The surface topography was characterized using a 3-D confocal laser microscopy and inspected with a scanning electron microscope. To assess residual stresses, the tetragonal peak shift at 147 cm−1 was traced using micro-Raman spectroscopy. Al2O3 sandblasting altered surface topographies (p < 0.05), although highly translucent zirconia showed more pronounced changes compared to conventional zirconia. 5Y-PSZ abraded with 110 μm sand showed the highest Sa value (0.76 ± 0.12 μm). Larger particle induced more compressive stresses for 3Y-TZP (p < 0.05), while only 25 μm sand induced residual stresses for 5Y-PSZ. Al2O3 sandblasting with 110 μm sand for 3Y-TZP, 90 μm sand for 4Y-PSZ, and 25 μm sand for 5Y-PSZ were considered as the recommended blasting conditions.This study investigated the effect of sandblasting particle size on the surface topography and compressive stresses of conventional zirconia  and two highly translucent zirconia . Plate-shaped zirconia specimens (14.0 × 14.0 × 1.0 mm Th. Th−1 watortions  or the ftortions , contrib−1 [−1 decreased with increasing particle size. This must be related to the low transformability of the cubic phase [−1, assuming that this peak broadening resulted from the lattice distortion of tetragonal zirconia [−1 exhibited a blue shift and the amount of shift increased up to 90 μm (p < 0.05), whereas no further peak shifting was observed (p > 0.05). This can be explained by the increased critical stress concentration associated with the crack growth behavior in a residual stress field [For 4Y-PSZ, a broad cubic peak was detected at 625 cm−1 . The intzirconia  or the pzirconia  as a conss field . The for−1 was blue-shifted with 25 μm sand (p < 0.05), indicating the presence of residual compressive stresses. However, the μRaman peak exhibited a shift toward a shorter wavelength after sandblasting using 50 μm sand or larger (p < 0.05), which was referred to as a red shift [For 5Y-PSZ, a larger particle created a bigger change in the surface topography, and the changes grew at a faster rate than the other zirconia grades . This fied shift   for 3Y-TZP. For 4Y-TZP, the use of 90 μm Al2O3 sand can be recommended in order to obtain a bigger change in the surface topography for a good bonding with minimal surface defects. For 5Y-TZP, sandblasting using 25 μm Al2O3 sand may be clinically preferred in order to prevent possible damage to the zirconia surface. However, there are some limitations to these recommendations. In this study, we focused on the changes in the Al2O3 particle sizes, while other parameters were kept constant. The effect of changes in other parameters on the surface topography and crystallography of three different zirconia grades should be added for future studies. In addition, the measurements of the flexural strength and bond strength of resin cement to zirconia for the tested groups were not performed here. Future research should investigate the influence of different sandblasting protocols on the mechanical properties or bonding performance of three different zirconia grades. In this study, we applied 3-D texture parameters  which were height parameters according to the ISO 25178 standard to evaluate the topography of abraded zirconia surfaces. Further studies on the characterization of the surface topography should include a complementary set of additional parameters, such as spatial, volumetric, or feature parameters. The use of multi-scale analysis can allow a more accurate evaluation of the surface topography. Within the range of sandblasting parameters investigated in this study, Al2O3 particle size in terms of surface topography, phase transformation, and internal compressive stresses.Three different dental zirconia grades showed different degrees of dependence on the Al2O3 sandblasting modified the surface topographies of three different zirconia grades, although highly translucent zirconia showed more pronounced topographic changes compared to conventional zirconia.AlA larger particle induced more compressive residual stresses related to the t → m phase transformation for 3Y-TZP, while only 25 μm sand induced residual stresses due to little potential for the phase transformation of the cubic grain in 5Y-PSZ.2O3 particle sizes under given pressure, time, distance, and impact angle  were 110 μm sand for 3Y-TZP, 90 μm sand for 4Y-PSZ, and 25 μm sand for 5Y-PSZ in order to obtain a bigger change in the surface topography for a good bonding with minimal surface damage.Therefore, the results of this study suggested that the recommended AlThis study presented a methodology for assessing the state of abraded zirconia surfaces using 3-D texture parameters , but a complementary set of new parameters can be beneficial for the accurate evaluation of surface topographies. Following sandblasting, a Raman peak shift was detected and residual stresses associated with phase transformations were calculated for three dental zirconia grades. Therefore, this analytic technique can also be used to quantify the residual stresses on the highly translucent zirconia surfaces. Based on the experimental and analytical results on the surface topography and residual compressive stresses of three different dental zirconia grades after sandblasting, the following specific conclusion was drawn:


The aim of this study was to compare the biomechanics of a four-strand hamstring graft with a tripled semitendinosus graft, with and without adjustable extra-cortical button fixation, in a cadaveric model.Four groups of 10 cadaveric hamstrings were tested: In group A, a tripled semitendinosus graft fixated with two adjustable extra-cortical buttons; in Group B, a four-strand semitendinosus and gracilis graft fixated with an adjustable extra-cortical button and a clamp; in group C, a tripled semitendinosus graft fixated to a steel hook and a clamp; in group D, a four-strand semitendinosus and gracilis graft fixated to a steel hook and a clamp. Each group was submitted to a cyclic loading test (1000 cycles between 50 and 250 Newton at a frequency of 0.5 hertz) and a load-to-failure test. Primary outcomes were ultimate failure load and stiffness. Secondary outcomes were graft elongation and graft diameter.p < 0.01). Group B showed more cyclic elongation (4.1 mm (3.4–5.7)) compared to group D (2.3 mm (1.9–3.0)), and also lower stiffness was noted (171 N/mm (139–204) vs 265 N/mm (227–305)). There was no difference in graft diameter among groups.There was no difference in ultimate failure load among groups. Group B achieved a median stiffness of 171 N/mm (interquartile range [IQR] 139–204) which was significantly higher than Group A (median 103 N/mm (74–119), The results of this study indicate that higher stiffness can be achieved using four-strand hamstring tendon grafts compared to tripled semitendinosus grafts when using femoral extra-cortical buttons, despite comparable graft diameters. Thereby, the use of adjustable extra-cortical fixation devices may result in more cyclic elongation and lower stiffness of the graft. A challenge in anterior cruciate ligament (ACL) reconstruction is to maximize functional outcome and to minimize donor site morbidity. In the history of ACL reconstruction, many graft options have been described, but graft choice remains a contested topic . The pasStill, there are various possibilities for hamstring graft preparation and graft fixation. One of the advancements in graft fixation techniques is the use of adjustable extra-cortical fixation devices. These devices allow the use of bone sockets instead of full tunnels, which reduces bone loss in the tibial tunnel and also reduces the required length of the graft. Because of this advancement, there has been an increasing interest in harvesting only the semitendinosus tendon (ST) and leaving the gracilis tendon intact. Several studies suggested that this approach may reduce donor site morbidity and nerve injury and that it may also result in a better postoperative stability and higher post-operative hamstring muscle strength , 17, 19.The tripled ST grafts are used in clinical practice alongside the more widely used four-strand hamstring (doubled semitendinosus and gracilis (STG)) grafts. However, there is a paucity of evidence on the biomechanical properties of tripled ST grafts compared to those of doubled STG grafts.Therefore, the aim of this study was to evaluate the differences in failure load and stiffness between the all-inside-technique using a tripled semitendinosus graft and a four-strand hamstring tendon (doubled semitendinosus and gracilis) graft technique in a biomechanical setting. Secondary outcome parameters were to determine graft elongation and failure mode of both the tripled versus four-strand construct with an adjustable extra-cortical fixation versus a fixed hook construct. Our hypothesis was that a tripled semitendinosus graft results in a lower failure load and a lower stiffness of the construct compared to a doubled semitendinosus and gracilis tendon graft.The study was approved by the ethical review board and all patients gave their informed consent.This biomechanical study was executed at the Erasmus MC, University Medical Centre Rotterdam department of Orthopaedic Surgery between January 2017 and January 2018. Forty fresh frozen cadaveric hamstring tendons were harvested by an orthopaedic resident (FH) and orthopaedic surgeon (DM). Each tendon was obtained via the National body donation program, and as such, the use of these tendons