The addition of RP improved the thermal stability of the starch and reduced the amount of leached amylose. Infrared analysis shows that strong hydrogen bonds formed between RP  and starch during co-gelatinization. In addition, RP improved the compactness of aggregated structure and played an important role in hindering the enzymatic hydrolysis of starch. These results enrich the theory of starch-protein interactions and have important implications for the development of carbohydrate-based foods with low digestibility and protein functional foods.Clarifying the interactions between food components is critical in designing carbohydrate-based foods with low digestibility. To date, the hindering effect of starch-protein interactions on starch digestion has attracted extensive attention. In this study, rice proteins were further hydrolyzed, and rice peptides (RP) with different molecular weights were obtained by ultrafiltration. The effects and possible mechanisms of RP with different molecular weights on the structure, thermal properties, and in vitro digestibility of cooked rice starch were investigated. All peptides slowed the digestion of rice starch in a concentration-dependent manner. A concentration of 10% RP Oryza sativa L.) is a staple food for half of the world’s population, and starch is the main carbohydrate in rice that produces blood glucose1. Starch-based foods are usually consumed after cooking, which rapidly and enzymatically breaks down in the body and causes a rapid rise in blood sugar in a short period2. Reducing the rate and extent of starch hydrolysis can help reduce the risk of diet-related diseases such as Type II diabetes, obesity, and cardiovascular disease3. Previous studies5 have shown that interactions between food components could affect the in vitro digestibility of starch.Rice  are defined as protein fragments that have positive effects on body function and health16. Many studies have found that protein hydrolysates were more effective in hindering starch digestion than intact proteins, and protein hydrolysates hydrolyzed by pepsin and pepsin-pancreatin (60 or 120 min) had different effects on starch digestion12, but the exact reason is unknown. Protein hydrolysates contain many peptide fragments with different molecular weights. Here, we hypothesized that peptide fragments of different molecular weights in protein hydrolysates played different roles in mitigating starch digestion. Therefore, in this study, peptides with different molecular weights in rice protein hydrolysates were obtained by ultrafiltration and their effects on the glucose release rate of gelatinized starch in vitro were explored. The effects of RP with different molecular weights on the digestibility of rice starch were investigated by determining the thermal properties, leached amylose amount of starch, starch structure , and starch-peptide interactions. Data from the current work contribute to further our understanding of the starch-protein interactions and have implications for the preparation of carbohydrate-based foods with low starch digestibility.Rice protein has been validated as a suitable protein source for the prevention of obesity and diabetes17 have found that some grain peptides can bind to amylase and inhibit its activity, which may explain why the RDS content of 10%-RP-starch is lower than that of 5%-RP-starch. Compared to RP1–5 and RP5–10, 10%-RP>10 formed less RDS (45.90 g/100 g) and more RS (36.54 g/100 g). This phenomenon might result from the large steric hindrance of peptide fragments with large molecular weights, which forms a physical barrier to starch and prevents the combination of partial digestive enzymes and starch. This might also result from the large number of amino acids in the peptide fragment with large molecular weight, which formed a strong bond with starch during the process of co-gelatinization, thereby inhibiting its hydrolysis.The in vitro digestion results for rice starch before and after the addition of RP are shown in Table k). The k value reflects the sensitivity of starch samples to amylase hydrolysis. As shown in Table −2 min−1. Except for RP1–5, both RP5–10 and RP>10 significantly decreased the kinetic constant of starch (6.20 × 10−2~8.50 × 10−2 min−1). These results show that both RP5–10 and RP>10 reduced the digestion rate of rice starch, which is consistent with the reduction in RDS content. The complexation of RP1–5 increased the kinetic constant of starch to 9.22 × 10−2 and 13.15 × 10−2 min−1, presumably because the complexation of RP1-5 resulted in the conversion of part of SDS to RS. To verify this conjecture, subsequent studies explored the effects of RP affecting starch digestion in terms of thermal characteristics, leached amylose amount during gelatinization, and starch structure .To further understand the mechanism of starch digestion, the data were fitted to a first-order kinetic equation to obtain the hydrolysis kinetic constant  and enthalpy changes (ΔH) were listed in Table 18. The To, Tp, and Tc values of rice starch were 64.47, 69.73, and 77.13 °C, respectively, and ΔH was 10.03 J/g. The gelatinization temperature and enthalpy of rice starch increased with the addition of peptides with different molecular weights. With an increase in peptide content from 0 to 10%, the gelatinization temperature of starch increased continuously, but the gelatinization enthalpy decreased.The effect of RP with different molecular weights on the thermal properties of starch was determined using differential scanning calorimeter (DSC). The gelatinization temperatures  and 12.47 J/g (10% addition). The results show that, in the presence of RP19. As shown in Fig. 12. The presence of rice proteolytic peptides prevents the diffusion of amylose during gelatinization. Within the range of 0–10% RP addition, the leached amylose amount of the starch/peptide system decreased with an increase in RP content. This may be because of the formation of complexes between amylose and peptide chains through intermolecular hydrogen bonds or hydrophobic interactions20, thereby improving the thermal stability of starch. In addition, the amount of leached amylose decreased in the order RP>10-starch < RP5–10-starch < RP1–5-starch. This is consistent with the thermal stability results (ΔH) obtained from the DSC analysis.During the gelatinization process, a large number of water molecules migrated into the starch granules, resulting in sufficient starch swelling and amylose leaching21. As shown in Fig. Native rice starch generally presents diffraction peaks at 15°, 17°, 18°, 20°, and 23° (2θ), showing a typical A + V-type crystal structure>10) and starch chains promoted the ordered degree of cooked starch and further enhanced