Infection of prosthetic vascular grafts can manifest as pain, pseudoaneurysms, or arterial insufficiency in the leg. We present the case of a female patient with a medical history of a right external iliac artery endofibrosis, with a persistently infected synthetic iliofemoral bypass graft, which we replaced with a bioengineered human acellular vessel. At the 12-month follow-up visit, the clinical and radiologic studies demonstrated adequate human acellular vessel patency, with no signs of infection, stenosis, or pseudoaneurysm. Subsequent to the initiation of hormone therapy and cessation of antiplatelet therapy, the patient developed graft thrombosis. She continued to do well after restoration of patency with lytic therapy. At 22 months, secondary patency has been maintained with continued anticoagulation therapy, and the patient has remained asymptomatic. She had presented with perigraft fluid collection and presumed infection of a right iliofemoral Dacron bypass graft and ipsilateral claudication. The patient’s medical history was significant for external iliac artery endofibrosis that had been bypassed 4 years previously at an outside hospital using an 8-mm Dacron interposition graft from the proximal external iliac artery to the common femoral artery.HAV implantation was performed through the patient’s previous incisions above and below the inguinal ligament. After exposure and control of the common iliac artery and common femoral artery, a tunnel was created lateral to the prior graft and running under the inguinal ligament for placement of the HAV. The Dacron graft was completely excised and the native artery debrided to healthy tissue. A 6-mm HAV was chosen and anastomosed end-to-end using 5-0 Prolene suture . After blood flow was restored, patency of the vascular reconstruction with the HAV was confirmed by intraoperative completion angiography, with the needle placed in the common iliac artery. Digital subtraction arteriography was performed in two planes with contrast enhancement and demonstrated wide patency of the graft and anastomoses proximally and distally .Fig 1A, The patient recovered well from surgery and was discharged on postoperative day 4. Intraoperative tissue cultures were negative for bacterial growth. Under the advisement of the infectious disease specialists, the patient received empiric antibiotic treatment (vancomycin and ceftriaxone) for 6 weeks and was then transitioned to oral doxycycline for an additional 6 months. She also started daily aspirin 81 mg.The patient underwent follow-up examinations at 1, 3, 6, 9, and 12 months postoperatively . At 1 moAt approximately 16 months postoperatively, the patient had developed progressive menometrorrhagia, leading to cessation of aspirin and initiation of hormonal therapy. However, at 18 months, she had presented with thrombosis of the HAV and right common iliac artery. Uterine embolization with subsequent initiation of transcatheter thrombolysis of the common iliac artery and HAV restored normal flow. No technical concerns were identified with the graft. The patient has received oral anticoagulation therapy after recovery from her hysterectomy and was doing well at 22 months postoperatively.,,18When considering the materials to use for replacement of an infected graft, the most widely used choices have been autologous, cryopreserved, and prosthetic grafts.The present report is an important early description of the use of a human tissue engineered vessel in a known infected field, in which a previously placed synthetic graft had become infected. Because of this infection, the lack of autologous graft options, and the risks associated with revascularization with another synthetic graft, the HAV was used as an alternative treatment option for our patient. Our patient was found to have had a culture-negative infection. Thus, the performance of a HAV graft in a culture-positive field, with more virulent bacteria, is unknown.At 22 months after implantation of the HAV, the patient had required one intervention to restore graft patency after thrombosis. This could have resulted from the initiation of hormone therapy or cessation of antiplatelet therapy in the setting of incompletely endothelialized tissue. Although nearly complete endothelial coverage has been confirmed in HAVs as early as 44 weeks, additional data regarding the risk of HAV thrombosis to guide the type and duration of antiplatelet or anticoagulant therapy is necessary when considering the risks of this approach.The present case has demonstrated how a bioengineered HAV can be used as an arterial bypass conduit in the setting of prior graft infection. This could provide a unique alternative to other graft options for the treatment of infected vascular prostheses. Further studies to demonstrate the utility of this approach are recommended.


Starch retrogradation is a complex process and in most food products is undesirable. Knowing and understanding the mechanisms and factors that influence this process may become the key to a better and innovative approach to food design. In this paper, we investigated the effect of 0%, 0.05% and 0.20% (w/w) xanthan gum (XG) addition on the short- and long-term retrogradation of 4%, 5% and 6% corn starch gels, depending on the amylose/amylopectin ratio in the starch. The changes were monitored throughout 90 days. The pasting characteristics of blends, rheological and texture analyses, as well as syneresis, revealed that XG stabilizes the starch in the short term, but it does not inhibit retrogradation caused by amylopectin. After 30 days of storage, the destabilization of the starch-hydrocolloid mixture was observed. Based on the obtained results, a probable mechanism for the retrogradation of corn starch process in the presence of xanthan gum was proposed. Retrogradation of starch is a complex process which depends on many factors. The amylose (AM) to amylopectin (AP) ratio seems to have dominant role . The greHydrocolloids used as thickeners, gelling agents and/or stabilizing starch systems may slow down/inhibit or, on the contrary, accelerate the process of starch retrogradation ,9. HowevHowever, for technical, technological and analytical reasons, there were numerous limitations that prevented an in-depth analysis of this issue. In the year of 2000, Karim et al.  describeNormal corn starches denoted as NCS1  and NCS2  were purchased from Cargill . Waxy corn starch (WCS)  was purchased from Cargill  and contained 0.75% amylose. The amylose contents of starch were presented in our previous paper, Sikora et al. 2018 [Xanthomonas campestris  was purchased from Sigma–Aldrich .Xanthan gum (XG) from The samples for rheological and textural measurements, as well as for syneresis, were prepared and measured according to Sikora et al.  in whichAnalysis was conducted according to our previous method  by the u3. They were left at RT for 2 h then placed in a refrigerator at 6 °C. Oscillation measurements were performed with a RheoStress RS 6000  rotary rheometer equipped with a plate–plate P35 Ti measuring system (35 mm diameter) and temperature control module based on the Peltier system. In order to adjust the gap, the distance between them was reduced with the 0.15 mm/s rate until the normal force reached 0.2 N. The estimations were performed after the samples were prepared (after 1 and 2 h) and then after 1, 2, 10, 30, 60 and 90 days storage. The oscillation measurements included running mechanical spectra in the range of 0.1 to 10 Hz at 25 °C and 0.3% deformation fitting the range of linear viscoelasticity. The estimations were run in duplicates.Freshly prepared pastes were transferred into disposable aluminum foils placed in nonventilated polystyrene Petri dishes in the amount of 4 cm3) were kept in polypropylene containers  firstly at room temperature for 2 h and then in refrigerated at 6 °C. Measurements of the hardness of gels with the Texture Analyzer TA.XT plus  were performed for freshly prepared samples (1 and 2 h) and after their storing for 1, 2, 10, 30, 60 and 90 days. The penetration test involved P/0.5 cylinder taken as a measurement probe. It was immersed 10 mm deep, and its velocity was 1.0 mm/s. The measurements were run in triplicates.The measurements for the gels were performed according to Krystyjan et al.  procedura—the weight of empty container with its cap (g),b—the weight of container with its cap after filling it with a sample (g),c—the weight of container with its cap and sample after the separated water was removed (g).The samples were prepared in the same way as for textural analysis  and keptStatistical analysis involved a Statistica 12.5  software employing mono- and bifactorial analysis of variance and the Duncan’s test for checking the significance of the differences at α = 0.05.The presence of hydrocolloid in the mixture affected the starch pasting. A significant decrease in the initial pasting temperature and an increase in the viscosity of the binary systems were noticed. A similar tendency was observed by Mandala and Bayas , who stuThe presence of the hydrocolloid in the mixture influenced the starch pasting characteristics and the setback parameter. XG decreased the value of the setback of native corn starch pastes in each concentration of the starch , which mThe rheological properties of starch gels without and with the addition of xanthan gum stored for 90 days are shown in G′ modulus over time for native starches  of the mixture, which proved a greater difference between G′ and G″. The difference was more pronounced with the lower starch concentration and decreased with increasing concentration of this polysaccharide in the mixture. Thus, the influence of the gum on the rheological properties of the mixture was more pronounced at a lower starch concentration. After 30 days of storage, tan δ values decreased, and thus, the differences between G′ and G″ were smaller. This behavior may suggest that XG stabilized the system for about 30 days, and after this time, intense changes in the viscoelastic properties of the mixtures, which indicate a destabilization of the system, were observed. Thus, the effect of gum on long-term starch retrogradation was small, which may suggest no interaction between amylopectin and gum. Our assumptions were confirmed by NMR studies performed by us on the similar systems [The addition of XG increased the value of the tan δ ( systems . AccordiThe hardness of starch gels increased with the storage time and the content of amylose. Native starch gels (NCS1 and NCS2) showed a much faster increase in hardness during their storage than the waxy starch gels . In the Based on the obtained data a,b, no sThe obtained results lead to the following observations. In the presence of xanthan gum, only the short-time retrogradation of starch was reduced. According to Gallant et al. , starch The way in which xanthan gum interacts with maize starch is a complex phenomenon. Various suggestions appear, but the most probable thesis was presented by Kim et al. . AccordiThe study confirmed that the process of starch retrogradation consists of two stages. In the first stage, which lasts 48 h, the amylose recrystallization takes place. In the next stage, changes caused by amylopectin retrogradation occurred. Xanthan gum controls short-term structural changes in corn starch through two possible mechanisms—phase separation and intergranular association. The speed of these changes depends on many factors but is mainly related to the amylose content in starch and the amount of hydrocolloid added to the mixture. Mechanical and textural measurement, syneresis studies confirmed destabilization of the starch–hydrocolloid mixture after about 30 days of storage.The achieved knowledge can be used in the design of long-term foods, such as sauces, dressings, mayonnaise and food concentrates. Thanks to the use of xanthan gum, unfavorable changes in the structure of products and the syneresis can be limited. However, the effectiveness of these action will depend on the gum/starch ratio and the type of product in which this binary system will be applied.