was approved by the ethical review board. The tendons were selected on adequate length (≥21 cm) and good quality on visual inspection. Each tendon was sharply trimmed parallel to the fibre orientations and cleaned of all adherent muscle fibres. Afterwards, the tendons were stored in airtight bags in a freezer at − 20 °C until the day of testing. Before testing, tendons were thawed in gauzes with saline and an independent researcher randomly assigned the tendons into four test groups. After graft preparation, the diameter and length of the grafts were measured. Tests were performed at room temperature (18 °C), and all tendons were kept moist with physiological saline during preparation and testing.The all-inside technique using a tripled semitendinosus tendon and the four-strand hamstring technique were simulated and tested with extra-cortical buttons in an isolated construction. For each technique that was tested, one group was added in which the adjustable extra-cortical fixation device was replaced by a steel hook at the femoral side and by a clamp at the tibial side. The steel hook should not allow elongation and the clamp should not allow for tendon slippage. In this manner, we could determine to what extent the extra-cortical fixation device influences the biomechanical properties. Thus, 4 test groups were compared with each other.For adjustable extra-cortical fixation, TightRopes RT  were used. To simulate the femoral cortex, a custom device was designed of stainless steel. In this device a tunnel was created with a diameter corresponding to the manufacturer’s recommendations (4.0 mm). The tibial side was secured with a clamp with small dents, thus assuring a firm grip on the tendon.A tripled ST graft technique was simulated in the first group, using TightRopes (TR) for fixation. First, the grafts were whipstitched six times up and down through both ends with insoluble sutures . Next, the semitendinosus graft was folded over the extra-cortical button loop at the proximal  side, and then it was folded over the extra-cortical button loop at the distal  side. Finally, the insoluble sutures of both ends of the graft were knotted via seven alternating knots to the loop of the adjustable extra-cortical button at the tibial side and the femoral side. The two TightRopes were attached to the metal fixation devices Fig. a. The leThe second technique was the commonly used four-strand hamstring graft technique, fixated with a TightRope at the proximal  side and fixated with a clamp at the distal  side. The STG tendons were symmetrically folded over the extra-cortical button loop at the proximal side, which was attached to the extra-cortical device. Then, the four strands were fixated into the clamp at the distal side. During fixation of the graft into the clamp, all the ends of the strands were whipstitched with sutures and then pulled through the clamp. In this way, it was attempted to divide all strengths equally over each strand Fig. b. Graft To determine the biomechanical influences of the TightRope, two groups were added in which we substituted the TightRope with a firm stainless steel hook at the proximal side.A tripled semitendinosus graft was combined with the steel hook. The ST graft was folded over the steel hook at the femoral side, then a loop was made at the tibial side, and the proximal end of the third strand was knotted to the steel hook with a whipstitched FiberWire. The distal end of the graft was fixated into the clamp at the tibial side Fig. c.A four-strand STG graft was folded symmetrically through the steel hook at the proximal side. Then it was fixated into the clamp at the distal side. Again, it was attempted to divide all strengths equally by pulling at the sutures, which were stitched through the ends of the graft Fig. d.Tests were performed with a tensile testing machine  that could apply a maximum pull-out force of 1000 Newton (N). The grafts were attached to the machine in such a way that the force on the grafts would have been in line with the long axis of a tunnel in clinical practice. In this way, worst-case testing was executed on the ACL reconstructions. All strands of the grafts were tied together in het center of the graft to create circumferential pressure and all grafts were pre-tensioned by hand before they were attached to the testing machine. The testing protocol of Aga et al. was used for cyclic testing and pull-to-failure loading . After tThe primary outcome was failure load (in Newton) and stiffness (Newton per millimetre). Failure load was measured at the maximum force before failure of the graft. Stiffness was calculated at the steepest point in the force-displacement curve before the maximum failure load. Secondary outcomes included graft elongation (in millimetre), mode of failure and graft diameter (in millimetre). Mode of failure was observed visually and recorded during and after testing. Failure was classified as tendon failure, failure of the adjustable extra-cortical fixation device, suture failure or clamp loosening. If the graft did not fail, this was also noted. Graft elongation was calculated as the difference between graft length after preloading and after 1000 cycles of our testing protocol. Graft diameter was measured with a graft-sizing block .Sample size calculation was performed and a minimum of 10 per group was sufficient to detect a 20% difference (α = 0.05%) in maximum failure load assuming an SD of 43 with mean maximum failure load of 859 N [Comparing the two grafts used in the clinical situation, group A and B were compared. Group C and D were compared to detect differences between a tripled ST graft and a four-strand hamstring graft without the influence of the adjustable fixation device. Group B and D were compared to observe the influence of the adjustable cortical button device.P < 0.05.Statistical analysis was performed using SPSS statistics version 24 . The significance level was set at The highest ultimate failure load was measured in the four-strand group D and group B, followed by the tripled strands in group C and group A, this however was not statistically significant Table . Also, tp < 0.01). Furthermore, our groups using a steel hook for femoral fixation, group C and D, achieved higher stiffness compared to group A and B respectively [Fig. Stiffness was highest in the four-strand group D, followed by group C (tripled graft), group B and group A Table . Stiffneely Fig. .No grafts failed during the cyclic testing from 50 N to 250 N. The lowest amount of total elongation after 1000 cycles was observed in group D with a median elongation of 2.3 mm (1.9–3.0), followed by group C with 2.8 mm (2.0–3.3), group B 4.1 mm (3.0–5.3) and group A with 5.2 mm (3.4–5.7)  as reported by Rupp and Shelburne , 15. TheThe most important clinically relevant finding of this study was that a higher stiffness was found in group B compared to group A. This is in line with the study of Mayr et al. who found that a four-strand graft with a femoral adjustable extra-cortical button and a tibial interference screw had a higher mean pull-out stiffness than a graft with dual cortical button fixation . HoweverSince stiffness is correlated with elongation, we observed greater elongation in the Tightrope groups than in the groups using a steel hook. These results match the ideas of Barrow et al., who found clinically significant increased loop lengthening for adjustable fixation devices during their cyclic loading tests . This leThe findings in our study may be limited by the in vitro biomechanical set up, that we chose for our tests. We have chosen for clamp fixation instead of an interference screw. In group B, only 1 clamp loosening was observed. In group C and D, clamp loosening was observed 7 times, at relatively high failure loads mean of 791 N) compared to the mean failure load reported by Mayr et al. (694 N ± 119 N). In eight out of 10 cases of their screw fixation group, these authors observed graft slippages from the tibial tunnel [1 N compaThe forces that were applied on the grafts were in an axial line, which is not comparable with the in vivo situation where the graft is placed into oblique bone sockets and the forces on the graft are not in an absolute axial direction. Nevertheless, we believe that the forces we applied correspond with the worst-case test scenario. Therefore, we think that in the clinical situation the elongation might be less than in our study.Another source of uncertainty is the abnormal distribution of our data. This is partly due to the small sample size and partly due to a few outliers. Our results therefore need to be interpreted with caution. Although our study design aimed to minimize confounding factors, we observed a larger width in our data than we expected. If we extrapolate this to the clinical situation, where biomechanical properties are subject to more influencing factors some unknown at the time of surgery, there may be more or less variation in fixation strengths, in distribution of forces on the strands and in stiffness of the construct and outliers could be interpreted as comparable to the clinical setting.In this biomechanical study, a four-strand semitendinosus gracilis graft is a stiffer construct compared to a tripled semitendinosus when using adjustable extra-cortical fixation devices, and might therefore be the preferred graft technique. The use of adjustable extra-cortical fixation devices negatively affects stiffness and cyclic elongation of the graft.


