBE and DBE genes were initially identified using HMMER3.0 with default settings  for each of the 74 proteome data sets62. The obtained alignments were then manually corrected in MEGA 7.0 software63. Phylogenetic trees were constructed using the maximum likelihood method with PhyML 3.0, the substitution model was assessed with the Akaike information criterion, and the reliability of internal nodes was evaluated by calculating Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT) values64. This evaluation method has been shown to be an accurate, powerful and robust tool for processing large data sets65. Finally, the phylogenetic tree was annotated and visualized using iTOL v366.Amino acid sequences of all identified candidate AGPase, SS, SBE and DBE genes were aligned in ClustalW v2.1 with default settingshttp://meme-suite.org/tools/meme). The following parameters were used: repetitions per sequence = zero or one occurrence per sequence and the number of motifs selected was based on an E-value less than 10e-10 and other parameters as default settings.All amino acid sequences of maize AGPase, SS, SBE and DBE genes were analysed with MEME (v4.11.4) to discover novel conserved patterns (https://swissmodel.expasy.org/) and RCSBPDB (http://www.rcsb.org/pdb/home/home.do) databases to search for the best reference model. Then, the PDB files corresponding to each starch-related enzyme isoform were analysed, visualized, and edited with PyMOL (0.99rc6) software (http://www.pymol.org/).The amino acid sequences of maize AGPase, SS, SBE and DBE isoforms were retrieved and submitted to the SWISS-MODEL (38. The expression levels of the four types of starch metabolism-related enzyme isoform-encoding genes were computed in RPKM (reads per kilobase of exon per million mapped reads) on maize gene models. Then, gene expression values were normalized ) for hierarchical clustering, and the complete linkage method as well as the Euclidean distance measure were used for hierarchical clustering of gene expression profiles with the R function hclust. Additionally, protein sequences of multiple enzyme isoforms were retrieved for protein-protein interaction analysis in string software (v10.5) (https://string-db.org/). The protein-protein interaction network was edited with Cytoscape software 67. All statistical analyses and drawings were performed using the R language (http://www.r-project.org).RNA-seq data were obtained from an article that we previously published and from openly published papers and corresponding libraries that were produced using the methods described in these papersSupplementary InformationSupplementary Dataset 1Supplementary Dataset 2


To develop biological approaches to restore vision, we developed a method of transplanting stem cell-derived retinal tissue into the subretinal space of a large-eye animal model (cat). Human embryonic stem cells (hESC) were differentiated to retinal organoids in a dish. hESC-derived retinal tissue was introduced into the subretinal space of wild-type cats following a pars plana vitrectomy. The cats were systemically immunosuppressed with either prednisolone or prednisolone plus cyclosporine A. The eyes were examined by fundoscopy and spectral-domain optical coherence tomography imaging for adverse effects due to the presence of the subretinal grafts. Immunohistochemistry was done with antibodies to retinal and human markers to delineate graft survival, differentiation, and integration into cat retina. We successfully delivered hESC-derived retinal tissue into the subretinal space of the cat eye. We observed strong infiltration of immune cells in the graft and surrounding tissue in the cats treated with prednisolone. In contrast, we showed better survival and low immune response to the graft in cats treated with prednisolone plus cyclosporine A. Immunohistochemistry with antibodies  revealed large number of graft-derived fibers connecting the graft and the host. We also show presence of human-specific synaptophysin puncta in the cat retina. This work demonstrates feasibility of engrafting hESC-derived retinal tissue into the subretinal space of large-eye animal models. Transplanting retinal tissue in degenerating cat retina will enable rapid development of preclinical in vivo work focused on vision restoration. Vision is by far the most dominant and most important sense to all primates, including humans, with 80% or more of all sensory information being perceived by means of sight . Loss ofarea centralis [Advances in regenerative medicine enabled generation of three-dimensional tissues (organoids) , partialentralis ,73,74, wentralis . Graftedentralis , also Brentralis , demonstThe focus of this article is to demonstrate the feasibility of grafting hESC-derived retinal tissue  into the subretinal space of a large-eye animal (wild-type cat) before moving this work to RD cat model. Therefore, in this study, we addressed the following challenges: (i) developing the surgical procedure of grafting tissue in a large eye, (ii) overcoming the immune rejection of the recipient, (iii) achieving maturation of the retinal tissue, and (iv) graft-> host connectivity. The establishment of these approaches leads the way to preclinical therapeutic studies utilizing cat RD models.2 in normoxic conditions (21% oxygen), with the addition of 20 ng/mL human brain-derived neurotrophic factor (BDNF)  and 20 ng/mL human bFGF. Approximately half of media was changed two to three times/week.The hESC line (HES03) was obtained from BioTime, Inc. The cells were maintained in feeder-free conditions using mTeSR1 ,77 with Immunohistochemistry of retinal organoids was done as described earlier . Briefly2. We extracted the temperature plots inside the incubator using the multiuse temperature probe . As a test, we shipped samples of mouse embryonic retina before shipping retinal organoids in exactly the same conditions by overnight FedEx from BioTime  to MSU  , fixed on arrival with freshly prepared 4% PFA for 30 min, processed for histology, and stained with antibodies to Cleaved Caspase 3 and Gamma H2AX [Retinal organoids were stored in Hibernate E medium containing 20 ng/mL human BDNF and 20 ng/mL human glial derived neurotrophic factor (both from R&D Systems)  durimma H2AX . We did All animal procedures