the ability of starch granules to resist enzymatic hydrolysis.The addition of different amounts of RP  and molecular weights  increased the RC value of rice starch (5.98–12.46%), showing that RP significantly enhanced the long-range ordered degree of cooked starch. This result can be attributed to the interaction between the side chain groups of RP and starch chains during the co-gelatinization process, which promotes the rearrangement of starch chains. Moreover, with an increase in the molecular weight of RP, the RC value of RP-starch increased gradually. These results indicate that the relatively strong interaction between RP with larger molecular weights  spectra of the cooked starch and RP-starch mixtures are shown in Fig. −1 were used to analyze the effects of RP addition on the short-range ordered structure of rice starch. The bands at 1022 and 995 cm−1 are related to the amorphous material and short-range order of starch. Usually, the ratio of the absorbance values at 995 and 1022 cm−1 (R995/1022) is used to characterize the change in the molecular order of starch25. As shown in Table R995/1022 value of cooked starch was 0.947, and the addition of RP increased the R995/1022 value of starch to 1.007–1.034. The results indicate that the presence of RP promoted the short-range arrangement of starch chains during thermal processing and improved the short-range-ordered structure. When the addition of RP increased from 5 to 10%, the R995/1022 value of the RP-starch mixtures showed an apparent decrease in R995/1022. In other words, the presence of excessive RP inhibited the rearrangement of starch chains during thermal processing. In addition, mixtures of starch and RP with relatively large molecular weights displayed a slight increase in R995/1022. More specifically, RP with large molecular weights were more likely to interact with starch than peptides with small molecular weights; thus, rice starch formed a more ordered short-range structure during the gelatinization process.Deconvoluted FTIR spectra of 1200–900 cm−1, but the scattering intensities on the SAXS curves of different complexes were different. Owing to the different structures of peptides with different molecular weights, the interactions between them and starch were also different, which induced the formation of different aggregated structures12. In the SAXS tests, the scattering peak intensity was determined by the number of well-organized semicrystalline structures and/or the difference in electron density between the crystalline and amorphous lamellae in the starch. The larger the electron density, the higher the intensity of the scattering peak on the SAXS curve26. Therefore, it can be seen that the presence of RP significantly enhanced the scattering peak intensity of rice starch, which resulted from the rearrangement of starch chains in the presence of peptides during hydrothermal treatment and enhanced the electron density difference between the two types of lamellae27. Combined with the XRD analysis, the increase in electron density is associated with an increase in the ordered structure. Meanwhile, the complexation with RP shifted the scattering peak of the starch paste to a lower q position (closer to 0.04 Å−1), showing that the addition of RP increased the size of the aperiodic structure of starch paste. The complex formed by starch and 5% RP>10 had the largest aperiodic structure.The aggregate structures of the rice starch-rice peptide complexes were further analyzed by small-angle X-ray scattering (SAXS). As shown in Fig. q2I(q) vs. q). As shown in Fig. q < 0.1 Å−1, indicating that the starch-peptide complexes had certain aggregation structures28. The intensities of the peaks had a similar trend to that shown in Fig. Furthermore, a Lorentz correction was performed to make the scattering peaks more distinct using a Kratky plot (I–q−α). As shown in Table 29. This is because the migration of water molecules breaks the hydrogen bonds between starch chains and induces the recombination of starch and peptide chains to form new aggregates during the gelatinization process, thus forming a higher-density aggregate structure. In addition, the complexes formed by RP>10 with starch were more compact than those formed by RP1–5 and RP5–10 when the peptide content was 5%. However, when the peptide content was 10%, compared to RP1–5 and RP5–10, RP>10 and rice starch formed a relatively loose aggregate structure. This result also indicates that the structural compactness of the aggregates formed by the peptide and starch was related to the molecular weight and addition amount of RP. In conclusion, the scatterers of RP-starch complexes were denser, and the surface smoothness was higher than that of rice starch. Combined with FTIR analysis, the results suggest that the changes in the fractal structure were related to the hydrogen bond network formed between the starch chains and RP.To investigate the surface smoothness and compactness of the rice starch-peptide structure, the D characteristics of the surface/mass fractal structure were further analyzed according to the scattering power-law equation . Pepsin , pancreatin from porcine pancreas , and glucoamylase  were purchased from Sigma-Aldrich . Glucose oxidase-peroxidase assay kits (GOPOD) were obtained from Leadman Biochemistry . All other chemicals and reagents used were of analytical grade.Rice protein (10 g) was weighed and dissolved in 200 mL of hydrochloric acid water (pH = 1.2) containing pepsin from the porcine gastric mucosa (2000 U/mL). After enzymatic hydrolysis for 120 min, the pH was adjusted to 7 with sodium hydroxide solution (1 mol/L) to inactivate the pepsin. The above enzymatic reactions were all carried out in a water bath at 37 °C with shaking at 150 rpm. The rice protein hydrolysates were then separated and fractionated by polysulfone ultrafiltration membrane with molecular cutoff values of 10, 5, and 1 kDa, successively, to obtain RP with molecular weights of 0–10, 5–10, and 1–5 kDa.1–5-starch, RP5–10-starch, and RP>10-starch.Rice starch  was accurately weighed and 50 mL of distilled water was added to prepare the starch suspension. Starch-based peptides  with different molecular weights  were added to the starch suspension. Then the mixtures were incubated in a water bath at 100 °C and a shear rate of 400 rpm for 30 min. Subsequently, the starch paste was cooled to 25 °C and then freeze-dried for further analyses. The blank group consisted of gelatinized starch without adding peptides. The mixtures were labeled as 0%, 5%, 10% RP7, 4.5 g of pancreatin was evenly dispersed in deionized water (40 mL) and centrifuged at 5000 × g for 10 min. Then, 27 mL of the supernatant was mixed with 3.2 mL of glucoamylase to