BioTM. We received our first impact factor of 6.091 this year. This is a massive accomplishment for all stakeholders of the journal. The journal was formally launched in 2015 and we published our first issue in 2016. Within a short period of 4 years, we were able to reach this significant milestone.2020 marks an important year in the evolution of BioTM authors who trusted the journal with their precious manuscripts in the early, unproven days. Submission of high‐quality manuscripts is the principal reason that led to an impressive launching impact factor. BioTM welcomes manuscripts in all aspects of bioengineering including drug delivery, drug discovery and development, tissue engineering, synthetic biology, biosensors, organoids and organ‐mimetic systems, stem cell therapies, gene therapies, immunotherapies, patient‐targeted therapies, medical devices, regenerative medicine, implant/patient interface, computational modeling, and bioinformatics.Huge thanks to all BioTM. This is expected because these fields represent some of the most active research areas at the interface of medicine and engineering, especially those that focus on developing/testing novel materials or devices, for example, nanoparticles and microneedles for drug delivery. Indeed, some of the recent BioTM papers in drug delivery have focused on the use of microneedles for vaccine delivery.Areas including drug delivery, gene therapy, sirRNA delivery, regenerative medicine, nanomedicine, biomedical devices, and cell therapy have been particularly well represented in the manuscripts recently submitted to BioTM papers, also consistent with the large community of researchers working at the interface of engineering and regenerative medicine. Covered tissue engineering topics included engineering of hydrogel and decellularized matrices and generation of tissue‐specific blood vessels.Tissue engineering was also a commonly discussed subject in recent BioTM are particularly focused on reporting translational advances and hurdles in the field.Consistent with general literature trends, a significant number of recent papers were focused on nanomedicine. Topics included translatable mouse models for cancer,BioTM. We feature one board member in each issue. You can read these biographies at https://aiche.onlinelibrary.wiley.com/journal/hub/journal/23806761/homepage/editorialboardprofiles.html.We are thankful for the strong support from our editorial board members. In 2020, we added six new editorial board members: Jennifer West , Xiaoyuan Chen (NIH), David Mooney (Harvard University), Molly Stevens , Maria Jose Alonso , and Andreas Lendlein . We look to our board for guidance and vision. We are especially thankful to the board members for routinely publishing their own research in BioTM. The support came in various forms including guest editing of special issues, submission of manuscripts, and reviewing manuscripts. Our special thanks to Prof. Aaron Anselmo of University of North Carolina, Chapel Hill, who started our feature BioTM Buzz, which highlights select papers from each issue. Follow BioTM_Buzz on Twitter for updates on our publications.We are pleased with the enthusiastic participation of the young generation of researchers in the launch and growth of BioTM has published several special issues dedicated to several topics, including nanoparticles in medicine, nucleic acid delivery, responsive materials, and bioengineered therapeutics as well as special issues dedicated to conferences in bioengineering and translational medicine and Engineering Conferences International (ECI) nanotechnology in medicine. We also published a special issue honoring two luminaries of the field: Robert Langer and Nicholas Peppas. These special issues were edited by our enthusiastic patrons, including Profs. Kaushal Rege, Kannan Rangaramajunam, Paolo Decuzzi, Zhen Gu, Kathryn Whitehead, Pankaj Karande, Josue Snitzman, and Millicent Sullivan. We are deeply thankful for their dedication. Without the enthusiastic participation of these committed individuals, we would not have gotten off to such a great start. We also thank Arthur Baulch for excellent management of the operation, Cynthia Mascone for leadership in publication policies, and the entire Wiley team for publication strategies.Having finished the 5‐year journey, I look forward to the next phase of the journal, which will include an increase in the number of publications each year, expansion of covered topics, and increased representation of industry and international authors in the journal. Thank you all for your strong support to the journal and look forward to your continued support in coming years.https://publons.com/publon/10.1002/btm2.10178.The peer review history for this article is available at


N-oxide (TMAO) in graft failure remains undetermined. Here, we investigated the clinical utility of TMAO and its dietary determinants for graft failure prediction in renal transplant recipients (RTRs). Methods. We included 448 RTRs who participated in the TransplantLines Cohort Study. Cox proportional-hazards regression analyses were performed to study the association of plasma TMAO with graft failure. Net Benefit, which is a decision analysis method, was performed to evaluate the clinical utility of TMAO and dietary information in the prediction of graft failure. Results. Among RTRs , the baseline median TMAO was 5.6 (3.0–10.2) µmol/L. In multivariable regression analysis, the most important dietary determinants of TMAO were egg intake , fiber intake , and fish and seafood intake . After a median follow-up of 5.3 (4.5–6.0) years, graft failure was observed in 58 subjects. TMAO was associated with an increased risk of graft failure, independent of age, sex, the body mass index (BMI), blood pressure, lipids, albuminuria, and the Estimated Glomerular Filtration Rate (eGFR) : 1.22; 2.14, p < 0.001)). A TMAO and dietary enhanced prediction model offered approximately double the Net Benefit compared to a previously reported, validated prediction model for future graft failure, allowing the detection of 21 RTRs per 100 RTRs tested, with no false positives versus 10 RTRs, respectively. Conclusions. A predictive model for graft failure, enriched with TMAO and its dietary determinants, yielded a higher Net Benefit compared with an already validated model. This study suggests that TMAO and its dietary determinants are associated with an increased risk of graft failure and that it is clinically meaningful.Background. Due to the critical shortage of kidneys for transplantation, the identification of modifiable factors related to graft failure is highly desirable. The role of trimethylamine- Renal transplantation is the treatment of choice for patients with end-stage kidney disease, being superior to dialysis in terms of quality of life, long-term survival, and healthcare costs . UnfortuDue to the scarcity of organs, approaches for lengthening graft and patient survival are highly desirable . Over thThe evaluation of the above-mentioned biomarkers should take into account the clinical utility of the biomarkers, in addition to their predictive accuracy. The application of decision theory analytical methods, i.e., the calculation of Net Benefit, has been proposed as an alternative for evaluating the clinical value of biomarkers and predictive models ,6.N-oxide (TMAO) is a methylamine osmolyte  was associated with an increased risk of graft failure, independent of age and sex  when included in either the crude or the sex- and age-adjusted models.The proportional hazards assumptions were not violated for any of the variables in the additive models. The interaction terms of the TMAO plasma concentration with age, sex, and eGFR with graft failure were not significant . The asp = 0.01). The Brier Score of the updated model containing TMAO and dietary information showed a discrete, albeit insignificant, improvement from 0.0879 to 0.0857 (p > 0.5)  .The Net Benefit analysis demonstrated a higher clinical utility of the model containing TMAO and dietary information over the traditional model for graft failure risk prediction .The TMAO and dietary enhanced model offered a greater Net Benefit, compared with the traditional model, for all the risk thresholds. The already validated model showed a Net Benefit of 0.466 at the 20% graft failure risk threshold, which is equivalent to detecting 46.6 RTR with future graft failure and suggesting no false positive results per 100 RTRs with future graft failure tested. At the same risk threshold, the TMAO and dietary enhanced model offered the greatest Net Benefit of 0.509, meaning that 51 RTRs with future graft failure could be detected per 100 RTRs tested, with no false positives. The TMAO and dietary enhanced model offered a double Net Benefit, compared with the traditional model at the 60% graft failure risk threshold, allowing the detection of 21 RTRs  with future graft failure. In contrast, the previously published model allowed the detection of 10 RTRs  .In this RTR cohort, the associations of plasma concentrations of TMAO with the risk of graft failure were investigated. Plasma concentrations of TMAO at the baseline were associated with reduced eGFR, and fish and seafood intake. The association of TMAO with an increased risk of graft failure remained significant when taking into account important risk factors, including the BMI, lipid profile, smoking, donor characteristics, diet, and renal function at the baseline.Furthermore, the addition of circulating TMAO and diet information to an already validated predictive model of graft failures enhanced its predictive ability in terms of NRI, as well as Net Benefit. To the best of our knowledge, the present study is the first to extensively evaluate the clinical utility of circulating TMAO and diet information in the context of graft failure prediction in RTRs.p < 0.001)). This result is in line with a previous study conducted in patients with end-stage renal disease where pre- and post-hemodialysis TMAO concentrations were measured, and showed a marked decline in TMAO concentrations, from 99.9 ± 31.9 to 41.3 ± 18.8 μM, after hemodialysis [p = 0.01). These findings are explained by the role of fish and seafood as sources of TMAO, given the fact that such foods are the only source of TMAO that do not require intermediate metabolism [p = 0.03). Such an association is in line with previous studies which have reported that an increased egg intake was associated with elevated concentrations of TMAO in both plasma and urine [p = 0.002). Notably, subjects in the highest tertile of TMAO consumed less vegetables and fruits and more eggs and fish and seafood than the average Dutch person . Notably, the prospective association of TMAO concentrations with graft failure was independent of CKD stage. The role of the gut microbiome in the context of chronic kidney disease has gained recent attention . HoweverIt is necessary to have reliable information about the clinical benefit of gut microbiome-related biomarkers and their modifiable determinants  for the prediction of graft survival in RTRs. Due to the fact that traditional measures, such as the sensitivity, specificity, or area under the curve, correspond to statistical abstractions which cannot provide direct information about the clinical value of biomarkers or prediction models, the use of decision-analytic measures, such as Net Benefit, has been proposed .n = 651, alive), and it was externally validated in independent cohorts from Tours, France (n = 736); Leeds, the United Kingdom (n = 787); and Halifax, Canada (n = 475). In our study, the clinical utility of the model was improved by the addition of TMAO measurements and dietary information, identifying more RTRs with future graft failure, without increasing the false positives cases. Such improvement is more marked in a threshold risk of graft failure < 40%. Interestingly, RTRs under such a threshold risk are those who obtain a greater benefit from more sensitive predictive biomarkers, given the fact that they have a better renal function at the baseline and presumably are those in which a dietary intervention may have a greater impact.Our results from Net Benefit analysis showed that the addition of TMAO measurements and dietary information increases the Net Benefit of an already validated graft failure predicted model . The preAccording to the authors who first introduced the decision curve analysis as a method to evaluate prediction models and diagnostic tests, in classical decision theory, the strategy with the highest expected utility should be chosen, irrespective of the size or statistical significance of the benefit . Therefon = 53), urinary TMAO adequately discriminated patients with a limited graft function from those with a prolonged graft function [This single-center study has several strengths. In our analysis, TMAO improved the reclassification of patients from a lower to a higher graft failure risk category. The performance of circulating TMAO is in line with the previously reported performance of urinary TMAO prediction of a prolonged duration of graft function in kidney transplant recipients. It is interesting to note that, in a relatively small study , represent modifiable factors that can be translated into nutritional recommendations in order to improve graft survival in RTRs. However, randomized clinical trials are required to demonstrate that the implementation of TMAO-lowering diets can actually prolong graft function in RTRs.Several limitations of the present study deserve to be mentioned. First, the present study was conducted in the north of the Netherlands, and mainly comprises individuals of Caucasian ancestry, which could limit the extrapolation of our findings. For instance, the previously validated model used to predict graft failure included ethnicity , which wIn conclusion, this study of stable RTRs revealed that high concentrations of circulating TMAO are associated with a higher risk of graft failure, independent of other risk factors, such as an impaired renal function. Furthermore, this study is the first to provide an extensive evaluation of the clinical utility of TMAO and dietary information for the risk classification and prediction of graft failure in RTRs. Further investigation is needed to unravel the complexity underlying the relationship between circulating gut microbiome-derived metabolites such as TMAO and CKD.


