Background: Although there are a few studies which compare fast and slow sintering in normal zirconia crowns, it is essential to compare the cracks and load-bearing capacity in zirconia screw-retained implant crowns between regular and speed sintering protocols. This research aimed to compare the surface structure, cracks, and load-bearing capacity in zirconia screw-retained implant crowns between regular sintering (RS) and speed sintering (SS) protocol with and without cyclic loading (fatigue). Methods: A total of 60 screw-retained crowns were fabricated from zirconia (Katana STML Block) by the CAD/CAM system. Then, 30 crowns were subjected to the RS protocol and 30 crowns were subjected to the SS protocol. Cyclic loading was done in half zirconia crowns (15 crowns in each group) using a chewing simulator CS-4.8/CS-4.4 at room temperature. The loading force was applied on the middle of the crowns by a metal stylus underwater at room temperature with a chewing simulator at an axial 50 N load for 240,000 cycles and lateral movement at 2 mm. Scanning electron microscopy was done to study the surface of the crowns and the cracks in the crowns of the regular and speed sintering protocols, with and without fatigue. Results: For the speed sintering group, the surface looks more uniform, and the crack lines are present at a short distance compared to regular sintering. The sintering protocol with a larger Weibull module and durability increases the reliability. It showed that the Speed group showed the maximum fracture load, followed by the regular, speed fatigue, and regular fatigue groups. The fracture load in various groups showed significant differences. Conclusions: It was found that the speed group showed the maximum fracture load followed by the regular, speed fatigue, and regular fatigue. The crack lines ran from occlusal to bottoms (gingiva) and the arrest lines were perpendicular to the crack propagations. Zirconia ceramics are the choice of materials for esthetic dental restorations and prosthetic crowns and they are also commonly used for the fabrication of implant crowns . RecentlTranslucent zirconia can be obtained by increasing translucency and decreasing opacity. Optimal translucency in zirconia can be obtained by increasing grain size, optimizing grain boundary region, and increasing yttria content to produce partially stabilized zirconia with increased amounts of nonbirefringent cubic phase ,16. In aZirconia is subjected to various processes such as milling, grinding, and polishing during the manufacturing process. From these processes, surface and volume flaws are formed which differ according to the location, size, and orientation, resulting in variable strength ,20,21,22Zirconia stabilized with 5 mol% yttria content (5Y-TZP) and possesses excellent mechanical properties and biocompatibility ,25. AlthThe study overview is shown in n = 30) and regular sintering (RS) protocol (n = 30).Scan post and scan body were utilized to transfer the information and position to the CAD software by using inLAb CAD Software 22.0  . Ti-baseThe zirconia screw-retained crowns were fabricated from zirconia block, then scanned by Cerec Omicam , designed by inLAb CAD Software 22.0 , and finally milled with an MCXL  milling unit . Then thAll crowns were cemented to Ti-base. Ti base and Zirconia crowns were sandblasted (50-micron 1 bar) for 5 s. Single Bond Universal  was utilized to apply both crowns and Ti-base surfaces. Rely X Ultimate  was utilized to cement following the manufacturer’s instructions. Screw-retained crowns were torqued at 25 Ncm to implant analog. Teflon tape and resin composite Z350  were used to close access screw holes of 2.6 mm diameter.The zirconia crowns (15 crowns in each group) were subjected to cyclic loading using the chewing simulator CS-4.8/CS-4.4  at room temperature. The technical specifications included traverse paths of the Z-axis approx. 120 mm, and X-axis approx. 50 mm, speed ranged between 1–60 mm/s in both axes, dimensions including thermocycling unit was 190 × 200 × 80 cm (length × height × depth), and weight load of maximum 10 kg per sample chamber. The loading force was applied on the middle of the crowns by a metal stylus underwater at room temperature with a chewing simulator at an axial 50 N load for 240,000 cycles (frequency of 1 Hz) and lateral movement at 2 mm A 29]..29].Then, the surface, cracks, and load-bearing capacity of the zirconia crowns were studied under regular and speed sintering protocols with and without fatigue.All zirconia crowns (fatigued and non-fatigued) were subjected to a single load-to-failure using the universal testing machine  according to ISO 6872 . An indeThe surface structures of the zirconia specimens were characterized using scanning electron microscopy (SEM)  of zirconia specimens (3 samples for each group). The specimens were submitted to sputter-coating with a gold-palladium alloy. The images were observed with an accelerating voltage of 20 kV in various magnifications. Selected crowns of each group were studied for the fractographic analysis with SEM .f = fracture probability, σ0 = characteristic strength, σc = fast-fracture strength without subcritical crack growth, and m = Weibull modulus which is the slope of the regression line in the lnσc − ln ln [1/1 – Pf)] diagram [0 relates to the stress point where 63.2% of the crowns would fracture. The fast-fracture strength of each crown was calculated by the formula shown in Equation (2) [For the zirconia crowns, a probabilistic approach was done to analyze the strength and lifetime. The fracture strength of zirconia is done by the Weibull formula as shown in Equation (1) :(1)Pf=1− diagram . σ0 relation (2) :(2)In Inp value = 0.05). One-way ANOVA with multiple comparisons using the Scheffe analysis was done to compare the results among various groups.Descriptive statistics were calculated, and the results were compared among the sintering groups. The statistical analysis was done using SPSS 20.0  to see the significant differences , whereas a high Weibull modulus signifies higher reliability of zirconia material. Weibull’s theory is based on the weakest link theory whereby failure is the result of the weakest line break in the chain [The zirconia restorations are subjected to the occlusal loads but are below the flexural strength of zirconia in the mouth. However, the pre-existing surface flaws may lead to the propagation of those cracks and eventually cause the fracture of the zirconia restoration ,33. We ahe chain .The crack propagation mechanism in zirconia materials differs according to the type of material composition and sintering protocols. For the two material types, it was found that an intergranular fracture is seen in 3Y-TZP and a combination of intergranular and intragranular crack propagation is seen in 5Y-TZP . When a p < 0.001). ZI (p < 0.001–0.004) and Zolid (p < 0.001–0.007) showed higher flexural strength after thermomechanical aging. In addition, the Weibull modulus of the three thermomechanically aged materials was negatively influenced by high-speed sintering; hence, they also mentioned that high-speed sintering is a valid alternative to conventional sintering protocols.Our results were similar to the results obtained by some previous studies ,35,36. MArcila et al.  studied Ordoñez Balladares et al.  comparedThe thickness reduction of zirconia and fatigue affects the failure load of monolithic zirconia crowns. Prott et al.  mentione2 might be higher if we cut out the samples or add more samples. This research is preliminary. Further research can be carried out to analyze of the chemical chain or strengthening phenomena in zirconia.Regarding the limitations of this research, we should note the limited sample size. For the regular group, the RSurface flaws are the failure origin, and the crack lines ran from the occlusal to the bottom. Handling of the zirconia plays an important role in the longevity of the zirconia restorations. It was found that the speed sintering group showed the maximum fracture load followed by the regular, speed fatigue, and regular fatigue groups. These values provide reference fracture load values for the implant crown and fixed partial denture and are used to assess the durability of zirconia in dentistry.