prepare the enzyme mixtures. Starch sample (~200 mg) was stirred with deionized water (2 mL). Pepsin (20 mg) was added to 4 mL HCl (0.05 mol/L) and stirred evenly. Then the pepsin solution was added into the sample centrifuge tube and shook for 30 min. After pepsin hydrolysis, 10 glass beads (3–4 mm in diameter) and 2 mL sodium acetate buffer  were added to the tube. The samples were placed in a water bath  for 30 min, and the enzyme mixtures (2 mL) were added to the starch samples. At some time interval , the enzymatic hydrolysates (0.1 mL) were removed and placed in 0.9 mL of ethanol solution (90%) to terminate the enzymatic hydrolysis reaction. The glucose content of the starch samples was determined using the GOPOD method. The RDS, SDS, and RS contents were calculated using the following formulas:According to Lu et al.G20: glucose released after 20 min; G120: glucose released after 120 min; Ws: weight of the sample.C∞ refers to the glucose concentration at the end of the reaction, Ct refers to the glucose concentration at time t, and k (min−1) is the reaction rate constant of the starch samples.The in vitro digestibility curve of the starch samples was fitted to the following first-order equation:32. Native starch and starch-peptide mixtures (4.0 mg) and distilled water (8 µL) were added to DSC crucibles. The crucibles were equilibrated at 25 °C overnight. Each sample was heated from 20 to 120 °C at a rate of 10 °C/min. An empty crucible served as the reference.The effects of RP with different molecular weights on the thermal properties of rice starch were investigated using differential scanning calorimetryg). The supernatant (0.5 mL) was then mixed with a sodium hydroxide solution . After incubation for 10 min in boiling water and rapid cooling, acetic acid  and I2-KI aqueous solution  were added. After incubating in the dark for 10 min (25°C), the absorbance values were measured at 620 nm using a UV-Vis spectrophotometer . Then the leached amylose amount was calculated by dividing the amylose content in the supernatant by the original weight of rice starch33.The iodine colorimetric method was used to determine the amount of amylose leached from the starch-peptide mixed system during gelatinization. The native starch or starch-peptide mixed system was incubated at 95 °C for 30 min and then kept at 37 °C for another 20 min. Subsequently, starch paste (1.0 g) was completely dispersed in 5 mL of distilled water and centrifuged for 20 min  was used to determine the effects of RP with different molecular weights on the crystalline and long-ordered structures of rice starch. The samples were scanned from 4 to 40° (2θ) at a scan rate of 5°/min. The relative crystallinity (RC) of the samples was calculated using the MDI Jade 6.0.−1. The FTIR spectra were processed using OMNIC 8.0 through automatic baseline correction, automatic smoothing, and Fourier deconvolution. The deconvolution half-band width was 21 cm−1 and the enhancement factor was 2.1.An ATR-FTIR spectrometer was used to determine the effects of RP with different molecular weights on the short-ordered structure of rice starch. An appropriate amount of the powder sample was taken and evenly distributed in the middle of the ATR accessory sample table. The acquisition range was 4000–400 cm26. Starch was suspended in deionized water at a mass ratio of 1:3 and shaken overnight at 25 °C to reach equilibrium. Before the experiment, the starch slurry was centrifuged for 5 min (5000 × g). The scattering patterns were then obtained using a Xeuss 3.0 C SAXS instrument. The wavelength (λ) was 1.542 nm, and the sample-detector distance was 1069 mm. The exposure time was 60 s and the q range was 0.01–0.2 Å−1.The starch samples were pretreated according to a previously described methodTo further investigate the lamellar structure of the starch samples, SAXS curves were transformed using the one-dimensional linear correlation function, γ(x):p < 0.05) and assessed with SPSS Statistics 20.0 software .The data were presented as mean ± standard deviation, with at least three replicates per experiment. Statistical significance of data was determined with Duncan’s test (


Poorer than expected, living donor liver transplant outcomes are observed after recipient graft artery thrombosis. At grafting, the risk for later thrombosis is high if a dissected hepatic artery is used for standard reconstruction. Surgeon diagnosis of dissection requires nonstandard management with alternative technique in addition to microvascular expertise. Intimal flap repair with standard reconstruction is contingent on basis of a redo anastomosis. It is a suboptimal choice for living donor transplantation. Achieving goal graft arterial perfusion at first revascularization is crucial for superior outcomes. Managing dissection at grafting with nonstandard left gastric artery reconstruction is unreported. Our experience is limited, but this is our preferred alternative technique to standard hepatic artery reconstruction complicated by dissection. Here, we describe our two-case experience with left gastric arterialized grafts for management of dissection. Our living donor graft recipients with alternatively arterialized grafts are now 6- and 2-years posttransplant. Implantation of the right lobe living donor liver graft begins with vascular reconstruction. The arterial anastomosis is last. Standard hepatic artery reconstruction (HAR) receives inflow from the recipient right, left, or proper hepatic artery (HA) to supply the donor artery. The recipient inflow is inspected for good wall integrity, a similar size, and available length to reach the donor artery for tension free anastomosis . The donSurgeon expertise and technique are key for a successful living donor liver transplantation. This is especially true at centers with living donor recipient candidates who are sicker at transplant with comorbid conditions that alone increase the estimated risk of HAT at or after grafting. Poor arterial circulation system health condition is revealing of candidacy and increased estimated HAT risk. Living donor graft recipient history of portal vein thrombosis (PVT) or hepatocellular carcinoma (HCC) is reported to increase HAT risk. A history of diagnostic or treatment mesenteric angiography, especially transarterial catheter embolization (TACE) for treatment of HCC, increases risk for HAT, and some authors advise avoiding endovascular procedures months prior to a recipient's scheduled living donor transplant. Studies report partial or occlusive HAT complicating TACE treatment that may take six months for recanalization –14. In aHere, we describe two cases of recipient HA intimal dissection (ID) diagnosed at HAR. Both recipients had ID with progression