were approved by the Institutional Animal Care and Use Committee of the MSU and conducted in accordance with the ARVO Statement for the Use of animals in Ophthalmic and Vision Research. The subretinal transplantation of retinal organoids was performed by a boarded veterinary ophthalmologist (S.P-J.). Cats were anesthetized with isoflurane and placed in dorsal recumbency. The eye was positioned in primary gaze and aseptically prepared for a routine two-port partial 23-gauge vitrectomy. Visualization of the posterior segment was by use of an irrigating vitrectomy lens .2-saturated tissue culture incubator in the culture medium until 10 min before implantation. Organoids were loaded into the injection cannula, a borosilicate tube 1.52 mm outer diameter (OD) and 1.12 mm inner diameter (ID) TW150–4 , directly in surgery room using a syringe attached to the cannula. Large organoids were cut in half (0.3–0.5 mm), while small organoids (which fit into the cannula) were transplanted as whole organoids (5–9 organoids/graft). The dish was placed on a 37°C surgical warming pad during cutting and loading steps. Organoids were injected into the subretinal space under direct visualization. Following placement of the organoids, the sclerotomies were closed using 6–0 Coated Vicryl suture . The conjunctiva and lateral canthus were closed in a routine manner. At the end of the procedure, a subconjunctival injection of a mixture of 0.1 mg dexamethasone , 2 mg methylprednisolone , and 1 mg gentamicin  was performed.Sclerotomies were 5 mm posterior to the limbus. A core vitrectomy  and detachment of the posterior vitreous face over the region of planned implantation were performed and with visualization from triamcinolone crystals  that were previously washed in Balanced Salt Solution . A subretinal injection of BSS was performed using a RetinaJect . The sclerotomy port was enlarged to accommodate the organoid glass injection cannula and a retinotomy performed into the detached retina with retinal scissors to allow entry of the injection cannula into the subretinal space. Organoids were kept at 37°C in 5% COImmunosuppressive therapy consisted of twice daily oral prednisolone  and in the second group of animals, also cyclosporine  starting 3 days before the procedure.Color fundus images were captured using the RetCam II  immediately following the transplantation and periodically thereafter. For sessions following the postoperative imaging, pupils were dilated with 1% tropicamide  and a topical sterile anesthetic applied . Standard light conditions were used for color fundus images.2) with the animals placed on a heating pad maintained at 37°C. A lid speculum and conjunctival stay sutures were used to maintain the globe in primary gaze. Both infrared and autofluorescent cSLO imaging were performed. High resolution line and volume scans were used to record graft and host retina appearance; enhanced depth imaging protocols were used as required.Scanning laser ophthalmoscope (cSLO) and spectral domain optical coherence tomography (SD-OCT), retinal cross-sectional images of the graft were captured  under general anesthesia  were euthanized using pentobarbitone according to the AUF protocol 05/17-075-00. The eyes were enucleated, incisions were made through the pars plana and the globes immersed in 4% paraformaldehyde (Electron Microscope Sciences) in PBS (Sigma-Aldrich Corp.) on ice for 3.5 h. The anterior chambers were then removed and the vitreous removed, and the eyecups were fixed for another 10–30 min in the same fixative depending how much vitreous was left. After three washes of 10 min each in 1× PBS, the cat eyecups were cryoprotected in sucrose solutions prepared in PBS, pH 7.8 . After two washes of 5 min each in 1× PBS, the cat eyecups were then snap-frozen in optimal cutting temperature embedding material  in a beaker partially filled with methanol on liquid nitrogen. They were then stored at −20°C until sectioned.The Microm HM550 cryostat  was used to produce 16 μm serial sections of cat eyes. Microscope slides were purchased from Fisher Scientific . Glass coverslips were purchased from Brain Research Laboratories . Eye cups were serially sectioned at 12 μm.The sections were first permeabilized with 0.1% triton X-100/PBS (PBS-T) at room temperature for 30 min, followed by 1 h of incubation in blocking solution  and 0.1% PBS-T at room temperature, and then incubated with primary antibodies diluted in blocking buffer at 4°C overnight . The folzonula occludens (ZO)-1 [In our earlier work, we differentiated hESC line H1 to retinal tissue using Noggin, DKK1, and IGF1 . Using as (ZO)-1  and pigms (ZO)-1 ,104 -1   60–70, was implanted into the subretinal space of five adult cats . The iniIn the second cohort of cats (subjects 3–5), we transplanted retinal organoids in subretinal space and maintained the cats for 36 days, while treating with a combination of cyclosporine A and prednisolone . ImmunosSD-OCT examination was performed on all transplanted eyes . The retWe first used pilot cohort (prednisolone only) to evaluate the extent of graft survival.In subject 1, the SD-OCT image showed small- to medium-sized graft, whereas in subject 2, one of the two grafts was large. Therefore, we euthanized subject 1 at 28 days, whereas subject 2 was euthanized at 66 days. Presence of grafts in the subretinal space of subjects 1 and 2 was detected by immunostaining with antibodies specific to human markers HNu (human nuclei)  and Ku80Next, we determined the infiltration of inflammatory or immunogenic cells in these grafts. Inflammatory and local immune response were detected by staining the retinal section with anti-ionized calcium-binding adaptor molecule 1 (Iba1)  ,106,107 On the contrary, we observed better graft survival and mild immune response in the subjects 3, 4, and 5, which were treated with a combination of prednisolone and cyclosporine A immunosuppression regimen .Immunostaining with anti-HNu antibody revealed well-defined nuclei with no evidence of nuclei fragmentation. Immunostaining with anti-Iba1 antibody showed low infiltration of Iba1-positive cells in the graft and surrounding area. The number of CD8-positive cells in the graft and surrounding cat retina was low . We obse-aminobutyric acid (GABA), and PMEL17 in