Finally, some strategies to improve cereal yield and quality were put forward based on the previous reports. This review would collectively help to design future studies on starch biosynthesis in cereal crops.Starch provides primary storage of carbohydrates, accounting for approximately 85% of the dry weight of cereal endosperm. Cereal seeds contribute to maximum annual starch production and provide the primary food for humans and livestock worldwide. However, the growing demand for starch in food and industry and the increasing loss of arable land with urbanization emphasizes the urgency to understand starch biosynthesis and its regulation. Here, we first summarized the regulatory signaling pathways about leaf starch biosynthesis. Subsequently, we paid more attention to how transcriptional factors (TFs) systematically respond to various stimulants  With the improvement of living standards, people have higher and higher requirements for food quality. As the main component of grain crops, especially cereal crops, the content, and quality of starch will directly affect the economic benefits of crops. The harvested heterotrophic parts of staple crop plants are usually starch-storing organs such as roots , tubers (potato), and cereal seeds  . Among oStarch is composed of amylose and amylopectin glucan polymers, which are packaged to form the insoluble semi-crystalline starch granules . There avia transferring linear glucan, and DBEs facilitate the formation of the crystalline amylopectin layers through removing α-1,6-linkages  were mostly focused here. More than eight hundred publications were retrieved, and article selection was conducted according to previous method  was supposed to be one of the most studied enzymes involved in the light-dependent signaling pathway, in which the regulation of AGPase was largely related to the modules of photosynthesis. For example, ferredoxin (Fdx), thioredoxins (Trx), and Fdx-dependent Trx reductase (FTR) participated in the regulation of AGPase in response to light . BesidesAs important forms of carbon budget, glucose and sucrose also function in the regulation of carbon allocation during starch biosynthesis. Nonetheless, this is a very complex process. More than that, glucose and sucrose are also greatly related to the balancing of growth and reproduction using the available carbon, which involves trehalose 6-phosphate (Tre6P) in higher plants .Tre6P, an intermediate of trehalose (TRE) biosynthesis, functions in the sucrose-Tre6P sensor nexus . It invoi.e., rice; i.e., Arabidopsis; In fact, in the endosperm of cereal crops, the deposition of starch grains is closely connected with Tre6P. In several cereal crops, a model for the role of Tre6P in carbon partitioning and yield was proposed and confirmed . Local sCollectively, the accumulation of sucrose was vital for plant growth through regulation of starch biosynthesis, and several sucrose-dependent signaling has been summarized here, including (i) sucrose→SnRK1→Tre6P→AGPase and (ii) sucrose→OPP→NADPH→NTRC→AGPase/SSIII/SSIV .β-amylase 3 (BAM3), the major starch degrading enzyme with a short half-life (α-amylase 3 (AMY3), and disproportionating enzyme 2 (DPE2), had long half-lives over the day/night cycle  to the developing grains . Three t+ leak .Viridiplantae plants, despite limited studies on the regulation of SUTs in cereal crops. The identification of sucrose transporters in cereal crops could be aided by the complete genome sequences of Arabidopsis and potato.Sucrose transporters appear to be relatively conserved within the genome uncoupled 4, which was revealed as one predominant regulator of chlorophyll biosynthesis, performed negative effects on the starch synthetic genes, such as GBSSI and AGPS1, in endosperm during early seed development, partially through mediating the accumulation of heme , OsSSIIa, BEI, OsBEIIb, and isoamylase-type debranching enzyme 2 (ISA2). Besides, GBSSI was reported to be regulated by several TFs, including bZIP33, bZIP34  were regulated by the interaction of FLO2 and bHLH . One novvia TaSPA and TaGAMyb to regulate SSIIa and sucrose synthase 1 (SuSy1) consequently affecting the starch accumulation in wheat  that play a key role in seed filling and plant hormone response to regulate their expression  in rice  and in bin wheat . This wain maize . Still, pression . Howeverpression . To sum pression .The function of starch biosynthetic enzymes usually depends on posttranslational regulation, including phosphorylation, the formation of a transient complex, and so on . For staImportantly, as an important rate-limiting enzyme for starch synthesis, AGPase subunits form a complex with different roles of large and small subunits to make ADP-Glucose, and thereby, the enhanced activity of AGPase would greatly increase the starch content . AGPase via the formation and segregation of transient complexes  consist of embryo, endosperm and seed coat, among which endosperm is a major storage organ for grain development , affects the endosperm starch synthesis and alters starch properties of, which is associated with defects in granule initiation , which are finally stored in the endosperm of seeds. Therefore, the modified synthesis of endosperm starch can be achieved at least by the following pathways . In rice, overexpressed GBSSI/Wx affected amylose content (AC) and gel consistency (GC) but had fewer effects on gelatinization temperature (GT) . Howeverg., SSIV .ISA1 via CRISPR/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system has affected GT and starch chain length distribution during endosperm development, which have potential implications for quality improvement in rice  Protein (CRCT), would significantly increase the starch content in the phloem of the leaf blade and leaf sheath during vegetative stages, which was supposed to be an alternative or potential approach to improve yields of food and biofuel  is the sum of starch that could not digest and absorb by the small intestine , and itsomplexes . Other fomplexes  and amylomplexes . Recentle chains . Interes in rice , which mStarch biosynthesis not only plays a critical role in the formation of grain yield and quality in cereal crops but also involves the coordination of different biological processes and various organs. These processes include sucrose synthesis and transport in source organs (such as leaves), sucrose and energy delivery from source organs throughout the vascular system (phloem) to sink organs (endosperm), conversion from sucrose to ADPG in the endosperm, and formation of amylopectin- and amylose-starch in the endosperm. The target-oriented improvement on yield and grain quality in cereal crops would optimize the current demand for starch to meet the living standard and be beneficial to overcome the problems in the availability of arable land by using the known mechanisms of starch biosynthesis.10.7717/peerj.12678/supp-1Supplemental Information 1Click here for additional data file.


Cystinosis, a rare lysosomal storage disease, is characterized by cystine crystallization and accumulation within tissues and organs, including the kidneys and brain. Its impact on neural function appears mild relative to its effects on other organs, but therapeutic advances have led to substantially increased life expectancy, necessitating deeper understanding of its impact on neurocognitive function in adulthood. We previously demonstrated intact auditory sensory processing, accompanied by mild sensory memory difficulties, in children and adolescents with cystinosis.We investigated whether further progressive decrements in these processes would be observed in adults with cystinosis, comparing high-density auditory-evoked potential (AEP) recordings from adults with cystinosis  to those of age-matched controls (N = 17). We employed a duration oddball paradigm with different stimulation rates, in which participants passively listened to regularly occurring standard tones interspersed with infrequently occurring deviant tones. Analyses focused on AEP components reflecting auditory sensory-perceptual processing (N1 and P2), sensory memory , and attentional orienting (P3a).Overall, adults with cystinosis produced highly similar sensory-perceptual AEP responses to those observed in controls suggesting intact early auditory cortical processing. However, significantly increased P2 and P3a amplitudes and reduced MMN at slower stimulation rates were observed, suggesting mild-to-moderate changes in auditory sensory memory and attentional processing. While cognitive testing revealed lower scores on verbal IQ and perceptual reasoning in cystinosis, these did not correlate with the AEP measures.These neurophysiological data point to the emergence of subtle auditory processing deficits in early adulthood in cystinosis, warranting further investigation of memory and attentional processes in this population, and of their consequences for perceptual and cognitive function. CTNS gene [Cystinosis is a rare autosomal recessive condition caused by a bi-allelic mutation in the 17p13.2-located TNS gene , but theTNS gene , has attTNS gene . It is nLittle is known about the long-term progression and sequelae of cystinosis and its treatments. Of particular interest is the characterization of brain function, given that central nervous complications  are among the additional concerns associated with disease evolution –9 and thStudies investigating cognitive and neural function in adults with cystinosis are scarce and tend to be inconsistent. For example, two behavioral studies using the same verbal and visual learning/memory paradigm reported conflicting findings: While one suggested an overall decline in performance with age in cystinosis, indicating progressive dysfunction over the lifespan , a laterIn a recent high-density electrophysiological study, our research group investigated auditory sensory-perceptual processing and sensory memory processes, specifically in children and adolescents with cystinosis. We used a simple auditory oddball paradigm whereby regular tones  were infrequently interrupted by a tone of the same frequency but of slightly longer duration . This allows for assessment of basic sensory-perceptual processing by interrogating the AEP componentry evoked by the standard stimuli, and of auditory sensory memory, by interrogating the so-called mismatch negativity (MMN) component evoked by the duration deviant. In our young cystinosis cohort, no differences were found in basic auditory processing between the groups, but a deficit in sensory memory appeared to be present in those with cystinosis . As suchHere, we employed the same duration oddball paradigm in a sample of adults with cystinosis and age-matched controls and focused on AEP components indexing basic auditory cortical processing , senConsidering earlier work suggesting associations between auditory sensory memory and working memory –32, we at tests were used to test for between-group differences. In cases in which the assumption of the homogeneity of variances was violated, Welch corrections were applied to adjust the degrees of freedom. Paired t tests were used to test for within-group differences. Statistical analyses revealed significant differences between the groups in IQ, with individuals diagnosed with cystinosis showing lower scores on verbal IQ and perceptual reasoning, but not on the Digit Span subtest (working memory). Both groups presented significantly lower perceptual reasoning scores when compared to verbal IQ.Table lmer function in the lme4 package [P values were estimated using Satterthwaite approximations [Figure  package  in R (Ve package ). The moimations .Fig. 1a ß = − 1.39, SE = 0.12, p = 0.01) and 1800  evoking larger responses than 450 ms. No effects of group or of the interaction between group and SOA were observed. In the P2 time window, individuals with cystinosis presented larger responses than the controls . There was also an effect of SOA, with both 900  and 1800  evoking larger responses than the 450 ms SOA.In the N1 time window, there was an effect of SOA, with both 900  when compared to controls. Post-hoc analyses revealed that this was mainly explained by a reduced MMN in the 1800 SOA in the cystinosis group . There was also an effect of SOA, with both 900  and 1800 ms  SOAs evoking larger responses than the 450 ms SOA. In the P3a time window, when compared to their peers, individuals with cystinosis presented an increased amplitude for the 900  and 1800 ms  SOAs. No effects of SOA were observed.Figure No significant correlations were found between cognitive function, number of renal