proximally without propagation to the left gastric artery (LGA). Therefore, the LGA was the most suitable alternate recipient vessel for primary anastomosis and inflow to the graft artery.A fifty-nine-year-old female with a history of hepatitis C virus (HCV) infection, liver cirrhosis, MELD score of 14, severe portal hypertension, and HCC underwent planned RLDLT. Her HCC history is significant for treatment with TACE using the right posterior HA and the inferior right phrenic artery. She had two prior abdominal surgeries making the recipient operation start more difficult.The donor operation was uncomplicated; the right lobe liver graft was delivered. The back table procedure was significant for a dual arterial blood supply and planned angioplasty. The two donor arteries measured 2 to 3 millimeters (mm) in diameter. After spatulating both lumens, a running 8-0 Prolene suture  was usedAfter hepatectomy, the implantation begins with standard vascular reconstruction of the hepatic vein outflow and portal vein inflow. The initial reperfusion is successful, and HAR begins with preparation of the recipient HA. Diagnosis of ID upon intraluminal inspection complicates and precludes final revascularization completion. The severely dissected HA is unusable and ligated. An alternative inflow technique is chosen for management. We free the LGA along the lesser curve for inspection. It has good arterial wall integrity, the lumen is without dissection, the orifice is 2.0 mm wide, and the length is estimated to be adequate for tension free anastomosis. The LGA mobilization then proceeds proximally until its celiac artery trunk takeoff is reached. Vascular shunts, gastric artery branches, and the left gastric vein are carefully controlled with suture ligature. Perigastric shunts are most prominent, requiring more time than usual for vascular control as dissection is carried into the lesser sac. We follow the gastropancreatic fold to the celiac trunk and inferior extent of our mobilization. Surgeon microvascular expertise is required to complete nonstandard reconstruction. The LGA is spatulated to size-match the graft artery common orifice . PrimaryOn postoperative day (POD) 1, the baseline Doppler ultrasound (US) confirms vascular patency and graft arterial inflow with typical waveforms . HoweverDiagnostic celiac angiography revealed a long segment stenosis at the anastomosis and this was simultaneously treated with balloon angioplasty . The comA sixty-four-year-old female with nonalcoholic steatohepatitis (NASH), decompensated liver cirrhosis with a MELD score of 25, portal hypertension, hepatorenal syndrome requiring dialysis, and HCC undergoes RLDLT. The donor operation and back table procedure proceed uneventfully until the graft is ready for implantation.Grafting in the recipient begins after hepatectomy in similar order as previously described to initial reperfusion. During HAR, the recipient HA is diagnosed with severe ID at intraluminal inspection, andstandard reconstruction is precluded. Following prior initial success, we continue with our preferred alternative strategy for managing the dissection with using the LGA instead. Simply spatulating, the recipient's 2.4 mm LGA matches it to the single 3.2 mm donor artery. Arterial anastomosis is with interrupted 8–0 and 9–0 nylon suture. Initial whole graft arterial perfusion is achieved, and final revascularization is completed. We proceed with biliary reconstruction using Roux-en-Y hepaticojejunostomy creation and end the recipient operation.On POD 1, the baseline Doppler US confirms vascular patency and appropriate waveforms . Acute rLiving donor graft artery diameter of 2 mm or less is used to exclude donor recipient pairs from RLDLT . Now, acDatabase-driven outcome studies report loss of expected posttransplant benefit in living donor recipients with history of graft artery thrombosis, regardless of redo anastomosis and salvage attempt status. Thus, dissection diagnosed at HAR should prohibit standard revascularization technique. Few report their nonstandard management at grafting, and outcomes are unknown. To our knowledge, this is the first report of observed long-term survival in recipients with left gastric arterialized right liver grafts as the alternative to standard HAR during RLDLT.RLDLT rates are rising, our technique is better, but graft HAT continues to challenge outcomes after living donation , 18. It Currently, described alternative techniques for HAR are done after the ischemic injury has occurred. Redo anastomosis at grafting with alternative inflow from the recipient left or right gastric , the rigOne author describes graft salvage in a recipient with TACE treatment-related HA injury and successful GDA reconstruction after redo anastomosis . One repMore distal inflow from the splenic artery or supraceliac aorta requires interposition grafting. Autologous vessel harvest of the sigmoid artery , or the Some assess intimal injury with severity grading at grafting to guide intimal repair. Other studies have also reported tacking or trimming surgical techniques for less severe hepatic artery injuries . AgarwalThe LGA is an attractive option for alternative inflow at reconstruction with a proximal celiac origin. The takeoff from the celiac is hidden from the pathologic cirrhotic environment in vivo, including splanchnic vessel remodeling. The LGA's winding course makes it challenging to access accidentally during scheduled TACE treatments . Its sizThe dissected recipient HA diagnosed at transplant clearly indicates its poor integrity and increased risk for HAT. Our management of ID involves ligating the HA and the immediate use of the LGA for inflow. Our experience with two RL-LDLT recipients supports its suitability and feasibility and, in our opinion, is the best alternate inflow for primary anastomosis. Successful final revascularization, posttransplant graft recipient recovery, and possible long-term survivability are benefits in addition to management dissection at grafting. Both living donor recipients continue to do well with overall survival of greater than 6- and 2-year posttransplant.Achievable expected living donor outcomes with left gastric arterialized grafts support using alternative left gastric artery inflow as the best choice for backup to standard HAR complicated by dissection at grafting.


Correction to: International Journal of Clinical Oncology (2020) 25:1977–1984 10.1007/s10147-020-01736-4In the original publication one of the funding numbers was incorrectly given as: JP19K08170. The correct number is: JP19K08179.The updated Funding statement with correct number is given in this Correction as:Funding This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI .


Nucleic Acids Research, Volume 43, Issue 3, 18 February 2015, Pages 1626–1636, https://doi.org/10.1093/nar/gku1368The Editors were first alerted in early 2020 that the bands in Figure 4C show unusual levels of similarity. The journal and institution investigated the matter at the time and did not find conclusive evidence to support the allegations.When similar allegations were brought to the Editors’ attention again in late 2021, the journal re-opened the investigation. With newer techniques, the journal also identified potential concerns with Figure 2D. While the corresponding author works with the institution where the research was conducted to retrieve the original data, the Editors advise readers to examine the details of this study with particular care.Julian E. Sale, Barry L. StoddardSenior Executive Editors


The HB flour treated at 15% moisture content had a higher storage modulus and loss modulus than native HB flour, indicating that HMT  favored the strengthening of the HB flour gels. X-ray diffraction and Fourier-transform infrared spectroscopy results showed that HMT HB flour retained the characteristics of an A-type crystal structure with an increased orderly structure of starch, while the relative crystallinity could be increased from 28.52% to 41.32%. The aggregation of starch granules and the denaturation of proteins were observed after HMT, with additional breakage of the starch granule surface as the moisture content increased. HMT could increase the resistant starch content from 24.77% to 33.40%, but it also led to an increase in the rapidly digestible starch content to 85.30% with the increase in moisture content and heating temperature. These results might promote the application of HMT technology in modifying HB flour.This study modified native highland barley (HB) flour by heat–moisture treatment (HMT) at different temperatures  and moisture contents . The effects of the treatment on the pasting, thermal, rheological, structural, and morphological properties of the native and HMT HB flour were evaluated. The results showed that HMT at 90 °C and 25% moisture content induced the highest pasting viscosity (3626–5147 cPa) and final viscosity (3734–5384 cPa). In all conditions HMT increased gelatinization temperature ( Hordeum vulgare L. var. nudum Hook. f) is mainly produced in the Qinghai Tibet Plateau and is the main food crop of the Tibetan people. It is widely used in the food industry in the form of alcoholic beverages, pasta products, and baked goods [Highland barley  and Fourier-transform infrared spectroscopy (FTIR) were used to examine the structure of starch and proteins to better understand the effects of different temperatures and moisture content of HMT on HB flour. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to observe the morphology of the flour granules. Additionally, the pasting, thermal, rheological, and digestibility properties of HB flour were determined, which are useful to understand the effect of HMT on the properties of HB flour.HB flour was obtained from Qinghai New Green Health Food Co., Ltd. . The chromatographic grade was required for potassium bromide (KBr). Other reagents used were of analytical grade.n (native HB flour), S90−15, S90−25, S90−35, S110−15, S110−25, S110−35, S130−15, S130−25, and S130−35 .An appropriate amount of distilled water was sprayed evenly and slowly on the surface of the HB flour , and constant stirring was carried out to avoid uneven mixing. The moisture content of the HB flour was adjusted to 15%, 25%, and 35%, respectively, and then equilibrated at a temperature of 4 °C for 24 h in sealed polyethylene bags. The samples were then placed into screw-capped glass containers (500 mL) and heated at 90 °C, 110 °C, and 130 °C for 2 h in a hot-air oven. After HMT, the HB flour was dried at 40 °C for 24 h. The obtained HB flour samples were marked as SThe pasting properties of starch were determined using a rapid visco analyzer  by the method suggested by Feng et al. . We tookThe thermal properties of HB flour were determined using a differential scanning calorimeter  by the method described by Ma et al. . We tookw/w) suspension, placed the suspension on the magnetic stirrer for 1 h, and then took 1 mL of the suspension on the rheometer disk. The rotor was PP35Ti with a gap of 1 mm. A proper amount of silicone oil was applied around the plate and then the plate was covered to prevent the sample from losing water during heating. The steady-state flow test was conducted at a shear rate of 0.1–100 s−1, and the dynamic viscoelastic test was conducted at an angular frequency of 0.1–100 rad/s at 1% strain within the linear viscoelastic region.The rheological properties of HB flour under different HMTs were measured using HAAKE RS6000 rheometer  by the method described by Wang et al. . We prep−1. The OMNIC 8.2 and PeakFit 4.12 software were used to process the experimental data.HB flour and KBr (chromatographic grade) were fully ground in agate mortar at the ratio of 1:99 and then pressed into transparent thin slices. The thin slices were scanned 32 times continuously using an FTIR spectrometer  in the wavelength range of 4000−400 cmHB flour was tested using an X-ray diffractometer  operated at 40 kV and 40 mA, at a scanning angular range of 5° to 40°, which was a function of 0.02° (2θ) at a scanning speed of 2°/min. The diffraction pattern was processed using the MDI Jade 6 software  to analyze the crystal type and relative crystallinity.A scanning electron microscope , under an accelerating voltage of 10.0 kV, was used to examine the granular morphology of HB flour at 1000× magnification. The HB flour was fixed on the sample table with conductive tape, and the microstructure of HB was observed under the accelerating voltage of 10 kV after spraying gold.Starch and protein were stained with fluorescein isothiocyanate (FITC), rhodamine B, respectively. About 60 mg of HB flour was dissolved in 700 μL of deionized water, then 30 μL of FITC (1 mg/mL) and 30 μL of rhodamine B (1 mg/mL) were added. Then, the sample was placed in the dark for 30 min and observed using CLSM . The excitation wavelengths of FITC and rhodamine B were 488 and 543 nm, respectively.0, G20, and G120 represent the amount of glucose released within 0, 20, and 120 min, respectively; and G0 is the amount of glucose in the sample before hydrolysis. TS represents the total starch content. The total starch content of HB flour was analyzed using α-amylase method and Total Starch Assay Kit  [The in vitro digestive characteristics of the samples were determined by the method described by Gu et al. with minor modifications . Each flIreland) .p < 0.05) for all experimental data obtained, and the results are shown as the mean ± standard deviation. The analysis of all the images was conducted using Origin 8.5.All data were analyzed at least in duplicate. The SPSS Statistical 20.0 software was used to conduct one-way analysis of variance and the test for statistical significance , indicating the disintegration and rearrangement of the double-helix structure of starch and the development of crystalline structures [90−15, S90−25, and S110−15 had higher peak viscosity than that of Sn. On the contrary, S90−35, S110−25, S110−35, S130−15, S130−25, and S130−35 had lower peak viscosity than that of Sn. This result indicated that HMT conditions of 90 °C and 15% moisture content caused the disordering of starch. The decrease in peak viscosity might be related to the decrease in the swelling capacity of the starch granules, which was caused by the decomposition and rearrangement of starch after HMT [130−15 did not show a statistically significant difference (p > 0.05) probably due to the lower moisture content during HMT. Pasting occurred only on the surface of the starch granules, and a hard shell was formed, preventing water from entering the interior of the starch granules, thus inhibiting the gelatinization of starch [90−35, S110−25, S110−35, S130−25, and S130−35 was lower than that of Sn. This result might be attributed to the fact that adequate water tended to promote the rearrangement of starch chains, which strengthened the amorphous region of starch and made the breakdown of starch granules difficult [90−15 stayed in the initial stage due to moisture or temperature restrictions, when mainly amylose was leached from the starch granules [The pasting properties of native and HMT HB flour determined using RVA are presented in ructures . The intructures . The HB fter HMT . At 110 f starch . The breifficult . The gelgranules . The decoT), peak temperature (pT), and conclusion temperature (cT) but decreased gelatinization enthalpy (ΔH) than the native counterpart. These changes in gelatinization temperature with increasing moisture content of the HB flour system at the same heating temperature were mainly because the increase in moisture content facilitated the enhancement of the mobility and flexibility of the crystalline and amorphous regions of the starch, promoting intermolecular interactions to