Ischaemia/reperfusion-injury degrades endothelial glycocalyx. Graft glycocalyx degradation was studied in human liver transplantation.To assess changes within the graft, blood was drawn from portal and hepatic veins in addition to systemic samples in 10 patients. Plasma syndecan-1, heparan sulfate and chondroitin sulfate, were measured with enzyme-linked immunosorbent assay.During reperfusion, syndecan-1 levels were higher in graft caval effluent  than in portal venous blood [101 (75–121) ng/ml], indicating syndecan-1 release from the graft. Concomitantly, heparan sulfate levels were lower in graft caval effluent  than in portal venous blood [112 (98–128) ng/ml], indicating heparan sulfate uptake within the graft. Chondroitin sulfate levels were equal in portal and hepatic venous blood. After reperfusion arterial syndecan-1 levels increased 17-fold (P < 0.001) and heparan sulfate decreased to a third (P < 0.001) towards the end of surgery.Syndecan-1 washout from the liver indicates extensive glycocalyx degradation within the graft during reperfusion. Surprisingly, heparan sulfate was taken up by the graft during reperfusion. Corroborating previous experimental reports, this suggests that endogenous heparan sulfate might be utilized within the graft in the repair of damaged glycocalyx. Glycocalyx is a layer of interacting proteoglycans, glycosaminoglycans and glycoproteins covering the luminal endothelial surface. It serves to sustains microvascular homeostasis and integrity. Intact glycocalyx regulates vascular tone and permeability, and inhibits blood coagulation. Glycocalyx also appears to control inflammatory responses as it regulates endothelial adhesion and transmigration of leukocytes and retains cytokines and chemokines within its heparan sulfate mesh ,2.Glycocalyx degradation occurs under pathophysiologic inflammatory conditions such as sepsis , trauma Although elevated levels of heparan sulfate  and syndWe hypothesized that glycocalyx degradation occurs within the liver graft during reperfusion, and therefore studied plasma proteoglycan (syndecan-1) and glycosaminoglycan  kinetics across the liver graft in human liver transplantation. To this end, blood samples were obtained both from the portal vein (ingoing blood) and hepatic vein (outcoming blood) during and after liver graft reperfusion. As the aim of this study was to investigate the effects of IR-injury on the hepatic glycocalyx, we sought to eliminate the effects of end-stage liver disease on glycocalyx ,12 by enThe study protocol was approved by the ethics committee in Helsinki University Hospital (Dnro 156/13/03/02/2014). After written informed consent, 10 PSC-patients undergoing orthotopic liver transplantation participated in the study . All patAll grafts were retrieved from brain-dead donors and preserved with University of Wisconsin solution at 4°C. The surgical technique of the graft implantation and reperfusion was standardized. All transplantations were performed with cross-clamping of the inferior caval vein. Neither "piggy-back" technique nor veno-venous bypass was used. Before the anhepatic period, i.e. clamping of hepatic artery and portal vein and native supra- and infrahepatic caval vein, all patients were anticoagulated with 5000 IU heparin. Additional doses were given to maintain activated clotting time (ACT) at 200–300 seconds before the anhepatic period. During graft implantation, suprahepatic caval anastomosis was completed first. The infrahepatic caval and portal anastomoses were then partially completed. With the suprahepatic caval vein clamped, the graft was flushed via the portal vein first with 1000 mL of Ringer’s solution, followed by flushing with approximately 400 mL of portal venous blood. Both Ringer’s solution and portal venous blood were wasted from the infrahepatic caval anastomosis, blood constituting the graft caval effluent. Portal and infrahepatic caval vein anastomoses were completed and the caval and portal vein clamps were removed. Then the hepatic arterial anastomosis was completed and artery declamped. Finally, the biliary tract was reconstructed with a Roux-en-Y hepaticojejunostomySurgery was performed under balanced inhalational anesthesia. Vasoactive agents used were phenylephrine boluses and/or noradrenaline-infusion. Fluid therapy during liver transplantation was left to the discretion of the attending anesthesiologist. For data analysis, we recorded total amount and type of the fluids and blood products used during surgery.During liver transplantation, systemic arterial blood samples were collected after induction of anesthesia but before surgery (time point 1), immediately before reperfusion of the graft with portal blood (time point 2), 5 minutes after portal vein declamping (time point 3), and 5 minutes after hepatic artery declamping (time point 4). To assess changes across the graft, blood samples were obtained by puncture from both portal and hepatic veins at the following time points: during reperfusion , 5 minutes after portal vein declamping (time point 3), and 5 minutes after hepatic artery declamping (time point 4).The volume of each sample was 10 mL. All samples were drawn into pyrogen-free syringes  and then transferred into sodium citrate tubes  on melting ice at 0°C. Plasma was separated within 15 minutes by centrifugation at 2000g for 10 minutes and stored in aliquots at -80°C until the analyses were performed.Plasma syndecan-1 (human sCD138) levels were measured with a commercial enzyme-linked immunosorbent (ELISA) assay . Reproducibility coefficient of variation was 10.2% and assay sensitivity 4.94 ng/ml.Plasma heparan sulfate levels were measured with a commercial ELISA assay (Elabscience Biotechnology Co.) for human heparan sulfate. Reproducibility coefficient of variation was <10% and assay sensitivity 0.188 ng/ml.Plasma chondroitin sulfate levels were measured with a commercial ELISA assay  for chondroitin sulfate. Reproducibility coefficient of variation was < 10% and assay sensitivity 0.188 ng/ml.Plasma alanine aminotransferase (ALT), international normalized ratio (INR), and bilirubin were measured daily as a part of the routine follow-up. For data analysis, we included the peak ALT value measured within 7 postoperative days, and INR and bilirubin levels measured at postoperative day 7 . We alsoAll statistical analyses were performed with SPSS version 22 software package . Due to the small number of patients, nonparametric tests were used. To calculate the transhepatic gradient, portal vein value was subtracted from the caval effluent or hepatic vein value. Unpaired data were compared with the Mann-Whitney U-test, whereas paired data were compared with the Wilcoxon signed rank test. Time-dependent changes were evaluated by Friedman’s test with post hoc Wilcoxon signed rank test with Holm’s correction for multiple comparisons. Bivariate correlations were evaluated by the Spearman rank correlation. The α-level was 0.05 for all statistical tests. Data are expressed as medians with ranges.Perioperative bleeding was relatively minor . In addiFive patients had significant postoperative complications. Four patients underwent relaparotomy, one due to biliary leakage, three due to postoperative bleeding. One of the patients with postoperative bleeding requiring laparotomy on postoperative day 5 had undergone dialysis for acute kidney injury before the bleeding. One patient had an unspecified infection, treated with antibiotics from postoperative day 8 onwards. Despite these complications, postoperative liver function was good in all recipients . Both paP 0.001 Friedman’s test; Plasma syndecan-1 levels in the systemic circulation increased during surgery, significantly so after graft reperfusion (P < 0.001, Friedman’s test; Plasma heparan sulfate levels in the systemic circulation decreased during surgery (Plasma chondroitin sulfate levels in systemic circulation decreased slightly during surgery, but the change was statistically not significant . ChondroNeither syndecan-1, heparan sulfate nor chondroitin sulfate levels correlated with graft steatosis, cold ischemic time, postoperative liver function or MEAF-score. Likewise, markers of glycocalyx degradation did not correlate with the amount or type of fluids administered perioperatively.The main finding of this study, somewhat surprisingly, is heparan sulfate sequestration within the liver graft during reperfusion. Also, concomitant decrease in systemic heparan sulfate levels occured after reperfusion. Still, this heparan sulfate uptake was accompanied by extensive release of proteoglycan syndecan-1 from the liver graft during reperfusion, together with a rapid increase in systemic syndecan-1 levels after reperfusion.In accordance with our hypothesis, changes in syndecan-1 concentrations indicate that glycocalyx degradation within the liver graft occurred during graft reperfusion. The results regarding the release of syndecan-1 from the liver graft are in line with those of Snoeijs et al , who demper se and not IR-injury [Concurrently with the increasing syndecan-1 levels in the systemic circulation, heparan sulfate levels decreased substantially during surgery. Our finding is surprising and contradicts the results from previous clinical studies. In patients undergoing vascular  or cardiR-injury . In our R-injury . HoweverWe can only speculate the pathophysiologic processes of hepatic heparan sulfate uptake. We propose that heparan sulfate might be utilized in the reperfused graft in an attempt to repair the damaged glycocalyx. Indeed, in human kidney transplantation, after the initial degradation, glycocalyx thickness increased significantly within 30 min after reperfusion . LikewisIn our highly selected patient cohort, either syndecan-1 release, that reflects damaged glycocalyx, or potential reparative heparan sulfate uptake, did not translate into clinical outcomes. However, preventing glycocalyx degradation during graft preservation and reperfusion could offer a strategy to improve clinical outcomes in liver transplantation, especially with regard to marginal grafts. The seemingly most feasible method for protecting graft glycocalyx during preservation is to supplement the preservation solution with substances known to inhibit glycocalyx degradation under inflammatory conditions, for example steroids –31, seriOur study has several strenghts. First, the surgeons took the blood samples by direct needle puncture from the portal and hepatic veins. Thus, the obtained results reflect real concentration differences across the reperfused liver. Second, our study cohort was homogenous. As patients with chronic liver disease have elevated syndecan-1  and hepaThere are limitations to this study. First, given the small number of highly selected patients and non-steatotic grafts with short cold ischemic times, and thus excellent postoperative liver function with a median MEAF-score of 2.84 , the lacTaken together, we demonstrated graft glycocalyx degradation during reperfusion in human liver transplantation in a selected patient cohort with normal liver and hemostatic function. The extensive syndecan-1 release from the graft was associated with concomitant heparan sulfate uptake into the graft, suggesting heparan sulfate can be utilized within the graft in pathophysiologic processes. To the best of our knowledge, this is the first in vivo observation of endogenous heparan sulfate uptake in a clinical context. In the light of previous experimental –28 and c