Therefore, it is conceivable that rapid reconstitution of the hILC compartment may be beneficial in HSCT, by promoting mucosal tissue repair/regeneration and providing protection from opportunistic infections. In this review, we will summarize the evidence for a role of hILCs in allogenic HSCT for the treatment of hematological malignancies in all its steps, from the preparative regimen to the immune reconstitution in the recipient. The protective properties of hILCs at the mucosal barrier interfaces make them an attractive target to exploit in future cellular therapies aimed at improving allogenic HSCT outcome.Helper Innate Lymphoid Cells (hILCs), including ILC1s, ILC2s, and ILC3s, are mainly localized at the mucosal barriers where they play an important role in tissue regeneration and homeostasis through the secretion of specific sets of cytokines. The recent identification of a circulating ILC precursor able to generate all ILC mature subsets in physiological conditions, suggests that “ILC-poiesis” may be important in the context of hematopoietic stem cell transplantation (HSCT). Indeed, in HSCT the conditioning regimen (chemotherapy and radiotherapy) and Graft  High exForm these evidences it can be concluded that ILC1 dysregulation, in terms of numbers and function, may be implicated in the pathogenesis and progression of diverse hematological malignancies, and that ILC2 induce a tolerogenic environment that favors tumor progression. However, it must be determined whether the phenotype and function reported for hILCs in these patients account for the disease, or they are a consequence of the malignancy itself.vs host disease (GvHD), and participate to the immune response that prevents leukemia relapse. Since hILCs have been discovered very recently, the actual contribution of hILCs in these steps has not been fully elucidated. We will summarize in the following paragraphs the evidence that came out of this recent field of investigation, and highlight some interesting aspects that suggest possible advantages of exploiting hILCs in HSCT.Because of their role both in anti-tumor response in hematologic malignancies and maintenance of epithelial barrier integrity, hILCs represent an attractive tool to exploit in the treatment of these tumors through allogenic hematopoietic stem cell transplantation . Indeed, they may play a protective role in the first phase of treatment, when the preparative regimen that precedes transplantation causes mucosal damages. Helper ILCs may then represent a cellular component of the graft and contribute to the defense from infections while the donor-derived immune system develops. Finally, hILCs may be important in the protection from graft In allo-HSCT, donor-derived HSCs engraft the BM of the recipient and differentiate into mature immune cells, thus reconstituting the recipient lympho/hemopoiesis compromised by either disease or myeloablative therapy. Allo-HSCT is used primarily for hematologic and lymphoid tumors. In adults, the majority of allo-HSCTs are performed for the treatment of acute leukemias, in particular AML. Other major indications include myelodysplastic syndrome or lymphoma (predominantly non-Hodgkin) and, to a lesser extent, MM, chronic myeloid leukemia (CML), and CLL . In pediHelper ILCs in graft vs host disease”).Prior to transplantation, patients receive conditioning chemotherapy and radiotherapy in order to kill malignant cells and to deplete non-malignant recipient immune cells to avoid rejection . Althoug+ HSCs in the PB (in vitro development of NK cells and hILCs is influenced by the source of CD34+ HSCs and G-CSF treatment (versus tumor effect), by recognizing recipient antigens, donor T cells also cause GvHD, as mentioned above. Another significant challenge in providing access to HSCT is represented by the availability of a suitable HLA-matched donor. HSCT from a haploidentical donor (haplo-HSCT) offers the option of immediate transplantation virtually to any patients in need of an allograft and lacking suitable HLA-matched donor. To prevent both GvHD and graft failure (+ HSCs and administration of CD34+ “megadoses” has been employed for many years (The predominant source of HSCs for hematologic transplantation is represented by granulocyte colony stimulating factor (G-CSF)-mobilized PB stem cells. In donors who fail to mobilize, G-CSF is administered in combination with plerixafor, a CXCR4 antagonist, inducing an increase of CD34reatment . Althoug failure , positivny years . Howeverny years –41. Throny years . The rolny years , 44. Theny years . Donor Nny years , 46–48. ny years , analogony years . MoreoveAlthough the number of circulating hILC is very low compared, for example, to NK cells, the presence and the function of mature circulating donor-derived hILCs in the graft in HSCT has not been investigated to date. It would be interesting to evaluate in retrospective studies whether there is a correlation between the abundance of infused hILCs in the graft and the clinical outcome of the patient in terms of both relapse and incidence of infections.More information is available regarding the reconstitution of hILC compartment following HSCT and the role of these cells in GvHD, which are the topics of the following paragraphs.+ NK cell progenitors to stage 4 CD56bright NK cells  capable of generating all human ILC subsets, including NK cells , and that maintain activated or dividing cells   . Any loc+KIR− cells, and the first appearance of KIR+, cytolytic, and potentially alloreactive NK cells requires 4–6 additional weeks  . These cvs Leukemia  and skin (CCR10 and CLA)  . Notablyto group , suggestin vitro from HSCs present in the graft might be adoptively transferred to patients receiving HSCT and suffering from GvHD with mucosal tissue lesions to facilitate epithelial regeneration. It would be interesting to verify whether tissue resident hILC reconstitution occurs from circulating ILCPs and study it in parallel with reconstitution in the PB. Indeed, although it has been shown that ILC3s in the thymus and GI tract are resistant to radiation injury  to accelerate in the recipient the differentiation of hILC subsets potentially useful for the treatment of severe mucosal damages. To do so, further studies on hILCs differentiation are clearly needed, especially to understand how the signals from the tissue microenvironment “tune” the generation of the appropriate ILC subset from a common HSC-derived precursor.In conclusion, the ability of hILCs to rapidly secrete an array of different cytokines in a subset-dependent manner makes them a promising tool to exploit to improve allogenic HSCT outcome, by protecting the recipient from GvHD and infections, and enhancing adaptive immune response reconstitution. The strategies aimed at exploiting the properties of these cells may be: (i) to preserve hILCs present in the graft and infuse them in the recipient; (ii) to generate and expand All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by grants awarded by Associazione Italiana per la Ricerca sul Cancro (AIRC)-Special Program Metastatic disease: the key unmet need in oncology 5X1000 2018 Id. 21147 (LM), AIRC IG 2017 Id. 19920 (LM); RC-2020 OPBG ; Ministero della Salute RF-2013, GR-2013-02356568 (PV). LQ has received funding from AIRC and from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no 800924. NT was supported by a AIRC fellowship for Italy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zeitschrift für die gesamte Neurologie und Psychiatrie. Today, he is rarely remembered, except mostly in the context of his research on the blood–brain barrier.In the first two decades of the twentieth century, there was probably no European neurologist who was not familiar with the name of Max Lewandowsky, German-Jewish neurologist, author of numerous works, including a handbook of neurology, and the editor of the neurological journal Friedrichsgymnasium in Berlin, graduating in 1893. Then he studied medicine in Marburg, Berlin and Halle. Among his teachers were Theodor Engelmann (1843–1909) and Hermann Munk (1839–1912). He defended his Ph.D. thesis in 1898 and immediately continued his scientific career, first in the physiology laboratory in Berlin. He attended courses of psychiatry led by Karl Bonhoeffer (1868–1948) and Franz Nissl (1860–1919) in Heidelberg and Theodor Ziehen in Berlin Charité clinic. In Paris he studied under Pierre Marie (1853–1940) at Bicêtre Hospital. In 1902 he became Privatdozent in Berlin, and in 1908 he was appointed extraordinary professor [Max Heinrich Lewandowsky was born on June 28, 1876 in Berlin, the son of Hermann Lewandowsky and Rose née Heymann. He attended Zeitschrift für die gesamte Neurologie und Psychiatrie. It was accompanied ‘by a publishing series “Monographs from the joint field of neurology and psychiatry” (Monographien aus dem Gesamtgebiete der Neurologie und Psychiatrie). In the same year, he began work on a multi-authored handbook of neurology, inviting dozens of renowned specialists from Germany and abroad [Lewandowsky was an efficient organizer. In 1910, together with Alois Alzheimer (1864–1915), he founded d abroad . Until 1d abroad . LewandoBluthirnschranke) [As a researcher, Lewandowsky was particularly interested in experimental work. His Ph.D. dissertation dealt with vagal control of lung function . Under tchranke) . His subchranke) . In 1907chranke) . He alsoMax Lewandowsky was married to a mezzosoprano singer Margarete (Gretchen) Gille in 1909; the marriage produced no children. After World War I broke out, Lewandowsky served as an army physician. He investigated neurological symptoms in head trauma and opposed inhuman methods of treating war neuroses . In the