the grafts  , human-STo investigate whether these cytoplasmic projections represented young axons projecting from the graft, we co-stained sections with antibody to STEM121 and the antibody to doublecortin (DCX), and performed high-magnification confocal analysis with z-stacking and compression of z-stack, to better visualize the projections in sections. DCX is a marker of early stages of axonal formation ,112. We To determine if STEM121-positive fibers found in the GCL migrate toward the optic nerve, we immunostained cat retina with NF200  and STEM121 antibodies. We found few STEM121-positive fibers running parallel to the host RGC layer . InteresTo determine if STEM121-positive fibers may be developing synapses with host neurons, we stained sections with antibody to human SYP, which can robustly decorate the presynaptic part of human  synapse ,78. We oIn this article, we outline the surgical procedures for grafting hESC-derived retinal tissue from organoids into the subretinal space of the cat eye, an immunosuppression protocol to allow graft survival, and the very promising outcomes of grafting, demonstrating robust survival and axonal connectivity (graft to host and host to graft). This work lays the foundation for developing retinal and vision restoration technologies in a large-eye model, relevant to human therapies.+/−Crx [−/−Aipl1 cat [area centralis, which is analogous to human maculae. The size of an adult cat's eye is very similar to the size of adult human eye [There are suitable spontaneous genetic models of early-onset RD in the cat, such as the Crx+/−  and Aipl1−/− cat , making uman eye , enablinIn this study, we chose to transplant retinal organoids at DD 60–70, which is in line with the work of others ,113. If Initially, we chose to test a mild immunosuppression regimen (prednisolone only) because the subretinal space is an immune-privileged site ,114–117,Although the grafts in the first cohort of cats were shown on IHC to have undergone rejection, the eyes did not show any gross evidence of inflammation. Two of the three grafts in this group decreased markedly in size, highlighting the rapid death of the human grafts in the xenogenic conditions, while the third graft was larger and retained patches of surviving HNu-positive cells. Interestingly, although the grafts in the prednisolone-only group could be seen by SD-OCT, funduscopy, and cSLO, the IHC data demonstrated very poor-to-no survival of human cells in grafts, and the pronounced presence of Iba1-positive microglia. CD8-positive infiltration was also pronounced in these grafts. We have previously observed similar results when describing the survival of the grafts of hESC-derived retinal progenitors in rodent work . Specifically, while some subretinal grafts were clearly visible by histology as a bulge, filled with cresyl violet-positive cells, such grafts had no, or only few, HNu-positive cells, but were filled with host-specific Iba1-positive microglia [Some of the difficult-to-explain differences in the efficiency of survival of human retinal cells in the xenogeneic grafts (even in the presence of immunosuppression) may be related to the animal model used rather than cells implanted. For example, if rabbits (which have merangiotic retina) are used , the retOther reported cases of graft rejection ,121 can In the second cohort of animals, we used a stronger immunosuppressive regimen, combining cyclosporine A with prednisolone ,124, to We did not observe any tumorigenesis in cohort 1 (subjects 1 and 2) and cohort 2 . However, we found the presence of some Ki67-positive cells in the graft, which is expected during retinogenesis , in lineAxonal and synaptic connectivity between the hESC-retinal tissue and recipient degenerating retina are needed to create a functional biological “retinal patch,” which can receive and transmit visual information from PRs of the graft to RGCs of the recipient retina . Our worWe observed bidirectional communication between the surviving grafts and the recipient cat retina . These fCollectively, our results indicate that the fibers connecting the cat retina and the grafts are young pathfinding axons and that the connectivity is bidirectional.boutons en passant, typical for neural grafts [terminaux boutons) can be seen in and around the grafts within a few weeks after grafting.We also observed patches of SYP-positive staining in the grafts and in the cat ONL and INL/IPL, and on CALB2-positive fibers . This mal grafts ,138, andCollectively, such connectivity, together with a robust immunosuppression protocol, should allow for rapid development of preclinical in vivo work focused on using hESC-retinal tissue for vision restoration in clinically relevant large-eye cat animal models ,65.Technologies such as retinal prosthetic devices  and feta


A review of the literature has indicated that lower body stiffness, defined as the extent to which the lower extremity joints resists deformation upon contact with the ground, may be a useful measure for assessing Achilles injury risk in triathletes. The nature of overuse injuries suggests that a variety of different movement patterns could conceivably contribute to the final injury outcome, any number and combination of which might be observed in a single individual. Measurements which incorporate both kinetics and kinematics (such as stiffness) of a movement may be better able to shed light on individuals at risk of injury, with further analysis then providing the exact mechanism of injury for the individual. Stiffness can be measured as vertical, leg or joint stiffness to model how the individual interacts with the environment upon landing. However, several issues with stiffness assessments limit the effectiveness of these measures to monitor athletes’ performance and/or injury risk. This may reflect the variety of common biomechanical stiffness calculations  that have been used to examine these three stiffness levels  across a variety of human movements (i.e. running or hopping) as well as potential issues with the reliability of these measures, especially joint stiffness. Therefore, the aims of this study were to provide a comparison of the various methods for measuring stiffness during two forms of human bouncing locomotion (running and hopping) along with the measurement reliability to determine the best methods to assess links with injury risk in triathletes.