Dear Readers and Nephrology Colleagues,Renal Failure. First of all, I would like to thank the previous Chief Editor, Dr. William F. Finn, for his years of dedicated work. Such transition will take place at a tumultuous time in academic publishing, but I hope to use this challenge as an opportunity to raise the Journal to the next level of academic exchange of information. Introducing myself, I am Board Certified Nephrologist and Internist and licensed to practice in both Mississippi and South Caroline (U.S.), as well as in the European Union (Hungary). Currently, I am a Full Professor of Medicine at the Medical University of South Carolina (MUSC) and Attending Physician for the MUSC’s Department of Medicine, Division of Nephrology and Ralph H. Johnson VA Medical Center and serving as Medical Director for Dialysis Units in the community and at the VA Medical Center. Earlier in my carrier, I have served on the faculty of several prestigious institutions, including the University of Mississippi, the University of Iowa, the University of Debrecen and the Semmelweis University in Hungary.As of January 1, 2019, I had the distinct privilege to assume the position of the new Editor-in-Chief for the journal Journal worldwide with an improved overall academic impact. OA publishing, while initially viewed with disdain by some of us, is now embracing ‘mainstream’ publications, increasingly becoming the norm of academic publishing. The success of journals such as PLoS One and Scientific Reports bears testimony to this transition. An increasing number of authors are now encouraged or required to publish OA by their funding bodies, institutions, or their employers. To that end, our Publisher will also offer significant support for authors from countries with lesser financial means: corresponding authors with primary affiliations in countries defined by the World Bank as ‘Low-Income Economies’ can apply for a 100% article processing change (APC) waiver; those from ‘Lower-Middle Income Economies’ can apply for a 50% APC discount. Additionally, corresponding authors with primary affiliations based in one of the EIFL (Electronic Information for Libraries) network countries may be eligible for a 100% or 50% discount as well.Over the last 2 years our Publisher, Taylor & Francis Online, and the dedicated personnel of the Editorial Office, have overseen the transition to an Open Access (OA) model of publishing and a dramatic increase of downloads for the Journal publish highly impactful papers on a wealth of issues, including meta-analyses on the link between proton-pump inhibitors and hypomagnesemia [Journal have explored the impact of AKI in donors on renal graft survival subsequent to renal transplantation [Journal have also explored the frequency and clinical characteristics of invasive fungal infections in renal transplant recipients [Journal over this period [Journal has published important works on animal models of AKI as well. These have included work on the potential protective role of N-acetylcysteine in oxalate-induced AKI in a rat model [Over the last couple of years, we have seen the gnesemia , on the gnesemia , on uricgnesemia  and a sygnesemia . Single-antation , the risantation  and qualantation . Additiocipients , the valcipients , the clicipients , post-pacipients  outcomess period ,12. Alonat model , the proat model  and its at model .Nephrology in the acute care setting is moving into an era of cooperation and interaction between several disciplines to address the complex needs stemming from the patients’ multi-faceted problems. We should remember that in most settings, renal replacement therapy in the Intensive Care Units is still associated with at least 50% in mortality rate ,16–18 – To stay relevant in the globally connected world, it is important to have a global representation and reverberations of our publications. In my role as Editor-in-Chief, I will look forward to identifying talented and motivated young clinician-scientists to assist us with the onerous work of peer reviews and to seeing them on our Editorial Board (EB) as the Journal grows. We will strongly seek to develop a pool of peer reviewers and EB members who are reflecting, in composition and interest, the Journal’s international readership. As an added benefit, our publication fee is already substantially reduced for the EB members. We also sincerely hope the next generation of young clinician-scientists will find it worthy not only to read the Journal but also to publish with us. We look forward to further attracting a vibrant and international readership who are likely to return to the journal’s website after their initial positive experiences. There is great competition going on in the modern world to attract quality papers and readers. Those who read papers are the most likely to organize their own studies and publish new ones. We will strongly consider the publication of novel ideas if the underlying science is sound, even if certain questions might remain unanswered in the study.Our impact factor is currently standing at 1.44 and we look forward to attracting exciting papers on a wealth of issues but focus predominantly on AKI and aspects of critical care nephrology ,28,29. N


Setaria italica), two cultivars of broomcorn millet (Panicum miliaceum) and three varieties of green bristlegrass (Setaira viridis). The residues from both used and unused facets of the stone tools were then extracted, and the starch grains studied for morphological features and changes from the native states. The results show that (1) Dehusking did not significantly change the size and morphology of millet starch grains; (2) After grinding, the size of millet starch grains increases up to 1.2 times larger than native grains, and a quarter of the ground millet starch grains bore surface damage and also exhibited distortion of the extinction cross. This indicator will be of significance in improving the application of starch grains to research in the functional inference of grinding stone tools, but we are unable to yet distinguish dehusked forms from native.Research on the manufacture, use, and use-wear of grinding stones (including slabs and mullers) can provide a wealth of information on ancient subsistence strategy and plant food utilization. Ancient residues extracted from stone tools frequently exhibit damage from processing methods, and modern experiments can replicate these morphological changes so that they can be better understood. Here, experiments have been undertaken to dehusk and grind grass grain using stone artifacts. To replicate ancient activities in northern China, we used modern stone tools to dehusk and grind twelve cultivars of foxtail millet ( Commonly cited plant food uses include dehusking and grinding cereals, and other proposed uses including paint processing, production of medicines, and tanning11.From the Late Paleolithic to the Neolithic period, the use of ground stone tools was commonplace throughout the world. In recent years, research including ethnoarchaeology13. These residues, however, frequently exhibit damage from processing with the tools that are being studied. Experiments that replicate these ancient processes have shown that both the size and morphology of starch grains can change after dehusking and grinding with stone tools15. Modern reference collections, however, predominantly consist of native, undamaged starch grains16. Some research has been completed on the morphological changes of starches from wheat (Triticum aestivum), foxtail millet , legumes and other crops after grinding17. Grinding causes damage to starch grains, and is observable in both the morphology (such as a fractured surface or irregular outline) and extinction cross17. However, we have found that, for the species of plants we encounter most frequently in our samples, the lack of precisely defined changes that are mathematically analyzed has hindered the identification of the damaged portions of our assemblages.As a method that extracts residues from the surfaces of used tools, starch grain analysis provides direct evidence for ancient processing methods and plant use18. At the early Neolithic sites of Nanzhuangtou, Donghulin and Zhuannian and the mid-Neolithic sites of Cishan, Xinglonggou and Peiligang, all in northern China, archaeobotanical assemblages of both macro- and micro-remains recovered from cultural deposits and ground stone tools are dominated by millets22. Because the archaeobotanical assemblages from these sites are key in understanding the transition from cultivation to domestication of these plants in this region, our studies focused on this suite of plants. We used stone implements to dehusk and grind samples from foxtail millet , broomcorn millet (Panicum miliaceum) and green bristlegrass (Setaria viridis), the three most commonly occurring species of millets at these sites. The starch grains in this simulation experiment were extracted via sonication, examined under the microscope, measured and then the statistical analysis was performed on the measured grains. Our statistical analyses of the sonication demonstrate that ancient starch residues from these plants can now be accurately identified in assemblages that include damaged grains.Of the archaeological sites thus far identified in northern China, 90 have yielded lithic grinding tools23.The morphological features of starch grains extracted from the modern millet seeds are consistent with results published previouslySurface morphology: The surface morphologies of all the recovered starches from the twelve different cultivars of foxtail millets are almost identical. The common features of the processed starches include a polyhedral shape, centric hilum, absence of lamellae, and smooth surfaces. Deeply crossed, or winged, astroid and Y-shaped fissures were noted in some grains. Radiating lines occur commonly from the center to the edge on the surfaces of processed grains Fig. .Figure 1Mean size: In total, 1704 starch grains from 12 foxtail millet samples were measured. The mean maximum length measurements fall between 9.0 μm and 12.2 μm  of the population. The remaining 73.0% of the starch grains fall into category A1 (n = 2926) within which 737 starches measure >14.0 μm   After dehusking, the morphology of millets does not change in a manner that would be identifiable in the archaeobotanical record. (2) After grinding, the size of millet starches increases up to 1.2 times larger than native grains, and a quarter of the ground millet starch grains bore