produce an ordered structure with higher thermal stability [oT, pT, and cT of HB flour after HMT could be due to the interactions between amylose and amylose, amylose and amylopectin, and amylose and fat molecules, which limited the flexibility of the internal amorphous structure of starch and hindered its swelling [H represents the quality and quantity of crystals in starch granules, which reflects the energy required to destroy the molecular interaction in the starch structure during gelatinization [H did not change significantly with the increasing heating temperature; however, this reduction in ΔH was pronounced when the system moisture content was 35%. The decrease in gelatinization enthalpy indicated that HMT promoted water molecules to enter the crystalline region from the amorphous region and destroyed the hydrogen bond in the starch, leading to the disintegration of the double helix in the crystalline region and the disruption of the amorphous region [The gelatinization parameters of native and HMT HB flour are presented in tability ,27. The swelling . Consequnization . When ths region . This efs region .110−35, S130−25, and S130−35  of all samples was higher than the loss modulus (G″), indicating that these samples had solid-like characteristics, except S S130−35 . In partweakened . Thermalweakened .90−15 was higher than the apparent viscosity of the native HB flour paste. We hypothesized that, with the increase in humidity and heat treatment temperature, the thermal movement of molecules accelerated, the chances of amylose movement in starch increased, the molecular chains entangled with each other, and the apparent viscosity of starch paste also increased.The apparent viscosities of HB flour paste under different HMT conditions decreased sharply first with the increase in the shear rate, and then tended to be flat, showing typical shear thinning and non-Newtonian behaviors . General90−15, S90−25, S110−15, and S130−15 showed similar A-type crystal structures as the native HB flour, indicating that the crystal type did not change. However, the peak at 19.88° of samples S90−35, S110−25, S110−35, S130−25, and S130−35 was higher than that of native HB flour, showing a V-type crystal structure, which represented the interaction between amylose and lipids due to complex formation [90−15, S90−25, S110−15, and S110−25 were higher than those of native HB flour, which indicated that new crystals were formed in the starch after HMT. The decrease in the diffraction peak intensities of samples S130−25 and S130−35 at 14.98° indicated the loss of crystal arrangement. This might be due to the damage of the hydrogen bond, leading to the displacement of adjacent double-helix and incomplete parallel rearrangement [130−25 and S130−35 began to decrease compared with that of the other HMT HB flour. The excessive heat and moisture during HMT caused a reduction in crystallinity. The result suggested that the relative crystallinity in starch granules was destroyed after HMT [The XRDs of HB flour were obtained to examine the effect of HMT on the crystalline structure . As showormation . The morangement . Also, tangement . Howeverfter HMT . The parfter HMT . Besides−1 are summarized in −1 bands were considered as the fingerprints of starch, which reflected the stretching vibration of starch C–C, C–OH, and C–H bonds [−1 were characterized by the structural features of the starch crystalline regions, corresponding to the ordered structure in the starch. The bands at 1022 cm−1 showed the structural characteristics of the amorphous region of starch, which belonged to the random curly structure of starch macromolecules. The absorbance ratio at 1047 cm−1/1022 cm−1 (R1047/1022) reflected the ratio of short-range ordered structures in starch. The higher the ratio, the more the short-range ordered structures in starch. The intensity of HMT HB flour at 1047 cm−1 and 1022 cm−1 decreased compared with that of native HB flour. The decrease in the absorption peak of the HMT HB flour might be attributed to the strengthening of the stretching vibration of the C=O, C–O–C, or C–C bond [1047/1022 of the treated samples was higher compared with that of the native HB flour. This result might be related to the retrogradation of the starch. During starch retrogradation, a part of the double helix structures re-formed by starch chains were arranged in order to form a long-range ordered structure, while another part of the new double helix structures were arranged in order to form a short-range ordered structure. FTIR is very sensitive to the determination of this short-range order structure [1047/1022 of sample S130−15 was lower than that of the native HB flour. This result might be attributed to the fact that HMT led to partial gelatinization of starch, which might destroy the double-helix structures and crystal domains. Moreover, when the sample was heated at high temperatures, the water evaporated and the water content of HB flour decreased, which was not conducive to starch retrogradation.FTIR is another analytical technique used to characterize the ordered structure of starch. The FTIR spectra of native and HMT HB flour are presented in –H bonds . The banC–C bond . After Htructure . The restructure ,42. Howe−1). We determined the proportion of the secondary structure content of HB protein (The secondary structure of proteins is usually studied via amide I corresponding band (1600–1700 cm protein . The HB 90−15, S110−15, and S130−15 showed no apparent change, which indicated that the changes caused by HMT under low-moisture conditions mainly occurred inside the HB starch granules. With the increase in HMT moisture content, the starch granules were destroyed more obviously, and the aggregation of starch granules was observed, which might be related to partial gelatinization. Excessive water in the HMT process caused the swelling and melting of HB starch granules, resulting in the interconnection of starch granules. During drying and crushing, the morphology of starch granules was also destroyed. Heating intensified the swelling and melting of starch granules, and finally made starch granules lose their original identity [The granule morphology of native and HMT HB flour is shown in identity . The suridentity ,46. Besin were 60.35%, 14.87%, and 24.77%, respectively. HB flour treated at 90 °C or 15% moisture content showed no significant change in the RDS content. The RDS content could reach 85.30% when both heating temperature and moisture content were increased. The SDS content increased with increasing moisture content at heating temperatures of 90 °C and 110 °C; however, it increased and then decreased with increasing moisture content at 130 °C. The RS content of HB treated at 15% moisture content increased to 33.40%. The formation of RS after HMT was mainly due to the interactions between starch chains and the rearrangements of starch molecules that could weaken the sensitivity to digestive enzymes [The contents of RDS, SDS, and RS for native and HMT HB flour are presented in  enzymes . HMT als enzymes . The inc enzymes ,49. When enzymes . These cThe pasting, thermal, rheological, and digestibility properties of HB flour after HMT changed significantly compared with that of native HB flour. These changes indicated the interactions between starch chains, interactions between starch and protein, or changes in the internal structure of the starch. The synergistic effect of heat and water caused the starch molecular chains to degrade and the double-helix structure to deconvolve during the HMT process. However, it also promoted the free movement of the starch molecular chains; the starch molecular chains were rearranged and reoriented, making the double-helix structure and the crystalline structure more perfect and increasing the ordered structure of the amorphous crystalline region and raising the RC of HMT HB flour to 41.32%. These changes ultimately improved the thermal stability of HMT HB flour. After the changes observed at 15% moisture content of HB flour, no significant changes were found in the morphology of the starch granules. The viscosity of the low-moisture content of the flour pastes increased. However, as the moisture content increased, the granules aggregated with each other, with tight junctions between the granules, forming agglomerates and damaging the starch surface. Protein aggregation mainly occurs with an increase in temperature. After the aforementioned changes, HMT induced changes in starch structure easily, such that HB starch bound to digestive enzymes, leading to an increase in RDS content. HB flour treated with low-moisture content could not easily bind to digestive enzymes, leading to an increase in the RS content. In general, HMT broadened the scope of the application of HB flour in the food industry, the effect of different HMT conditions on HB flour was variable, and in particular, the effect of moisture differences was the most significant. The conditions of HMT could be adjusted to obtain the desired HB flour properties.