−1 and hopping (2.2 Hz) tasks.Vertical, leg and joint stiffness were estimated in 12 healthy male competitive triathletes on two occasions, 7 days apart, using both running at 5.0 msInter-day reliability was good for vertical (ICC = 0.85) and leg (ICC = 0.98) stiffness using the time method. Joint stiffness reliability was poor when assessed individually. Reliability was improved when taken as the sum of the hip, knee and ankle (ICC = 0.86). The knee and ankle combination provided the best correlation with leg stiffness during running (Pearson’s Correlation = 0.82).The dynamic and time methods of calculating leg stiffness had better reliability than the “true” method. The time and dynamic methods had the best correlation with the different combinations of joint stiffness, which suggests that they should be considered for biomechanical screening of triathletes. The knee and ankle combination had the best correlation with leg stiffness and is therefore proposed to provide the most information regarding lower limb mechanics during gait in triathletes. Overuse injuries are characterised by progressive onset of symptoms with no specific causal event. The causative mechanisms of overuse injuries have been suggested to be related externally to training loads and equipment or internally due to the biomechanics of the individual . The proIt is believed that the study of movement patterns combining both kinetics and kinematics into single risk factor measures may provide greater insight into risk of injury than isolated movement components. Stiffness, of the lower limb ‘springs’ provides a means of assessing the influence of kinetics and kinematics on the tissues of the lower limb during running. Review of the literature indicated that five risk factors were associated with risk of Achilles injuries in running athletes . All fivRunning as a bouncing gait has been modelled using the spring-mass model, a point mass balanced on a massless, compressible, linear spring . The stiThe basic model of lower extremity stiffness has been represented in a number of mathematical forms . VerticaThe vertical displacement of the centre of mass used in calculations for vertical stiffness occurs due to changes in hip, knee and ankle angles, resulting in the leg becoming effectively shorter. Modelling leg stiffness where the leg is a spring, and there is compression of the ‘leg spring’ in response to contact with the ground, estimates stiffness taking into account the angle of leg swing (from ground contact to mid-stance), accounting for horizontal movement . During The compression of the ‘leg spring’ is achieved by rotation around the hip, knee and ankle joints, to dissipate landing forces and store energy for elastic return in the next step or hop. The individual joints have been modelled as rotational springs which have associated stiffness due to muscular activity controlling the speed and magnitude of the joint rotations, and mechanical behaviour of tendons, ligaments and other structures comprising the joints . Joint sTriathletes and other endurance running athletes typically sustain near constant running velocities over a large portion of their training and races. The ability to maintain running velocity may require relatively consistent (reliable) vertical and leg stiffness. However, as vertical and leg stiffness may be controlled by the coordinated action at the ankle, knee and hip during the first half of stance to absorb impact and store energy for return during toe off, subtle differences in the body positions at foot strike may require step to step adjustments at the level of the ankle, knee and hip joints, thereby altering the individual joints stiffness. Similar to other biomechanical outputs that demonstrate that the variability of measurements may become greater as the measurement becomes more specific or isolated , it is tAnother issue affecting the potential applicability of using a variety of stiffness measures to monitor performance and injury risk concerns the challenges in assessing running in ecologically valid situations. Currently, lower extremity stiffness  have been measured for over ground running and treadmill running as well as hopping, with the method of measurement largely dictated by space and equipment . In ordeTo investigate whether stiffness can be used routinely to monitor running performance and injury risk, the reliability of the various measures of stiffness needed to be assessed. Where increased variability is present due to the inherent nature of human movement, understanding the variability that can be expected and to what extent the variability is minimised elsewhere in the kinetic chain is fundamental to understanding the results. It is also important to quantify the comparability of the various methods of measuring stiffness to determine the most appropriate measures to use for an investigation and to provide insight into the mechanism for any stiffness changes that may be observed.The complexity of the human neuromusculoskeletal system allows for a relatively infinite combination of joint moment and activation patterns to be utilised when performing repeated activities, such as running and jumping. Joint compensation is supported by the observation of stable ground reaction forces and joint kinematics within subjects but highly variable hip and knee joint moments during both walking and running . For exaThe aim of the study was to provide a comparison of the various methods for measuring stiffness during two forms of human bouncing locomotion (running and hopping) along with the measurement reliability to determine the best methods to assess links with injury risk in endurance athletes such as triathletes. Vertical and leg stiffness were hypothesised to show better reliability than the individual joints, even though the hip, knee and ankle all appear to play a role in the control of leg stiffness during running and hopping. Combining the joints in different combinations is hypothesised to reduce the measurement variability to allow interday assessment. Hopping is proposed to not be a good surrogate for running in triathletes when assessing injury risk.Twelve well-trained, male