transplants and cysteamine dosage, and neural responses across or within groups.High-density EEG recordings during a passive oddball paradigm characterized basic auditory processing, sensory memory, and involuntary attention in a sample of adults with cystinosis. Although statistically significant differences were observed in the P2, MMN, and P3a amplitudes between the groups, these differences were quite subtle, and by-and-large adults with cystinosis presented highly similar neural responses compared to those of age-matched controls.In the N1 time window, no detectable differences were found between groups, suggesting intact sensory transmission through the auditory system in individuals with cystinosis. Further indicative of intact basic auditory processing in cystinosis was the modulation of the N1 as a function of SOA (i.e. rate of presentation). This modulation, consistently described in the literature for the neurotypical population  as refleIn the P2 time window, individuals with cystinosis presented increased amplitudes when compared to their peers, which was not observed in children and adolescents in our earlier study . An enhaThough often conceptualized in the context of the N1/P2 complex, P2 can be dissociated from the N1 experimentally, developmentally, and topographically . The fiIn the MMN time window, despite showing robust MMN responses across SOAs, individuals with cystinosis showed a slightly decreased response to the 1800 ms SOA when compared to their age-matched peers. In children and adolescents, such reductions were likewise observed, not only for the 1800 ms, but also for the 900 ms SOA, which we interpreted as a sign of reduced short-term auditory sensory memory , 56. HenOne of the express purposes of employing three rates of presentation in the current study was to parametrically tax the auditory sensory memory system. That is, when the tones are presented at a rapid rate , this rapid presentation establishes a strong auditory sensory memory trace for the standard tones such that the longer duration deviants strongly “pop out”. As the rate slows , the sensory memory representation of the ongoing standards tends to weaken, and the general thesis is that the longer deviants are less perceptible at these slower rates . As can been seen from the MMN data herein, even at the slowest rate, a robust MMN was generated for both groups. In contrast, in data collected from individuals with Rett Syndrome using the same paradigm, an MMN was only detected at the fastest rate and was completely absent at the two slower rates . ClearlyLastly, in the P3a time window, and similar to what was observed for the P2, individuals with cystinosis presented increased amplitudes for the 900 and 1800 ms SOA conditions. There are several interpretations of the role of the P3 component (see ). One coIn summary, this study provides the first neural evidence of auditory sensory memory and involuntary attention differences in adults with cystinosis and pinpoints areas that warrant more research. It is nevertheless important to stress that despite the differences found between the groups, the adults with cystinosis tested here presented overall similar neural responses to controls. Some limitations should be noted. First, larger samples would have allowed for more detailed analyses focused on the associations between neural, cognitive, and behavioral outcomes and further investigation of the effects of cysteamine and its dosage on brain function. Given that compliance with cysteamine has been linked to better clinical outcomes , it coulWe used high-density EEG recordings and a passive duration oddball paradigm to investigate basic auditory processing, sensory memory, and involuntary attention in a sample of adults with cystinosis. Our findings suggest intact early auditory processing, but mild-to-moderate changes in auditory sensory memory and attentional processing in cystinosis. Given that group differences appear to be stronger in our prior study in children, such changes do not seem to be due to chronicity and/or time taking medication. This work contributes to the understanding of how brain function is affected in cystinosis, and suggests future directions of research to advance such understanding. The knowledge that we gain through this work can be used to inform the development of appropriate intervention strategies designed to positively impact everyday functioning for individuals with cystinosis.Fifteen adults with cystinosis  and 17 age-matched controls were recruited for this study. Hearing difficulties and current neurological problems were exclusionary criteria for the cystinosis group. Hearing problems, developmental and/or educational difficulties or delays, and neurological problems were exclusionary criteria for the control group. A hearing test  was performed on both ears using a Beltone Audiometer (Model 112). All individuals presented typical hearing thresholds. One individual with cystinosis was tested at an off-site location and, therefore, no hearing test was conducted. This individual reported normal hearing. All participants signed an informed consent approved by the Institutional Review Board of the Albert Einstein College of Medicine. Participants were monetarily compensated for their time. All aspects of the research conformed to the tenets of the Declaration of Helsinki.Tests of cognitive function and EEG data were collected over a 2-day period. The Wechsler Abbreviated Scale of Intelligence, WASI-II  was usedtm, The Netherlands; 10-20 montage). Preprocessing was done using the EEGLAB toolbox  for MATLAB  .EEG data were acquired continuously at a sampling rate of 512 Hz from 71 locations using 64 scalp electrodes mounted on an elastic cap and seven external channels  .Data were downsampled to 256 Hz, re-referenced to TP8 and filtered using a 1 Hz high pass filter  and a 45 Hz low pass filter . Both were zero-phase Hamming windowed sinc FIR filters. Bad channels were automatically detected based on kurtosis measures and rejected after visual confirmation . Artifacts resulting from eye blinks and saccades were removed by running an Independent Component Analysis (ICA). After ICA, the previously excluded channels were interpolated, using the spherical spline method. Data were segmented into epochs of − 100 to 400 ms using a baseline of − 50 to 0 ms. These epochs went through an artifact detection algorithm (moving window peak-to-peak threshold at 120 µV). All subjects had trial rejection rates below 30%. No significant differences were found between the number of trials included in the analyses per group , number of transplants , and MMN amplitude  were different from normal distribution and thus Spearman correlations were instead used for these variables. All p-values (from post-hoc tests and correlations) were submitted to Holm-Bonferroni corrections for multiple comparisons [p.adjust of the stats package in R [The definition of the time windows of interest was based on the typical time of occurrence of the components, and on visual confirmation that amplitudes were maximal in these intervals. Mean amplitude for the N1 was measured between 90 and 130 ms, P2 was measured between 150 and 180 ms, MMN between 200 and 260 (100 to 160 ms post deviance onset), and P3a between 310 and 350 ms. Mean amplitude measures  were taken at FCz, where signal was at its maximum for both groups. These amplitudes were used for between-groups statistics and correlations. Correlation analyses were computed across and between groups per component. Pearson correlations were performed for most of the variables, given that their distributions were not significantly different from the normal distribution, as tested by the ity test , implemeage in R : verbal parisons , using tage in R .


International Journal of Women's Dermatology (IJWD), everyone agreed that making our reach as inclusive and international as possible was critically important. The WDS has members from five continents and, to that end, both the IJWD’s editorial board and advisory board also have international representation across the globe, including Argentina, Australia, Brazil, Denmark, France, Iran, India, Israel, Germany, Greece, the Philippines, and the United States. What does international actually mean? According to the Oxford English dictionary, international means “across several countries”; hence, the inclusion of articles from North America among articles from South America, Europe, the Middle East, Asia, and Oceania does not mean that articles contributed from the United States are not international. Otherwise, the term international would be misrepresented in a U.S.-centric view of the world.When the board of the Women's Dermatologic Society (WDS) voted in 2013 to approve the proposal by board members Drs. Dedee Murrell and Jane Grant-Kels to explore the feasibility of developing the Within the IJWD, our focus is not just on the skin and hair disease of women and their families, but also on issues faced by women in the field of dermatology in their careers. Dermatology is one of the most competitive fields of medicine to gain entry into and requires top grades in medical school, research and publications, and connections to go beyond just training and into a successful academic, public hospital, or private practice career, along with hopefully a good personal life. The IJWD's January 2020 issue focused on the gender gap in academic dermatology and the supporting the personal and professional lives of female dermatologists. The IJWD March 2021 issue focused on skin of color and diversity and inclusion initiatives. Both issues highlighted many of these aspects for patients and dermatologists alike .Another way that the IJWD and WDS foster the careers of women in dermatology is to enable them to submit and publish interesting case letters, research letters, clinical pearls, editorials, review articles, and original research to our journal. In this issue, our signature piece, the Women's Health Highlight, comes from India regarding an issue very important to women: hirsutism . We alsoInternational Journal of Dermatology, whose editor is also an Egyptian woman, Dr. Rokea el Azhary, from the Mayo Clinic. Cindy was born in Princeton, New Jersey, and was a Fulbright scholar in French-speaking Guinea, Africa. There, she met her future husband, who also became a Fulbright scholar in the United States. Cindy ran the ISD almost single handedly for many years. She died tragically in a car accident in Maryland  Froehlich . For morMaryland .The ISD's mentorship program, currently co-chaired by IJWD editorial board members Prof. Rashmi Sarkar from India and Dr. Martin Kassir from Dallas, has supported both female and male dermatologists from poor countries to learn from experts in other countries for periods of up to 3 months . Cindy w