surface damage and also exhibited distortion of the extinction cross. (3) The number of starch grains deposited on the surfaces of stones was much higher after grinding than after dehusking. While taphonomic processes will certainly affect starch assemblages, the results of this study can be helpful in distinguishing tool function in assemblages derived from similar contexts.Our data demonstrate that an assemblage approach, taking into account the proportions of damaged and large-sized starch grains, may be necessary in understanding the underlying composition of the millet population, and, thus, will assist in documenting the transition from wild to domesticated forms in the archaeobotanical record.Setaria .Twelve cultivars of foxtail millet Fig. , one varria Fig. , and onecum Fig.  were colcum Fig.  and one ria Fig.  were colria Fig.  was collria Fig. .Figure 72. Resources available to us that best mimicked the material characteristics of the grinding stone tools excavated from the sites in northern China were sandstone and shale. We selected 17 sets of grinding stones and slabs from the area surrounding the farmland in the village of Xindian, Machikou town, Changping district of Beijing .The same seventeen sets of stone tools were cleaned in the manner described above. The stone from each group was used to break the millet seeds, which were put on the flat stone, by rolling the stone back and forth repeatedly, until the dehusked millet grain was reduced to flour (~17 min/millet lot).23.Ten mature millet seeds selected from each sample of the seventeen millets were dehusked using clean tweezers and moved immediately into new, sterile test tubes in which they were soaked with ultrapure water for 12 h. The soaked seeds were then gently crushed with clean, glass, stirring rods to release the starches. The starch/water suspensions were then pipetted on to a clean glass slide, mounted in 10% glycerine and 90% ultrapure water, and then the cover glass was sealed with nail polish. For detailed protocol for extraction of starch grains from native millet seeds, please refer to ref.2O2 was used for the oxidative breakdown of some of the larger particles. Next, the starch grains were isolated using a heavy liquid flotation with CsCl . The recovered residue was mounted on a slide in 10% glycerine and 90% ultrapure water, then sealed with nail polish.The used and unused facets of each stone were cleaned with ultrapure water and sonicated for 10 min at a power of 40 kHz/200 W. A solution of 6% H31. Therefore, observations and statistical analyses were only performed on starch grains that measured >5 μm. Because domesticated foxtail millet is the only plant in our previous experiments that has starch grains measuring >14.0 μm23, we also made note of the millet starches that measured >14.0 μm in this experiment. All slides prepared from all samples in this simulation experiment were examined using compound light microscopy and transmitted light microscopy. If the number of starch grains (>5 μm) in the slide was less than 100, we recorded, measured and counted every starch grain. If the number of starch grains (>5 μm) in the slide was more than 100, we examined the samples in the microscope first, and then found a clear view with unobscured and non-overlapping starch grains. Next we recorded, measured and counted every starch grain in the clear view. The numbers are somewhat variable due to the nature of the slides and the large amounts of starchy residues. To try to correct for this issue, if the number of starch grains in a field of vision under microscope did not reach 100, we then moved to the next field of view and counted until more than 100 starch grains were analyzed.Previous studies have demonstrated that starch grains less than 5 μm long are difficult to detect via compound light microscopy at 400×, are rarely diagnostic for taxa, and occur in many plant tissuesSPASS statistical software Version 20.0) was used to analyze the starch data from both the grinding and seed extractions (Figs 0.0 was u23: shape, surface features, position and form of the hilum and fissures, number and characteristics of pressure facets, presence or absence of demonstrable lamellae and mean maximum length averaged from the measurement of more than 100 grains. The main variables recorded include changes in shape and size of grains, surface modifications, visibility of lamellae and change in the extinction cross. Details of the millet starch grain classification are presented in our previous study in ref.23.Our starch classifications for modern millet starches emphasize attributes demonstrated by previous studies to be useful in identification


The aim of this study was to measure and compare the biomechanical properties of two different graft link preparation techniques for anterior cruciate ligament reconstruction. We hypothesised that there would be differences in elongation, load at failure and failure mode due to the different graft link preparation techniques. Thirty fresh-frozen anatomical specimen knees were used. Both tendons (semitendinosus and gracilis) were harvested and randomly assigned to two groups. Graft links prepared with a continuous loop technique were allocated to group 1, whereas those prepared with a buried-knot technique were allocated to group 2. The mechanical properties of both techniques were measured. A mean load to failure of 731 N and an overall graft elongation of 6 mm was found in the continuous loop group. In the buried-knot group, a higher load to failure (848 N) and a lower mean overall elongation (5 mm) was found. The buried-knot technique showed better results with significantly higher load to failure and significantly less elongation compared to the continuous loop technique. It is essential in clinical practice to choose the most accurate technique for graft link preparation to ensure graft stability, especially in the early phase of recovery. It requires special graft preparation techniques from the operating surgeon. The graft source, graft length and fixation method need to be considered for each patient2. Especially for the All-Inside ACL-R technique2, several graft preparation techniques have been described using different suture techniques5.Anterior cruciate ligament – reconstruction (ACL-R), utilizing autologous hamstring tendons, has become one of the most frequently performed orthopaedic proceduresDepending upon the preferred reconstruction technique special graft preparation is necessary.According to surgeon experience and clinic standards different techniques are used, all with the same goal of creating the “ideal” graft link.9. During daily activities, the anterior cruciate ligament (ACL) is exposed to forces of approximately 156 Newton (N) to 170 N, this can increase to 448 N by going downstairs11 and up to 700 N during stumbling, which can cause graft failure in an early stage after reconstruction. These forces need to be considered when using different graft link preparation techniques.The suture technique for graft link preparation is of great importance since it binds the graft together, is required for passing the graft through the tunnels and needs to withstand the tension forces during implantation as well as in the first months of implant ingrowth. Forces affecting the graft, either during graft implantation or in the early postoperative phase, until complete ingrowth into the bony tunnel, may cause clinical failure, which is of significant concern12, and the number of used suture throws9. An increase in the number of sutures across the tendon graft may provide more contact area between the thread surface and the tendon to ensure security of the construct. However, using more sutures results in a higher amount of material near the joint, which could irritate the joint or increase the risk of infection14. Successful ACL reconstruction with a tendon graft requires solid healing of the tendon graft in the bony socket, which itself requires adequate graft fixation initially as well as tight press fit between graft and bone socket.Several factors have been shown to potentially affect graft elongation, e.g. tissue quality, stitch technique, suture strength15.To enhance graft-tunnel healing, tissue-engineering approaches, including the use of growth factors, mesenchymal stem cells, and periosteum graft augmentation, have been used showing promising results in terms of enhancement of bone-graft healing rateHowever, literature lacks comparisons of different suture techniques for the All-Inside ACL-R technique.The purpose of this study was to measure and compare the biomechanical properties  of two different ACL graft link preparation techniques. We hypothesised that there would be differences in elongation, load at failure and failure mode due to the different graft link preparation techniques. The graft link preparation using more sutures through the tendon construct will result in a more stable graft with regards to tendon strength and elongation.Thirty fresh-frozen anatomical specimen knees  were obtained from the Department of Anatomy and Cell Biology, Medical University of Vienna. The donors had a mean age at death of 68 years . The biomechanical testing was carried out at the Institute of material science and technology, Technical University of Vienna, under the guidance of a specialist in material science.All included knee specimens were stored until harvesting at 4 °C in a designated refrigerator at the Department of Anatomy and Cell Biology, Medical University of Vienna. Through a medial incision semitendinosus- and gracilis tendons were harvested from all included knees to ensure protection of the tendons. All tendons were measured and included if they provided a minimum length of 270 mm and reached final diameter of minimum 8 mm after quadrupling. To measure tendon length a standard ruler was used. To measure tendon diameter the tendon-measuring block  was utilized. Finally, a total of 24 grafts , with 12 in each group of equal lengths were randomly assigned. Prior to inclusion, macroscopically degenerative or pathological changes were ruled out.After harvesting, included tendons were fresh frozen (−20 °C) until final testing. Before testing they got moved from the freezer to the refrigerator to ensure slow thawing. Finally they were kept at room temperature (21 °C). The whole procedure takes 36 hours before testing. Slow thawing ensures that there is no dehydration. Before and during the preparation all tendons were humidified with 0.9% sodium chloride to counter dehydration.2  solely performed all graft link preparations. Each graft link preparation was timed. The graft preparation and tension device  was utilized for preparation of all tendons. Two different preparation techniques were performed in this anatomic specimen study: Group 1: the buried-knot technique presented by Lubowitz J. in 2012In each graft link a femoral and tibial diameter was defined to be able to name the area of graft failure. In the buried-knot