The V‐shaped structure characteristic peaks occurred obviously in CS‐SI complexes detected by XRD patterns, which formed a new crystalline structure. The thermal stability was improved in CS‐SI complexes revealed by TGA and DTG curves that the thermal cracking temperature increased from 315°C to 320°C. The enthalpy (ΔH) of CS‐SI complexes decreased from 2.34 J/g to 1.75 J/g showed by DSC data, which indicated that the ordered structure of starch was destroyed. Furthermore, the content of resistant starch increased from 10.53% to 21.78% and predicted glycemic index (pGI) reduced in CS‐SI complexes. In conclusion, the digestibility and pGI of starch can be improved by complexed with soy isoflavone.Rapid starch digestion rate is negative for the normal level of human blood glucose. This study investigated the protective effects of corn starch (CS) complexed with soy isoflavone (SI) on the control of starch digestibility and glycemic index (GI). The structure of the corn starch–soy isoflavone (CS‐SI) complexes was characterized by Fourier transform infrared (FT‐IR), X‐ray diffraction (XRD), Thermogravimetric analysis (TGA), and Differential scanning calorimetry (DSC), and the complexes digestibility was evaluated using in vitro digestion model. The results of FT‐IR spectrum showed that, compared with corn starch, new characteristic peaks were not occurred in CS‐SI complexes, and the value of R Soy isoflavone entered the spiral cavity of amylose through hydrophobic force, and a V‐shaped crystalline structure was formed in CS‐SI complexes, which increased SDS and RS content, and the starch digestibility and predicted glycemic index were reduced. Starch has a positive effect on maintaining human life and movement, which play a key role obtaining energy from the diet for human. However, long‐term consumption of high glycemic index food was positively associated with obesity, hyperglycemia, diabetes typeⅡ, and other metabolic complications  was purchased from Shanghai Jinsui Biological Technology Co., LTD company. Soy isoflavone  was purchased from Xian Tianfeng Biotechnology Co., LTD company. α‐amylase from porcine pancreas (8 × USP) and glucosidase from aspergillus niger (≥260 U/ml) were purchased from Sigma‐Aldrich. D‐glucose assay kit was purchased from Megazyme International Ireland. Other reagents were of analytical grade.2.2g for 5 min, washed three times with ethanol solution , and centrifuged to remove soy isoflavone that were not complexed with corn starch, and the samples were freeze‐dried. The samples obtained are marked as SSI‐0, SSI‐1, SSI‐2, SSI‐3, and SSI‐4.The preparation of CS‐SI was slightly modified with reference to the published method  curves of samples were recorded.2.70T, the peak temperature of gelatinization pT, the final temperature of gelatinization CT, and the enthalpy value ΔH were known.Using DSC to determine the thermal properties of samples, refer to published methods for minor modifications , slowly digestible starch (SDS), and resistant starch (RS) of CS‐SI complexes was calculated by the following formula:Based on the Englyst method with slight modification ] was applied to investigate hydrolysis kinetics and pGI of the CS‐SI complexes .The hydrolysis index (HI) is the ratio of the area under hydrolysis curve (AUC) of samples to the AUC of fresh white bread. According to the equation, pGI = 8.198 + 0.862HI, get pGI , and the variances are calculated using SPSS 21 software (SPSS Inc). Analysis measurement, the difference is statistically significant. The significant differences were evaluated and displayed by different letters.33.12 present in starch molecule. The band at 1642/cm is attributed to the C = O stretching vibration in a carbohydrate group. Strong absorption peak appeared in all samples. Compared with SSI‐0, in the spectral range of 4,000 ~ 400/cm, the shape and position of infrared absorption peaks were similar and offset not obviously, new characteristic peaks not appeared or disappeared, which indicated that new group not generated in CS‐SI complexes and they were not combined by a covalent bond.FT‐IR spectroscopy was performed to investigate the structure of CS‐SI complexes, as Figure 1045/1022 and R995/1022 decreased, shown in Figure FT‐IR spectroscopy has been widely applied to investigate the short‐range molecular structure changes in starch double helices, and the differences in the crystalline and amorphous regions were observed between complexes. The ratio of FT‐IR spectrum (1045/1022)/cm and (995/1022)/cm absorption peak intensity can reflect the change of ordered structure in starch. The characteristic of ordered structure in aggregated structure of starch crystalline region is attributed to absorption peak near 1,054/cm, the characteristic of random coil structure of starch in the amorphous region is represented to absorption peak near 1,020/cm, and the absorption peak near 995/cm is an ordered structure of hydrogen bonds formed by the hydroxyl groups of anhydroglucose units between starch molecules Shingel, . Phenoli3.27 helical‐type diffraction ; pT and cT both showed an upward trend. The increase in gelatinization temperature could be attributed to interaction between amylose–amylose or amylose–amylopectin chains, and the formation of complexes between amylose and other substances. In our present research, ‐OH groups in SI might bind to starch granules, the redistribution of water between soy isoflavone and corn starch, which in turn affected the starch gelatinization transformation and hydration of amorphous region in starch granules, resulted in starch gelatinization temperature increased.Starch gelation and phase change can be quantitatively analyzed by DSC. Table ΔH) reflects the energy required for destruction of starch double helix during starch gelatinization, and before gelatinization of starch, measures the order of molecular chains inside starch granules. The destruction of ordered structure in starch granules will lead to a decrease in enthalpy value. The data in Table ΔH of CS‐SI complexes decreased significantly (p < .05). Similar to the result of Han , indicated that digestibility of corn starch after complexing with soy isoflavone was significantly reduced. The maximum hydrolysis degree C∞ of CS‐SI complexes ranged from 69.482 to 82.098, which was significantly lower than SSI‐0 (92.875). It is further shown that starch hydrolysis rate was reduced complexed with soy isoflavone, thereby digestibility of starch was reduced. In addition, the hydrolysis kinetic constant K value was decreased gradually. V‐shaped crystal structure of starch shows more resistance to α‐amylase hydrolysis, and the rate of starch hydrolysis can be reduced , as the concentration of soy isoflavone increased, pGI of CS‐SI complexes decreased gradually, and SSI‐4 had the lowest pGI value (68.09). Relatively higher SDS and RS content and lower pGI values in SSI‐1, SSI‐2, and SSI‐3, however they were still considered high GI (GI > 75) foods, while SSI‐4 was considered as medium GI food (Babatola et al., 4In this experiment, structural and in vitro digestion characteristics of corn starch incorporated with soy isoflavone were analyzed systematically, and glycemic index was predicted. The change mechanism of digestibility in CS‐SI complexes was revealed, and the non‐covalent interaction between corn starch and soy isoflavone was verified. CS‐SI complexes lost its original crystal, and a unique V‐shaped crystal structure was formed. By complexed with soy isoflavone, short‐range ordered structure and enthalpy value of starch were reduced, thermal stability of starch was improved, the content of SDS and RS in starch was increased, and pGI was decreased. From the structure characterization and digestibility results of CS‐SI complexes, it could be concluded that soy isoflavone entered amylose spiral cavity through hydrophobic force, and digestibility of starch was reduced by the change of CS‐SI complexes crystal structure. Therefore, complexed with soy isoflavone will open a pathway to modulate digestibility of starch.The authors declare no conflict of interest.


Nanoindentation technology with high spatial resolution and force sensitivity is widely used to measure the mechanical properties of hard biomaterials and tissues. However, its reliability to analyze soft biomaterials and organs has not been tested. Here, we evaluated the utility of nanoindentation to measure the passive mechanical properties of soft biological specimen. Kidney, liver, spleen and uterus samples were harvested from C57BL/6 N mice. We assessed test–retest repeatability in biological specimen and hydrogel controls using Bland–Altman diagrams, intraclass correlation coefficients (ICCs) and the within-subject coefficients of variation (COVs). The results were calculated using Hertzian, JKR and Oliver & Pharr models. Similar to hydrogels, Bland–Altman plots of all biological specimen showed good reliability in stiffness test and retest examinations. In gels, ICCs were larger than 0.8 and COVs were smaller than 15% in all three models. In kidney, liver, spleen and uterus, ICCs were consistently larger than 0.8 only in the Hertzian model but not in the JKR and Oliver & Pharr models. Similarly, COVs were consistently smaller than 15% in kidney, liver, spleen and uterus only in the Hertzian model but not in the other models. We conclude that nanoindentation technology is feasible in detecting the stiffness of kidney, liver, spleen and uterus. The Hertzian model is the preferred method to provide reliable results on ex vivo organ stiffness of the biological specimen under study. Cyanotic patients with congenital heart diseases are often characterized by increased arterial stiffness in comparison with healthy population2. There is a direct relationship between aortic stiffness and left ventricular systolic and diastolic dysfunction in patients with inflammatory bowel disease3. Eradication of hepatitis C virus infection causes a significant decline in liver stiffness -particularly in patients with high baseline level of inflammation or patients who received direct-acting antiviral agents5. Increased arterial stiffness can occur in parallel with the decline of glomerular filtration rate in patients with mild-to-moderate chronic kidney disease6. Type 2 diabetes had greater impact on pulse wave velocity of the central arteries than peripheral arteries7. In clinical settings, a number of various techniques have been introduced to detect changes in stiffness of human organs, such as transient elastography, ultrasonography, acoustic radiation force impulse elastography, point shear wave elastography and magnetic resonance elastography15, which are helpful and meaningful in diagnosis of organ fibrosis. Presently, a variety of testing techniques have been developed and utilized widespread from bulk scale to the micro/nano-scale for characterizing some biomaterials ex vivo21. Piuma nanoindenter is one of these technologies to be used to study elastic property