Anterior cruciate ligament (ACL) reconstruction (ACLR) has higher failure rates in young active patients returning to sports as compared with older, less active individuals. Augmentation of ACLR with an anterolateral procedure has been shown to reduce failure rates; however, indications for this procedure have yet to be clearly defined.The purpose of this study was to identify predictors of ACL graft failure in high-risk patients and determine key indications for when hamstring ACLR should be augmented by a lateral extra-articular tenodesis (LET). We hypothesized that different preoperative characteristics and surgical variables may be associated with graft failure characterized by asymmetric pivot shift and graft rupture.Case-control study; Level of evidence, 3.Data were obtained from the Stability 1 Study, a multicenter randomized controlled trial of young active patients undergoing autologous hamstring ACLR with or without a LET. We performed 2 multivariable logistic regression analyses, with asymmetric pivot shift and graft rupture as the dependent variables. The following were included as predictors: LET, age, sex, graft diameter, tear chronicity, preoperative high-grade knee laxity, preoperative hyperextension on the contralateral side, medial meniscal repair/excision, lateral meniscal repair/excision, posterior tibial slope angle, and return-to-sports exposure time and level.Of the 618 patients in the Stability 1 Study, 568 with a mean age of 18.8 years  were included in this analysis. Asymmetric pivot shift occurred in 152 (26.8%) and graft rupture in 43 (7.6%). The addition of a LET  and increased graft diameter  were significantly associated with lower odds of asymmetric pivot shift. The addition of a LET  and older age  significantly reduced the odds of graft rupture, while greater tibial slope , preoperative high-grade knee laxity , and greater exposure time to sport   were significantly associated with greater odds of rupture.The addition of a LET and larger graft diameter were significantly associated with reduced odds of asymmetric pivot shift. Adding a LET was protective of graft rupture, while younger age, greater posterior tibial slope, high-grade knee laxity, and earlier return to sport were associated with increased odds of graft rupture. Orthopaedic surgeons should consider supplementing hamstring autograft ACLR with a LET in young active patients with morphological characteristics that make them at high risk of reinjury. Age has been a significant predictor of ACLR failure in multiple studies.53 The Multicenter Orthopaedic Outcomes Network (MOON) knee group showed that the odds of ACLR failure decrease by 9% for every year increase in age. Webster et al also showed that patients <20 years old had a 30% cumulative risk of ACL reinjury or contralateral knee ACL injury in the first 2 years after reconstruction. Other risk factors for ACLR failure have included increased posterior tibial slope, meniscal deficiency, graft size, and graft choice.There are many studies detailing the successful outcome of anterior cruciate ligament (ACL) reconstruction (ACLR); however, there are also many reports of unsatisfactorily high rates of failure, particularly in the younger athlete. and more recently Marom et al, the addition of a LET results in reduced graft forces after ACLR. This may provide some protection during the graft healing and maturation phases. Unfortunately, there is a paucity of literature regarding the indications for adding an anterolateral procedure during ACLR. Two recent consensus articles outlined potential indications for the augmentation of ACLR with an anterolateral procedure.50 The first included revision ACLR, increased posterior tibial slope, generalized ligamentous laxity (or knee hyperextension >10°), young age, and return to contact pivoting sports. The second targeted revision ACLR, high-grade pivot shift, Segond fracture, participation in pivoting sports, and hyperlaxity as primary criteria and contralateral ACL rupture, Lachman test displacement >7 mm, deep lateral femoral notch sign, and age <25 years as secondary criteria. However, both studies were based on level 5 evidence and would suggest that nearly all patients undergoing ACLR should have an anterolateral procedure augmentation. Consequently, given the lower level of evidence behind these position statements and therefore the potentially erroneous conclusions that have been made regarding the addition of anterolateral procedures, studies with higher evidence levels are necessary to determine the appropriate indications for lateral augmentation during ACLR. It is clear that more robust evidence is required to guide surgeons and patients to decide when an anterolateral augmentation of ACLR is required.Anterolateral-based procedures, such as lateral extra-articular tenodesis (LET) or the newer anterolateral ligament reconstruction techniques, have emerged as surgical methods to attempt to reduce persistent anterolateral rotatory laxity and ACL graft failure, particularly in patients who may be at high risk of graft failure. A number of theories have postulated why an anterolateral procedure can reduce graft failure. The reduction of persistent rotatory laxity is the most obvious. However, as shown by Engebretsen et al At 2 years after surgery, the addition of the LET resulted in a 60% relative risk reduction of graft failure as compared with the ACLR alone. The purpose of the present study was to (1) identify preoperative variables associated with persistent rotatory laxity or graft rupture in high-risk patients from the Stability 1 Study and (2) determine key indications to inform surgeons when hamstring autograft ACLR should be supplemented with a LET. The hypothesis of this study was that different patient characteristics and surgical variables may be associated with graft failure characterized by asymmetric pivot shift or graft rupture.We recently performed a multicenter randomized clinical trial in which patients aged ≤25 years who were deemed as being at high risk of reinjury were treated with hamstring tendon autograft ACLR with or without a LET (Stability 1 Study).1214Data for this analysis were obtained from the Stability 1 Study . An overall 618 patients between the ages of 15 and 25 years were recruited from 9 centers  and then followed for 2 years postoperatively. Patients underwent clinical assessment and completed patient-reported outcome measures at postoperative 3, 6, 12, and 24 months. The study was approved by the Western University Research Ethics Board and the local board at each institution. A detailed study protocol and results have been published. The pivot-shift test, which has been shown to be specific  for detection of ACL rupture, was completed at each visit by a trained, blinded member of the surgical team and graded according to the International Knee Documentation Committee (IKDC) form. A positive pivot-shift test finding correlates with worse functional outcome26 and is widely accepted as a marker of failed ACLR.3335The primary outcome in the Stability 1 Study was clinical failure of the ACLR, defined as (1) persistent grade 1 pivot shift at multiple visits, (2) grade ≥2 pivot shift at any visit, or (3) graft rupture confirmed arthroscopically or on magnetic resonance imaging.49 sex, treatment group , preoperative knee hyperextension on the contralateral side, graft diameter, posterior tibial slope, time from injury to surgery (months), preoperative high-grade knee laxity,30 and meniscal treatment status .We selected 12 predictors for our analyses based on previous findings and clinical hypotheses: age, The presence of a grade 3 Lachman sign (>10-mm difference vs the other side) or grade 3 pivot-shift result (+++ gross) was used to define high-grade knee laxity, similar to previous research by the MOON group.30 The posterior tibial slope for all patients was measured on a true lateral radiograph taken preoperatively by 1 fellowship-trained orthopaedic surgeon according to the technique used by Webb et al. Tibial slope was included as a continuous variable to assess the effect of a 1° increase in tibial slope.Patients reported age, sex, and date of injury on a preoperative questionnaire. Age was included as a continuous variable to assess the effect of a 1-year increase in age. Tear chronicity was calculated as the interval in months between the date of injury and date of surgery. Passive hyperextension >10° was measured on both knees preoperatively as part of the Beighton score. Because the degree of hyperextension between uninjured knees is highly correlated and the surgical knee in some patients was locked or otherwise unable to demonstrate preinjury extension, we used the measurement from the nonoperative knee. Pivot-shift and Lachman tests were performed with the patient under anesthesia by the operating surgeon at the time of surgery according to the IKDC guidelines.Patients were randomized intraoperatively to ACLR alone or ACLR + LET. The type of procedure performed, the ACL graft diameter, and the presence and treatment of meniscal tears were documented on the standardized surgical report forms by the operating surgeon.23Patients reported their primary sports and participation levels  preoperatively. Postoperatively, patients were given an RTS questionnaire and asked to indicate when they returned to sports and whether at a higher, similar, or lower level. We classified those who returned to competitive, varsity, or elite sports as high level; those returning to recreational sports as low level; and those who did not return at all. Patients were also classified by their primary sports as high or low risk. High-risk sports were defined as those that required cutting, pivoting, or landing from jumps  while low-risk sports did not . Exposure time—the number of months that patients were playing sports during the study period—was determined by subtracting the postoperative RTS month from the total follow-up period of 24 months. For example, a patient who returned to sports 6 months postoperatively would have an exposure time of 18 months, and a patient who did not RTS would have an exposure time of zero months. Exposure time was included as a continuous predictor to assess the effect of a 1-month increase in exposure time. Early return to knee-strenuous sports and higher activity level have been shown to be related to increased risk of graft failure.The primary outcome variable was separated into 2 groups by distinct event: asymmetric pivot shift  and graft rupture. Different factors may be associated with each event. We performed multivariable logistic regression to predict asymmetric pivot shift and graft rupture using the all-enter method with 11 of 12 predictors. Radiographs were not available for 55 patients; therefore, tibial slope was added in a second step to show the effect of removing these patients and adding slope to the analysis. The graft rupture model was adjusted for the number of postoperative months in which patients returned to sport and the level at which they returned. Potential interactions were evaluated between treatment group and other predictors to determine whether they influenced the effectiveness of adding a LET. Outliers and influential points were identified using DFbeta values >0.10, with planned sensitivity analyses to determine whether removing influential points changed the contribution of each predictor.To check the assumptions for logistic regression, locally weighted scatterplot smoothing curves were used to assess linearity between continuous predictors and the log odds. The variance inflation factor was used to detect multicollinearity between predictors. A variance inflation factor >2 required investigation, and a variance inflation factor >10 required the removal of predictors causing multicollinearity.P > .30 and presenting odds ratios (ORs) and 95% CIs for each model. The Hosmer-Lemeshow goodness-of-fit test and area under the curve (AUC) for a receiver operating characteristic (ROC) curve were used to assess model fit and its ability to correctly classify individuals. To inform our second objective to determine patients for whom the addition of a LET may be most beneficial, predictors that could not be measured pre- or intraoperatively were removed . The relationship between these predictors and the addition of a LET were explored by calculating the risk ratio of graft rupture for different thresholds of each variable by treatment group. Last, the predicted probabilities of graft rupture for patients from the Stability 1 Study were determined and presented in graphical form for ease of interpretation. All statistical analyses were performed using Stata 15.1 (StataCorp).We pared the models down to include only important predictors, removing variables where A total of 618 patients were recruited for the Stability 1 Study, and 587 patients (95.0%) had outcome data available at postoperative 2 years. Nineteen (3.2%) of the remaining patients were missing predictor variables and were removed from this analysis. The characteristics of the 568 patients that we included are presented in Of the available patients, 152 (26.8%) had asymmetric pivot shift and 43 (7.6%) had graft rupture. Adding tibial slope to the 11 predictors in the asymmetric pivot-shift model removed 55 patients from the analysis owing to missing data; the effect of this decision on predictor estimates and precision in the full model is provided in The model met all assumptions, and 3 outliers were identified; however, removing them did not change the model estimates or statistical significance. All variance inflation factor values were <2, indicating no multicollinearity among predictors, and no significant interaction terms were identified between treatment and other predictors. Adding a LET  and increasing graft diameter  significantly decrease the odds of an asymmetric pivot shift.P > .05). Knee hyperextension, medial meniscal repair, medial meniscal excision, and greater tibial slope were associated with increased odds of asymmetric pivot, although none of these variables were statistically significant (P > .05). The Hosmer-Lemeshow test returned a nonsignificant result (P = .52), indicating adequate model fit, and the AUC for the ROC curve was 0.64.Increasing age remained in the model and was associated with decreased odds of positive pivot-shift