technique the femoral part was defined as the part where only one handle of the tendon was brought through the TightRope Fig. . In the 2. All hamstring grafts were fixed with the TightRope  system at the material testing machine, between two metal plates through a hole representing the button flipped over the cortex.In both groups a No. 2 FiberWire  suture was used. An additional No. 2 FiberLoop  suture was used in the buried-knot techniqueDuring the preparation all tendons were humidified with 0.9% sodium chloride as a routine procedure at the Department of Orthopedics and Trauma Surgery, Medical University of Vienna. A total of 24 tendons, after ruling out macroscopically degenerative or pathological changes, and final grafts of equal length ±10 mm and a diameter ±1.5 mm, were prepared. The final graft link  was measured with a sliding calliper. For graft link measurement a standard ruler was used.A digital single lens reflex camera, type Nikon D500, was used to take photographs. All grafts got pre-tensioned at the graft preparation system by Arthrex Naples, FL, before loading at the testing machine. Both ends of each single graft were secured with the ACL TightRope  system, fixed through a metal plate, which was attached at the Zwick Z050, universal material testing machine . The Zwick Z050 was loaded with a 1 N load cell and a video extensometer  onto the Zwick Z050 a photograph was taken to measure the primary length of the graft link. 50 N at a rate of 200 mm/min for 10 cycles was applied to the graft link to mimic the tension of sutures tied over a cortical bone bridge. The strength of the graft link was tested by applying 50 to 250 N cyclic loading for a total of 500 runs. This testing procedure is a standard protocol utilized in previous studiesThe statistical analysis was performed using Excel 2014  and SPSS  software packages. Regions of interest (ROI) were defined before final testing. (1) A1 to A3 - area of pretensioning, (2) B1 to B3 - total elongation during the cycling load, (3) C2 - load to failure and (4) mode to failure. . For comparison of two metric variables, a student’s t-test was used.Normal distribution of metric variables was verified by application of the Shaphiro-Wilk test. If the Shaphiro-Wilk test was rejected, the Mann-Whitney U test was used. Tests were used to compare (1) suture and tendon percentage elongation with pre-tensioning, (2) cyclic loading, (3) load to failure and (4) stiffness. A significance level was set at p ≤ 0.05. As this study was a pilot study no power analysis was performed.Prior to study begin the corresponding ethic review board, Medical University of Vienna, approved the study (EK 1214/2015). All patients signed informed consent that they donate their body to science after death. All used methods were carried out in accordance with the relevant guidelines and regulations of the institutions involved.The suture material, TightRopes and the working station were provided by Arthrex for free . Parts of the presented data (group of continuous loop technique) have been published previously in a paper from the first author.  10.1016/j.knee.2018.03.010).The total graft preparation time (mean 7 minutes) was comparable in both groups, not showing significant differences (p = 0.873). After applying 1 N for measuring initial tensioning, no differences were found between tested techniques (p = 0.7298). Detailed overview of data for the continuous loop technique and the buried-knot technique is presented in Table The measured load to failures showed significant differences, revealing the buried-knot technique to be stronger (p = 0.002). There were also differences found in the overall elongation with the buried-knot technique showing a significantly lower elongation rate (p = 0.045). However, the tibial diameter of the grafts was significantly lower p = 0.037) in the buried-knot technique group; differences regarding the femoral diameter were not found. With regard to measured stiffness there was no significant difference found (p = 0.4631), however the continuous loop technique presented with better values regarding stiffness  in the buried-knot technique was rupture of the TightRope “crossing” after an average load of 864 N ± 40 N . The TightRope “crossing” was defined as the part of the TightRope where tightening is possible  compared to the continuous loop technique. This might be due to the fact that all tendons in the buried-knot technique were stitched three to four times more often, with baseball stiches, compared to the continuous loop technique. In the continuous loop technique, tendons were only stitched once and the graft was then surrounded by FiberWire . This could also be the reason for easier cutting through of the tendon compared to the buried-knot technique.et al.18 showed that by using greater graft diameters a higher strength could be reached, leading to the conclusion that even a tendon with a diameter of 6 mm produces an average load to failure of 2358.8 N. This is in line with our results where tendon failure was rarely seen.Interestingly, as we found the buried-knot technique to present with a significant higher load to failure, in this technique only one handle of the tendon is loaded through a TightRope (defined as the femoral site, Fig. 21, or number of used knots6, however, this is the first study investigating biomechanical differences of two ACL graft link preparation techniques in human specimens. By using the ACL all-inside ACL-R technique a detailed knowledge of the preparation technique is necessary and therefore should always be performed by an experienced surgeon or under direct supervision.Regarding the current literature, several studies investigated different knot techniques2. However, from our clinical experience, graft diameter should be at least 8 mm to avoid early graft failure.Before graft link preparation, suturing and tensioning, graft specifications need to be followed in both techniques. Graft length should not be greater than 270 mm in length to avoid an overlengthening of the graft after pretensioning. Lubowitz recommended a graft diameter with a maximum of 8.5 mm23. Ruling out the most crucial ones is very hard and further work is needed. In the MARS cohort, technical faults were presented in 24% of the patients leading to ACL revision23. Reasons for this are diverse also including inadequate graft link preparation.In summary, there are many factors presented in literature influencing final graft link stability and the risk of revision surgery in ACL-RThere are limitations to any biomechanical study. Firstly, although all tendons were checked macroscopically for any damage or failure, microscopical differences in tendon quality could not be ruled out and might influence outcome. Furthermore, only fresh frozen anatomic specimens were used for this study. Consequently, it would be advisable to repeat this study with fresh human anatomical specimen semitendinosus and gracilis tendons. Despite this limitation, there was unanimity amongst the authors with regards to the included tendons, which were finally used for the experiment. All tendons were randomly assigned to one of the two groups. The grafts were not implanted into bone, as done during actual surgery, so it is not possible to know how these preparation methods perform after fixation in bone, which is a critical issue.Secondly, this study focused on tendon elongation and load to failure without considering the physiological healing of the tendon and the tendon-bone construct, which could influence to the success of tendon graft reconstruction surgery. This needs to be considered when interpreting the results.ex vivo biomechanical setting. Nevertheless, this is the first study using human graft link constructs in an ex vivo setting examining the performance of graft link constructs by comparing two different preparation techniques.Thirdly, for graft link testing cyclic loading was used only in an axial direction, not considering any rotational movement, which would be normal in physiological conditions. Reconstruction of the ACL using tendon grafts often leads to compacting and shear forces between the tendon and the bone, which is sophisticated to stimulate in an in-vivo studies are needed, including bony ingrowth of the tendon, physiotherapy protocols, e.g. influencing early graft link stability.The buried-knot technique yielded significant improvement of the load to failure and significant reduction of elongation compared to the continuous loop technique. It is essential in clinical practice to choose the most accurate graft link preparation technique to ensure the highest graft stability, especially in the early phase of recovery. However, further


Sociedade Brasileira de Urología (SBU)) was founded in 1926 and in 1974 was launched the Brazilian Journal of Urology , a journal dedicated to publish the research from the Brazilian Urological community.The Brazilian Society of Urology , now a journal to where 2130 authors of 542 institutions from 62 countries have submitted 891 papers in the last 12 months.IBJU is an unique journal because it is an open-access journal which does not charge any fee for submission and/or publication; it is produced inside SBU headquarters and every expenses are supported by the society. Furthermore, it is possible to Brazilian and other Portuguese-speaking urologists to submit their papers in Portuguese and, once approved after peer-review, get a free English version..Luciano A. Favorito, MD, PhD was chosen to be the next Editor-In-Chief and we have been working together in the last two issues.I was elected as Editor-in-Chief in 2012 and this is my last issue since my term will end next December. Last August, 2019, It has been an honor to serve SBU and to conduct this very prestigious journal. It has been a great experience. During the last eight years IBJU's impact factor has risen, the review time has decreased significantly and the journal has attracted higher quality papers.Morais, Bruno A. Nogueira and Patricia Gomes.Editorial process is a team work and I need to thank the support of the successive SBU Board of Directors, to my Associate Editors and to the group responsible for our journal production: Ricardo de Finally, neither peer-review journal is produced without the reviewers; in 2019, 1536 reviewers helped us voluntarily to make an even better journal. As my last act I would like to announce the five most effective reviewers of 2019, based in their review quality, number and time length of review:Alexandre Danilovic, MD, Departamento de Urologia, Hospital das Clínicas da Faculdade de Medicina da USP, SP, Sao Paulo, Brasil; David Hernandez, MD, Department of Urology, University of South Florida, Tampa, FL, USA; Elcio Dias Silva, MD, Clínica Dr. Élcio Dias Silva, Campinas, SP, Brasil; Hubert Swana, MD, Pediatric Urology, Nemours Children's Hospital Orlando, Orlando, FL, USA; Trushar Patel, MD, Department of Urology, University of South Florida, Tampa, FL, USA.See you.