Proteus mirabilis) or particle accumulation, which may compromise stent's drainage performance. Limited research has, however, been conducted to evaluate the relationship between flow dynamics and accumulation of crystals in stents. Here, we employed a full-scale architecture of the urinary system to computationally investigate the flow performance of a ureteric stent and experimentally determine the level of particle accumulation over the stent surface. Particular attention was given to side-holes, as they play a pivotal role in enhancing urinary drainage. Results demonstrated that there exists an inverse correlation between wall shear stress (WSS) and crystal accumulation at side-holes. Specifically, side-holes with greater WSS levels were those characterized by inter-compartmental fluid exchange between the stent and ureter. These “active” side-holes were located either nearby ureteric obstructions or at regions characterized by a physiological constriction of the ureter. Results also revealed that the majority of side-holes (>60%) suffer from low WSS levels and are, thus, prone to crystals accumulation. Moreover, side-holes located toward the proximal region of the ureter presented lower WSS levels compared to more distal ones, thus suffering from greater particle accumulation. Overall, findings corroborate the role of WSS in modulating the localization and extent of particle accumulation in ureteric stents.Ureteric stents are clinically deployed to restore urinary drainage in the presence of ureteric occlusions. They consist of a hollow tube with multiple side-holes that enhance urinary drainage. The stent surface is often subject to encrustation (induced by crystals-forming bacteria such as  The urinary tract is a critical system of the human body, since one of its primary functions is to regulate the composition of body fluids, which involves filtration and elimination of waste products.et al.et al.et al. developed a numerical model to investigate urinary flow in the stented ureter, whereby the ureter geometry was varied to encompass tubular, funnel-shaped, and undulated architectures.et al., in the presence of a ureteric constriction, the role of side-holes was enhanced in close proximity to the constricted region. In two separate CFD studies, they also demonstrated that increasing the number of side-holes throughout the length of the stent resulted in greater urinary drainage (within an undulated ureter model),et al.et al.et al.In 1970, Lykoudis and Rooset al.Proteus mirabilis), but it rather correlates with the physical and chemical characteristics of the local urine environment. It should be noted that numerical CFD models could also be developed to simulate particle dynamics and accumulation in ureteric stents. Upon experimental validation, these models could provide a predictive tool to evaluate particle accumulation in urological devices during pre-clinical development.In these previous models, however, an evaluation of the correlation between stent flow performance and the progression of encrustation or particle accumulation was not carried out. In this regard, a study by Waters et al.et al.et al. investigated the performance of different stent designs  using an in vitro model of the urinary system, comprising a flexible tubing to simulate the ureter and glass vessels to replicate the renal unit and bladder.In addition to mathematical and computational models, experimental systems have also been developed to investigate the flow field in the stented ureter and its relationship with stent encrustation or particle accumulation. Choong Building upon these earlier investigations, we recently developed a microfluidic-based model of the stented and occluded ureter (referred to as “stent-on-chip”).Despite previous research has investigated the mechanistic aspects of encrustation development in stents, there appears to be a limited body of work that has attempted to quantitatively correlate urine flow metrics  with the accumulation of encrusting particles within a full-scale model of the stented urinary tract. Therefore, in the present study, we performed a combination of numerical and experimental simulations to assess whether our previous observations in simplified microfluidic-based models are applicable to a more clinically-relevant, full-scale scenario. This would also allow us to assess whether particle accumulation at side-holes is dependent upon their location in the stented ureter, which could not be fully achieved using a microfluidic-based approach. We specifically investigated whether there exists a correlation between particle accumulation and the magnitude of WSS over the stent surface with a focus on the functionality of side-holes. Findings from this study can further our understanding of flow-mediated mechanisms of stent failure, as well as inform the design of stent architectures with increased lifetime.supplementary material Table S1 for a list of maximum velocity values at these side-holes). These results, thus, likely indicate that, in the case of an unobstructed and stented ureter, distal side-holes of the stent experience a greater degree of inter-compartmental flow exchange compared to the more proximal side-holes. This may be due to a reduction in the diameter of the ureteral lumen in the region closer to the UVJ (and a corresponding increase in the associated hydraulic resistance), which diverts part of the flow from the ureteral lumen into the stent. These findings are also consistent with earlier numerical investigations showing that, in the absence of occlusions of the ureter or stent, the total flow rate partitions between the stent and ureter lumens on the basis of their relative cross-sectional areas.supplementary material Table S2 for a list of numerical WSS values at these side-holes). Results show that the lowest WSS magnitude corresponds to side-holes located in the renal pelvis, ranging between 3.5 × 10−5 and 5.0 × 10−4 Pa (holes 1–4). This appears to support earlier qualitative clinical observations, as well as quantitative analyses on stents retrieved from patients, showing enhanced encrustation over the stent coils.−2 Pa that, in our previous study, was shown to correlate with increased particle accumulation rates.