of biomaterials. This technique has been widely used to test passive mechanical properties of hard biosamples, such as bone and cartilage26. However, so far there are only very few studies31 which used the Nanoindentation technology to study organ stiffness of soft biomaterials, such as the kidney, liver, spleen and uterus samples. There is uncertainty on suitable ways in analyzing ex vivo organ stiffness by this technique.Several diseases lead to changes in stiffness in certain organs, which could give rise to illnesses. Potentially, there is also a possibility that a variation of stiffness in tissue or cells influences the function or structure in other organs in the body. For example, patients with heart failure and a preserved ejection fraction can exhibit increases in passive myocardial stiffnessEff) by considering the Hertzian contact model37, follows the fit of the loading curve , h0 and a0 are fitting parameters  and Young’s modulus (E) by the following equation:For elastoplastic materials, the unloading part of the curve is often fitted by the so-called Oliver & Pharr modelmentclass2pt{minimEff, in all models the Piuma software also enabled to directly calculate E, for which a Poisson’s ratio of 0.5 was pre-defined . We determined both Eff and E for each biomaterial because the material property of tissues and the Poisson’s ratios are unknown.Except Eff and E  levels26, in the knee joint22, in articular cartilage23. Other examples are human donor cornea27, fibrotic intestinal tissue30, pancreatic acellular scaffolds31, soft plates28 and particularly calcified aneurysmal abdominal aortas29. The feasibility and reliability of this technology in measuring the stiffness of soft biological materials, particularly organs ex vivo, is unknown. Compared with hard biomaterials, certain properties of soft biomaterials, such as viscoelasticity and adhesion, are more prone to deviations in nanoindentation. Our study is the first to use this technology to test the stiffness of soft biological organs ex vivo, particularly from mice, which are widely used for modeling human and animal diseases. We applied the Piuma nanoindentation technology, which is easy to use and utilizes a specific probe to measure Young’s Moduli to match with the specific sample properties of the tissue44. Different tissue contains different mechanical properties, therefore, different protocols should be applied in tissues with some special characterizations45. Since there is no way to judge the feature of tested samples before an experiment, we analyzed both the loading and unloading parts for their elastic behavior detection. In addition to the operation of the system and the development of measurement strategies, the preparation and fixation of tissue is expected to be important. An irregular tissue is impossible to be tested as the device only recognizes flat and stable surfaces, and the calculation of stiffness would be affected by the condition of sample. For example, if the tested surface is a slope , the contacted area would not be fully indented by the tip of probe, which means the losing depth and force could led to a mistake in stiffness measurement. A globose organ  is also untestable because it cannot be stabilized during the process of measurement. Furthermore, it is impossible for this technology to test a sunk surface  due to the obstacle at the edge of the tissue. A lumpy surface  would not only affect the accuracy of the measurement results but also cause the probe cantilever to be damaged due to jamming. Together, the tested tissue needs to be prepared testable in an appropriate shape and size. We did overcome these possible limitations for the feasibility and reliability of the nanoindentation technology to measure soft organ stiffness by using isolated kidneys, liver, spleen and uterus dissected in an appropriate manner. We confirmed the feasibility and reliability of the results by a comparative study of this technology to Matrigen hydrogels. Matrigen hydrogels at a given stiffness, stable shape, appropriate thickness and flat surface were taken as a quality control, although their given stiffness is not considered golden-standard. Nevertheless, our Bland–Altman plots, ICCs and COVs demonstrated a good reliability of the gels used. Therefore, we conclude that nanoindentation technology works well and is reliable in our laboratory settings and on this material.Piuma nanoindentation technology has been widely used in research on biomaterial stiffness of hard animal organs, for example in boneWe next tested the technology in measuring the stiffness of four organs ex vivo, namely the kidney, liver, spleen and uterus. Although the Bland–Altman plots did not give us numerous out-of-qualification results, the reliability of the results from the four organs hardness in different models can only be validated by comparing the ICCs and COVs. In the four organs, all Hertzian model’s results followed the quantified criteria of ICCs and COVs showed reliable results. The results in JKR or Oliver & Pharr models did not always meet the high quality criteria. The reason for the observed differences may rely on differences between specimens in stickiness under unloading state.29, while other studies did not report the model used31. In addition, comparing the results of the uterus with the three other organs, we found that even in the Hertzian mode, the COV value of the uterus is 11.6893% in the case of calculating the hardness according to Eff and 14.1841% in the case of calculating the stiffness according to E, which were very close to the threshold and much higher than the COV values of the other three organs in the Hertzian model. This indicates that the variability between repeated measurements of the uterus is greater than that of the liver, kidney and spleen. A possible reason is that the uterus is smaller and thinner than the other three organs, and in the course of the experiment, we found that the edge of the smaller and thinner organ uterus was more likely to be rolled up causing a similar situation shown in Supplement 1 Fig 1B, which is expected to affect the results of the measurements. Therefore, the reliability of the method is better in relatively large and thick soft organs ex vivo.For example, in the JKR model, even on the same sample, some spots are sticky, while some single indentations show no adhesion, as shown in Fig. Moreover, the successful application of nanoindentation is highly depended on material features such as the shape of the tissue being measured; measurement of biological materials with complicated rough surface tends to be difficult. When a tissue is manually transformed into a material that can be tested, it is unknown that whether its elasticity keeps the same property as the elasticity of the original organ, and whether part of the elasticity of the organ can represent its overall elasticity. Accordingly, for certain organ studies, in vivo testing may be a better or sometimes even the only option to provide detailed insights into the organ’s mechanical properties. However, in vivo tests are likely interfered and affected by other factors during the measurement process, so perhaps it is an advantage that the nanoindenter can be directly contacted with the target material for measurement. At present, there are more limitations and shortcomings in the application of this technology. For example, its validity and authenticity still need to be further verified, and the standardized routine for biomaterial nanoindentation has not yet been established. Therefore, we cannot be certainly sure whether it will become an indispensable tool in the research of mechanical biology and biomechanics of soft organs and tissues. However, as the research and development of this technology is going more and more in-depth, we expect it will have great opportunities to be applied to research in multiple fields such as physiology and pathology of soft organs.Piuma nanoindentation technology is an easy and feasible method to test the stiffness of ex vivo organs, such as kidney, liver, spleen and uterus. In small and thin tissues with disorder surface, we expect that the variation of results will increase. The Hertzian model is the most reliable method to measure the passive mechanical property of soft organs and biomaterials ex vivo. JKR and Oliver & Pharr models did not thoroughly provide reliable results.Kidneys, liver and spleen were dissected from 5 week-, 10 week-, 20 week- and 30 week-old C57BL/6 N mice. For kidneys and spleen, 2 mice of each sex and age were used. For liver, 2 to 4 mice of each sex and age were used. For uterus, eight mice were used, all these mice were around 100 days old. The experiments were approved by the regulations of local animal care committee  and the animal welfare officers of the Max Delbrück Center for Molecular Medicine (MDC) (No. X 9011/19).Hydrogels with different stiffness  were purchased from Softwell, Matrigen, Matrigen Life Technologies, Brea, CA and used for quality control ., the four parts were all taken into indentation. Liver samples were taken from left lobe .Left and right kidneys were respectively divided into two from the middle line of side Fig. A, the foobe Fig. A. Spleenobe Fig. A. Left uobe Fig. A,B. All 33 to apply load and simultaneously measure indentation depth using a fiber optic based readout . The device utilizes a ferrule-top cantilever probeout Fig. A, 5A, 6Bout Fig. B, 5B. In35, intraclass correlation coefficients (ICCs) and within-subject coefficient of variations (COVs) were used to analyze the reliability of test–retest results. If the difference value of test–retest results is between 95% limits of agreement in the Bland–Altman plots, it means the reliability is good. If the value of ICC is greater than 0.8, there is good reliability between the measurement and re-measurements. If COVs are smaller than 15%, it is considerable that the test–retest result is reliable.Bland–Altman plots All analyses were performed using SPSS 19.0 , GraphPad Prism 7.0  or MedCalc 19.3 software (Belgium).The usage of mice was abided by the regulations of local animal care committee  and the animal welfare officers of the Max Delbrück Center for Molecular Medicine (MDC) (No. X 9011/16). There are no ethical concerns.Supplementary Information 1.Supplementary Information 2.