Conclusions: In symptomatic patients with renewed angiography, venous bypass grafting of the RCA showed acceptable long-term patency rates. Single bypass grafting of the RCA was superior to sequential grafting, which needs to be further investigated. Objective: Little is known about the fate of bypass grafts to the right coronary system. To investigate the long-term patency of venous bypass grafts directed to the right coronary artery (RCA) based on postoperative angiograms and to identify predictors of graft occlusion. Methods: In this single-center study, all patients who underwent coronary angiography from 2005 to 2021 after previously undergoing isolated coronary artery bypass grafting (CABG) were included. The primary endpoint was graft occlusion over a median follow-up of 9.1 years. Results: Among a total of 1106 patients  years median age), 289 (26.1%) received a sequential vein graft and 798 (72.2%) a single graft. Multivariate regression revealed age , the urgency of CABG , and severely impaired left ventricular function , but not gender and chronic total occlusion (CTO) as predictive factors for graft occlusion. Single conduits were found to be a predictor of graft patency (HR 0.575 CI 95% 0.449–0.737). The angiographic outcome showed an overall 10-year freedom from graft occlusion of 73.4% ± 1.6%. The 5-year (10-year) freedom from graft occlusion was 76.9% ± 2.8% (57.8% ± 4.0%) for sequential grafts and 90.4% ± 1.1% (77.8% ± 1.7%) for single grafts (log-rank  Coronary artery bypass grafting (CABG) remains a standard procedure for revascularization of three-vessel disease/coronary artery disease (CAD) ,2. DespiDespite standard recommendations preferring arterial revascularization, even the largest randomized controlled trial has not been able to show benefits in terms of survival when bilateral internal thoracic artery (ITA) CABG was performed instead of single ITA . In addiThis study aims to analyze the long-term patency of venous conduits utilized for the revascularization of the right coronary artery and determine possible risk factors associated with graft occlusion. The cohort comprises a total of 1106 patients. All patients underwent isolated CABG  with revascularization of the RCA utilizing a saphenous venous graft (SVG) at our institution. However, “sequential” RCA grafts also included bypasses coming via the left coronary system. Angiographic investigations were performed between 2005 and 2020. All patients underwent CABG between January 1993 and January 2021. During this period, 16,194 isolated CABG procedures were performed at our institution. Of those, 7898 patients received an RCA bypass graft. We excluded all patients who underwent coronary angiography within the first 30 postoperative days . Graft occlusion was detected in fifteen patients  and five patients deceased within 30 days) since the need for early cardiac catheterization is usually due to periprocedural adverse events, which does not reflect the objective of our investigation. Angiographic investigations for patients with a history of CABG performed at an external institution were also excluded. The study design is depicted in the CONSORT diagram .Due to the retrospective nature of the study, the need for informed consent was waived by the local Research Ethics Committee of the University of Heidelberg, and the study was subsequently approved .Data sets were collected from medical records consisting of over 5000 variables for each patient. Included were demographic characteristics, cardiovascular risk profile, diagnostic methods, patients’ preoperative clinical status, details of the surgery, postoperative outcome, and angiography findings. The maximum of institutional follow-up was included. All available preoperative and postoperative angiographies and their reports have been analyzed and verified by experienced interventional cardiologists and cardiac surgeons. If multiple angiographies were detected for a patient, the earliest described graft occlusion was documented in this study. Graft occlusion was defined as the complete cessation of blood flow in the respective vessel. Furthermore, later interventions concerning the native coronary arteries (CA) or grafts were also included. t-test and Pearson’s χ2 were used for continuous and categorical variables, respectively. Continuous variables were expressed as mean ± standard deviation or median and interquartile range (IQR) in data not normally distributed, and categorical variables as absolute and relative frequencies. Student’s p values are of descriptive nature. A p value < 0.05 was considered significant. All analyses were performed using SPPS Statistics 28  and GraphPad Prism 9.3.1 .To investigate the influence of different patient characteristics and procedural factors associated with graft occlusion, a cox hazard univariate logistic mixed effect model was performed, containing factors that have been proven to influence outcomes after CABG. Aiming to investigate the influence of additional factors on graft patency, we analyzed parameters such as measured graft blood flow, coronary diameter, and the number of sequential anastomses, utilizing a cox hazard logistic mixed effect regression, after dividing the cohort into a single graft and sequential graft subgroups. The influence of blood flow was not analyzed for the total cohort since flow rates of sequential grafts are not comparable to flow rates of single anastomosis grafts if not measured within the respective sequence. Nineteen patients who received a T-/Y-venous graft were included in the overall patency analysis but not further analyzed due to sample size limitations. Due to the exploratory character of this analysis, all A total of 1106 patients were included in the study after exclusion according to the above-mentioned criteria. The median patient age was 64 (IQR 57–71) years at the time of initial CABG, of whom 188 (17%) were female, with a median logistic EuroScore of 6.05% (IQR 3.14–10.43). Thirty-five percent of the patients underwent previous percutaneous coronary intervention (PCI), and 3.5% had previous cardiac surgery. Cardiovascular risk factors were distributed as shown in A mean number of 2.81 ± 0.65 grafts were utilized for revascularization. From 1106 patients, 289 (26.1%) received sequential vein grafting, 798 (72.2%) single vein- and 19 (1.7%) a T-/Y-venous graft. Chronic total occlusion (CTO) of the RCA was preoperatively detected in 174 (15.7%) patients. Visible collateralization of the RCA with distinguishable targeting vessels  was observed in 95 of 174 (55%) CTOs in preoperative angiography.The median period between CABG and the latest angiography was 9.1 (IQR 4.5–14.3) years. The overall graft occlusion rate was 33.3%. The native RCA intervened in 15.8%, and the SVG directed to the RCA intervened in 11.8% of the cases during follow-up. The coronary angiography findings, as well as graft characteristics, can be found in p < 0.001)  B.p < 0.001, data not shown).Of note, an additional analysis comparing bypass graft patency of single versus sequential conduits without censoring patients that underwent coronary angiography within the first 30 postoperative days  also showed superior patency rates of single RCA bypass grafts in comparison to sequential grafts . A combined endpoint analysis  also revealed lower event rates in patients with single bypass grafts .In addition, when comparing single and sequential bypass grafts, analysis of “graft failure” (defined as occlusion or severe stenosis ≥75%) also showed the superiority of single RCA bypass grafts  patients, only the RCA segment was sequentially targeted with detected total graft occlusion in 28 (40.0%) patients during follow-up. In 215 (75.4%) patients, both the right and left coronary system was targeted 215 (75.4%). Thereof total occlusion was detected in 73 (34.0%) patients. Of 73 occluded sequential grafts targeting both RCA and LCA, 22 grafts were patent within the LCA system and 51 occluded on the level of the neo-ostium at the proximal aortic anastomosis . p < 0.001). CTO showed no influence on patency. Impaired LV function and atrial fibrillation have been detected as possible risk factors, as well as BMI. Age was found to be a significant risk factor for early graft occlusion . The grade of RCA-stenosis did not correlate with graft patency in this investigation (p = 0.255).Univariate regression analysis revealed several influencing factors on RCA conduit patency, which are listed in After including significant influencing univariate factors in a mixed effect model, multivariate analysis revealed age, grafting technique, impaired LV function, and urgency of CABG procedure as significant risk factors for long-term graft occlusion . p = 0.56), whereas the measured blood flow (61.37 ± 34 mL/min) was detected as a risk factor for graft occlusion in univariate analysis . To distinguish whether multiple sequential anastomoses affect graft occlusion, we performed a regression analysis for sequential grafts , showing no significance for multiple anastomoses in a sequential graft (p = 0.335).Furthermore, within the subgroup of single bypass conduits, the lumen diameter of the target vessel (mean 1.578 ± 0.3 mm) did not show a relevant influence on graft occlusion  targeting the RCA of 73 % after ten years in a cohort that has undergone repeated angiography, mainly for symptomatic reasons rather than for study purposes . The venous conduits target the RCA proof of acceptable long-term durability, despite controversial findings from previous studies ,11. HoweIn this study, the grafting technique proved most influential, resulting in a significantly higher patency rate for single bypass conduits to the RCA compared to sequential vein-grafting. This finding is in keeping with an analysis from the PREVENT IV trial, demonstrating a higher one-year venous graft failure for sequential conduits compared to single SVGs . On the First, the RCA—proximal to the crux cordis—usually provides larger vessel diameters compared to the typically targeted LCA branches. Consecutively, the RCA system is more susceptible to competitive blood flow than the LCA system. Hence, arterial revascularization of the RCA should only be considered in the presence of severe native vessel stenoses to avoid competitive blood flow, which in turn elevates the risk for early graft occlusion of arterial conduits, such as the RA as well as gastroepiploic artery (GEA) ,16,17. NSecond, compared to the LCA, which is mainly supplied during diastole, the RCA perfusion occurs in up to 50% of cases during systole . ThinnerThird, the optimal positioning of the venous conduit is of great importance. Technical challenges are an underestimated factor for the long-term patency of sequential grafts . On the n = 112) and SVG (n = 304) for grafting of the RCA. No significant differences were found between patency rates and clinical events [p = 0.0046) after 15 years [According to current guidelines, arterial conduits should be chosen as the second and even the third conduit, predominantly based on systematic reviews of RCTs ,23,24. Hl events . The gra15 years .Interestingly, Glineur et al. analyzed data from two randomized controlled trials and found significantly higher patency rates for the SVG compared to the gastroepiploic artery and RA in a protocol-driven 3-year coronary angiography follow-up. The lumen diameter was found to be a relevant predictor for graft patency in arterial conduits to the right coronary system assessing a threshold of under 1.1 mm beneficial for arterial conduit patency. The venous conduits were not affected by lumen diameters . In lineBesides age, we found impaired left ventricular function and urgency of CABG procedure both to be relevant risk factors for graft occlusion. Despite outcome improvement for patients with impaired LV function undergoing CABG over medical therapy alone, heart failure remains a relevant risk factor for early graft occlusion and survival ,29. SurpInterestingly, the presence of diabetes did not correlate with venous graft occlusion to the RCA, like the findings reported by Raza et al. concerning outcome differences in diabetic and non-diabetic patients for long-term patency of the ITA- and the SVG . The revascularization of the RCA will remain controversial, especially regarding the decision between sequential- or single grafting as the technique of choice as graft occlusion has a deeply multifactorial etiology, and our analysis is not representative of the general CABG population. However, with growing evidence leaning towards total arterial revascularization for the left coronary system, single venous bypass grafting to the RCA should retain its value for several reasons: First, in the case of bilateral ITA for complete arterial revascularization, including the RCA is often limited due to graft length. On the other hand, the use of RA as a possible third arterial conduit may not seem feasible if RCA stenosis is not of the highest grade. Moreover, if T/Y-grafted arterial conduits are used for revascularization of multiple vessels, steal phenomena may occur, especially if the radial artery is utilized and anastomosed to the left ITA. Thus, targeting the RCA with the SVG will remain part of surgeons’ armamentarium in addition to arterial revascularization of the LCA.This is a retrospective observational study and is thus susceptible to the inherent limitations of this type of analysis. It comprises a heterogenic patient cohort without a comparator group, yet it focuses on outcome analyses in a comparatively large group of patients. When the baseline characteristics of patients with single or sequential bypass are compared, significant differences between the cohorts are apparent in several respects. Missing values are consecutively endogenous and are addressed for each variable. In addition, information on the presence of ICD devices was not available in our analysis, and ICD implantation in a CABG population may potentially impact long-term follow-up in multiple aspects ,32. Furt