Most previous studies on plasmid conjugal transfer employed experimental setups with two strains: one as a donor and the other as a recipient. However, the results obtained sometimes failed to agree with observations obtained under natural environmental conditions or in a model microcosm using natural soil and water samples. Therefore, we consider that there is a “gap” in our understanding of plasmid behavior in the context of bacterial consortia that exist under the actual environmental conditions. In this study, we clearly showed that the conjugation selectivity of a plasmid can be affected by the recipient candidates existing around the donor strain by the use of a simplified experimental setup with one strain as the donor and two strains as recipients. These phenomena could not be explained by factors known to affect plasmid transfer as suggested by previous studies. Therefore, we suggest the presence of novel elements regulating plasmid transfer within consortia. Pseudomonas putida KT2440 and Pseudomonas resinovorans CA10dm4 as model hosts and pCAR1 (IncP-7), NAH7 (IncP-9), pB10 (IncP-1β), and R388 (IncW) as model plasmids. As expected, in most cases these plasmids were generally transferred more frequently to a recipient of the same species than to a recipient of a different one under conditions of liquid and filter mating, although NAH7 was transferred from P. resinovorans more frequently to P. putida than to P. resinovorans during filter mating. With the exception of pCAR1, which was less affected, the coexistence of other recipients enhanced the preferences of conjugative transfer to the same species. In particular, preferences corresponding to transfer from P. putida to a different recipient (P. resinovorans) were reduced by the presence of a coexisting same recipient (P. putida) during transfer of NAH7 in liquid and transfer of R388 in filter mating. We determined that large cell aggregates and substances secreted into culture supernatant were not responsible for this phenomenon. Overall, the results of this study suggest the existence of unknown factors determining optimal plasmid transfer to native recipients.Understanding the mechanisms underlying plasmid behavior under conditions of various environments is important to predict the fate of plasmids in nature. Most previous studies on plasmid transfer employed two strains: one as a donor and the other as a recipient. However, in natural environments, there are usually different recipient cells available to which plasmid can be transferred. In this study, to reveal the underlying mechanisms, we assessed the transferability of plasmids from one donor strain to either of two recipient candidates as the most simplified model. We used IMPORTANCE Most previous studies on plasmid conjugal transfer employed experimental setups with two strains: one as a donor and the other as a recipient. However, the results obtained sometimes failed to agree with observations obtained under natural environmental conditions or in a model microcosm using natural soil and water samples. Therefore, we consider that there is a “gap” in our understanding of plasmid behavior in the context of bacterial consortia that exist under the actual environmental conditions. In this study, we clearly showed that the conjugation selectivity of a plasmid can be affected by the recipient candidates existing around the donor strain by the use of a simplified experimental setup with one strain as the donor and two strains as recipients. These phenomena could not be explained by factors known to affect plasmid transfer as suggested by previous studies. Therefore, we suggest the presence of novel elements regulating plasmid transfer within consortia. Ntr) in Pseudomonas putida is involved in inhibiting the conjugation efficiency of the naphthalene degradative IncP-9 plasmid NAH7  , which does not reflect the actual natural environmental conditions, in which there are several types of strains present around the donor strain at the same time. Therefore, in this study, we used two different species as possible candidate recipients and one donor strain (1:2 mating) as a most extensively simplified conjugation design under natural conditions. We employed a KT2440  and P. rof CA10)  as modelof CA10) , and antP. resinovorans were lower at 16 h than at 3 h. Higher growth rates of donor cells than of transconjugant cells during the mating procedure might be responsible for the observed lower TFs at 16 h than at 3 h. Therefore, we concluded that the longer mating time is not suitable for comparison of TFs. Since 1 h was too short a time to perform experiments in triplicate, we adopted 3 h as the optimal mating time for all mating experiments in this study. In addition, we also counted the cell number after the mating assays and confirmed that there was no effect of the viability or growth of each strain on the TF of plasmids during the 3-h mating assay procedure  P. resinovorans was used as the donor, and P. putida or P. resinovorans (or both) was used as a recipient in 1:1 (or 1:2) mating assays. Mating assays were performed for 1, 3, and 16 h. Experiments were performed in triplicate for the 3- and 16-h matings, and bars show mean numbers of TFs (transconjugants/donor) calculated from triplicate spots of serially diluted mixtures. White diamonds show the frequencies calculated from each spot. For 1-h mating, experiments were performed at a single line three times and bars show results representative of technical triplicate experiments. White bars show the TFs of the plasmid to P. putida, and black bars show those to P. resinovorans. Download FIG S1, TIF file, 0.7 MB.Transfer frequency (TF) of each plasmid in 1:1 and 1:2 liquid mating assays. Plasmid-harboring (A) Copyright © 2018 Sakuda et al.2018Sakuda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00490-18.4TABLE S1Table S1, DOCX file, 0.03 MB.Cell numbers of donors, recipients, and transconjugants (CFU per milliliter). Download Copyright © 2018 Sakuda et al.2018Sakuda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. putida as the donor are shown in P. putida were higher than those for P. resinovorans during 1:1 mating. In 1:2 mating, although the TFs detected were generally lower than those detected in 1:1 mating, higher TFs were detected in P. putida as recipient than in P. resinovorans as recipient. In particular, transfer of NAH7 from P. putida to P. resinovorans was markedly reduced during 1:2 mating  indicate significant differences between plasmids in statistical analysis . Download Table S2, DOCX file, 0.02 MB.Ratios of TFs and kin indices (KIs) of respective plasmids. Asterisks (*) indicate ratios of TFs to different species/ratios of TFs to the same species during 1:1 mating. Daggers (†) indicate ratios of TFs to different species/ratios of TFs to the same species during 1:2 mating. Double daggers (‡) indicate KIs, which were calculated by the following equation: KI = Copyright © 2018 Sakuda et al.2018Sakuda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00490-18.6TABLE S3P values of multiple comparisons by Conover-Iman test for data in P < 0.05; **, P < 0.01. Download Table S3, DOCX file, 0.02 MB.Copyright © 2018 Sakuda et al.2018Sakuda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. putida strain as the donor. On the basis of this result, it could be concluded that the coexistence of P. putida (same species) as a candidate recipient in the same location had no or negligible effect on the TF of pCAR1 to P. resinovorans (different species) during 1:2 mating. In contrast, there were larger differences between those ratios when the other plasmids were used, and the KIs were less than 1. The comparison of KIs among the four plasmids clearly showed that transfer of NAH7 to a different species recipient was reduced dramatically by the presence of same species recipient (KI = 1.7E−03 ± 2.3E−03) (As shown in 2.3E−03) .P. resinovorans as a donor during liquid mating were assessed  and P. resinovorans (green), showed a free-living state, and no large aggregation of cells was detected in the mating mixtures for each plasmid. Similar results were observed when P. resinovorans was used as the donor. These results were markedly different from those seen with the aggregated control sample. Although we cannot rule out the possible existence of small aggregations of 2 to 3 cells in the mating mixture, these observations suggested that formation of large cell aggregates was not the cause of the effect of the presence of coexisting recipients on plasmid transfer.Because cell aggregation may enhance plasmid transfer , we asseP. putida culture or of the mating mixture of NAH7-harboring P. putida and P. putida to the cell mixture of NAH7-harboring P. putida and P. resinovorans (as a 1:1 mating experiment). If the compound(s) secreted into the culture supernatant affected NAH7 conjugation to P. resinovorans, the TF of NAH7 to P. resinovorans would be decreased by adding the supernatants. However, the TF of NAH7 was not affected, as shown in P. resinovorans was secreted into the P. putida culture supernatant or into that of the mating mixture of NAH7-harboring P. putida and P. putida.We evaluated whether the candidate recipient strain(s) secreted a substance(s) into the culture supernatant that affected plasmid transfer. In this study, we added the supernatant of a Pseudomonas strains as hosts in this study. Therefore, it will be necessary to confirm this phenomenon in other host strains and with other host/plasmid combinations.Our results suggest that plasmids were transferred generally more frequently to the same species recipient than to different species in 1:1 combinations under the liquid and filter mating conditions. The genome modification system of the host, such as