The high morbidity and mortality associated with clinical cases of Lassa fever, together with the lack of licensed vaccines and limited and partially effective interventions, make Lassa virus (LASV) an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine. +) T cell epitopes, including epitopes common to both Nigerian and Sierra Leonean survivors. These data should be useful for the identification of dominant Lassa virus-specific T cell responses in Lassa fever survivors and vaccinated individuals as well as for designing vaccines that elicit cell-mediated immunity.Early and robust T cell responses have been associated with survival from Lassa fever (LF), but the Lassa virus-specific memory responses have not been well characterized. Regions within the virus surface glycoprotein (GPC) and nucleoprotein (NP) are the main targets of the Lassa virus-specific T cell responses, but, to date, only a few T cell epitopes within these proteins have been identified. We identified GPC and NP regions containing T cell epitopes and HLA haplotypes from LF survivors and used predictive HLA-binding algorithms to identify putative epitopes, which were then experimentally tested using autologous survivor samples. We identified 12 CD8-positive  an important health concern in its regions of endemicity in West Africa. Previous infection with LASV protects from disease after subsequent exposure, providing a framework for designing vaccines to elicit similar protective immunity. Multiple major lineages of LASV circulate in West Africa, and therefore, ideal vaccine candidates should elicit immunity to all lineages. We therefore sought to identify common T cell epitopes between Lassa fever survivors from Sierra Leone and Nigeria, where distinct lineages circulate. We identified three such epitopes derived from highly conserved regions within LASV proteins. In this process, we also identified nine other T cell epitopes. These data should help in the design of an effective pan-LASV vaccine. Lassa fever (LF), a hemorrhagic illness caused by infection with the Old World mammarenavirus, Lassa virus (LASV), is an important health concern in its regions of endemicity of West Africa due to high morbidity and mortality among hospitalized patients. Limited health care infrastructure in rural West Africa makes the study of LF in humans difficult. This difficulty is compounded by the classification of LASV as a biosafety level 4 pathogen.+) and CD8+ T cell responses, a prolonged CD8+ T cell response, and a delayed LASV-specific IgG response (+ T cell responses in response to a nucleoprotein (NP) antigen. Six CD4+ T cell epitopes with variable cross-reactivity to Lassa lineage-specific sequences were described for one of these patients  found that NHPs vaccinated with a measles virus vector encoding both the glycoprotein (GPC) and NP induced nearly sterilizing immunity after a single dose, while control of viremia was not as effective when the NP was excluded . This fiMastomys natalensis, the major natural reservoir of LASV  with rscVSV-expressing antigens for which only mRNA was readily detected.To identify LASV-specific CD8 T cells, we created a library of recombinant single-cycle vesicular stomatitis virus (rscVSV) encoding LASV antigens  and 60-amino acid (aa) polypeptides (herein fragments) derived from these antigens  from the+ T cell responses to NP and GPC as well as responses to regions within these viral proteins to identify sequences that harbor T cell epitopes. We use antigens from lineage IV viruses which circulate in Sierra Leone to stimulate T cell responses from Sierra Leonean and Nigerian survivors, despite the latter likely being infected with lineage II and III LASV. We previously described results indicating comparable CD8+ T cell responses between Nigerian and Sierra Leonean LF survivors to lineage IV antigens , while we observed CD8+ T cell responses to rscVSVs encoding LASV GPC but not those encoding NP in LF survivors N-07 and 3568610  occurs in the overlapping sequence. If we observed IFN-γ- and TNF-α-expressing CD8+ T cells in response to one rscVSV and no response to rscVSVs encoding adjacent sequences, we deduced that the epitope-containing region occurs in the nonoverlapping region plus an additional seven amino acids in the overlapping regions on either side. These additional amino acids account for most epitopes that would have been only partially encoded by rscVSVs encoding adjacent sequences. Most deduced epitopes were different between Nigerian and Sierra Leonean survivors  to identify putative epitopes for further testing. To reduce the number of peptide candidates and to limit potential false-positive epitopes that might be generated if we were to use entire LASV NP or GPC sequences, we restricted this analysis to the deduced epitopes identified earlier that were specific for HLA expression of each individual. We tested peptides with <2.0 percentile rank based on predicted affinity unless there were less than three candidates under 2.0. In such cases, we tested at least the top three candidates regardless of percentile rank -binding prediction algorithms hosted by the Immune Epitope Database (ile rank . We stim508-569), while no response was observed to rscVSVs encoding the adjacent fragment 12   among HLA alleles expressed by these two individuals. HLA-A*74:01 is the only allele shared by these two LF survivors and is the most likely restriction for NP552-561.Nigerian LF survivor N-13 and Sierra Leonean LF survivor 2848950 both responded to NP fragment 13   and B, iurvivors  and 3D. 557-566 and NP558-567. CD8+ T cell responses to NP557-566 and NP558-567 were similar   . This re+ T cells from Nigerian survivor N-07 and Sierra Leonean survivor 3568610 coexpressed IFN-γ and TNF-α in response to rscVSVs encoding GPC f6 (GPC194-256) but not to surrounding fragments f5 (GPC153-212) or f7 (GPC240-299), suggesting the existence of an epitope within GPC206-246  and HLA-B*07:06 (expressed by 3568610) were predicted as having the highest affinity for GPC235-244 .We also interrogated the carboxy terminus of GP1 based on the conservation of deduced epitope regions between Nigerian and Sierra Leonean survivors. CD8C206-246  and B. Wividuals  and D, w 3568610 . Though + T cells from Nigerian survivor N-13 and Sierra Leonean survivors 2889600 and 2848950 responded to rscVSVs encoding GPC f6 (GPC194-256) and f7 (GPC240-299) (+ T cells from N-13 and 2889600 (+ T cell response to f7 from survivor 2848950 (206-259), whereas the pool of peptide epitope candidates tested in PBMCs from N-13 (three peptides) and 2889600 (four peptides) were limited to sequences within the deduced epitope (GPC240-259). All three LF survivors responded to GPC248-257, while 2889600 and 2848950 also responded to GPC247-256 .CD8240-299)  to C. Re 2889600  and B, w 2889600 . A weak  2848950  led us tC247-256  to F. SiC247-256  and F su552-561, GPC235-244, and GPC248-257 were more conserved than areas outside these regions, we analyzed the extent of amino acid conservation using 600 LASV NP and GPC sequences . We found a high degree of amino acid conservation within epitopes shared by Nigerian and Sierra Leonean survivors  compared to nonepitope protein regions . Individually, amino acid conservation is 99.5% for NP552-561, 99.4% for GPC235-244, and 99.0% for GPC248-257.To assess if epitopes common between Nigerian survivors, NP+ T cell responses to T cell epitopes derived from NP and GPC sequences, with the greatest responses to NP155-164, NP453-462, and GPC235-244 , f11 (GPC404-463), and weakly to f1 (GPC1-58), which contain the previously identified epitopes GPC60-68, GPC441-449, and GPC42-50, respectively (REF). Sierra Leonean LF survivor 3568610, who also responded to GPC60-68, did not respond to GPC f2 or f11 and only very weakly to f1. This result could indicate a lack of sensitivity of our assay, a subdominant response from these previously identified epitopes, a cross-reactive response of using the peptide alone, or a combination thereof.LASV epitope identification efforts have been focused on epitopes that may be presented by common HLAs , 35. ThrLASV GPC . Two LF + T cell responses in diverse populations across West Africa, vaccines should deliver either whole antigens or encode those regions within the NP and GPC that we have identified as regions that elicit LASV-specific responses in the majority of survivors studied.Multiple factors are responsible for the selection and dominance of epitope-specific T cells and associated responses during acute viral infections, including (i) precursor frequency , (ii) stThis study was approved by the following institutions: Human Subjects Committees of the Broad Institute, The Scripps Research Institute, Tulane University’s Human Research Protection Program, the Sierra Leone Ethics and Scientific Review Committee, ISTH Research and Ethics Committee, and the Oyo State Research Ethical Review Committee. All subjects have a documented history of Lassa fever. Because of the manner in which samples were coded at the time of collection, no demographic, disease, or virus sequence information is available for the Nigerian participants. Demographic information and time from disease onset to an analysis of memory responses for Sierra Leonean patients are listed in Blood was collected in EDTA vacutainers at the Kenema Government Hospital  or the Irrua Specialist Teaching Hospital . PBMC isolation was performed according to standard protocols. Briefly, blood was diluted three times with phosphate-buffered saline (PBS) and layered onto Lymphoprep (StemCell Technologies) or lymphocyte separation medium  and centrifuged at 1,450 rpm without break. Buffy coat layer was isolated and washed twice with PBS before being resuspended in CryoStor CS10 (StemCell Technologies), slow frozen at −80°C for 3 h, and stored in a liquid nitrogen dry shipper for shipment to the United States for analysis.2 before staining with the following antibodies: anti-human brilliant violet 421 CD4 , fluorescein isothiocyanate (FITC) CD8a , anti-human PE TNF-α (BD Biosciences), PE/Cy7 IFN-γ , and APC IL-2 . BD fixation/permeabilization kit was used for intracellular stains. Positive controls were treated similarly except for incubation with unlabeled anti-human CD3  and CD28  instead of rscVSVs.Plasmids used to rescue rscVSVs expressing LASV antigens have been described , 42, 43.2.Peptide stimulations were performed at a concentration of 10 μg/ml . After 1 h stimulation, brefeldin A (4 μg/ml) was added, and cultures were incubated for an additional 4 h at 37°C in 5% COwww.iedb.org) to generate a list of putative epitopes. We used the recommended settings (consensus and NetMHCpan) and selected all 10-aa peptides below the 2% prediction ranking for testing.Deduced epitope sequences and HLA profiles  were ent6 PBMCs using the Quick DNA miniprep plus kit (Zymo Research) and quality assessed by electrophoretic separation on an 0.8% agarose gel. For long-range amplification and library preparation, we used the TruSight HLA version 2 sequencing panel (CareDx) followed by sequencing of barcoded libraries on the Illumina MiSeq platform. Lastly, TruSight HLA Assign software (version 2.1 RUO) was used to analyze results and compared with sequences stored in the International ImMunoGeneTics Information System/HLA database (version 3.37). A reference DNA sample, IHW09263, was used for each sequencing experiment.As previously described , genomicwww.iedb.org; submission IDs 1000831, 1000832, and 1000836). In addition, more information on our subjects, including HLAs, is stored online by the Center for Viral Systems Biology (www.cvisb.org).All epitopes, including longer deduced epitopes and 10-aa peptide epitopes along with associated HLA alleles, have been submitted to the Immune Epitope Database (


The journal hereby retracts the above cited article. This follows the recommendations of aninvestigation by Leiden University's Committee on Scientific Integrity which foundthat the paper contained gross data manipulation. These issues could not have been detectedduring review but nevertheless invalidate the study's findings. The retraction hasbeen approved by the journal Chief Editors.