Variations in starch pasting properties, considered an alternative potential quality classification parameter for rice starches, are directly controlled by the diverse starch molecular composition and structural features. Here, the starch characteristics of four rice cultivars  differing in pasting properties were assessed, and their relationship was determined. The results revealed that protein and moisture contents and their crystalline type were similar among the four rice starches. However, their molecular compositions and structures  significantly varied among different genotypes, which resulted in distinct swelling, solubility, gelatinization, retrogradation, and hydrolytic resistance properties. The swelling power and gelatinization enthalpy (∆H) were positively correlated with C-type granule and relative crystallinity, but were negatively correlated with amylose content, B-type granule and median particle size (d(0.5)). Conversely, the water solubility and resistant starch content negatively correlated with C-type granule, but positively correlated with amylose content, B-type granule, and d(0.5). The gelatinization onset temperature (To(g)), and retrogradation concluding temperatures (Tc(r)), enthalpy (∆H(r)), and percentage (R%) were positively impacted by the amount of protein, amylose, and B1 chains (DP 13–24), while they were negatively correlated with short A chains (DP 6–12). Collectively, the starch physicochemical and functional properties of these Thai rice starches are attributed to an interplay between compositional and structural features. These results provide decisive and crucial information on rice cultivars’ suitability for consumption as cooked rice and for specific industrial applications. Rice starch is one of the world’s most important sources of food energy . In ThaiStarch chemical composition and molecular structure can affect starch physicochemical properties and in vitro digestibility, which ultimately determines the final starchy-food product quality. Due to the wide genotypic diversity, starches from various Thai rice cultivars have been the focus of increasing research interest ,8,10. RiThailand possesses a large collection of elite rice cultivars with considerably high variations in starch properties ,5,8,9,19Therefore, four rice varieties were selected based on their differences in pasting properties. Their starch chemical composition, molecular structure, physicochemical properties, and functional characteristics were characterized. The relationships between these features were then inferred to pinpoint the characteristics that putatively play an important role in defining its functionality and potential end-use applications. The obtained information would provide potential selection criteria for consumers and the industrial sectors to select suitable rice varieties, for consumption as cooked rice with sensory attributes and health benefits and for particular industrial applications, respectively.Seeds of the 11 Thai certified rice cultivars, namely RD6, RD21, RD29, RD37, RD41, RD43, RD57, RD61, KDML105, Phitsanulok80 and Pathumthani1, were supplied by the Pathumthani and Khon Kaen Rice Research Stations . Seeds were germinated and transplanted into 8″ plastic pots and placed in a greenhouse under natural light conditions at the Faculty of Science, Khon Kaen University from September 2019 to January 2020. A total of 4 replicates per cultivar were grown for their starch pasting property analyses. The three key starch pasting properties, including PT, PV, and SB , and thed-glucose as standards .® statistics v.23 software . A one-way analysis of variance (ANOVA) and Tukey’s post hoc test were conducted for comparison of the significant differences (p ≤ 0.05) among the mean values. The data were expressed as mean ± standard error of the mean (SEM) . Thθ: an unrp ≤ 0.05) less than that of the low-amylose rice KDML105 and very low-amylose rice RD6 starches, respectively (p ≤ 0.01) between amylose content and SP were observed (p ≤ 0.01), while it was negatively correlated with S  , but they were positively correlated with S   (p ≤ 0.05) higher than that of KDML105 and RD6 starches with low and very low amylose content, respectively  . Similarctively) . Furtherctively) . The preectively . Therefop ≤ 0.01), but a significantly negative correlation with ∆T(g)  (p ≤ 0.01), Tp(g) , and Tc(g)  (p ≤ 0.01) and CL , while strongly positive correlations were found between RC and Tc(g)  and between RC and ∆H(g)  ; peak, Tp(g); and conclusion, Tc(g)), temperature range (ΔT(g)), and enthalpy change of gelatinization (ΔH(g))) of the four Thai rice starches with different pasting properties revealed that RD57 starch had the highest gelatinization temperatures and the lowest ∆T(g) . This co ≤ 0.01) , which w ≤ 0.01) . It was  ≤ 0.01) . We also ≤ 0.01) . Among t ≤ 0.01) . In casep ≤ 0.01, r = 0.750, p ≤ 0.01, and r = 0.746, p ≤ 0.01, respectively), and the proportion of DP 13–24 had a positive correlation with Tp(r), Tc(r), ∆T, ∆H, and R%  (p ≤ 0.01), whereas a significantly negative correlation was observed with Tp(r) , Tc(r) , ∆T , ∆H , and R%  (The retrogradation properties of the starch gels upon storage at 4 o°C for 14 days showed significant variations among the four rice starches . Interesctively) . Additio ≤ 0.01) . Interes ≤ 0.01) . This co ≤ 0.01) . The hig ≤ 0.01)  2,46].,46.p ≤ 0p ≤ 0.01), and the proportion of B-type granules also showed a positive correlation with the RS content   contents of all four rice starches were in the range of 0.025 to 0.153% , which i ≤ 0.01) . Starch  ≤ 0.01) . However ≤ 0.01) . It is, Starches from the four representative Thai commercial rice cultivars with different pasting properties exhibited significant differences in their chemical compositions and molecular structures. The four rice starches possessed varied amounts of amylose, from 1.65 to 23.60%. The A-type crystalline polymorph starches with different amylopectin chain length distribution showed distinct median granule sizes ranging from 4.62 to 6.08 μm, and also differed in relative crystallinity. The diversity in the chemical composition and structural features of Thai commercial rice starches is intrinsically linked to their wide variations in swelling power, water solubility, gelatinization and retrogradation properties, and resistant starch content. As supported by the correlation coefficients, SP and ∆H increased with the declining amounts of amylose and B-type granule and the increasing amount of C-type granule. However, the opposite effects were found on S and RS content. Interestingly, the increases in the amounts of amylose and DP 13–24 and the decrease in the amount of DP 6–12 were, mainly, concertedly responsible for the increases in To(g), Tc(r), ∆T(r), ∆H(r), and R%. On the other hand, the Tp(g) and Tp(r) declined with increasing proportions of DP 6–12 and DP 25–36 and the decreasing proportion of DP 13–24, while the opposite effects were observed on To(r). Concludingly, starch physicochemical and functional properties of the four Thai rice genotypes are attributed to an interplay of many factors, such as amylose, amylopectin fine structure, granule size distribution, and crystallinity. These representative Thai rice cultivars were selected based on their distinctive pasting properties, and they also provided suitable materials for the study on the relationship between starch compositional, structural, physicochemical, and functional attributes. The results of the present study could further the understanding of the key factors affecting starch functionality, and may provide useful information for their possible industrial end uses.


Psychologische Forschung started as a journal “für Psychologie und ihre Grenzgebiete” and became strongly associated with the Berlin school of Gestalt psychology. Parallel to the fate of that school, the Journal was discontinued after 1938 and re-appeared only 1949. A number of years with variable and broad editorial boards and without a clear profile followed. In 1974 the Journal switched to English as the first German psychology journal and became Psychological Research. Gradually and without any abrupt changes—as indicated e.g. by analyses of citing and cited journals—the current profile as “An International Journal of Perception, Attention, Memory, and Action” was developed. During the last one to two decades the number of papers, the number of contributing countries, and the impact increase. Psychological Research has a history which reflects not only the evolution of psychology throughout the century, but also the political upheavals in Germany. In short: the Journal is a survivor of both political and scientific darkness.Why should one write a paper on the history of a journal? When I pondered this question, two reasons came to my mind. The first one is a somewhat personal one, the person-journal relationship. In his first editorial, the current Editor-in-Chief wrote: “this is not just any journal, but my first love—journal-wise” Hommel, . There mPsychological Research. So this is a biased (or opinionated) report. And it is not the first one of the history of the Journal. The first 50 volumes, with volume 49 as the last one included in the data reported, have been reviewed by Scheerer , and the scope—as outlined on p.1 of the first issue—emphasized that “papers will be accepted on the basis of achievement rather than school affiliation” , so this fact may have strengthened the tendency to associate the Berlin-based journal with Gestalt psychology.Despite its broadly defined scope, the image of the early d by Ash , after WPsychologische Forschung as the journal of Gestalt psychology became more and more justified by the published papers. This is evident from a list of pre-war papers that still received citations between 1971 and 1980 , and he served on the editorial board for only one volume. In addition to the psychiatrist Gruhle, who also served for the next volume, the first post-war editorial board consisted of five psychologists , and an anthropologist . Among the psychologists, both Heiss and Lersch had a non-empirical orientation and published primarily on “Charakterologie”, a kind of personality research. Only Düker and Metzger were experimental psychologists. Metzger was one of the few non-emigrated Gestalt psychologists  According to Scheerer , the iniMetzger, , p. 203.Metzger, , p. 61, The first post-war issue (volume 23/1–2) appeared on January 1st, 1949, with five papers; the following three issues of volume 23 with ten papers in total appeared 1950 and 1951. Of the first five papers, three were of an experimental nature, two with a Gestalt background by Rausch and Cymbalistyj, a student of von Allesch. The third one was by Düker. The final two papers were nonexperimental, written by Gruhle and by Thurnwald. Thus, there was a mixture of topics and theoretical as well as methodological approaches. As noted by Scheerer  and EngeThe year 1967 (vol. 31) saw major changes of the editorial board. Two of the new editors were from the USA: Richard Held of MIT, who had spent some time with Köhler at Swarthmore College, and Herschel Leibowitz, also a prominent researcher in the field of visual perception. Three of the new editors from Germany were post-war psychologists: Theo Herrmann, who moved to Marburg in 1968, Hans Hörmann, who moved to Bochum in 1969, and Karl-Hermann Wewetzer from Giessen. Neighboring disciplines were represented by Otto-Joachim Grüsser, a neurophysiologist from Berlin, and three ethologists, Paul Leyhausen, Detlev Ploog, and Franz Sauer. There was also a new subtitle of the Journal in two languages: “Empirische Untersuchungen von Grundproblemen der Psychologie, Basic Research in Psychology”, which seems to imply the inclusion of findings from neurophysiology and ethology.The year 1967 is memorable for two more reasons. First, it is the year that Wolfgang Köhler died. The obituary in volume 31/1 had contributions by Rudolf Bergius, who represented the Deutsche Gesellschaft für Psychologie, Hans Hörmann, and Hans-Lukas Teuber, then head of the psychology department at MIT (and born in Berlin). Teuber reports a statement by Köhler that I repeat here because it has some relevance for a debate that came a few years later: “You know, Lukas, it’s really amazing, how many unclear things you cannot say in English.” The obituary is followed by Köhler’s last paper, the manuscript for an invited address to the APA. Second, in the same volume (31/1 and 31/4) four papers appeared which grew out of oral presentations at a symposium held in Boston, Mass. They are by Schneider, Ingle, Trevarthen, and Held and espouse the notion of two visual mechanisms , one for WHAT we see and one for WHERE we see it. This notion became quite popular (and modified) later on , also to attract authors on certain topics and from certain regions. These are outward-directed functions of an editorial board. But there is also routine work to be done. According to Scheerer , these ePsychologische Forschung, we not only decided to change the subtitle to reflect the emphasis found in editorial material, but also to translate its original title, so that henceforth the journal will be known as: Psychological Research, An international Journal of Perception, Learning and Communication, Founded as Psychologische Forschung. For the same reason we invited several distinguished colleagues from abroad to join the Editorial Board.” It is emphasized that the change in title does not imply a change in publication policy, and that neuropsychological, psychophysiological, and ethological contributions are welcome. “It is this very open-mindedness that seems to us so necessary for the advancement of psychology as a science.” Whereas vol. 37 still had four papers in German and vol. 38 one, from vol. 39 on all papers were in English.The next major step in the comeback of the Journal was vol. 37 (1974/1975). Robert B. Freeman was appointed as what was now called “Coordinating Editor”, and in vol. 39 he was listed as such. He had moved from Penn State to Konstanz University in 1972. In an unsigned editorial in vol. 37/1 it is said: “To accentuate the international character of Psychologische Forschung was the groundbreaker among German psychology journals in the process of anglicization. Other journals followed with a delay, most of them only after the turn of the millennium. The change of language stirred a lively debate, briefly reviewed by Scheerer , the bulk of the work—with the exception of occasional support—rested on him. Only about 10 years later, in 1988, there was a major change in the editorial board that was followed by a more distributed processing of manuscripts: only Broadbent and Engelkamp remained on the board, and Bridgeman, van der Heijden, Heuer, and Mewhort came in.From volume 41 (1979) on, Eckart Scheerer served as the Coordinating Editor which he had actually started in 1978. For him the person-journal relationship was a quite close one  Is there anything that has been accomplished during these years? Future historians of science will have to decide. In my own view, the most important thing is that the journal was kept alive.”  was used to make it clear that Psychological Research is the same Journal as Psychologische Forschung. The success of this measure appears somewhat mixed. For example, in the Journal Citation Reports  the compound name of the Journal appears, whereas for a Google Scholar search with Harzings’s Publish or Perish only the German and English names used separately produced meaningful results. Beginning in 1991, there was also a modification of the subtitle: “An International Journal of Perception, Cognition, and Action”. After Johannes Engelkamp resumed the Coordinating Editorship in 1996, the subtitle was made more specific in 1998 (vol. 61): “An International Journal of Perception, Attention, Memory, and Action”. This covered quite well the topics of the papers in the Journal  on, the compound name There is one aspect of the publication strategy that was initiated by Eckart Scheerer in 1980, continued throughout the consolidation phase, and is still present. This is a series of Thematic Issues, with manuscripts being subjected to a normal review process. A kind of precursor, though published across separate issues of a volume, are the four papers on two visual systems mentioned above. Thematic Issues often, but not always, grew out of conferences and were mainly handled by guest editors. On average, there was almost one Thematic Issue per volume since vol. 51, with the majority of them on topics in the general field of perception and action.Psychological Research and APA journals such as the Journal of Experimental Psychology series, this strategy for receiving excellent submissions may have failed occasionally as a manuscript finally submitted upon invitation could be of a kind the author thought appropriate for a low-level journal; I have to admit that my memory here may actually refer not to real events, but only to risks discussed at one or the other occasion.)Thematic Issues serve three functions at least. In the comeback and consolidation phases, there could be problems with too few submissions. Thematic Issues would help, but it could happen that more submissions arrived than expected, leading to an extra volume at least once . The trend stopped for the next dozen years or so, but the target length of 8 pages that Engelkamp . At least in terms of impact factors, the huge reputation gap of the past between Psychological Research and APA Journals such as Journal of Experimental Psychology: Human Perception and Performance has been reduced considerably (and in some years even be reversed).The current phase of the Journal is not only distinguished by the increasing number of published papers, but also by its increasing impact factor as illustrated in Fig. Psychological Research has always been an international journal, even when it was Psychologische Forschung and most papers were in German. Nevertheless, the decision to publish in English only had also been motivated by the objective to boost contributions from other than German-speaking countries. Therefore both Scheerer  and two journals of the Frontiers series (since 2010 and 2007). . Second, higher ranks in 2019 can be observed for Neuroscience journals such as Neuropsychologia, Experimental Brain Research, and Journal of Neuroscience ; among the new journals is the Journal of Cognitive Neuroscience. Third, journals dedicated to perception such as Attention, Perception, & Psychophysics, Vision Research, and Perception have lost ranks. Fourth, the German-language journals that also switched to English (Zeitschrift für Psychologie and Psychologische Beiträge) have lost considerably; the former Zeitschrift für experimentelle und angewandte Psychologie, later Zeitschrift für experimentelle Psychologie and now Experimental Psychology did not appear in the 1986/87 ranking, and has now rank 60.A few trends in Table Psychological Research and an increasing weight of neuroscience findings. The latter might reflect the increasingly tighter link between the traditional functional analyses of experimental psychology and the identification of the relevant neural correlates. In fact, strengthening of the cross-disciplinary links to the neurosciences is an explicit objective for the Journal . There is a clear positive relationship, indicating a family of journals with similar mutual citation frequencies. The Spearman rank correlation is 0.61. Of course, this correlation is not perfect. Deviations may be chance, but there might also be systematic asymmetries. What distinguishes journals that are more often cited by Psychological Research than citing it from journals that cite Psychological Research more often than being cited by it?What is the relation between the journals cited in Citing appears to be a somewhat delicate process that becomes even more delicate as the number of citable papers increases. When several papers could be cited in principle, a selection has to be made. Sometimes there are “standard references”, such as Fitts and Posner  for the Psychological Research are indeed those with a higher impact factor. Figure Psychological Research . Journals with an above median excess frequency of being cited in Psychological Research have a somewhat higher impact factor, indeed, but this difference is not impressive. . A statistical comparison between the impact factors of the two groups of journals failed to approach statistical significance—thus the evidence for a citation asymmetry between journals with respect to the impact factor is weak at best. Given that papers in high-impact journals are cited more frequently than papers in low-impact journals, the absence of a clear citation asymmetry between journals may appear counter-intuitive. However, what applies to individual papers might not apply to journals, for example because the larger number of papers in lower- than in higher-impact journals can compensate for the difference in citation rates of individual papers.Based on such considerations, I checked whether those journals that are more often being cited than citing Psychological Research  were all journals that did not yet exist in 1986, namely PLoS ONE, Consciousness & Cognition, Psychological Science, Frontiers in Human Neuroscience, Trends in Cognitive Sciences, and Frontiers in Psychology. However, another broad open-access journal, similar to PLoS ONE, namely Scientific Reports, marked the strongest asymmetry against Psychological Research , followed by Human Movement Science and Journal of Motor Behavior. Close to that extreme was also a high-impact neuroscience journal, namely Trends in Neuroscience. In short, I was not able to identify any conspicuous feature of journals that at least in part could account for the observed citation asymmetries.In search of other factors that could possibly shape the citation asymmetry, I listed the journals with the strongest asymmetries. The six journals with the strongest asymmetry towards Psychological Research/Psychologische Forschung has survived a century of crises of various kinds. Notorious are the crises of Psychology, which have even found an entry in the current 19th edition of the traditional German “Psychologisches Wörterbuch”, the “Dorsch”. This dictionary, which is now called “Lexikon der Psychologie”, started with the first edition in 1921 and thus shares the century with the Journal. According to Fahrenberg (Psychologische Forschung/Psychological Research has never been strongly influenced by behaviorism. Thus, for the Journal the so-called cognitive revolution was more a smooth continuation than a revolution. As it appears to me, the Journal has published a steady flow of scientific advancements, and in the post-war period, at least after a certain level of recovery, it has not been shaken by crises or paradigm shifts. As was the case from the very start, the Journal—as stated in the “Aims and Scope”—is still “devoted to the dissemination of knowledge based on firm experimental ground, but not to particular approaches or schools of thought”.hrenberg , the authrenberg . In Germhrenberg . In the Psychological Research. The reason is that the crisis seems to apply primarily to “groundbreaking” research and less so to research of a more “incremental” nature. These are buzz words, and at least the word “incremental” is usually used pejoratively in contrast to “novel” (cf. Marder, Psychological Research is a journal for incremental research—perhaps better called “cumulative research” to avoid the pejorative connotations—along a straight road that leads to the horizon and beyond. Thus, new findings generally should be in line with previous findings and/or plausible theories. Conspicuous deviations from the straight road, findings that are unexpected from the perspective of previous findings and theorizing, that are at variance with what is reasonable for the common sense or an ‘experimentally trained mind’, invite the suspicion of future replication problems.The current crisis of psychology—among other sciences—is the replication crisis which might shake the “firm experimental ground” on which the Journal is built. As it turns out, the experimental ground in general is not really firm but somewhat muddy. The main reason is the uncertainty of statistical inferences—there is a probability above zero that they are wrong, and with an increasing number of researchers and studies the number of wrong inferences does necessarily increase. The problem with wrong inferences is aggravated by the available computational power that allows trying different analyses quickly to identify a reportable one. This is not the place to enter into a discussion of the replication crisis, but it should not cause major problems for The buzz words discussed by Marder  illustraThe changes of submission and review procedures certainly have to do with the increasing load placed on editors by an increasing number of submissions, with the increasing number of review requests, and with attempts to keep instances of non-replicable findings and scientific misconduct away from a journal. Somehow the system differs from what appears to me as a sort of ideal: editors who are knowledgeable in the field for which they handle manuscripts, know which reviewers to invite and how to weigh their comments and to evaluate the revision of the manuscript; reviewers who accept review invitations and can expect to write one review in due time, and not two or more reviews of successive revisions. This clearly is not the world we live in, but wanting editorial systems to run like this may not just be an old man’s longing for the past, but a desideratum for the future.