result, although it was not statistically significant  no RTS; (2) low risk, low level; (3) low risk, high level; (4) high risk, low level; and (5) high risk, high level. Very few patients returned to low-risk sport, so categories 2 and 3 were condensed to 1 low-risk category. The effect of adding tibial slope and deleting 55 cases from the full model is shown in All assumptions were checked, identifying 10 potential outliers; however, removing these observations did not change the model estimates or affect statistical significance. The pared-down model with ORs and 95% CIs for important predictors of graft rupture is presented in P = .91), and the AUC for the ROC curve was 0.78.The LET procedure was significantly associated with 60% lower odds  of graft rupture, and a 1-year increase in age was associated with 17% lower odds of rupture . A 1° increase in posterior tibial slope was significantly associated with 15% higher odds  of rupture. Patients with high-grade preoperative knee laxity were at 3.27-times higher odds  of graft rupture. Exposure time remained in the model as a significant predictor, with each additional month of exposure time  increasing the odds of rupture by 18% . The Hosmer-Lemeshow test returned a nonsignificant result  and thus may be indications for a LET based on the goal of the procedure in young high-risk patients.Last, we performed a sensitivity analysis using an ROC curve to determine the optimal threshold of the posterior tibial slope variable that best identified those at greater risk of graft failure. The optimal threshold for tibial slope was 9.4° , and patients with a slope >9.4° were at 2.7-times greater odds  of graft rupture than patients with a slope below this threshold.This multivariable analysis has identified preoperative patient characteristics and surgical variables that are associated with persistent rotatory laxity and graft rupture within the Stability 1 Study randomized clinical trial. The most important finding was that augmentation of a hamstring tendon autograft ACLR with a LET reduces the odds of graft rupture by 60% and postoperative asymmetric pivot shift by 46% after adjusting for other confounding factors.59 Webster et al observed that the incidence of ACL failure in patients aged <20 years was 30% at 2 years when ipsilateral and contralateral injuries were combined. In a study from the MOON group, Kaeding et al demonstrated that young age was associated with ACL reinjury, with a 9% decrease in risk for every year gained in age. In a more recent study from the MOON group, college athletes aged <23 years had a 19.7% incidence of ACL rupture of either knee by postoperative 6 years. Furthermore, patients treated with a hamstring autograft had 2.1-times higher odds of rupture than those treated with a bone–patellar tendon–bone autograft.Age was tightly constrained within our cohort, as only skeletally mature patients who were ≤25 years old at the time of surgery were included. Regardless, younger age was associated with higher odds of asymmetric pivot shift and significantly higher odds of graft rupture after adjusting for RTS time (exposure) and level. Previous studies have also shown that younger patients are at higher risk of graft failure. , which found an association between slope and reinjury. The same group studied the association of tibial slope and age, demonstrating the “catastrophic” effect of young age and increased posterior tibial slope, with an 11-times increase in risk of graft rupture at postoperative 2 years if <18 years old with a tibial slope >12°. Our data showed that patients with a tibial slope above approximately 9.5° had over twice the odds of graft rupture as compared with patients with a slope below that threshold, although the risk difference between patients receiving ACLR alone and ACLR + LET was similar at all levels of tibial slope . If the effectiveness of a LET does not change with tibial slope , this suggests that a LET may contribute to a different mechanism than tibial slope. While tibial slope is modifiable through osteotomy procedures, surgeons may first want to consider using less aggressive procedures known to reduce risk of graft rupture, such as a LET,52 or a different graft choice, such as bone–patellar tendon–bone autograft, even if these factors work independently.In the Stability 1 Study, posterior tibial slope was a significant predictor of graft rupture and contributed to the model for asymmetric pivot shift. The mean tibial slope in our study was 9° , which is similar to the mean slope from the case-control study by Webb et alAppendix Tables A1 and A2, available online) demonstrate a similar relative risk at all values of slope and age, with the addition of a LET being 3 to 4 times more protective than a hamstring autograft ACLR alone.To understand the significantly increased risk of ACLR failure related to age and tibial slope, we investigated the effectiveness of adding a LET visually. We plotted the predicted probability of graft rupture with 95% CIs across the range of tibial slope values and patients’ ages within the Stability 1 Study, adjusting for other important predictors. The graphs suggest that adding a LET to a hamstring tendon autograft ACLR significantly reduces the probability of failure in patients aged <23 years and with a tibial slope >6°, as the 95% CIs no longer overlap at these proposed thresholds. Tables showing graft rupture between groups  was performed as compared with meniscal repair  in relation to no treatment of the medial meniscus. The menisci are understood to behave as secondary stabilizers to anterior translation and anterolateral subluxation in the ACL-deficient knee. this evidence supports the need for meniscal preservation during ACLR, as a deficient medial meniscus may be more problematic. Robb et al prospectively followed 124 patients undergoing primary ACLR over the course of 2 years and performed a survival analysis to determine prognostic factors of graft survival. Eighteen patients (14.5%) in that study experienced graft failure. In addition, medial and lateral meniscal deficiency was associated with the risk of failure, and the risk of failure was >4 times higher for those with medial meniscal deficiency. Research from the MOON group on concomitant meniscal tears at the time of ACL surgery revealed that while medial and lateral repairs fail at a similar rate by postoperative 6 years, medial retears occur earlier than lateral retears . The SANTI Group found that in 383 patients followed for 2 to 5 years, the addition of an anterolateral ligament reconstruction at the time of ACLR was protective of medial meniscal repair, as the failure rate was 2 times lower in the group undergoing ACLR and anterolateral ligament reconstruction than ACLR alone. As such, anterolateral procedures such as LET or anterolateral ligament reconstruction may be protective of not only ACL graft rupture but also meniscal repair failure, which in turn may have a combined effect on ACLR outcomes. In contrast, there are concerns that the addition of an anterolateral procedure may overconstrain the knee joint, potentially leading to an increased risk of osteoarthritis (OA) development in the long term. In a 2017 systematic review of 8 studies and 421 patients, Devitt et al showed a low incidence of OA in the first 11 postoperative years after combined ACLR and LET; however, 2 studies with >24-month follow-up demonstrated OA rates >50%. Further evidence of long-term outcomes is required; thus, surgeons should weigh the potential risks and benefits when deciding whether to augment ACLR with a LET.Medial meniscal excision, while nonsignificant, was associated with 1.9-times higher odds  of graft rupture in our model. Despite suggestions that repair of a medial meniscal tear may lead to poor outcomes,38 This may be secondary to reduced neuromuscular conditioning as well as the lack of ACL graft maturity. In a recent narrative review, Nagelli and Hewett posed the question of whether RTS should be delayed until at least 2 years postoperatively to allow for appropriate healing and rehabilitation. While this may not be a plausible option for many young athletes, it does highlight the need for better RTS assessment and functional testing before release of patients back to full activity. Understanding these factors warrants a conversation with patients regarding the addition of a LET but also to determine their postoperative goals, particularly surrounding their desire and intended timing of return to high-risk sports. Despite the lack of retention in the model, our analysis showed that return to a higher-risk sport carried greater risk than return to a lower-risk sport  was associated with graft rupture. Multiple studies have demonstrated the negative effects of an early RTS.port see . demonstrated that a graft size <8 mm was associated with failure, while a follow-up study by Spragg et al reported that a 0.5-mm increase in graft diameter was associated with a 14%-18% reduction in the likelihood of revision surgery. Graft diameter was not related to graft rupture in our study, likely in part because of the specific efforts made to control for graft size intraoperatively by tripling the semitendinosus tendon if a 4-strand semitendinosus/gracilis construct was <8 mm. However, even with these measures in place, we did find that a 1.0-mm increase in graft diameter was associated with 38% lower odds of asymmetric pivot shift.Graft diameter has been shown to be predictive of failed hamstring ACLR. Snaebjörnsson et al superficial laceration, and increased tension on the ACL, it is not surprising that hyperextension is predictive of rotational instability but not rupture. Several biomechanical and cadaveric studies have assessed the effect of knee hyperextension on impingement and graft tension. Jagodzinksi et al performed magnetic resonance imaging scans of 15 knees and found strong correlation  between the degree of hyperextension and graft impingement. Goss et al took 5 fresh-frozen cadaveric knees and assessed contact pressure and graft tension for 3 tibial tunnel positions: they found (1) higher contact pressures between the ACL graft and the intercondylar notch as hyperextension increased and (2) increasing graft tension as the degree of hyperextension was greater, regardless of tunnel placement. Clinical research studies have shown conflicting results. Several studies have shown an association between knee hyperextension and preoperative instability,43 postoperative instability, and graft failure, while others report no relationship between hyperextension and risk of laxity or rupture.Knee hyperextension contributed to the model for asymmetric pivot shift but was not predictive of graft rupture. Because hyperextension is linked with mechanisms that can increase graft laxity, such as impingement, including 62% of patients with grade 2 Lachman test and 76% of patients with grade 2 pivot-shift test. Thus, the low- vs high-grade variable is largely a comparison of grade 2 vs 3 laxity. Our findings suggest that patients with high-grade laxity have similar odds of residual rotational laxity as those with low-grade laxity, while high-grade laxity does contribute to the risk of graft rupture. In contrast, Jacquet et al recently showed that high-grade preoperative laxity was predictive of residual laxity in a cohort of 266 patients, although they were older  and predominantly male (71.3%). Magnussen et al30 showed an association between high-grade laxity and graft rupture in the MOON cohort at postoperative 2 and 6 years. They also showed that high-grade laxity was not associated with the risk of contralateral ACL tear, suggesting that high-grade laxity was related to injury-specific rather than patient-specific factors.High-grade preoperative knee laxity, as determined by a grade 3 Lachman or pivot-shift test result, was significantly associated with 3-times greater odds of graft rupture within the Stability 1 Study, although it was not retained in the asymmetric pivot-shift model. The Stability 1 cohort was intentionally based on high-risk patients with significant preoperative laxity, and this issue is under investigation in our ongoing Stability 2 Study . Second, while the pivot-shift test was scored by experienced members of the surgical team , this was a multicenter study; thus, various members of the surgical team performed the pivot-shift assessment. While the assessment was performed according to the IKDC scoring system and the trial methodology required multiple grade 1 pivot-shift test results for the primary outcome to reduce the effect of