triathletes  volunteered for the study. All triathletes were currently competitive as top level age group athletes in either Olympic or long distance events. Personal best times in the previous season of under 2 h 20 min for Olympic distance or under 10 h for Iron distance and the ability to run for over 2 min at 4.0 min/km was required. Triathletes were excluded if they currently had a lower limb injury or had not been back to full training for at least 6 weeks following a previous lower limb injury. To avoid the possible effects of maturation  and ageiA test–retest between-day protocol on two separate days, seven days apart was performed. Triathletes were requested to keep training the same in the week prior to each testing session  to minimise changes in running biomechanics which might occur because of fatigue due to different training volumes and intensities. Triathletes were not asked to refrain from exercise at any point to minimise disruption.2. In training, triathletes use pace to monitor running speed rather than horizontal velocity. Therefore, running pace was used to provide a familiar measure and to ensure that future injury outcome data can be accommodated to the triathletes training environment. Data were collected for the final 20 s of each 2 min block to ensure gait had stabilised following pace change. Triathletes were unaware of when recording was taking place.A treadmill graded run was performed initially on an instrumented treadmill, followed by bent knee and straight knee hopping tasks. Thermal effects on stiffness measures as a result of repeated energy dissipation are unknown, therefore tasks were not randomized. As the hopping task required practice, this was scheduled after the run trial. Running pace was not randomized to reduce the injury risk that may be associated with accelerating to the faster paces from an initial slow pace. Due to the racing level of the triathletes and the distances and speeds experienced in training, this protocol was unlikely to fatigue the triathlete and therefore non-randomisation was deemed acceptable . FollowiAfter a 5 min rest, triathletes were given as much time as needed to familiarise themselves with hopping in time with the metronome. Triathletes performed single leg hopping, first on the right leg and then on the left, on the stationary treadmill (in-ground design with the treadmill belt in line with the laboratory floor). Hopping was carried out first with no instructions other than to keep in time to the metronome set at 2.2 Hz. Hopping was repeated with triathletes instructed to keep the knee as straight as possible. Ten hops were recorded once rhythm had stabilized to match the metronome frequency, based on visual inspection.During the running and hopping tasks, all triathletes wore spandex shorts or trisuits and their own regular training shoes. Height (mm), mass (kg) and bilateral trochanterian height (mm) were recorded according to International Society for the Advancement of Kinanthropometry (ISAK) protocols . RetroflOptimization Toolbox, Mathworks Inc.; Natick, MA, USA) detailed by Visual 3D, C-motion, Inc.; Germantown, MD, USA). Anatomical co-ordinate systems were defined according to specifications reported by x-axis was the line joining the two calcaneal markers. The y-axis followed the longitudinal axis of the foot from the proximal calcaneal marker, to the forefoot midline marker. The z-axis was orthogonal to the x and y axes.A 9-camera VICON motion analysis system  combined with a Bertec instrumented treadmill  were used for kinematic (200 Hz) and vertical, horizontal and lateral ground reaction force  collection, respectively. Full analysis of the Bertec instrumented treadmill force performance, both static and dynamic, has been reported by Variables were averaged over ten steps per leg for each individual for the running trials to allow for step variability  taken frLabview, National Instruments Corp.; Austin, TX). Stiffness was calculated for the first half of stance from initial heel contact to maximal vertical ground reaction force for all stiffness measures (leg∕brake as absolute change of force and leg length between ground contact and Fmax. The greater trochanter marker (GTR) was used as reported by leg∕GTR. Improved repeatability is reported from using functional hip and knee joints for defining the leg segments (leg∕HJC) was compared to kleg∕GTR. Joint stiffness combinations, ksumjoints, khip+knee and kknee+ankle were calculated using Stiffness values were calculated using a custom written Labview program  was <5% and the effect size (ES) was trivial (0–0.2) or small (0.2–0.6) . If one Measurement variability was assessed from typical error, reported as coefficient of variation percentage (CV%)  and intrFor overall reliability, all four variables  were assessed. ‘Good’ reliability required all four criteria to be met. ‘Moderate’ reliability resulted from one criteria outside the limits, while if two or more criteria were outside the limits, a ‘poor’ overall reliability was recorded .Biomechanical stiffness models were checked for comparability using Pearson’s correlation coefficient both within the stiffness type and between stiffness types . Leg stiffness and joint stiffness have different units, therefore results were converted to unitless values prior to comparison using A Pearson’s correlation coefficient > 0.90 was interpreted to show ‘very large’ correlation between the stiffness models or stiffness types. Correlations between 0.70 and 0.90 indicated ‘large’ comparability, moderate was considered to have been obtained with correlations between 0.50–0.69 while anything below 0.50 indicated a poor correlation between the two variables of interest . HoppingData for the left leg only are presented in leg∕brake (Equation L3) estimation than for kleg∕dynamic (Equation L1) or kleg∕time (Equation L2). However, the magnitude of the kleg∕brake (Equation L3) estimate was closer to the combined joint stiffness. Ankle stiffness was greater than knee stiffness during running but this relationship was reversed for both hopping conditions. The variation (% of mean) of knee stiffness was larger than the other two joints and this variation was greatest with straight leg hopping (60%). Variation of the stiffness