Enriched bread had a hard texture and required a lot of chewing force compared to the control. Furthermore, all samples yielded a shelf-life of 5 days when monitoring the mold growth. From the results, the bread enriched with 20% cricket powder yielded the best consumers’ acceptance score of 77%. It was predominantly high in indispensable amino acids such as leucine, phenylalanine, lysine, and arginine. Therefore, cricket powder could be a novel alternative protein source and successfully utilized in whole wheat bread to enhance its protein content and indispensable amino acids with consumers’ acceptance responding to the current market trend.The current market trends in modern sedentary lifestyles drive the development of new functional products able to fulfill consumers’ demand for a healthy diet. Whole wheat bread contains more protein and fiber than white bread; however, it could be improved in terms of protein content and quality. Cricket powder, which contains high protein , could be used as an alternative source to tackle those deficiencies in such bread. Hence, the study aimed to apply cricket powder in the whole wheat bread formula to enrich protein content, indispensable amino acids and determine their physico-chemical properties, consumers’ acceptance, and shelf-life storage. The results showed that all enriched bread presented high protein , fat , and ash  with the increment of the cricket powder than those of the control bread. Enriched breads’ crust colors were not significantly different ( The steadily increasing global population will reach 9.8 billion people in 2050  resultinBread is a vital staple food and is widely accepted and consumed throughout the world. Increasing bread consumption was reported as a 1.43% Compound Annual Growth Rate (CAGR) between 2019 and 2024 in the world market due to its convenience, portability, nutrition, and taste . The staThe research and development on the application of edible insects is being actively pursued and can be observed by a large number of patents and research articles. Currently, 167 patents and 488 articles related to the applications of edible insects have been filed . In prevAcheta domesticus) was selected due to it being a good source of protein  [There are some species of insects that are very popular to consume in Thailand such as house crickets, bamboo caterpillars, and grasshoppers ,18. Cons70, wt%) , its aff70, wt%) . Additio protein 5–70, wt%70, wt%) . Moreove70, wt%) . These aAcheta domesticus) was purchased from the cricket farm . Pandan leaves were purchased from the local market of Ban Du, Chiang Rai, Thailand. The ingredients for the production of whole wheat bread were purchased from a baking supply shop  including the main ingredients; whole wheat flour , wheat flour  for 12 h before the production of cricket powder. Cricket powder was prepared using the method according to Lucas-González et al.  with sliDeterminations of chemical compositions including moisture, protein, fat, and ash content followed American Association of Cereal Chemists (AACC) approved methods of analysis . MoisturDetermination of physical properties including color using a slightly modified method according to Bawa et al. . The criWater holding capacity (WHC) and oil holding capacity (OHC) were determined by using a slightly modified method according to da Rosa Machado and Thys . One graFoaming capacity (FC) and foaming stability (FS) were conducted by a slightly modified method by Stone, Tanaka and Nickerson . Two graFor foam stability, the samples were allowed to stand for 30 min at room temperature and the remaining foam volume was measured. The following formula was used to calculate FS:®, St. Joseph, MI, USA) and mixed at speed No. 4 for 15 min. while vanilla flavor and milk were gradually added until the dough was obtained. Then, the mixer bowl was covered with plastic wrap, and the dough left to ferment at 40 °C for 1 h. After dough fermentation, the dough was removed by a rolling pin and molded-in aluminum bread mold tray (6.7 cm width × 13.7 cm length × 5.5 cm height) then covered with plastic film wrap and let stand for 20min. The dough was baked at 175 °C for 30 min then the loaves removed from the bread mold tray. Loaves were left to cool down at ambient temperature for 2 h. All loaves were packed in polyethylene bags and stored at −40 °C until analysis.Whole wheat bread was prepared following a formula presented in Determinations of chemical compositions including moisture, protein, fat, ash, and carbohydrate content were performed as previously described above.Amino acid profile was performed only in the cricket powder enriched bread with the best acceptance score and determined using outside analysis service by Central Laboratory  Co., Ltd. employing the in-house method TE-CH-372 based on the Official Journal of the European Journal of communities, L257116 by amino acid analyzer technique.Physical properties in terms of the color of crust and crumb and texture profile including hardness, cohesiveness, springiness, and chewiness were determined. The color of crust and crumb were determined followed the method described by Bawa, Songsermpong, Kaewtapee and Chanput , Loave sDetermination of the shelf-life of loaves was conducted following the method described by Kamaljit . Loaves Sensory evaluation was conducted at the Food Sensory Lab, Department of Food Science and Technology, School of Agro-Industry, Mae Fah Luang University. Ethical approval for the consumer testing and evaluation of the products was granted by Mae Fah Luang University, Chiang Rai, Thailand. The consent of all participants was achieved. Participants for the consumer testing and evaluation of the bread samples comprised 30 untrained panelists, 15 females and 15 males. The panel was asked to rate their preference of how they like the appearance, texture, flavor, taste, and overall acceptability of the products by indicating a numerable value to rate their preference from one (expressing dislike extremely) to nine (like extremely). They were also asked to rate their acceptability of the products via nine points hedonic from one (dislike extremely) to nine (like extremely). To obtain a good understanding of consumer opinions, this stage was evaluated by 100 untrained panelists in the sample which provided the best score from the previous evaluation and commercial whole wheat bread. The panelists were also requested to provide comments and suggestions regarding product development.p < 0.05 was considered significantly different.All data analyses were investigated triplication and expressed as mean ± standard deviation. The different formulas were analyzed using one-factor analysis of variance (ANOVA) by the Statistical Tool for Agricultural Research (STAR) software program . The significance level of The chemical composition of the cricket powder was presented on a dry weight basis  including moisture 2.77 ± 0.11, protein 55.11 ± 3.10, lipid 22.83 ± 0.40, ash 4.80 ± 0.10, carbohydrate 17.26 ± 0.23, respectively. Cricket powder predominantly consists of protein and lipid. The same results were reported by Rumpold and Schlüter , who repThe color of cricket powder showed L* (40.81 ± 0.61), a* (4.9 ± 0.53), b* (15.13 ± 2.02), respectively. The resulting value indicates that the cricket powder has a dark color due to the luminosity (L*) which indicates the lightness is quite low. The same result was reported by other researchers including Lucas-González, Fernández-López, Pérez-Álvarez and Viuda-Martos  who statThe applicability of such protein ingredients depends upon their potential to fulfill one or more functional requirements, viz. solubility, water holding capacity (WHC), and oil holding capacity (OHC) or foam and emulsion stabilization . WHC andThe foaming capacity (FC) and foaming stability (FS) of cricket powder were 8.1 ± 1.20% and 32.43 ± 9.89%, respectively. It was reported that foamability is related to the rate of decrease in the surface tension of the air/water interface caused by the absorption of protein molecules. Low foamability could be related to highly ordered globular proteins, which resist surface denaturation . Ndiritup < 0.05) from the control bread. The moisture content seemed inconsistent with the linear increase in cricket powder. During kneading, protein absorbs water to enable it to flex into gluten. The more protein in the flour, the more water is required to effectively hydrate the dough [The results of moisture, ash, protein, lipids and total carbohydrate on a weight dry basis (g/100 g) are depicted in he dough . Howeverhe dough . Crickethe dough .Amino acid compositions were determined only in the bread enriched with 20% cricket powder which yielded the highest acceptance score from panelists and is depicted in Texture profile analysis (TPA) showed that all cricket powder enriched loaves have a higher value of hardness  and chewiness  than control . This caImages of the cross-section of bread slices are depicted in Generally, homemade bread has a shelf-life of 4–6 days under ambient conditions . In thisBacillus licheniformis, Bacillus subtili and Brevibacillus borstelensis. The first two species are associated with ropy spoilage of bread and B. licheniformis is also potentially pathogenic to humans. Another point is that allergens are a major safety concern surrounding edible insects. Several research groups have identified potential antigens and IgE binding proteins in various insect species, which may correlate to an allergic reaction after consumption [The limitation of edible insects can be perceived by biological hazards associated with the rearing environment and insect’s gut. The presence of spore-forming bacteria in insect-based foods represents a serious threat to consumers’ health since these microorganisms are food-borne pathogens . Howeversumption . The guisumption  stated tp < 0.05) from that of enriched bread with 20% cricket flour, the enriched bread with 20% cricket flour was selected for the further step because of the higher protein content. In the comparison among all bread, it was found that enriched bread with 20% cricket flour yielded a higher score on the overall acceptability. In terms of the food industry and marketing of the product, sensory evaluation with a high overall acceptability score could be used as the key point due to one of the most significant factors influencing the consumer purchase decision and consumption [The result showed that consumers favor the 20% cricket formula the most. Although the results of the 10%, 15%, and 20% cricket formulas were similar in some characteristics such as appearance, texture, color, flavor, taste, and overall acceptability which are demonstrated in sumption . As reposumption , insect-Commercial whole wheat bread was reported to contain total energy , total fat (4%), protein (2%), total carbohydrate (44%), and dietary fiber (4%). It was found that the acceptance score of 77% was derived from the appearance, texture, color, flavor, taste, and overall acceptability of enriched bread with 20% cricket flour compared to those of commercial bread which is 100%. Nevertheless, consumers suggested improving the quality of the product in terms of texture since cricket powder contains high lipid content, thus, making the bread quite crumbly. Balancing the ratio of ingredients and reducing fat content should be improved in future studies.Cricket powder can be utilized as a novel alternative protein source in terms of protein quality, and as an environmentally friendly resource. All enriched bread formulas presented high protein content and the one enriched with 20% cricket powder had the best acceptance without any effect on shelf-life. The derived products successfully combine the compositional and sensory characteristics with some peculiar nutritional properties of cricket. Cricket powder can constitute a novel source of innovative ingredients to be used for the protein enrichment of indispensable amino acids and minerals. In future studies, fatty acid profile analysis is advisable to confirm the functional fatty acids contained in the starting material and final product. Future investigation should also consider improving the texture as well as insect bioactive properties within the food matrix and the effect on allergenicity. Lastly, the current work is not only in response to the current trends in modern sedentary lifestyle with the development of the new functional products able to fulfill consumers’ demand for a healthy diet, but the economics of the country as well. The edible cricket industry will be expanded by adding up the value of processed products. Moreover, bakery industries and entrepreneurs also yield the outcome from this study with the invention of functional health products. The world is also impacted in terms of world food security and environmental issues because insect farming is more pre-eminent in terms of lower greenhouse gas emissions, water, and soil use, but with higher feed conversion and edible mass compared to domestic livestock. Moreover, making consumers respond with positive attitudes toward innovative insect-based foods is one of the most challenging issues facing the industrial sector.