measurement variability, there may be some differences in how the pivot shift was graded. Third, surgical variables such as tunnel placement have been identified as predictors of ACLR failure in previous studies but were not controlled for in the Stability 1 trial. Furthermore, given the relatively low number of graft ruptures (n = 45), we are at risk of overfitting the rupture model, as logistic regression requires at least 10 events per predictor to be adequately powered. Validation outside the study sample is particularly important for a model with few events; however, the predictors identified by this analysis do coincide with clinical hypotheses for graft rupture.50This study has limitations, particularly in relation to the patient sample included in the analysis. First, the sample was part of a randomized controlled trial of young active patients at high risk of retear who underwent ACLR with a hamstring autograft, exhibiting specific criteria that put them at higher risk of failure. As such, this cohort is not representative of the overall ACLR population or those receiving other graft types for ACLR. It is also not clear whether the addition of a LET to a bone–patellar tendon–bone autograft would provide the same level of protection, as found in the previously mentioned MOON study,The addition of a LET to hamstring autograft ACLR was significantly associated with 60% lower odds of graft rupture, while younger age, increased tibial slope, high-grade preoperative knee laxity, and early RTS were associated with higher odds of graft rupture. Younger age, knee hyperextension, increased tibial slope, and medial meniscal repair or excision were all related to higher odds of asymmetric pivot shift, while adding a LET and increasing hamstring autograft diameter significantly reduced asymmetric pivot shift in our high-risk cohort from the Stability 1 Study. Orthopaedic surgeons should consider supplementing hamstring autograft ACLR with a LET in young active patients with morphological characteristics that make them at high risk of reinjury, as the LET was protective when adjusted for other variables in this analysis.Andrew D. Firth, MSc, Dianne M. Bryant, MSc, PhD, Alan M.J. Getgood, MD, Robert Litchfield, MD ; Robert G. McCormack, MD ; Mark Heard, MD ; Peter B. MacDonald, MD ; Tim Spalding ; Peter C.M. Verdonk, MD, PhD ; Devin Peterson, MD ; Davide Bardana, MD ; Alex Rezansoff, MD ; and the STABILITY Study Group .Click here for additional data file.Supplemental material, sj-pdf-1-ajs-10.1177_03635465211061150 for Predictors of Graft Failure in Young Active Patients Undergoing Hamstring Autograft Anterior Cruciate Ligament Reconstruction With or Without a Lateral Extra-articular Tenodesis: The Stability Experience by Andrew D. Firth, Dianne M. Bryant, Robert Litchfield, Robert G. McCormack, Mark Heard, Peter B. MacDonald, Tim Spalding, Peter C.M. Verdonk, Devin Peterson, Davide Bardana, Alex Rezansoff, STABILITY Study Group, Alan M.J. Getgood, Kevin Willits, Trevor Birmingham, Chris Hewison, Stacey Wanlin, Ryan Pinto, Ashley Martindale, Lindsey O’Neill, Morgan Jennings, Michal Daniluk, Dory Boyer, Mauri Zomar, Karyn Moon, Raely Moon, Brenda Fan, Bindu Mohan, Gregory M. Buchko, Laurie A. Hiemstra, Sarah Kerslake, Jeremy Tynedal, Greg Stranges, Sheila Mcrae, LeeAnne Gullett, Holly Brown, Alexandra Legary, Alison Longo, Mat Christian, Celeste Ferguson, Nick Mohtadi, Rhamona Barber, Denise Chan, Caitlin Campbell, Alexandra Garven, Karen Pulsifer, Michelle Mayer, Nicole Simunovic, Andrew Duong, David Robinson, David Levy, Matt Skelly, Ajaykumar Shanmugaraj, Fiona Howells, Murray Tough, Pete Thompson, Andrew Metcalfe, Laura Asplin, Alisen Dube, Louise Clarkson, Jaclyn Brown, Alison Bolsover, Carolyn Bradshaw, Larissa Belgrove, Francis Milan, Sylvia Turner, Sarah Verdugo, Janet Lowe, Debra Dunne, Kerri McGowan, Charlie-Marie Suddens, Geert Declerq, Kristien Vuylsteke and Mieke Van Haver in The American Journal of Sports Medicine


The saphenous vein is the most commonly used conduit for coronary artery bypass grafting. Arterial grafts are harvested with the outer pedicle intact whereas saphenous veins are harvested with the pedicle removed in the conventional graft harvesting technique. This conventional procedure causes considerable vascular damage. One strategy to improve vein graft patency has been to provide external support. Ongoing studies show that fitting a metal external support improves conventionally harvested saphenous vein graft patency. On the other hand, the no-touch technique of harvesting the saphenous vein provides an improved graft with long-term patency comparable to that of the internal mammary artery. This improvement is suggested to be due to preservation of vessel structures. Interestingly, many of the mechanisms proposed to be associated with the beneficial actions of an artificial external support on saphenous vein graft patency are similar to those underlying the beneficial effect of no-touch saphenous vein grafts where the intact outer layer acts as a natural support. Additional actions of external supports have been advocated, including promotion of angiogenesis, increased production of vascular-protective factors, and protection of endothelial cells. Using no-touch harvesting, normal vascular architecture is maintained, tissue and cell damage is minimized, and factors beneficial for graft patency are preserved. In this review, the significance of external support of saphenous vein grafts in coronary artery bypass grafting is discussed. Furthermore, the SV in humans differs from other veins that generally remain under fairly constant flow and pressure, because the SVs of the lower limbs are subjected to variable orthostatic pressures caused by alterations in posture and movement.3 Apart from structural differences compared to other veins,4 being subjected to these pressure variations may precondition segments of SV grafts when subjected to the hemodynamics of the coronary arterial system.The saphenous vein (SV) was first introduced as a conduit for coronary revascularization five decades ago.5,6 compared to the high incidence of early graft occlusion, progressive intimal hyperplasia, and late graft atherosclerosis of conventional (CT) SV grafts  and radial artery has been encouraged because of their good long-term patency, long-term survival, and freedom from reinterventions,V grafts .7 Althou removed . This tr12 Here, placement of a monofilament knitted tube increased the patency of jugular vein grafts compared to controls in a dog bypass model. Fifteen years later, using the same experimental model, placement of an external Dacron stent was shown to reduce intimal hyperplasia and increased the density of the vasa vasorum in the vein graft wall.13 Both processes played an important beneficial role in vein graft performance. Although other forms of support have been described, including external mesh14 and fibrin glue,14,15 it is the external stents that have received most attention recently. A pig model of arteriovenous bypass grafting was developed and used extensively to study the potential effect of external stent placement on SV graft patency  formation and angiogenesis.17 Furthermore, increases in prostacyclin I2, cyclic adenosine monophosphate, and cyclic guanosine monophosphate formation were noted in both stented and nonstented grafts compared to ungrafted SV, with the production being greater in the stented compared to the nonstented graft. Also, in the adventitia of stented vein grafts, nitric oxide synthase was associated with microvessels as well as inflammatory cells. Taken together, these data are suggestive of a role for prostacyclin I2 and nitric oxide in promoting micro-angiogenesis in the adventitia of stented vein grafts, which may in turn minimize graft hypoxia, an established contributory factor to neointima formation. While an inflammatory response to external supports has been demonstrated in the pig and other animal bypass models, as far as we are aware, this has not been studied in sections of human SV grafts where external support has been used.The potential use of external supports in improving vein graft performance was first proposed over 50 years ago. patency .16,17 Th19 The results of a small randomized trial of the Extent device (a macroporous Dacron sheath reinforced with polytetrafluoroethylene ribs) were disappointing.20 Here, 20 patients received an Extent-supported graft to either the right or the left coronary system. However, in all 17 patients returning for follow-up, the Extent-supported grafts were found to be thrombosed on angiographic assessment (6 and 19 months postoperatively). On the other hand, all of the LIMA grafts and non-stented SV grafts remained patent. The authors attributed this to a combination of stent rigidity, oversizing, and incomplete tube design leading to graft kinking either at the anastomoses or in the middle of the graft. Since potential problems associated with stent rigidity and testing of biodegradable stents in the pig model had been recognized earlier, it seems surprising that Dacron stents were used in CABG patients. These bench-to-bedside/translational studies failed, most likely due to various limitations of the pig experimental model that was used.10Since there were concerns regarding potential long-term complications of a permanent polyester stent, a subsequent study was undertaken. This study investigated the effect of a biodegradable external stent on vein graft thickening at one month and the longer-term effects at six months. The loose-fitting biodegradable polyglactin external stent reduced porcine vein graft thickening at one month and this persisted in the long-term, even after degradation of the stent itself. The authors concluded that biodegradable external stents have potential advantages over permanent stent material in clinical application.21 Here, 14 sheep underwent CABG with one SV graft supported and a non-supported SV serving as a control. Early and late quantitative angiography was performed with histological evaluation carried out on graft sections on completion of the study. Ten animals survived with 3 of the control grafts thrombosed and one occluded. Although there was no difference in uniformity between grafts immediately after CABG, there was significant (p < 0.002) nonuniformity in the control versus supported grafts. This was confirmed by histological evaluation showing that the mean intimal area of the supported grafts was significantly lower than that in the control grafts (p < 0.02). The authors admit the limitations of their study, in particular the small sample size and short follow-up period. Also, the quality of the histological sections provided in the figures casts doubt on the accuracy of the morphometric data. Furthermore, ongoing venous external support trials (VEST) are in progress on patients undergoing CABG. Despite the positive data from VEST,22,23 the patient numbers are low and the follow-up period short. In a recently published study, Taggart and colleagues23 reported the results of VEST IV with comparable vein graft failure rates between stented and nonstented grafts  at a mean time 4.5 years. On the other hand, intimal hyperplasia and the development of luminal irregularities were significantly reduced by external stenting.More recently, an external support made from braided cobalt-chromium-nickel-molybdenum-iron alloy fibres, forming an expandable external SV support, has been developed and tested in a sheep bypass model .21 Here,7,10 In contrast to CT harvesting, the no-touch (NT) technique of SV harvesting introduced by Souza24 preserves the cushion of surrounding tissue , pulsatile flow, and shear stress of ∼3–6 dyne·cm−2.31 Within a month, these conditions result in SV graft remodelling and the development of diffuse intimal hyperplasia. Over time, intimal thickening and atherosclerotic lesions are formed specifically at regions where disturbed flow occurs , nonpulsatile flow, and a shear stress of ∼0.2 dyne·cmw occurs .32 Vein 35 potentially even reaching 2000 mm Hg.36 These pressures cause serious endothelial damage and exposure of the intimal basement membrane. With the NT technique, the SV is handled by its outermost cushion and as there is no direct contact with the SV by surgical instruments, spasm rarely occurs. Hence, there is no need for distension and endothelial damage is minimized.25,26,3739 An additional beneficial effect of preserved outer layers of the SV is the protection conferred on the luminal endothelium once inserted as a graft and subjected to arterial hemodynamics. A