measurements increased when moving from the “global” vertical stiffness to the more focused joint stiffness.Hopping with a straight knee resulted in a reduction in knee stiffness compared to bent knee hopping  to give an average value closer to running knee stiffness. Ankle stiffness was lower in both hopping conditions compared to running and did not significantly differ between the two hopping conditions. The combined stiffness of the hip, knee and ankle was also less in straight knee hopping than running. All joint variables were closer to running for straight knee than bent knee hopping. Leg stiffness was more than two-fold greater when using the kleg∕brake (Equation L3) estimates. Good reliability was achieved when the top of the ‘true’ leg was measured from the modelled hip joint centre (kleg∕brakeHJC). When measured from the single trochanterion marker, overall reliability was moderate. Individual joint stiffness ranged from poor to good with the hip having the poorest and ankle having the best overall reliability. Combining the joints as hip, knee and ankle, hip and knee or knee and ankle tended to improve the reliability to between moderate and good. For bent knee hopping kleg∕time (Equation L2) and kleg∕brakeGTR (Equation L3) methods showed good reliability, with all other leg stiffness measures showing only moderate reliability. All joint stiffness estimates, including joint combinations had poor overall reliability for bent knee hopping. Straight leg hopping improved the kleg∕dynamic (Equation L1) estimate reliability but gave only poor to moderate kleg∕brake (Equation L3) reliability. Overall reliability of the ankle was improved with straight leg hopping.The reliability and measurement variability are reported in leg∕brakeGTR (Equation L3) which was poor. Conversely, comparisons between straight knee hopping and running were moderate for the individual knee and ankle (Equation J), hip+knee  was with kleg∕time (Equation L2). Confidence limits for all joint measurement correlations were large, with this variation smallest between kknee+ankle (leg∕time (Equation L2). The time (Equation L2) and dynamic (Equation L1) methods had large correlations with knee stiffness (Equation J), however ankle and hip stiffness alone showed poor correlations with leg stiffness. All kleg estimates had large correlations with both kvert∕dynamic(Equation V1) and kvert∕time (Equation V2).As the middle level of stiffness, leg stiffness estimates were compared with vertical and joint stiffness estimates . The higee+ankle  and klegUnderstanding the mechanism of overuse injuries is confounded by the insidious nature of the injury with lack of a single defining injurious event. Biomechanical research into injuries has tended to focus on individual joints or muscles to isolate risk factors for injuries. It is possible that it is the interaction between multiple segments and structures within the kinetic chain that results in progressive disruption of the structure and function resulting in overuse injuries . The varAnalysis of joint stiffness gives greater information regarding the mechanics of the lower limb, however the poor reliability of isolated joint stiffness estimates  has currvert∕dynamic (Equation V1) was similar to results reported for treadmill running , stiffness was similar to kvert∕dynamic (Equation V1) but substantially smaller than the average stiffness reported by Mean k running . When coleg∕dynamic (Equation L1) and kleg∕time (Equation L2) for the triathletes were similar to results for treadmill running (leg (leg (leg∕dynamic (Equation L1) than the current data for over ground running in middle distance runners. Due to the small sample sizes in the present study and those studies reported above, population differences would alter the mean stiffness estimates  for the current data was similar to results for sprint running (knee and kankle (Equation J) was reported for thirteen runners, running over ground at a range of velocities  were acceptable for ankle, knee and hip, the knee and hip had large measurement variability (%CV and ICC). When the joints were combined as sumjoints , hip +knr = 0.66) (r = 0.61) (leg∕time (Equation L2) were lower than for the knee and ankle combination   (Equation L2) was similar to the knee and ankle combination   and the  = 0.61)  with kle2–0.94]) . Knee cobination  indicatiThe current data suggests that in triathletes, hopping at 2.2 Hz, either with normal knee bend or a straight knee, does not correlate well with treadmill running for leg and joint stiffness estimates. Interestingly, bent knee hopping correlates moderately with running for leg stiffness but straight knee hopping has better correlations than bent knee for joint stiffness. Gait has been divided into two models based on the trajectory of the centre of mass, the ‘inverted pendulum’ for walking  and ‘bouBesides biomechanical differences in the task demands between hopping and running, the poor stiffness correlations between these two tasks could be due to the lack of familiarisation the triathletes had for hopping. It should also be noted that stiffness is altered as a result of changing contact time . Therefoleg∕time (Equation L2) method is recommended for assessing leg stiffness. However, kleg∕dynamic (Equation V2) also appears to be a good measure and when combined with joint angle recording also allows for calculation of joint stiffness. Joints should be assessed as a system in relation to each other rather than in isolation to gain acceptable reliability of the measure. The knee and ankle combination appears to be the most important when assessing changes in leg stiffness for triathletes running at the paces assessed in the present study. Hopping at 2.2 Hz is not a good substitute for running when estimating stiffness in triathletes. Further analysis is required to determine if hopping correlates with running when frequency is constrained to the triathletes, preferred stride rate or assessed in sub-groups of athletes who differ in their foot strike patterns or familiarity with vertical hopping.Due to the superior reliability and correlations with both combinations of joint stiffness and vertical stiffness, the k10.7717/peerj.5845/supp-1Supplemental Information 1Click here for additional data file.