Human respiratory syncytial virus (RSV) infection is the most important cause of acute lower respiratory tract infection in infants, neonates, and young children, even leading to hyperinflation and atelectasis. Oxymatrine (OMT), originating from natural herbs, possessed potential antivirus activity against influenza A virus, Coxsackie B3 virus, and RSV, whereas the absence of an in vivo study indicated the difficulties in overcoming the physiological obstacles. Since RSV basically replicated in lung tissue, in this study, we fabricated and characterized a chitosan (CS)-coated liposome with OMT loaded for the treatment of lethal RSV infection via inhalation. The results uncovered that OMT, as a hydrophilic drug, was liable to diffuse in the mucus layer and penetrate through the gas–blood barrier to enter systemic circulation quickly, which might restrict its inhibitory effect on RSV replication. The CS-coated liposome enhanced the distribution and retention of OMT in lung tissue without restriction from mucus, which contributed to the improved alleviative effect of OMT on lethal RSV-infected mice. Overall, this study provides a novel inhalation therapy for RSV infection, and the CS-coated liposome might be a potential inhalable nanocarrier for hydrophilic drugs to prevent pulmonary infections. RSV presents a high tropism to the pulmonary system of preterm infants, neonates, and young children, it being the number-one cause of acute lower respiratory tract infection (ALRTI), i.e., pneumonia or bronchiolitis, and severe conditions such as asthma . RSV inf T cells ,4. Conse T cells .Sophora flavescens Ait  by a Japanese group in 1895 [S. flavescens Ait, as investigated in several reports, including against the influenza A virus (IAV), by inactivating the promoters of TLR3, TLR4, TLR7, MyD88, and TRAF6 of host cells and deregulating IAV infection-induced activations of NF-κB, Akt, ERK1/2, and p38 MAPK pathways [Matrine-type alkaloids are a group of monomeric compounds that were isolated from  in 1895 . They ar in 1895 . Oxymatrpathways ; howeverPulmonary administration has the priority in the treatments of pulmonary infectious diseases, due to it being free from first-pass effect and direct delivery of a loaded drug to lung lesions . HoweverEnlightened by the constitution of endogenous pulmonary surfactants, we prepared OMT liposomes (Lipo/OMT) by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), hydrogenated soybean phosphatidylcholine (HSPC), and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG) in this study instead of soyabean lecithin, in order to improve its stability in vivo. To further enhance the pulmonary delivery, a surface modification strategy was used to prevent the degradation and immune recognition of liposomes and facilitate the mucus penetration . 1,2-disIn this study, we developed inhalable CS-coated OMT liposomes for the treatment of RSV-infected mice. The optimal CS-coated OMT liposomes were evaluated in vitro and applied on the RSV-infected mice model in which the anti-RSV mechanisms of OMT might be attributed to its preventive effects on RSV . This st50 assay result, measurements of 150 μg/mL, 75 μg/mL, 37.5 μg/mL, and 18.75 μg/mL OMT were found to significantly decrease viral titers and viral gene copies indicating the intervention effects of OMT on the virus biosynthesis , cholesterol (Chol), polyoxyethylene stearate (SPEG) and octadecyl amine (ODA) for pulmonary emphysema treatment . Howeverw/w) and sharply decreased to 234 nm at 1:5 (w/w), and then a slightly variation was observed. Given that the high ratio of HCS resulted in a high viscosity of the solution that failed in effective inhalation, the LCS was used in the following study. The zeta potential was decreased until the LSC:liposome ratio reached 1:2.5 (w/w), and a larger SD value was measured at a higher LSC:liposome ratio, which suggested that LCS reached saturated adsorption on liposomes. Therefore, LCS was coated on the surface of Lipo/OMT at the weight ratio of 1:2.5 to receive Lipo/OMT@CS.CS coating was suggested to confer muco-adhesivity to nanoparticles and mediate cellular uptake ,29,30,31The physiochemical properties of OMT liposomal preparations were characterized and the stability was evaluated for one week . Lipo/OMThe high stability of OMT-loaded liposomes in pulmonary-relevant fluids is the prerequisite of subsequent cellular uptake and drug release by the epithelial cells, since the aggregation or dissociation of liposomes would seriously restrict the pharmacological effects of OMT. As mucin play an important role in the mucus, the size variation of OMT-loaded liposomes in mucin media was monitored. As shown in For further assessment on the potential diffusion of liposomes through the airway mucus, two in vitro models were used to evaluate the penetration of rhodamine B (RB) liposomal preparations and OMT liposomal preparations. A two-layer model that consisted of a mucin upper layer and a gelatin sublayer was used to directly evaluate the penetration capability of the hydrophilic drug and its liposomes D,E. BothFor lung infection treatment, pulmonary distribution and retention closely related to the therapeutic effects and systemic escape of drugs might lead to undesirable side effects. To discover the effects of liposomal carrier and CS coating on the biodistribution of hydrophilic drug, RB liposome (Lipo/RB) and CS-coated Lipo/RB (Lipo/RB@CS) were prepared, and the concentration of RB in lung, heart, liver, spleen, kidney, bronchoalveolar lavage fluid (BALF) and plasma were quantified at 0.5 h, 1 h, 2 h, 4 h and 8 h after inhalation . Half anGiven that faster mucus penetration behavior of hydrophilic drug was observed in vitro , liposomIn order to evaluate the potential alleviative effects of OMT liposomal preparations in vivo, we introduced the lethal RSV infection mice model developed by our group . As showCombining the results of 2/CO2 exchange, and fewer leukocytes and cytokines infiltrations were shown, resulting in a lower possibility of fatal cytokine storm. The H&E staining slides were further evaluated by pathologists  was purchased from Xi’an ruixi Biological Technology Co., Ltd. .Oxymatrine , rhodamine B , and chitosan  were offered by Macklin Biochemical Co., Ltd. . Cholesterol  was purchased from VetecTM reagent grade . 1,2-dipalmitoyl-sn-glycero-3-phosphocholine  was provided by Corden pharma Switzerland LLC . Hydrogenated soybean phosphatidylcholine  and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol)  were supported by A.V.T Pharmaceutical Co., Ltd. . 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)  were cultured under 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) supplemented with DMEM/F12 GlutaMax-I . GZ08-18, a highly virulent mouse-adapted RSV strain, was developed and stocked in our lab , and it Eight-month-old retired breeder BALB/c female mice (25–35 g) were supplied by the Experimental Animal Center of Guizhou Medical University. All animal care and experiments have been approved by the Animal Welfare and Ethics Committee of Guizhou Medical University (No: 1800020). Mice were raised at room temperature (23 ± 2 °C) and constant humidity (45 ± 10%) in an SPF condition. One week before starting experiments, animals were placed for adaptation.To evaluate the intervention of OMT on virus biosynthesis, GZ08-18 dilution with a multiplicity of infection (MOI) of 0.05 was added to HEp-2 cells for 1 h at 37 °C, then we discarded the virus dilution, washed the cells once with PBS, removed unadsorbed viruses, and added cell maintenance solutions containing different concentrations of OMT to culture for 72 h. To evaluate the inhibitory effects of OMT on virus penetration, GZ08-18 dilution with MOI of 0.05 was added to Hep-2 cells and cultured, at 4 °C, for 2 h, then we discarded the virus solution, washed the cells once with pre-cooled PBS, added different concentrations of OMT solutions, and cultured at 37 °C for 2 h. The OMT solution was discarded, and the cell maintenance solution was added to incubate cells for 72 h. To evaluate the inhibitory effects of OMT on virus attachment, pre-cooled OMT solutions and GZ08-18 diluent with MOI of 0.05 were added to Hep-2 cells, for 2 h, at 4 °C, then we discarded the OMT solution and virus diluent, washed the cells once with PBS, and removed unabsorbed OMT/virus. The cell maintenance solution was added to the cell and cultured for 72 h. To evaluate the preventive protective effects of OMT, different concentrations of OMT were added to the cells, incubated at 37 °C for 4 h, then we washed the cells once with PBS, added GZ08-18 dilution with MOI 0.05 for 1 h, at 37 °C, and then we washed the cells with PBS once and added the cell maintenance solution to incubate cells for 72 h. Meanwhile, ribavirin was used as the positive drug to coculture with virus and HEp-2 cells according to above experiments as the substitution for OMT.OMT liposomal preparations were prepared using the pH-gradient method as reported previously . In brieThe RB-encapsulated liposomes were prepared via a film dispersion method. Briefly, a thin lipid film was cocultured with 4 mL RB solution at 0.5 mg/mL and ultrasonicated. The Lipo/RB was obtained after passing through 0.45 μm filter. Lipo/RB@CS were obtained after modification of CS via the same methods described above.v/v) at a flow rate of 1 mL/min and detecting at the wavelength of 210 nm. The loading efficiency (LE) and drug loading capacity (DL) were calculated according to Equations (1) and (2).drug loaded is the drug encapsulated into