study comparing sections of CT versus NT harvested SVs showed marked differences in endothelial integrity.26 Here, certain SV segments were subjected ex vivo to intraluminal saline distension at 300 mm Hg pressure, well within the range of manual pressure used in CT SV harvesting.40 While there was marked endothelial damage in CT SVs, the endothelium of NT SVs that were not distended was virtually intact. An additional two treatments were performed in this study where CT SVs  were not distended and NT SVs (segments with the cushion of surrounding tissue intact) were distended. The endothelium of SVs in both groups remained relatively undamaged (26 There are three conclusions to be drawn from these results: it is mainly the distension, not the surgery, that causes endothelial damage to CT SVs; the cushion of surrounding tissue acts as a buffer and protects the endothelium against high-pressure distension of NT SVs; if the cushion of surrounding tissue in NT SVs protects against 300 mm Hg distension at harvesting, it will protect the endothelium once implanted as a graft where it is subjected to systolic arterial pressure usually ranging from 100 to 180 mm Hg.10,26 Another mechanical aspect of external support is protecting long vein grafts from kinking (The perivascular fat also plays an important, indirect role in protecting the luminal endothelium from the deleterious consequences of damaging this important single layer of cells. When using CT SV harvesting, a high percentage of SVs go into spasm that is overcome using high-pressure manual distension. Such pressures have been shown to be as high as 560 mm Hg,ndamaged .26 There kinking . This fe20 In earlier pig studies, end-to-end SV-to-carotid artery grafting was performed, a criticism raised in relation to grafts used in CABG.41 Subsequently, the more recent pig studies have used end-to-side grafts. Altered hemodynamics were investigated in a small study comparing stented vs. nonstented SV grafts in which resin casts were used to assess changes in graft geometry and the potential effect on blood flow.42 At 1-month post-implantation, unstented graft geometry was irregular, with nonuniform dilatation, substantial axial lengthening, curvature, kinking, and possible long-pitch helical distortion. In contrast, stented grafts showed a reduction in these changes although fine corrugation, occasional slight kinking or narrowing of segments, and possible long-pitch helical distortion were observed in some instances. In a later review, the effect of external stents or sheaths on the symmetry of grafts in response to arterial hemodynamics were discussed. Here, vein graft thickening was described as markedly asymmetric, a condition causing marked alterations in turbulent blood flow, which can elicit thrombosis and NIH. External stents or sheaths were said to keep the graft contained in a way that prevents turbulent blood flow and imposes laminar and symmetric blood flow, thereby reducing NIH.17 While these studies were performed using the pig vein graft model, more recent hemodynamic studies in patients have been described using VEST. A method for analyzing and comparing SV grafts following CABG was used to study flow dynamics inside vein grafts with and without support using VEST. Specific computational fluid dynamic measurements were used to characterize the relevant hemodynamic parameters and flow conditions in a small number of patients in whom stented and nonstented SV grafts were assessed at 12 months post-transplantation. SV grafts surrounded by VEST were associated with similar time-averaged wall shear stress, yet had fewer lesions, a lower oscillatory shear index and relative residence time index, and more uniform flow with fewer flow discrepancies. Based on these data, the authors concluded that VEST improved hemodynamic factors that correlate with graft failure following CABG.22Certainly, high-pressure distension at harvest and altered turbulence/shear both stimulate various mechanisms of graft failure, particularly those associated with NIH and thrombus formation. This may account, at least in part, for the detrimental effects described in the Extent trial where all Extent SV grafts were thrombosed but non-stented SV and internal mammary artery grafts remained patent.18 In the questions and answers section, Professor Wallwork asked: “do you not think that one of the issues about looking at a chronic ejection model is that you strip the outer layer of the vessel, therefore you are making it ischemic? Would you not get the same effect if you took the vein out with a lot of fat around it instead of stripping it? It would be a nice experiment to see whether that would give you the same results as putting the stent around it.”43 This would indeed have been “a nice experiment”. If acted upon, over 30 years ago, supporting evidence may have shown that preserving the perivascular fat contributes to improved performance of SV grafts as subsequently described in SVs harvested by the NT technique.10,27,28Interestingly, early beneficial aspects of the pig external stent model were proposed in a review based on a conference presentation where issues regarding stent size, porosity, and fit were discussed.44 Of particular relevance to conduits used for CABG are results from in-vitro studies showing that perivascular adipose tissue has an anticontractile action on both the internal mammary artery and SV.45,46 The potential role of perivascular fat-derived factors in SV graft performance is discussed in detail in a number of recently published research studies and reviews.47,48It is now recognized that perivascular fat is not merely a means of mechanical support for blood vessels, and there has been much interest in the beneficial effects of so called adipocyte-derived relaxing factors; for a review, see Gollasch and Dubrovska.49 Furthermore, removal of the adventitia of the carotid artery induced NIH, but intimal hyperplasia regression was observed on the appearance of a neoadventitia associated with the restoration of medial oxygenation.50 Interestingly, in tissue-engineered blood vessels developed as potential substitutes for autologous bypass grafts, the appearance of “adventitial” vasa vasorum has been described after implantation of the engineered graft.51There is experimental evidence from a variety of animal models for a role of the adventitial vasa vasorum in maintaining a healthy vessel. For example, rapid development of atherosclerotic lesions in rabbit carotid artery is induced by perivascular placement of a close-fitting collar. This resulted in NIH caused by obstruction of the adventitial vasa vasorum and creation of mural ischemia.17 Based on these observations, it has been proposed that, by means of angiogenesis, the regeneration of the vasa vasorum may constitute an important adaptation of vein grafts to arterial conditions and to the repair and reintegration of the microvascular system.17 The question arises, why promote angiogenesis in damaged SVs artificially when, in patients undergoing CABG, the SV vasa vasorum remains intact when harvested by the NT technique? The vasa vasorum is a microvessel network providing the adventitia and media with oxygen and nutrients and is more pronounced and penetrates deeper towards the lumen in veins than in arteries. When harvesting the SV conventionally, the adventitia and associated structures are often damaged, including damage to the vasa vasorum. Scanning electron microscope studies have identified various aspects of damage to the vasa vasorum, including severing of these microvessels at the adventitia-media border (52 In addition, ultrastructural studies have shown constriction and collapse of numerous vasa vasorum microvessels and clumping of erythrocytes.5355 Such features would ultimately reduce transmural blood flow, potentially leading to NIH and thus affecting graft patency.49,50 By contrast, the vasa vasorum of NT SV grafts remains intact with normal endothelial and vascular smooth muscle cell morphology.53 An unexpected finding in this study was the observation of small openings within the luminal endothelium, suggested to represent terminations of the vasa vasorum that extend from the adventitial-medial network.52,5456 These luminal terminations may play an important role in the improved performance of NT SV grafts because filling of the adventitial vasa vasorum has been observed in such grafts perfused during CABG.39,54 This has also been confirmed ex vivo in isolated segments of NT SV grafts perfused with blood from the cardiopulmonary bypass machine.39,54 In a recent ex-vivo study using India ink perfusion of SVs from CABG patients, sections from these veins showed staining of the luminal endothelium and the vasa vasorum throughout the vessel wall, extending to the capillary network within the perivascular fat.57Regarding the suggested mechanisms underlying the pig vein graft/external stent model, an exudate was observed in the gap between the stent and the graft within a week. This exudate was rich in fibrin and fibrinogen which are both potent angiogenic factors. Over the following weeks, microvessels appeared in the space between the graft and the stent, which proceeded to organize into a well-defined structure, much of which was comprised of microvessels. The formation of “neo-vasa vasorum” within the exudate in the graft-stent interface is suggested to play an important beneficial role in the pig external stent model.a border .52 In ad4 The potential importance of the vasa vasorum in SV grafts has been investigated, comparing their regional distribution and density.54 The density, size, and total area of the vasa vasorum in the media and adventitia of transverse histological sections was assessed by computer-assisted morphometry comparing NT with CT SV grafts. Although there was no significant difference between density and size in these vessel layers, there was a significantly greater total area in both the media and adventitia of NT vs. CT SVs.54 A subsequent study revealed striking differences between SV and arterial grafts. The density of the vasa vasorum was not only far higher in SV vs. arterial grafts but this study provided quantitative support for the previously described penetration of vasa vasorum in veins vs. arteries.4 The vasa vasorum was virtually absent in the media and significantly lower in the adventitia of both the LIMA and radial artery compared to the SV.58 Also, of particular relevance, is the significant reduction in the total number of vasa vasorum when stripping the adventitia, as occurs in CT SV harvesting.54 Therefore, in CT grafts, there is a high risk of developing hypoxic or ischemic conditions in the SV wall and initiating detrimental processes that affect graft patency.55As oxygenated blood passes through the lumen of arteries, the endothelium and vessel wall receive sufficient oxygen and nutrients by diffusion. However, the vasa vasorum in veins represents a microvessel network with the principle role of supplying blood to the wall as luminal oxygen and nutrient levels are low. Hence, the vasa vasorum of veins penetrate closer towards the intima than those of arteries, and are seen to advantage in the thick walls of the SV.59We agree with the concept and strongly advocate that SV grafts will perform better if equipped with external support. However, it is surprising that while recognizing the damage caused to the SV when harvesting conventionally, various strategies that are potentially harmful, complicated, time-consuming, or costly are pursued. Nevertheless, using the relatively simple surgical technique of NT harvesting preserves normal SV architecture, providing a superior graft for patients undergoing CABG. The recent success of artificial external stents for the SV in CABG provides yet another argument for harvesting this vein with its natural stent of perivascular fat. Ongoing and future studies will determine whether the benefit of synthetic external stents will translate into superior SV graft patency over the longer term. The discrepancy between how most arterial and venous grafts are harvested has been challenged since the development of the NT SV graft harvesting technique. The technique was recommended to be used in open harvesting of the SV by the European Society of Cardiology and the European Association for Cardiothoracic Surgery in 2018. We unpretentiously leave the decision to our surgical colleagues to determine which technique is most suitable for each individual patient, with a pertinent quotation from Benjamin Franklin: “An ounce of prevention is worth a pound of cure”.