South African Journal of Physiotherapy (SAJP) is important and why it is worth putting in so much effort to continuously improve the standing of the journal. Many people are involved in producing our journal, and before going further, thanks need to be given to the Editorial Board, the reviewers, those who review with such good grace, the hard-working and supportive publishers and an increasingly broad readership. Do you read scientific journals all of you professional physiotherapists out there? Hope so!At the end of the year, we all tend to reflect on what the year has been like for us and we also consider what we may be planning for the year ahead. Part of my reflection this year has been to think about why the We now are in the fortunate position of having many international physiotherapy journals, many of which are very good and publish cutting-edge and interesting findings that continue to propel our profession to constantly improve. Think about where we are now in our international professional standing and where we were not that long ago! So if there are so many of these journals, why is our journal important?Our journal is important because we showcase our own research; we give a voice to our own researchers and have as our audience our own physiotherapy profession. We offer unique studies that are not considered by the large international journals that are mostly published in the developed world. We have unique and challenging health and rehabilitation issues that need not only to be aired but also to be considered by our profession. We need to constantly think about these unique challenges and how they can be managed by such an important rehabilitation profession as ours. We straddle the developed and developing world and because we have such a strong profession in South Africa and an increasingly strong group of researchers, we are in a position to start finding answers to the rehabilitation challenges that we and the rest of Africa experience. We have the means and the ability to consider and provide appropriate solutions to many of these challenges. We all need to encourage our researchers to do this; more and more researchers from other African countries, other developing countries and the developed world should join us in finding answers to the rehabilitation challenges faced by all of us.SAJP by publishing their strong evidence to advance our profession here in South Africa. Increasingly, the SAJP must be one of the international journals of choice for our researchers. A process of inviting our top researchers and clinicians and/or researchers to contribute to our journal has begun with a favourable response from them.As we move towards more and more of our physiotherapists in South Africa having PhDs, we must start considering large multi-centre trials that can start providing answers to some of our rehabilitation issues. We have worked hard to get to the point where we have this increasingly large cohort of established researchers and I call on them to do big studies and strengthen the How can we attract these researchers to publish their good studies in our journal? The only way to do so is by publishing good studies so that all our researchers can be encouraged to send their findings to our journal. This is a ‘chicken and egg’ scenario but, in reality, the only way that a journal improves. Getting onto international scientific lists of journals is a very important step and one that will encourage established researchers to publish in our journal; however, we must have good-quality articles to get there. The fact that we are now on PubMed is a huge step in the right direction. All who were involved in the arduous process to achieve this milestone must be congratulated!SAJP and I am sure with all who support and care for this journal, we can get to the point where our journal will be recognised as one of the many top international physiotherapy journals.We must now move towards the next steps in international recognition for the


RSM) and artificial neural network (ANN) were studied. All the independent variables  were of significant (p < .05) importance in achieving esterified starch of Amaranthus viridis. Optimum conditions of 152.46 g of starch, 11 ml of acetic anhydride and time of 2.92 min with corresponding acetyl content and degree of substitution (DS) of 1.74% and 0.06, respectively, were established for ANN. The RSM gave optimum conditions of 149.57 g (starch), 10.38 ml (acetic anhydride) and 3 min (time) with corresponding acetyl content and DS of 1.61% and 0.06, respectively. The order of priority of the process variables was established as acetic anhydride (42.59%), starch solids (33.90%), and reaction time (23.51%). The results provided useful information on development of economic and efficient acetylation process for modification of A. viridis starch.The optimum reaction conditions for the derivation of acetylated (esterified) starch using response surface methodology ( AmarantStarch is an important food ingredient in the food industry. It is reported that about 53% of starch total production is used in the food sector ; 28% is used for production of paper, cardboard and corrugated board, 13% is used for fermentation, and others 5%  technique. It is worthy of mentioning that the Design of Experiments (DOE) approach was not used in most of these reports on Amaranthus starch.The starch properties of the seeds of some amaranth cultivars have been characterized , which defines the effect of the independent variables, alone or in combination in a process, is nowadays being applied in modeling and optimization studies , Kano, Nigeria and matured grains were collected after 14 weeks . The grains were wet cleaned and dried in a hot air oven  at 50°C for 8 hr. All chemicals were of analytical grades and obtained from Fisher Scientific  and Sigma Chemicals .2.2Starch was extracted as described by Kong, Bao, and Corke  with som2.3The orthogonal central composite design (CCD) of RSM was used for this work. The three independent variables considered for the modeling include starch solid 50–150 g), acetic anhydride (5–15 ml) and time (3–13 min). The number of experimental conditions , adjusted (Adj. R2), probability value at 95% confidence interval, predicted R2, coefficient of variation, lack‐of‐fit, and analysis of variance (ANOVA). The modeling and optimization of the esterification process were carried out, using RSM of the Design Expert software version 8.0.7.1 . Pareto chart was developed using Statistica software, version 12.0 .The following statistical indicators were