liposomes, Wtotal liposome is the mass of liposome with drug encapsulated, and Wdrug added is the drug added in the preparation of liposomes.The morphology of Lipo/OMT and Lipo/OMT@CS were observed by transmission electron microscope . The particle diameter, polydispersity (PDI), and zeta potential of Lipo/OMT and Lipo/OMT@CS were measured by NanoBrook 90Plus PALS . The concentration of OMT in liposomal preparations was measured by high performance liquid chromatography (HPLC), which used Shimadzu LC-16  with a UV detector  using a reversed phase column , at 30 °C. A mobile phase consisted of acetonitrile and 0.2% triethylamine  holding 1 mL OMT liposomal preparations were immersed in 15 mL of PBS in a capped bottle and incubated, at 37 °C, with stirring at a speed of 50 rpm. At predetermined time point, 500 μL medium was withdrawn and replenished with fresh PBS at the same volume. The amount of OMT released into the medium was determined using the HPLC method described above. To determine the stability of formulations, OMT liposomal preparations were kept in an incubator, at 4 °C, for one week. The particle size, PDI and zeta potential were measured over the storage periods.g for 10 min to receive a 0.08% (w/v) mucin supernatant solution. For the analyses, 200 μL of OMT liposomal preparations were added to 2 mL of medium and incubated, at 37 °C, for 72 h. At selected time intervals, the particle size of OMT liposomal preparations were monitored. The variation on particle size were considered as indicative of disassembly/aggregation state.Stability of OMT liposomal preparations in mucin solution was evaluated. Mucin powder was dispersed in PBS and stirred overnight, then the dispersion was centrifuged at 3500× w/v) and OMT liposomal preparations were mixed at volume ratio of 1:1 and vortexed for 1 min. The absorbance at 650 nm of the mixtures was measured at 0, 30 and 60 min after incubation, at 37 °C, by the ultraviolet spectrophotometer . The mucin solution and the liposomal preparations dispersions in PBS were analyzed as controls. For the measurement of zeta potential, the mucin solutions  and mixture samples were diluted in PBS  and measured.The mucoadhesive tendency of OMT liposomal preparations were assessed by turbidimetric measurement as previously reported ,40. Briew/v) hot gelatin solution was prepared, placed into a centrifuge tube, hardened at room temperature, and placed in a refrigerator, at 4 °C, until use. For the mucus penetration study, 0.5 mL of mucin solution  was added above the gelatin solution and 125 μL of free RB solution and RB liposomal preparations were added on the mucus layer and maintained, at 37 °C. After 0.5 h, 1 h, 2 h, 4 h, 8 h, and 12 h incubation, the mucus layer was transferred to 1 mL volumetric flask. RB in the mucus layer was extracted with methanol and measured by a fluorescence spectrophotometer  at λEx/λEm = 550/580 nm. The percentage of RB penetrated through mucus was calculated according to Equation (3).Mucus penetration of RB-encapsulated liposomal preparations were evaluated by the in vitro mucus model ,41. One w/v) was transferred to the insert of transwell . Then, 100 μL of free OMT and OMT liposomal preparations were gently added above the mucus layer. The insert of transwell was immersed in the acceptor chambers that were filled with 1 mL of PBS and incubated, at 37 °C. At the scheduled time intervals, 100 μL of samples were collected from acceptor chambers, and an equal volume of fresh PBS were replenished. The amount of OMT in the acceptor chambers was determined using the HPLC method, and the percentage of OMT penetrated through mucus was calculated according to Equation (3).The transwell multiwall plates were applied to evaluate the penetration capacity of OMT liposomal preparations across mucus layer . Briefly®. I. V. Catheter, Terumo Corp, Biñan, Philippines) was used for the inhalation administration of free RB and RB liposomal preparations. Briefly, at specific time points, mice were anesthetized by isoflurane via inhalation anesthesia. Free RB solution and RB liposomal preparations were inhalation administered at an equivalent RB dose of 0.75 mg/kg, respectively. At 0.5 h, 1 h, 2 h, 4 h and 8 h after treatment and under anesthesia, blood samples were taken from the aorta abdominalis, and the plasma was obtained by centrifugation  and stored, at 4 °C, until analysis. After the euthanasia treatment, BALF and organs  were obtained. The tissues were homogenized in PBS. Afterwards, ethanol was added to the plasma, BALF and organ homogenates at certain ratio to extract RB. Thereafter, the samples were centrifugated at 3000× g for 10 min, and the supernatants were used to measure the concentration of RB via a fluorescence spectrophotometer.The distribution of RB liposomal preparations in organs were investigated according to the method reported previously . Trachea10TCID50 virus) was inoculated into mice via trachea, and three regimen  should be inoculated into pulmonary system of mice to establish the lethal RSV-infected mouse model. A total of 63 BALB/c mice were involved in the body weight change and survival experiments. They were randomly divided into 7 groups (n = 8–11 for each group): Mock, Model (virus control), Blank Carrier (blank preparation plus virus), Free OMT (High) (free OMT 20 mg/kg plus virus), Free OMT (Low) (free OMT 5 mg/kg plus virus), Lipo/OMT (5 mg/kg Lipo/OMT plus virus), and Lipo/OMT@CS (5 mg/kg Lipo/OMT@CS plus virus). The Mock mice were administered with saline as negative control. The Model mice were administered with saline plus virus. The Blank Carrier mice were administered with Lipo@CS solution.Based on lethal RSV-infected mouse models established by our laboratory as one regimen, 100 μL GZ08-18 culture medium . We performed the RSV qRT-PCR absolute quantitative detection method established by our laboratory to quantitatively measure the viral copies via a standard curve of RSV N gene. Since N is the most conserved RSV gene across all known genetic clades, and is also expressed most abundantly during viral replication [At 2 DPI, the mice were properly anaesthetized and euthanized. Subsequently, the right lobe of lung was fully perfused with 800 μL DMEM/F12 GlutaMax-I , and the BALF was obtained. After centrifugating the BALF sample under 4 °C, at 12,000 rpm for 10 min, the cell-free supernatant is transferred to sterilized enzyme-free 1.5 mL centrifuge tubes for subsequent qRT-PCR assay for viral load analysis. Briefly, the viral RNA was firstly extracted by using the TaKaRa MiniBEST Viral RNA/DNA Extraction Kit  from BALF-derived cell-free supernatant. cDNA was generated by PrimeScript™ 1st Strand cDNA Synthesis Kit . The qRT-PCR was then performed with the TB GreenAt 2 DPI, the mice were properly anaesthetized and euthanized. The left lung lobe was collected and fixed immediately in 4% formaldehyde under 0.01 M phosphate buffer (pH 7.4). After dehydration and embedding in paraffin, the lung tissue was cut into 5.0 μm-thick sections and stained with hematoxylin and eosin (H&E) for evaluation of histopathology and of the severity of pneumonia. The slides were scored with a semiquantitative system according to the relative degree of inflammation and tissue damage .The left lung lobe harvested at 2 DPI was processed into paraffin sections, and then the sections were then deparaffinized and rehydrated. After treatment, immersed the slides in EDTA antigen retrieval buffer for antigen retrieval. Because F protein is one of viral membrane proteins and the function of F protein is fusing RSV and cell membrane or infected cell membranes together. So its expression would demonstrate the attachment and entry capability of virus. Therefore, slides were blocked by 1% BSA and incubated with primary anti-RSV F antibody . Then, slides were incubated with fluorescent secondary antibody. 4’,6-diamidino-2-phenylindole (DAPI) counterstained nucleus for 15 min, and then images were detected and collected by fluorescent microscope  and confocal microscope . Nucleus is blue by labeling with DAPI, and positive signals are red according to the fluorescent labels used.p values of less than 0.05.Data are presented as mean ± or + SD. The one-way ANOVA followed by post hoc Tukey test was used in the statistical analysis among multiple groups using GraphPad Prism 6. Comparisons between Mock and Model groups were assessed by unpaired, two-tailed Student’s t test. Variance test (F test) was implemented before the one-way ANOVA analysis and t-test to check whether the data met the normal distribution. The Kaplan–Meier method was used for survival analysis, and the Log-rank test was performed to analyze the differences among the survival curves. The Mann–Whitney test was applied for the pathologic scores analysis. Significance was defined as In this study, we developed a CS-coated liposome to facilitate the alleviative effect of OMT on lethal RSV infection via improving the lung accumulation and retention of OMT to exert its anti-virus effects by inhibiting the penetration and biosynthesis of virus. We revealed that effective penetration through the blood–gas barrier leads to fast systemic distribution and short exposure at lung, instead of pulmonary mucus being the main barrier for OMT in the treatment of RSV infection. OMT liposomal preparations, especially Lipo/OMT@CS, reduced the virus replication and increased the survival rate of RSV-infected mice. Further investigation is warranted to elaborate the exact mechanisms of OMT on the alleviation of lethal RSV infection and to optimize the liposomal carriers.