methylation, a restriction enzyme system, and a CRISPR-Cas system, might affect plasmid behavior under 1:1 mating condition. The presence of the same species recipient candidate affected plasmid transfer to different species during 1:2 mating, and the ratio of TF to different species were lower in most combinations of donor/recipient strains and plasmids. This tendency was clearly shown by the fact that the KIs detected in almost all experiments were <1 , where e” motif . These de” motif . Furtherble pili . BecauseWe have succeeded in revealing that there is a novel but unknown mechanism that determines the fate of conjugation by selecting recipient cells among the bacterial consortia. The phenomena observed in this study cannot be explained by factors reported in previous studies, which were found in a mating assay performed only with one strain as a donor and another strain as a recipient. We have not succeeded in understanding the molecular mechanisms or clarified whether the factor(s) involved in these phenomena is encoded on the plasmid or on the host or on both. However, further forthcoming analyses might close the gap between laboratory conditions and actual environmental conditions in the study of plasmid behavior.E. coli and Pseudomonas strains were grown overnight in lysogeny broth (LB) (−1 for kanamycin (Km), 30 μg ml−1 for gentamicin (Gm), 25 μg ml−1 for rifampin (Rif), 100 μg ml−1 for ampicillin (Ap), and 12.5 μg ml−1 for tetracycline (Tc). Solid media were prepared by adding 1.6% (wt/vol) agar to liquid LB medium. Derivative plasmids of pCAR1, NAH7, pB10, and R388 PA1/04/03DeRedExpress-a] (E. coli SM10/λpir(pUX-BF13) (E. coli DH5α(pRK2013) (A1/04/03-gfp cassette was inserted into the chromosome of the P. resinovorans CA10dm4 spontaneous Rifr strain by filter mating with E. coli K-12 [miniTn7(Gm)PA1/04/03gfp-a] (E. coli SM10/λpir(pUX-BF13) (E. coli DH5α(pRK2013)   containiAR1::rfp , NAH7K2 AR1::rfp , pB10::rrfp [rfp , and R38d method . Each plP. putida strains as donors, P. putida SM1443(pCAR1::rfp) (P. putida SM1443(NAH7K2) (P. putida SM1443(pB10::rfp) (P. putida SM1443(R388::rfp) , P. resinovorans CA10L(NAH7K2), P. resinovorans CA10L(pB10::rfp), and P. resinovorans CA10L(R388::rfp) were used as donors of pCAR1::rfp, NAH7K2, pB10::rfp, and R388::rfp, respectively. Strains P. putida KT2440RGdr and P. resinovorans CA10dm4RGgfp were used as recipients. Overnight cultures of donor and recipient cells were harvested and washed with fresh LB. The resulting cells were suspended in fresh LB to an optical density at 600 nm (OD600) of 2 × 10−1 for the donor and 2 × 100 for the recipient. Equal volumes (200 μl) of donor and recipient cell suspensions were mixed for 1:1 mating. A 200-μl aliquot of the donor cell suspension and 100 µl of each recipient cell suspension were mixed for 1:2 mating. Donor and recipient cells were mixed in 2-ml microtubes sealed with a gas-permeable adhesive seal  and incubated for 1, 3, or 16 h at 30°C for liquid mating. A mixture of donor and recipient cells was transferred onto a 0.22-μm-pore-size membrane filter  using glass microanalysis filter holders and filtering flasks (Millipore) for filter mating. Each filter was placed on an LB agar plate and incubated at 30°C for 1, 3, or 16 h. After incubation, 10 or 100 μl of a diluted mixture from each tube was spotted or spread on selected agar plates. The number of donor cells was calculated by determination of the number of CFUs grown on Km-added agar plates. While the transconjugants could also be grown on the Km-added agar plates, the numbers of transconjugants were completely different from the number of donors , so we ignored them, in accordance with a previous study  , P. puti(NAH7K2) , P. puti10::rfp) , and P. 88::rfp)  were use10.1128/mSphere.00490-18.1TEXT S1Text S1, DOCX file, 0.04 MB.Confirmation of the GFP correlation with the two strains. Download Copyright © 2018 Sakuda et al.2018Sakuda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00490-18.3FIG S2P. putida was used as the donor and P. putida and P. resinovorans as the recipients in the 1:2 mating assay. (A to D) Green fluorescent protein (GFP) fluorescence of colonies on a selected agar plate of transconjugants (A) and membranes of colony hybridization (B to D). The GFP fluorescence of colonies was detected with a Dark Reader DR46B Transilluminator . The membranes were hybridized using different oligonucleotide probes. The probes were prepared from the 0.8-kb fragment of the repA gene on pCAR1 (B), the 1.0-kb fragment of the parI gene on the P. putida KT2440 chromosome (C), and the 0.5-kb fragment of the PCA10_13490 gene on the P. resinovorans CA10dm4 chromosome (D), as specific probes for the pCAR1 and recipient strains. The inset shows a magnified image of the square area. The colonies with GFP fluorescence (arrows) were colonies of P. resinovorans harboring the plasmids, and the colonies without GFP fluorescence were colonies of P. putida harboring the plasmids. The experiments were performed twice. Download FIG S2, TIF file, 1.2 MB.Fluorescence imaging and colony hybridization for colonies on selected agar plates of transconjugants. Copyright © 2018 Sakuda et al.2018Sakuda et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the t tests (P < 0.05). Differences in the KIs of each plasmid in liquid or filter mating assay were analyzed using the Kruskal-Wallis test (P < 0.05). As a result, the P values corresponding to the results of comparisons of liquid mating using P. putida as the donor, liquid mating using P. resinovorans as the donor, filter mating using P. putida as the donor, and filter mating using P. resinovorans as the donor were calculated as 0.02451, 0.516, 0.01723, and 0.02488 from P values of <0.05 by the Conover-Iman test . The results are shown in The data used to determine the effect of the different recipients on the conjugation frequency of the different plasmids were assessed using Student’s P. putida SM1443 or P. resinovorans CA10L were used as donors, while P. putida KT2440RGdr and P. resinovorans CA10dm4RGgfp were used as the two recipient strains. The 1:2 mating mixtures were prepared as described under “Mating assay” above and were incubated in 2-ml microcentrifuge tubes at 30°C for 3 h. After the incubation, each mating culture was stained with 50 μg ml−1 DAPI  for 15 min at 25°C and 2 μl of each sample was observed with fluorescence microscopy . The resulting images were analyzed using DP2-BSW software (Olympus). The recipient (P. putida KT2440RGdr) was grown in the medium with succinate as the sole carbon source at 30°C for 4 h to act as a positive control for aggregation.Plasmid-harboring strains of P. resinovorans CA10dm4RGgfp or P. putida KT2440RGdr or of 3-h mating cultures of P. putida SM1443(NAH7K2) and P. putida KT2440RGdr was prepared. The mating mixtures of P. putida SM1443(NAH7K2) and P. resinovorans CA10dm4RGgfp were prepared as described under “Mating assay” above, and the cells were collected from the mating mixtures by centrifugation . After suspension of the cells in the culture supernatants, the mating mixtures were sealed in 2-ml microtubes with a gas-permeable adhesive seal (Thermo Fisher Scientific) and were incubated at 30°C for 3 h. TFs were calculated as described under “Mating assay” above.Culture supernatants were prepared by centrifuging the cell cultures  and filtering the supernatants with 0.22-μm-pore-size filters (Millipore). A 400-μl aliquot of the filtered supernatants of


Hypothermic oxygenated machine perfusion (HOPE) was introduced in liver transplantation (LT) to mitigate ischemia-reperfusion injury. Available clinical data mainly concern LT with donors after circulatory-determined death, whereas data on brain-dead donors (DBD) are scarce. To assess the impact of end-ischemic HOPE in DBD LT, data on primary adult LTs performed between March 2016 and June 2018 were analyzed. HOPE was used in selected cases of donor age >80 years, apparent severe graft steatosis, or ischemia time ≥10 hours. Outcomes of HOPE-treated cases were compared with those after static cold storage. Propensity score matching (1:2) and Bayesian model averaging were used to overcome selection bias. During the study period, 25 (8.5%) out of 294 grafts were treated with HOPE. After matching, HOPE was associated with a lower severe post-reperfusion syndrome (PRS) rate  and stage 2–3 acute kidney injury (AKI) . Furthermore, Bayesian model averaging showed lower transaminases peak and a lower early allograft dysfunction (EAD) rate after HOPE. A steeper decline in arterial graft resistance throughout perfusion was associated with lower EAD rate. HOPE determines a significant reduction of ischemia reperfusion injury in DBD LT. In this regard, various machine perfusion techniques have emerged as valuable alternatives to static cold storage (SCS), allowing a safer utilization of ECDs15.The unmet gap between organ demand and supply in recent years has pushed the liver transplant (LT) community to make increasing use of so-called extended criteria or marginal donors (ECD). Although criteria to define marginality are arbitrary, extended criteria grafts are most frequently those with moderate or severe graft steatosis or from donors of an advanced age or after circulatory death (DCD). Utilizing these grafts has been associated with an increased rate of primary non-function (PNF), early allograft dysfunction (EAD), graft cholangiopathy (GC), and with reduced graft survival18. Amongst these, end-ischemic hypothermic oxygenated machine perfusion (HOPE) has been associated with improved post-transplant outcomes, especially with grafts from DCD donors. Although use of machine perfusion may also be beneficial for grafts from donors after brain death (DBD)19, clinical data in this specific area