We also proposed two new concepts: a rejection tolerance threshold (RTT) and an associated rejection range (RR), based on a binomial acceptability question and a probit regression model. These new methods provide more realistic interpretations of rejection-type thresholds based on user-defined allowable rejection levels, or “rejection tolerance” (for RTT), and a model-derived stimulus range (RR) to capture the rejection level of interest while providing flexibility for product formulation. All thresholds were investigated separately for color, flavor, texture and overall perception, elucidating differential sensorial effects of our novel ingredient on the food matrix. We suggest that conducting all demonstrated techniques in a single testing session gives a more expansive view of rejection thresholds while requiring minimal additional resources.Recently, sensory threshold concepts have been expanded to include affective perceptions of foods and beverages, especially measures of product rejection; however, each threshold interpretation depends largely on the methodology employed. By substituting cricket powder for whole-wheat flour in snack crackers , we proposed modifications to two existing threshold methodologies: a modified consumer rejection threshold (M-CRT) using a 2-alternative choice (2-AC) preference test with “no preference” option; and a modified hedonic rejection threshold (M-HRT) based on less-than-neutral hedonic scores according to a one-sample  Sensory thresholds represent the limits of auditory, visual, tactile, olfactory and/or gustatory capacities. Conceptually, a threshold is a value on a stimulus continuum (a boundary of sorts) where a perceptual change occurs . This po2s)) .To facilitate the following discussion, The CRT was the first affective threshold developed and originally used to find the level at which consumers would “reject” cork-tainted white wine . The autThe currently proposed modification, which separated M-CRT from existing CRT methodology, is the use of a “no preference” option, where, as opposed to a forced choice scenario 2-AFC) ,2, M-CRT-AFC ,2, ® may provide added value [We know from existing literature that, when given the option, consumers do report ties, although their readiness to do so depends on (among other factors) the product category ,24,25. Ped value . Within Instead of relying on a fixed critical value to assign the threshold as with the CRT, M-CRT used Thurstonian 2-AC modelling ,28, whicA precise estimate for the color threshold could not be determined, as it fell short of our lowest cricket powder substitution level of 5% a. PrelimNot surprisingly, the affective dimension of preference proved to be the most sensitive, among those addressed, to the effects of cricket powder on snack cracker quality. However, these discontinuities in preference should not be confounded with conclusions of acceptance or rejection. Proceeding hedonic data will show that cricket-containing crackers were still liked at levels well exceeding their M-CRT estimates.2s [The gap in interpretation between comparative ratings of inferior preference and independent measures of acceptance or rejection was noted by Filjo et al. when they proposed two additional affective thresholds, the CAT and HRT  . Both we2s , were ap2s . To thist-test to evaluate a one-sided hypothesis . The associated question of interest is then related to the point where “liking drops significantly below 5”. Following HTM protocol, which asked respondents to evaluate their liking of each product monadically, we estimated the HRT and M-HRT for cricket powder in snack crackers.Although the words “liking” and “acceptance” are often used interchangeably, the present discussion describes data from a labeled 9-point hedonic scale (anchored at 1 = dislike extremely and 9 = like extremely) in terms of liking, and later, responses to a binomial yes/no questions about product acceptability in terms of acceptance (for “yes” responses) or rejection (for “no” responses). Using the 9-point hedonic scale, Filho et al.  designatcolor = 17.2% cricket powder; Whereas preference-based M-CRT estimates ranged from <5% to 15.6% cricket powder depending on the attribute evaluated , the meat-value of −1.67, . Again, color was the only attribute for which the hedonic threshold was obtained  as a function of % cricket powder (x) in snack crackers. The new M-HRT was interpolated at the intersection of the regression line with the critical t-test may deliver added confidence that the M-HRT dependably predicts stimulus levels (based on hedonic scores) consistent with rejection. By plugging the calculated M-HRTcolor value of 19.97% cricket powder into the SLR equation (Equation (6), R2 = 0.98) used to determine HRTcolor:Intuitively, the extra statistical rigor from a significant a et al.  evaluateFor each sample, consumers were also asked to report (“yes” or “no”) whether the sample was acceptable overall and in terms of color, texture, and flavor. By compiling all these responses  and pairing them with their associated hedonic scores, it became evident that consumers held different standards. If we consider a “no” acceptability response as self-reported rejection, then 3.2% of 6 scores and 6.3% of 5 scores were associated with rejection, and at the hedonic category of 4, the observed rejection rate was 45%. This implies that, even with a “dislike slightly” score, samples were still deemed acceptable 55% of the time (data not shown).Admittedly, consumers’ perceptual responses to food and beverages are neither uniform nor consistent . This prTo estimate the RTT, probit regression was used to model the probability of rejection as a function of increasing cricket powder. Fechner first applied a cumulative distribution curve to explain variability in perceptual responsiveness . The cur50) could be estimated by the mean of the distribution [In one classical approach to thresholds ,2, the pribution . Other o25 was determined for flavor at 14.6%, overall acceptability at 15.3%, and color at 16.8% whole-wheat flour substitution with cricket powder  and overall acceptability (13.0%–18.4% cricket powder)  and precision  in characterizing a pragmatic RR, which may inform profitable decision making. For an ingredient such as cricket powder, which to some extent hinders affective quality, the first reaction may be to simply choose the lower bound of the RR. Instead, the RR should direct strategic adjustments of the product formulation to achieve goals related to cost, quality, health or marketing claims, or nutritional value—all of which decidedly influence consumers’ food choice  and ultiBy narrowing down the stimulus region of interest, more focused testing  withi® and internal preference mapping, may be explored in future studies to reveal product attributes which drive acceptance/rejection across multiple segments of consumers.The present research presented two modified approaches to existing rejection-type threshold methodologies: the M-CRT and M-HRT, as well as two newly proposed thresholds: the RTT and RR. Measures of preference, liking, and acceptance/rejection produced different threshold estimates for cricket powder in whole-wheat snack crackers, as these represent clearly different affective constructs. Terminology used to interpret each threshold should be considered accordingly. As all of the discussed rejection-type threshold methods present value to food research and industry, the authors suggest employing them in the same testing session. Doing so should typically require minimal additional time, sample, cost, and effort on the part of panelists, and provide a more complete view of stimulus effects. Tracking the differential impact of cricket powder on multiple sensory attributes was also valuable to exploring sensory-based limits of a novel ingredient. Other nonlinear approaches, such as Landscape Segmentation AnalysisThese results supported existing criticisms of thresholds pertaining to inconsistency in consumers’ perceptual evaluations and lack of congruence between theoretical intent and objective threshold estimates. Rather than relying on statistical significance or some arbitrary boundary between acceptance and rejection, the RTT and RR offered threshold strategies based on practical significance to the investigators or food companies. These concepts are suggested as more realistic approaches to modeling rejection of a food product and offer adaptable methodologies based on a new application of the probit regression model. The RTT can recommend a stimulus level based on an allowable rejection tolerance, and the RR can suggest a stimulus range that will satisfy such a tolerance with a quantifiable level of reliability. These protocols can be used to guide product formulation strategies to meet specific targets in terms of price, quality, nutrition, or functionality and are relevant to various food processing, ingredient, and contaminant effects. Future validation studies should investigate the repeatability of these threshold estimates within a given population.


Emerging Themes in Epidemiology, I would like to take this opportunity to thank the previous Editor-in-Chief of the journal, Dr. Clarence Tam, as well as the current members of the Editorial Board and reviewers for their many contributions to the journal. The reviewers and Editorial Board members have important roles in ensuring the quality of the peer review process and the overall success of the journal.As the new Editor-in-Chief of New epidemiological concepts and methodsNovel ways of presenting and providing insights into existing epidemiological concepts and methods, including in a teaching environmentUse of epidemiological research methods in non-medical settingsCausal inference in epidemiologyApplications of new technologies in field studies Ethical issues in epidemiological researchNew statistical methods, or novel uses of existing methods, in epidemiological studiesMethodological developments in molecular and genetic epidemiology and novel applications in these areasHistorical articles and re-assessments of classic papersEmerging Themes in Epidemiology is an open access, peer-reviewed journal that publishes original research papers to promote debate and discussion on practical and theoretical aspects of epidemiology. We welcome primary research and discursive articles. We particularly encourage submissions in the following areas:The journal considers the following types of article for publication: research papers, analytic perspectives, commentaries, debate articles, hypotheses, methodology articles, and reviews.The articles published in the journal encompass virtually all epidemiology content areas and sub-disciplines including chronic disease epidemiology, infectious disease epidemiology, genetic epidemiology, occupational and environmental epidemiology, and public health practice. Due in part to the journal’s global readership and contributors, published articles also address important topics in global health.To ensure the continued success and growth of the journal over time, it is important for reviewers and Editorial Board members to offer their recommendations and critiques in a timely fashion and for the journal to strive to reach decisions about publications in a reasonable timeframe. The continued success of the journal depends upon the timeliness of editorial decisions and the thoughtful contributions of many reviewers, as well as the productivity and insights of contributing authors. I look forward to contributing to the journal’s continued success and reading the many articles submitted to the journal for consideration for publication.