Conditional marketing authorization (CMA) facilitates timely access to new drugs for illnesses with unmet clinical needs, such as late graft failure after kidney transplantation. Late graft failure remains a serious, burdensome, and life-threatening condition for recipients. This article has been developed from content prepared by members of a working group within the European Society for Organ Transplantation (ESOT) for a Broad Scientific Advice request, submitted by ESOT to the European Medicines Agency (EMA), and reviewed by the EMA in 2020. The article presents the rationale for using surrogate endpoints in clinical trials aiming at improving late graft failure rates, to enable novel kidney transplantation therapies to be considered for CMA and improve access to medicines. The paper also provides background data to illustrate the relationship between primary and surrogate endpoints. Developing surrogate endpoints and a CMA strategy could be particularly beneficial for studies where the use of primary endpoints would yield insufficient statistical power or insufficient indication of long-term benefit following transplantation. The guideline CHMP/EWP/263148/06 of the European Medicines Agency (EMA) Committee for Medicinal Products for Human Use (CHMP), issued in 2008, identifies the primary composite endpoint for clinical trials in organ transplantation as recipient death, graft failure, biopsy-confirmed acute rejection, and graft (dys)function  graft failure as a composite of two important primary endpoints: loss of graft function , and recipient death with a functioning graft.We consider that using 1 year post transplantation as the border between early and late graft failure reflects current clinical research standards and epidemiological data. These illustrate a fundamental difference in general improvement of graft outcome within and beyond 1 year after transplantation .In addition, a 1-year threshold for the definition of late graft failure could be appropriate, given that research standards usually consider primary endpoints at 6 months to 1 year following transplantation. This was the case for pivotal trials that supported the approval of immunosuppressive drugs (reviewed in ). The 1-• Aimed at treating, preventing, or diagnosing seriously debilitating or life-threatening diseases• Intended for use in emergency situations • Designated as orphan medicines, i.e., for the diagnosis, prevention, or treatment of a life-threatening or chronically debilitating condition that is rare .The European Medicines Agency (EMA)-initiated concept of CMA  is an imIn kidney transplant patient populations, late graft failure is a common, seriously debilitating, and life-threatening condition; no specific measures are available for its prevention. Immunosuppressive drugs were primarily approved for prevention of early acute rejection, with limited impact on (late) graft failure . In EuroSeveral aspects make late kidney graft failure a serious condition for which there is an unmet medical need. First, there is the requirement for dialysis reinitiation, which carries a heightened risk of mortality, comorbidities, and impaired health-related quality of life. Second, there is a high risk of human leukocyte antigen antibody (HLA) sensitization, which is associated with prolonged waiting time for repeat transplantation and further increased risk of dialysis complications. Third, increased risk of graft failure is observed after re-transplantation, which is related to heightened risk of antibody-mediated rejection (AMR) because of preformed antibodies against the first donor kidney , 14. In As of December 31, 2019, at the time ESOT was discussing this issue, ∼55,000 patients were on the transplantation waiting list in Europe , the vasWhile increasing longevity of kidney grafts could decrease the need for re-transplantation, importantly, the >20% of patients waitlisted for re-transplantation on Eurotransplant databases represents only those who are eligible for such procedures. Among European and US patients who experienced death-censored graft failure, 48% were waitlisted (median time 7.7 months) and 61% had HLA antibodies; most of the sensitized patients were not relisted for transplantation and remained on dialysis until death , of whomp = 0.033), with most deaths happening within 3 years of relisting  will have worse outcomes on dialysis. In addition, none of these analyses considers the burdens of returning to dialysis after failed transplantation, such as the costs associated with treatment , decreasIn addition, late graft failure could be considered as an orphan indication, when its occurrence is calculated in absolute terms with the general population as reference. A hypothetical steady-state situation, where the same number of grafts are failing as are being transplanted, would result in ∼21,000 graft losses per 512 million inhabitants in the EU, equivalent to four graft losses per 100,000 people, per year. This may fulfill the definition of an orphan indication and would do so even if twice as many graft losses were to occur.Death of the recipient with a functioning graft is the most important reason for graft loss, and is usually a primary safety endpoint in studies of interventions that aim to prolong kidney transplant function. The main causes of death with a functioning graft are cardiovascular disease (CVD) and over-immunosuppression resulting in adverse events such as malignancy or infection –35. The Relative contributions of different pathological processes to graft failure have been evaluated , 40–42. Increasing evidence suggests a continuous alloimmune response to the donor graft, despite modern immunosuppression, unrelated to the patient’s level of adherence to immunotherapy. The incidence of acute cellular  rejection in the early months after transplantation is ∼10% and rarely leads to immediate graft loss if treated appropriately, but TCMR is also an important, relevant, risk factor for long-term graft loss , 44. By As histologic studies show that progressive fibrosis is a major cause of late graft loss, and because calcineurin inhibitors (CNIs) are known to cause fibrosis, it was proposed that late graft loss might be partly attributable to CNI nephrotoxicity , 48, cauAfter alloimmune-mediated injury, recurrence of native kidney disease in the transplanted organ is another common cause of graft loss , 51. SomNonimmunologic factors that contribute to post-transplantation nephron damage include brain death of the donor, poor donor management, and cold and warm ischemia times –54; delaOther factors that contribute to graft failure are reflux nephropathy or obstruction due to ureteral stenosis . FinallyClearly, late graft failure is often a multifactorial process: active/acute diseases are additive and coincide with cumulative chronic injury , 56, 57.Several studies highlight the importance of progressive fibrosis as a key pathway to graft failure and a target for intervention, independent of the recognized role of late AMR in graft failure , 44. BioProgressive IFTA in the absence of inflammatory disease is a process once known as “mysterious dysregulated fibrosis” , 58. NewCurrent immunosuppressive agents were approved for marketing based on studies with follow-up periods of <1 year. The approval of drugs that improved these short-term outcomes was based on research focusing on TCMR inhibition, which led to an important decline in early graft failure rates , 9 but dThe impact of older immunosuppressive agents  is not limited to short-term endpoints, however. Studies with ≥5-years follow-up periods, including cyclosporine withdrawal regimens, have demonstrated the effect of immunosuppressive drugs on long-term graft outcomes , 64. Thide novo DSA development) are more important than TCMR for long-term transplantation success.Very few randomized controlled trials (RCTs) have evaluated newer immunosuppressive agents  with long-term graft survival as an endpoint. Extensions of the BENEFIT studies, reported at 7 and 10 years post transplantation , 67, demOther studies had extended follow-up (beyond 1 year) after transplantation, comparing regimens of immunosuppressive agents that were approved based on short-term data. Although graft function sometimes improved over time, this did not reduce the rates of long-term graft failure , 69. SomIf CMA applications for novel drugs aiming at preventing or treating late graft failure are admissible to the EMA, the next discussion relates to the choice of the endpoints to be used for the required clinical trials. Graft failure is a highly relevant hard endpoint in clinical studies, but it is a late endpoint. This hampers the feasibility of using graft failure as an endpoint in clinical trials that aim at improving late graft failure rates.Surrogates for late graft failure are therefore needed but require robust definitions. A good surrogate endpoint should fulfil four criteria: 1) The disease process is sufficiently understood; 2) The surrogate endpoint has biologic plausibility; 3) The strength of the consistency supports the relationship between the surrogate marker and outcome; 4) Treatment effects on the surrogate endpoint predict treatment effects on the clinical outcome of interest.Kidney graft function and combined functional markers, donor-specific HLA antibodies and composite scores could be considered as surrogate endpoints, but do not fulfill all these criteria. For a detailed discussion on the potential acceptability of these surrogate endpoints for late graft failure, we refer to another manuscript in this Special Issue .After successful application for CMA of a product aimed at improving long-term graft survival, conversion to full marketing authorization is necessary, based on a post-marketing confirmatory commitment.ESOT sees different options for this conversion of CMA to full marketing authorization. For example, applicants could consider requests for full marketing authorization based on long-term registration studies with accepted primary endpoints relating to graft rejection –4 functiAlternatively, applicants could base the comprehensive evidence for full marketing authorization requests on good-quality data from registration studies, utilizing real-world data. Indeed, the EMA has already considered data from two other registries suitable for their decision-making processes: the European Cystic Fibrosis Society patient registry and the Cellular Therapy module of the European Blood and Marrow Transplant registry. The EMA Patient Registries Initiative  offers gA final option could be to use data from a qualified surrogate endpoint as a source of comprehensive evidence for a full marketing authorization request. Although CHMP/EMA has suggested to initiate a formal Qualification of Novel Methodologies procedure for e.g. the finalized iBox model  as a surEach of the above options for post-marketing commitments seems unsatisfactory at present, in the field of kidney transplantation. This may hamper the current admissibility of CMA applications for therapies aiming at reducing the incidence and burden of late kidney transplant failure. The results of formal qualification procedures are eagerly awaited and will hopefully change the landscape in future.• Late graft failure (loss of graft function >1 year post transplant) is a condition with unmet medical need. Therefore, CMA should be considered for interventions that demonstrate potential benefits: ○ Late graft failure is a seriously debilitating, life-threatening disease for which no specific preventive or treatment options are available. ○ CMA of therapies aimed at preventing late graft failure could be based on trials that show benefit on a validated surrogate endpoint for graft failure.• For drugs aimed at reducing late graft failure, applying for CMA could be considered. ○ CMA procedures facilitate timely access to new therapies. ○ Confirmatory post-marketing commitments will be needed to convert CMA to full marketing authorization.• The CHMP agreed that improving long-term outcome after kidney transplantation is an area of unmet medical need; arguments for orphan designation of late graft failure were not followed.• Should a novel therapy be proposed for CMA, the product will need to fulfil all of the following four criteria at the time CMA is considered: 1) positive benefit/risk balance; 2) it is likely that the applicant will be able to provide comprehensive data later; 3) unmet medical need is fulfilled; and 4) the benefit to public health of the medicinal product’s immediate availability on the market outweighs the risk due to need for further data.a priori for any given product or indication.• Criteria for CMA will be reviewed for specific data submitted; CMA cannot be granted