Increased fibroblast growth factor 23 (FGF23) is a risk factor for mortality, cardiovascular disease, and progression of chronic kidney disease. Limited data exist comparing the association of either c-terminal FGF23 (cFGF23) or intact FGF23 (iFGF23) in kidney transplant recipients (KTRs) with overall  graft loss.We conducted a prospective observational cohort study in 562 stable kidney transplant recipients. Patients were followed for graft loss and all-cause mortality for a median follow-up of 48 months.p < 0.0001 and 45.24 [18.63–159.00] versus 29.04 [15.23–60.65] pg/ml; p = 0.002 for cFGF23 and iFGF23, respectively). Time-dependent ROC analysis showed that cFGF23 concentrations had a better discriminatory ability than iFGF23 concentrations in predicting overall  graft loss. Cox regression analyses adjusted for risk factors showed that cFGF23  but not iFGF23  was associated with the overall graft loss.During a median follow-up of 48 months, 94 patients had overall graft loss (primary graft loss or death with functioning graft). Both cFGF23 and iFGF23 concentrations were significantly higher in patients with overall graft loss than those without (24.59 [11.43–87.82] versus 10.67 [5.99–22.73] pg/ml; Elevated cFGF23 concentrations at baseline are independently associated with an increased risk of overall graft loss. iFGF23 measurements were not independently associated with overall graft loss. The cFGF23 ELISA might detect bioactive FGF23 fragments that are not detected by the iFGF23 ELISA.The online version contains supplementary material available at 10.1186/s12882-021-02329-7. Kidney transplantation is the preferred replacement therapy for end-stage kidney disease (ESKD) –3. The b2- VD3 [2D [Fibroblast growth factor 23 (FGF23) is an osteocyte-derived hormone and is involved in mineral-bone homeostasis by regulating serum phosphate, parathyroid hormone (PTH), and 1,25-(OH)2- VD3 –9. The k VD3 [2D . Two typ VD3 [2D , whereas VD3 [2D .Prior clinical studies revealed a close relationship between FGF23 and the cardiovascular morbidity and mortality in both the CKD populations and the general population , 14. In Limited data exist with regard to head-to-head comparisons cFGF23 and iFGF23 measurements in kidney transplant recipients with respect to graft loss and all-cause mortality. Thus, we carried out this study to compare the predictive power of both iFGF23 and cFGF23 with composite outcomes of graft loss and all-cause mortality in KTRs.This prospective observational cohort study comprised prevalent kidney transplant recipients who had kidney transplantation before 15th October 2012 and came for routine check-ups at the transplant outpatient clinic Charité-Mitte, Berlin, Germany.Patients with active infections, malignancy, acute rejection, a recent cardiovascular event at the time of study inclusion or those unwilling to participate were excluded. Patients were prospectively followed up for a median of 4.0 years (IQR 3.93–4.04). Prespecified endpoint was defined as overall renal graft loss included both functional grafts lost due to recipient death and death-censored graft loss. Graft loss was defined as return to dialysis, graft removal, re-transplantation, based on the judgment of the treating physicians. The primary outcome was defined as overall  graft loss comprising primary renal allograft loss and death with functioning renal allograft. The study protocol was approved by the ethics committee of the Charité - Universitätsmedizin Berlin  and informed consent was obtained from all participants. All methods were carried out in accordance with relevant guidelines and regulations.Baseline characteristics of KTRs and kidney donors, such as age, gender, recipients’ primary kidney diseases and post-transplant duration, cold ischemia time, human leukocyte antigen mismatches, panel reactive antibodies (PRA) were extracted from hospital records.Blood and urine samples were collected from patients during routine visits from April 2012 until December 2012. Baseline laboratory measurements, such as creatinine, serum albumin, total cholesterol, fasting blood glucose, calcium, phosphorus, as well as urinary protein excretion were measured in the central clinical laboratory of the Charité Universitätsmedizin Berlin, Germany. Baseline estimated glomerular filtration rate (eGFR) was calculated using the CKD-Epidemiology Collaboration equation.https://www.bmgrp.com/wp-content/uploads/2019/03/bi-20702-fgf23-elisa-validation-data-150306.pdf), and ≤ 8 and ≤ 6% for iFGF23 assay . All samples were measured in duplicate and all assays were subject to regular quality control.cFGF23 and iFGF23 were measured with commercially available ELISA [FGF23  multi-matrix ELISA, cat. no. BI-20702, Biomedica, Austria and FGF23 (intact) human ELISA, cat. no.BI-20700, Biomedica, Austria) according to the instructions of the manufacturer. The average intra-and inter-assay coefficients of variation were ≤ 12% and ≤ 10% for cFGF23 assay , model 5 was adjusted for the same factors as model 3, plus iron status markers (mean corpuscular volume and ferritin), model 6 was fully adjusted model, including all adjustments. The Bland-Altman plots were used to evaluate the agreement of mean difference between cFGF23 and iFGF23. P value < 0.05 was regarded statistically significant. Statistical analyses other than time-dependent ROC were performed using SPSS version 25.0 . Time-dependent ROC curves was conducted with R software , in addition, timeROC (version 0.4) and ggplot2 (version 3.3.3) packages were included.Descriptive statistics of baseline characteristics are presented as means (standard deviation) for normally distributed data or as medians (interquartile ranges) for not normally distributed variables. Categorical data are presented as numbers (%). Statistical differences between baseline characteristics were analyzed using a Student’s t test or Mann Whitney U test, or χDetails of the data sources, search strategy, study selection, eligibility criteria, data extraction, quality assessment and analysis method were described in supplementary file.n = 546) and iFGF23 (n = 502) were 12.57 pg/ml  and 30.94 pg/ml , respectively. Both median intact and c-terminal FGF23 levels were significant lower in the survival group compare to the patients who had graft loss or died (p = 0.002).Demographic and clinical data of the cohort are shown in the Table During a median follow-up of 4.0 years , 94 patients suffered from overall graft loss. Time-dependent ROC analysis showed that cFGF23 concentrations had a better discriminatory ability than iFGF23 concentrations in predicting overall  graft loss. Plots of AUROCs (area under the ROC) at 1-year, 2-year, 3-year, 4-year of cFGF23 and iFGF23 for overall graft loss are shown in Fig. P < 0.001)  graft loss and explored possible variations in this association. We used Cox regression models with a stepwise selection of variables that associated with death or graft failure. cFGF23 remained a significant predictor of overall  graft loss, even after full adjustments Table . The effSame analyses as cFGF23 were conducted in iFGF23. Plots of AUROCs (area under the ROC) at 1-year, 2-year, 3-year, 4-year of cFGF23 and iFGF23 for overall graft loss are shown in Fig. P = 0.011) . However, when adjusted for eGFR, the association was no longer statistically significant (P = 0.473). log transformed iFGF23 did not consistently show a significant association with composite endpoint after additional adjustments were added , which indicate that cFGF23 may capture risk of reduced kidney function, which is not similarly captured by iFGF23.This prospective cohort study is the first study in KTRs simultaneously analyzing baseline iFGF23 and cFGF23. Our study demonstrated - after adjusting for risk factors – that only cFGF23 was independently associated with the overall graft loss, and this was consistent with the results of a meta-analysis analyzing all so far reported cFGF23 studies –19 and oP = 0.97), however, this study did not analyze cFGF23.Only one published study investigated the association between iFGF23 and the overall graft loss in KTRs so far . In lineFGF23 is a 251 amino acid peptide with a 24 amino acid signal peptide and 227 residues forming the mature FGF-23 structure   to assess the association between FGF23 and overall graft loss. We acknowledge several limitations of this study. First, iFGF23 as well as cFGF23 concentrations were only measured once at study entry, hence it is impossible to investigate the alteration over time in this cohort. Second, concerning the limited number of endpoint events, we did not conduct subgroup analyses of graft loss and death separately. Third, the binding sites of the antibodies in the used cFGF23 ELISAs were not identical, because they came from different companies (Immutopics versus Biomedica). Although data coming for studies using either the cFGF23 Immutopics Elisa or the cFGF23 Elisa from Biomedica yielded qualitatively identical results, the differences in the binding sites might have influenced the ability of the individual ELISA to detect breakdown-products of FGF23 (see supplementary Figure Consistent with prior studies, we also demonstrated that baseline serum cFGF23 was independently associated with the overall graft loss in KTRs. Simultaneously measured iFGF23, however, was not associated with the composite outcome after adjusting for risk factors. The cFGF23 ELISA might detect bioactive FGF23 fragments that are not detected by the iFGF23 ELISA.Additional file 1.