are very limited.The term ‘machine perfusion’ covers a variety of techniques characterized by different settings, timing and technology. The potential benefits and indications of each of these factors are still under investigation20, HOPE was employed in selected cases of DBD liver transplantation following an established protocol since November 2017  HOPE at our center, which began in April 2016. After an initial favorable experienceth, 2016 and June 12th, 2018, including baseline patient and recipient features, prognostic scores23, transplant operation details, preservation time, use of HOPE, and postoperative outcomes, were prospectively collected and retrospectively analyzed. Retransplants and DCD LTs were excluded from the analysis. Minimal follow-up was six months. At the entry on the waiting list, all patients signed a consent form agreeing to the possibility of receiving a graft treated with preservation techniques other than static cold storage, including machine perfusion. Due to the retrospective observational design, no specific approval was sought from the local Institutional Review Board; by Italian law (n° 91/1999), regional transplant centers are custodians of recipient biomedical data, including for research purposes.Data on all consecutive primary adult (>18-year-old) DBD LTs performed between March 162 associated with apparent severe graft steatosis as assessed by the retrieval surgeon; (3) logistic issues determining expected ischemia time ≥10 hours. As our center practice does not include pre-transplant routine histologic assessment of the graft by liver biopsy and frozen section examination, graft macroscopic appearance and donor BMI were used as surrogates of graft steatosis. Assessment of graft macro- and microsteatosis was carried out on the biopsy obtained after graft reperfusion into the recipient.After the first four cases performed in April-May 2016, machine perfusion use was considered from November 2017 in the following scenarios: (1) donor age ≥80 years; (2) donor body mass index (BMI) ≥30 kg/met al.24); (5) moderate-to-severe acute kidney injury (AKI) rate; (6) significant post-reperfusion syndrome (PRS) rate; (7) acute rejection rate; (8) grade ≥3 complications rate ; (9) biliary complications rate at 6 months; (10) patient and graft survival. AKI was defined and graded according to 2012 KDIGO guidelines26. Severe PRS was defined according to Hilmi et al. as a persistent drop in arterial blood pressure (>30% of the anhepatic level), asystole or hemodinamically significant arrhythmias27. Biliary complications were classified as anastomotic (including strictures and fistulae) and extra-anastomotic. Non-anastomotic strictures were defined according to Weeder et al.28 as a combination of narrowing and dilatations  of the larger intra- and extrahepatic donor bile ducts, either with or without intraluminal sludge and cast formation, and in the presence of a patent hepatic artery. As proposed by deVries et al.29 we used the term’post-transplant cholangiopathy’ (PTC) to identify patients with non-anastomotic strictures. Each type of biliary complication was further classified according to the presence of symptoms and the need for operative treatment.Endpoints included: (1) peak levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT); (2) lactate level in the recipient at the end of transplant operation; (3) duration of hospital and intensive care unit stay; (4) rate of EAD  and resistance (mmHg/ml/min) values obtained from both portal vein and hepatic artery circuits at the end of the first hour of HOPE. For analysis, we considered both absolute values and values normalized to liver graft weight (kg). Finally, for each perfusion circuit, flow and resistance values measured at 10-seconds interval during the first HOPE hour were plotted against time to calculate slopes, intended as mean flow and resistance variation per 10-second interval Fig. .Figure 2® solution  during preservation and backtable preparation, until they were flushed with chilled 5% albumin before implantation in the recipient. The bile duct was accurately flushed at the start of cold perfusion and before packaging with chilled saline. Liver graft was weighed at the end of backtable preparation. In HOPE cases, liver graft was connected to the perfusion device immediately after backtable preparation without being flushed. Livers preserved by static cold storage were flushed with a supplementary 1 L of preservation solution through the hepatic artery at the end of backtable preparation. Standard transplant technique was piggyback cavo-cavostomy with retro-hepatic vena cava preservation, portal reperfusion first, and end-to-end biliary anastomosis with a 2.5 Fr T-tube. In selected cases in which it was not possible to use the piggyback technique, inferior vena cava replacement was carried out; veno-venous bypass was never employed. Biliary reconstruction by hepatico-jejunostomy was selectively used in patients with primary sclerosing cholangitis, or if the recipient bile duct was deemed unsuitable for biliary reconstruction. Immunosuppression was based on tacrolimus (target through level 8–10 ng/ml), mycophenolate mofetil (500 mg b.d. in patients with no leukopenia or thrombocytopenia) and steroids , which were tapered and stopped 3 months after transplant. Induction with basiliximab was administered to patients with autoimmune or cholestatic liver disease.After retrieval, liver grafts were stored in cold CelsiorIn patients with a T-tube, a cholangiogram was routinely obtained 3 months after transplant, after which the T-tube was removed. Other investigations (e.g. magnetic resonance cholangio-pancreatography) were performed where clinically indicated.7. Briefly, in HOPE cases, standard graft backtable preparation was initiated upon arrival at our transplant center, while the LiverAssist® device  was primed with 3 L of Belzer MPS® solution . The coeliac trunk and superior mesenteric vein were subsequently cannulated leaving a side branch open  to purge air from the circuit before starting perfusion. When an aberrant hepatic artery was encountered , we carried out the appropriate vascular reconstruction at the end of backtable preparation, before machine perfusion. Pressure was set at 5 (continuous) and 25 mmHg (pulsatile) in the portal vein and hepatic artery circuit, respectively; gas analyses were obtained every 30 minutes to check if target oxygen pressure in the perfusate (~600 mmHg) was reached. The reservoir temperature was ~10 °C throughout perfusion. Dual-HOPE was performed in all but two cases, in which only the portal vein was perfused due to a the hepatic artery pump unit malfunction. In these cases, during the priming of the device, we observed a malfunction of the rotary pump with no flow in the hepatic artery unit. The hepatic artery unit was then shut down and HOPE was carried out through portal vein circuit only. Based on the study by Schlegel et al.30, the start of the transplant operation was coordinated to allow for a minimum HOPE time of 90 minutes. HOPE was stopped at the end of recipient hepatectomy, when the graft was disconnected from the device and transferred to recipient’s table for implantation.End-ischemic HOPE was applied as previously described31. Variables used to calculate the propensity score were donor age, macrovesicular steatosis ≥15%, donor BMI, laboratory model for end-stage liver disease (MELD) at LT, donor risk index (DRI) and balance of risk (BAR) score. The matched cohort was built by selecting two patients for every HOPE-treated patient in the control group who had the nearest propensity score. Kaplan-Meier survival estimates were used for survival analysis in the unmatched and matched cohorts. Finally, the association between early outcomes (AST and ALT post-LT peak levels and EAD onset) and the flow and resistance values measured during or at the end of the first hour of machine perfusion was analyzed using linear or logistic regressions as appropriate, and Mann-Whitney U test.Results are expressed as mean and standard deviation (SD) or as a number and percentage. HOPE cases were first compared with those in which liver was preserved by SCS by using non-parametric Mann-Whitney U test, Chi-squared or Fisher’s exact tests, as appropriate. To overcome the selection bias due to the allocation of HOPE to marginal grafts, propensity score matching with a 1:2 ratio was carried out32. In short, this approach allows for averaging among all possible statistical models, which are supported by the data, to obtain an unbiased estimate of the effect size of the different variables on the analyzed endpoint33. The percentage of inclusion (PI) expresses in which percentage of all possible models the variable has been included. The number of possible models is given by all possible combinations of baseline covariates that have a posterior model probability of at least 1/20. PI can be directly interpreted as the probability of a variable being included in the model and suggests which variables have a confounding effect on the outcome and should be used for adjusting. The effect on the outcome is expressed as an odds ratio (binary outcome) or as a mean variation of the outcome variable per unit variation of the variable in the model (continuous outcome). Finally, the probability of a significant result, i.e. the probability odds ratio is less than 0 or the probability the mean variation is less than 0, is provided as a measure of the size of the association. All analyses were performed with R version 3.3.3.The propensity score analysis does not take account of the uncertainty in the selection of variables in the propensity score equation. Moreover, residual imbalance between treated and untreated subjects may still be present due to the