employed: coefficient of determination  group was calculated as shown in Equation The methods described by Medinav, Pardo, and Ortiz  with somwhere, blank is ml of HCl used in the native starch titration, sample is the ml of HCl used in the esterified starch titration, 0.043 is milliequivalents of the acetyl group, 162 is the molecular weight of glucose, 4300 is molecular weight of the acetyl group multiplied by 100, and 42 is the molecular weight of the acetyl group −1.2.5E), and absolute average deviation (AAD).A commercial software, NeuralPower version 2.5 (CPC‐X Software), was used for the ANN modeling and optimization. The dependent variables (%acetyl and DS) were predicted by using multilayer full feed forward (MFFF) and multilayer normal feed forward (MNFF) neural networks, which were trained by different learning algorithms such as incremental back propagation (IBP), quickprob (QP), genetic algorithm (GA), batch back propagation (BBP), and Levenberg‐Marquardt algorithm (LM). Each ANN was trained using a default stopping criteria of 100,000 iterations. The dataset in Table n is the number of experimental data, ap,i is the predicted values, ap,ave is the average predicted values ae,i is the experimental value, ae,ave is the average experimental values, and j is the number of input variables  are presented in Table The DS observed in this work ranged from 0.01 to 0.10. The results were similar to a range of 0.05–0.17 reported by Saartrat et al.  for kidn3.2p‐value (p < .05) indicates that the model is considered to be statistically significant.In order to describe the relationship between the dependent variables (acetyl content and DS) and the independent variables , the dependent variables were fitted by second‐order polynomial quadratic regression models. By applying multiple regression analysis on the experimental data obtained for the dependent variables Table , the anaR2 and significance of lack‐of‐fit. The R2 is defined as the ratio of the explained variation to the total variation, a measure of the degree of fit  strengthened the reliability of a model.The fitness and adequacy of the models were judged by the coefficient of p‐value (<.0001) and F‐value (125.27) indicate the statistical significance of the quadratic model. The R2 and adjusted R2 were .9912 and .9833, respectively, which illustrate that there were excellent correlations between the independent variables; and the fitted model could describe the independent variables adequately , which also shows a good fit between experimental data and the model.The coefficient of variation, CV, which is independent of the unit is defined as the ratio of the standard deviation of estimate to the mean values of the observed response. The CV is a measure of reproducibility and repeatability of the model , lack‐of‐fit (0.1331), R2 (.9586), Adj. R2 (.9213), CV (9.60%) and Ad. Pred. (16.39) confirmed the suitability of the generated model. The R2 value indicates that the model could explain up to 95.86% of the total variation in the esterification process. It, therefore, shows that the developed models for the esterification process could adequately define the real behavior of the process and could be used for the prediction of the acetyl content and DS. The mathematical models in coded forms to predict the acetyl content and DS are presented in Equations In the case of DS, the model 1; acetic anhydride, x2; and reaction time, x3 have significant (p < .05) influence on the amount of acetyl group present in the starch. Starch solids and reaction time were inversely related to the acetyl content of the esterified starch while the quantity of acetic anhydride was directly proportional. There was significant (p < .05) interactions among the independent variables. The DS was significantly (p < .05) influenced by amount of starch solids and acetic anhydride. There was positive correlation between acetic anhydride and DS. The DS was also significantly (p < .05) influenced by the interactions between starch solids and acetic anhydride; acetic anhydride and reaction time. The quadratic term of all independent variables had significance effect (p < .05) on DS. The graphical representations of the regression equations for the optimization of the esterification process are shown in Figure Equations p < .05) influenced the amount of acetyl group in the esterified starch. Acetic anhydride was identified as the most significant process variable on acetyl level of the esterified starch. Figure p < .05) and linear model term of acetic anhydride and interaction model term of starch solid x acetic anhydride were significant (p < .05). A positive coefficient shows favorable interaction effect on the dependent variable while a negative coefficient shows an inverse relationship between the model term and dependent variable  was estimated as 149.57 g, acetic anhydride (x2) was 10.83 ml, and reaction time (x3) was 3 min; the corresponding acetyl content and DS were 1.61% and 0.06, respectively. The confirmatory experiments conducted under the optimal condition showed acetyl content of 1.68% and DS of 0.06, which were consistent with predicted values. The validity of the predicted optimum points was done using AAD and mean E. The AAD values of 4.17% and 0% were recorded for acetyl content and DS, respectively; the values of E for acetyl content and DS were 0.17% and 0%, respectively. The small values (<10%) of AAD and E for the dependent variables indicated the validity of the model and the developed models are suitable for describing derivation of esterified starch from native starch of A. viridis.According to United States Code of Federal Regulations  connection type and incremental back propagation (IBP) network with hyperbolic tangent as both hidden and output layers function was identified as the most suitable ANN model for acetyl content and DS synthesis  effects on the modification process. The optimal condition of 152.46 g of starch, 11 ml of acetic anhydride, and reaction time of 2.92 min yielded acetyl content of 1.74% and degree of substitution (DS) of 0.06 for the ANN method while optimal condition of 149.57 g (starch), 10.38 ml (acetic anhydride), and 3 min (time) yielded acetyl content of 1.61%, and DS of 0.06 for RSM method. ANN was demonstrated to be a superior modeling tool than RSM. The work provides useful information regarding the development of economic and efficient process for modification of A. viridis starch via acetylation method.The work examined the acetylation (esterification) of The authors declare no conflicts of interest.Not applicable.