small sample size of the matched cohort. To address these issues, a further analysis using Bayesian Model Averaging (BMA) was carried out on the whole cohort2, n = 11; logistic issues associated with expected ischemia time ≥10 hours, n = 2. In these last two cases we had to call for a back-up recipient after the scheduled one was deemed unsuitable due to a previously undiagnosed severe pulmonary hypertension discovered during anesthetic preparation in one case, or to extra-hepatic dissemination of hepatocellular carcinoma discovered at laparotomy in the other. Total preservation time was 10 hours and 10 hours 33 minutes, respectively.End-ischemic HOPE was employed in 25 (8.5%) out of 294 primary adult DBD LTs performed during the study period. Mean (SD) perfusion time was 186 (49) minutes. No adverse event related to machine perfusion was observed. Indications for HOPE were distributed as follows: donor age ≥80 years, n = 12; macroscopic steatotic appearance of the graft at retrieval + donor BMI ≥30 Kg/mAs summarized in Table There were no cases of PNF. Outcome analysis in the whole cohort Table  showed aTo obtain more robust estimates of the effect of HOPE on analyzed outcomes, a further analysis using propensity score-based Bayesian model averaging was carried out, which confirmed the association between HOPE use and a 71% and 79% reduction of stage 2–3 AKI and severe PRS, respectively Table . TogetheAfter a minimal follow-up of six months, there were 49 18.2%) and 6 (24%) patients who presented with a biliary complication in SCS and HOPE group, respectively (p = 0.659)  or post-transplant cholangiopathy rate . The difference remained not significant even when only symptomatic cases were considered. Incidence of symptomatic post-transplant cholangiopathy was 2.2% and 0% in SCS and the HOPE group, respectively (p = 0.99) , symptomatic anastomotic complications , post-transplant cholangiopathy  or symptomatic post-transplant cholangiopathy  Table .Bayesian model averaging analysis confirmed that HOPE use was not associated with the development of overall, anastomotic or non-anastomotic biliary complications Table .Table 5BMedian (IQR) follow-up was 367 (196–525) and 264 (215–288) days in controls and the HOPE group, respectively. In both groups, median survival time was not reached at the end of follow-up. There were no significant differences between HOPE and SCS groups concerning patient and graft survival in both the unmatched and matched cohorts; however, 100% patient and graft survival was observed in the HOPE group . Flow and resistance values showed poor correlation with post-LT ALT peak , leading to the production of other reactive oxygen species, like hydrogen peroxide (H2O2). Reverse electron transfer through mitochondrial complex I is the main mechanism of superoxide production as a result of the high protonmotive force that is created upon reperfusion35. This leads to aspecific inflammation, Kupffer cells activation, dendritic cells maturation, endothelial expression of adhesion molecules, neutrophils infiltration, platelet aggregation and, finally, impairment of the microcirculation36. In the experimental setting, HOPE has shown to improve liver graft preservation as compared to SCS through continuous shear stress over sinusoidal endothelial cells, washout of metabolites produced during ischemia, increase of cellular adenosine triphosphate content, better peribiliary vascular plexus perfusion, and decreased mitochondrial release of reactive oxygen species at reperfusion by reverse electron transfer through mitochondrial complex I43. These effects would be particularly beneficial in steatotic grafts, which suffer from a baseline impairment of microcirculation and are therefore particularly vulnerable to the decreased synthesis of protective molecules  determined by the lack of mechanical shear stress over the endothelium during SCS44. Clinical data concerning HOPE use are still limited and mainly focused on grafts from DCD donors. In their pioneering papers, Guarrera et al.10, using hypothermic machine perfusion (HMP) without active oxygenation (O2 pressure was around 230 mmHg due to passive oxygenation), observed lower transaminases, bilirubin and creatinine peak after HMP, and no cases of post-transplant cholangiopathy. The Zurich group found that 1–2-hours end-ischemic HOPE in DCD LT is associated with lower transaminases peak, better synthetic function, lower EAD and post-transplant cholangiopathy rate, and better patient and graft survival45. From the same group, Kron et al.11 published a study in which six recipients of HOPE-treated steatotic livers (20–40% macrovesicular steatosis), 5 of which from DCD donors, were compared to 12 recipients of steatotic grafts (20–60% macrovesicular steatosis) from DBD donors, matched for donor and recipient age and total preservation time. HOPE was associated with lower AST peak, lower requirement of renal replacement therapy, shorter intensive care unit stay and better 1-year patient survival. The Groningen group has observed that end-ischemic dual-HOPE of grafts from DCD donors is associated with reduced biliary injury and better preservation of peri-biliary glands compared to SCS, and with a lower incidence of post-transplant cholangiopathy47. In Italy, combined use of normothermic regional perfusion49 and HOPE has allowed for the successful implementation of DCD LT despite the 20-minute no-touch period imposed by Italian law50.In last years, the necessity to cope with the detrimental effects of ischemia-reperfusion injury on grafts from extended criteria donors prompted a renewed interest towards machine perfusion techniques. The basics of ischemia-reperfusion injury, as applied to different organs and settings, have recently been reappraised. The first event in the induction of pathological ischemia-reperfusion injury appears to be a burst of mitochondria-derived superoxide .In this study, we also sought to explore HOPE’s potential as tool for graft quality assessment. At present, the inability to assess graft quality during hypothermic machine perfusion is one major limitation of the technique. Therefore, we investigated whether the value of vascular graft resistance and flow patterns during the first hour of machine perfusion can be used as predictors of ischemia-reperfusion injury and postoperative graft function. In kidney transplantation, higher vascular renal resistance at the end of hypothermic machine perfusion is a risk factor for delayed graft function and 1-year graft loss, but its predictive value is loweak Figs  and 5. H61, whereas graft steatosis is a risk factor for EAD and early graft failure63, we chose to employ HOPE in case of advanced donor age or graft steatosis, using donor BMI and direct graft inspection as surrogates of pre-transplant histologic graft assessment. This was dictated by the difficulty of obtaining routine pre-LT evaluation of graft biopsies in our setting. Unfortunately, donor BMI and graft evaluation by the retrieval surgeon had a poor predictive value for graft steatosis, resulting in only 5 out of 11 grafts identified as steatotic actually harboring a macrovesicular steatosis ≥15%. This led to the selection of a rather heterogeneous donor population mainly characterized by older age  and, consequently, by higher DRI , but with a comparable rate of macrosteatosis ≥15%  as compared to the reference group. We acknowledge that these features would hardly be defined as “extended criteria” in present days. It should be noted, however, that this limitation is shared by many published studies in which different machine perfusion techniques have been applied to discarded livers, which are far from being a homogeneous population17.This study represents the retrospective evaluation of a pilot experience at a single center and it has therefore many limitations, including its retrospective nature, limited numerosity and arguably a lack of power to achieve statistical significance for many analyzed outcomes, in particular patient and graft survival. The main limitation is certainly represented by the criteria that were used to consider HOPE use. As donor age is the main risk factor for biliary complications64. At present, there are several ongoing randomized controlled trials  evaluating the clinical impact of HOPE in different settings, which will hopefully help drawing the line beyond which HOPE use is associated with a relevant and economically sustainable clinical benefit.Although our results can be considered promising, the lack of a clear advantage in terms of patient and graft survival and biliary complications is questioning. Apart from the limited sample size, it is likely that our use of HOPE in a rather heterogeneous and low-risk setting hampered our ability to show a major benefit of the technique. This highlights the necessity to identify the areas in which application of machine perfusion techniques would yield a significant clinical advantage over static cold storage. As recently proposed, future studies will have to focus on relevant clinical endpoints and include a health economics analysisIn conclusion, in our experience end-ischemic HOPE in DBD LT was associated with a significant reduction of severe PRS, stage 2–3 AKI rate and EAD, and with a lower transaminases peak. These findings suggest a reduction of ischemia-reperfusion injury in HOPE-treated grafts. The slope of arterial graft resistance during HOPE may represent an indicator of graft quality, to be integrated with other prognostic factors. This study represents the report and analysis of a real-life attempt at improving the outcome of our patients and, given the scarcity of data in this context, we believe it could be of interest to other Centers that are planning to adopt this technology.