−3 Pa until hole 31 and increased only in the more distal region, ranging between 2.3 × 10−2 and 6.9 × 10−2 Pa (at holes 33–36). The distal coil of the stent also suffered from low WSS levels, as for the proximal coil, which supports observations of enhanced encrustation at both coils of the stent.supplementary material Fig. S1 shows that the maximum, minimum, and mean WSS magnitude over the internal wall of the stent were 1.4 × 10−2, 2.0 × 10−5, and 2.0 × 10−3 Pa, respectively. Taken together, the spatial distribution of the WSS and velocity magnitude confirms that side-holes characterized by inter-compartmental flow exchange (herein referred to as active side-holes) experience greater levels of WSS, coherently with the findings from our previous study using a microfluidic-based model.supplementary material Fig. S2], the maximum, minimum, and mean values were equal to 1.1 × 10−1, 1.2 × 10−6, and 1.3 × 10−2 Pa, respectively. Importantly, values were lower than those observed over the internal wall of the stent, at corresponding locations, suggesting that the external surface of the stent may be more susceptible to particle accumulation. This could be due to changes in the diameter of the ureter lumen along its length (for a constant stent diameter), leading to fluid exchange through side-holes being mainly directed into the stent lumen  to 1.1 × 10−1 Pa (hole 37), the corresponding mean coverage area significantly reduced from 91.5% to 1.1%. Results also indicate that the large majority (>60%) of side-holes likely suffer from significant accumulation of crystals with only a limited number of active side-holes remaining unobstructed.In order to evaluate whether there exists a correlation between WSS and particle accumulation in a full-scale model of the stented ureter, an experiment was conducted employing the ureter model shown in −5, 3.5 × 10−2, and 1.1 × 10−1 Pa. These findings, thus, suggest that the proximal segment of a ureteric stent may be more susceptible to failure due to the accumulation of crystals.μCT) and image segmentation to quantify the volume of stent encrustation.In summary, our findings using a full-scale unobstructed and stented ureter model demonstrate an inverse correlation between the WSS magnitude (determined from CFD simulations) and the extent of particle accumulation. It was also shown that the large majority of side-holes in a stent are inactive and are, thus, prone to particle accumulation, and this is more enhanced for side-holes located toward the proximal region of the stented ureter. These findings concur with recent observations by Zheng and co-authors on stents retrieved from stone patients, which were analyzed using micro-computed tomography  compared to those located at the proximal region. Greater velocity magnitude at side-holes located just before and after the obstruction was also observed and was equal to 27.5 mm/s at hole 11 (before the obstruction) and 22.2 mm/s at hole 22 (after the obstruction). Results, thus, show that there is a greater degree of flow exchange through side-holes located in the vicinity of a ureteric obstruction, allowing the urine to by-pass the source of obstruction, and also in correspondence to the physiological narrowing of the ureter lumen located nearby the UVJ (as for the unobstructed model). These observations are consistent with those reported in earlier modeling studies.The CFD model was employed to investigate the effect of a ureteric obstruction on the WSS field acting over the stent surface. supplementary material Table S5.) As for the unobstructed model, the lowest WSS magnitude was at the lateral walls of side-holes located in the kidney pelvis . The WSS over both the internal wall of the stent and side-holes significantly increased from holes 6 to 18 (located before and after the obstruction) within the range between 5.2 × 10−3 and 1.7 × 10−1 Pa. It then reduced to about 9.5 × 10−3–9.9 × 10−3 Pa until hole 31 and increased only in the more distal region . As for the unobstructed model, the distal coil of the stent suffered from low WSS levels. The box plot in supplementary material Fig. S3 shows that the maximum, minimum, and mean WSS magnitude over the stent internal wall were 1.9 × 10−1, 3.0 × 10−5, and 2.3 × 10−2 Pa, respectively. The WSS and velocity distributions, thus, show that side-holes characterized by inter-compartmental flow exchange (referred to as active side-holes) experience greater levels of WSS, which is coherent with our earlier microfluidic-based findingssupplementary material Fig. S4 shows a box plot to illustrate the distribution of WSS values. The maximum, minimum, and mean WSS were 1.8 × 10−2, 1.9 × 10−6, and 1.3 × 10−1 Pa, respectively. As observed in the unobstructed model, values were lower than those acting over the internal wall of the stent at corresponding locations [see ions see , suggestIn summary, findings obtained using the obstructed model align with those discussed above for the unobstructed model. The primary difference between the two models is in the increased inter-compartmental flow exchange (and corresponding WSS levels) at side-holes located in the close vicinity of the ureteric obstruction. From these findings, it can, therefore, be inferred that—even in the occluded ureter—the majority of side-holes experience low WSS levels and are, thus, likely to suffer from particle accumulation.μCT as a means to quantify encrustation in stents.The study described in this paper presents several limitations relating to the model and methods employed, which are discussed below. Future work should, therefore, assess whether findings are applicable to conditions that are more physiologically and clinically relevant. (a) The ureter model in this study had a straight centerline, and both stent and ureter were axisymmetric. The physiological ureter instead shows some characteristic curvatures with the stent touching the inner ureter wall in multiple regions. These factors may likely influence flow exchange at side-holes. Based on previous modeling studies,Ureteric stents are endourological devices consisting of a hollow tube with multiple side-holes along its length and two J-shaped coils at both ends. The demonstrated function of side-holes is to promote urinary drainage in the case of a ureteric obstruction or other pathological conditions. Despite their proven clinical efficacy, a large proportion of stents suffer from encrustation (often occurring at stent side-holes and coils),in vitro experiments and CFD simulations conducted in this study revealed an inverse correlation between wall shear stress and accumulation of crystals at side-holes of a full-scale stent model. Results also demonstrated that the majority of side-holes (>60%) in a stent suffer from low WSS levels and are, thus, prone to the accumulation of crystals. Moreover, side-holes located toward the proximal region of the ureter present lower WSS levels compared to the more distal ones. Side-holes with greater WSS levels are those characterized by inter-compartmental fluid exchange between the stent and ureter lumens (referred to as active side-holes). These side-holes are located either nearby pathological ureteric obstructions or at regions characterized by a physiological reduction of the ureteral lumen . Previous studies have shown that occlusion of side-holes has only limited impact on renal pressure but also concurred that maintaining side-hole patency is important to facilitate fluid transfer between the stent and ureter lumens in the presence of pathological or physiological constrictions.The combination of Findings from this study, thus, corroborate our earlier microfluidic-based observationsProteus mirabilis), which results in elevated urine pH levels and subsequent formation of crystalline deposits over the stent surface.Encrustation of ureteric stents is a complex and multi-step process. Upon deployment, a stent is rapidly coated with a conditioning layer that comprises different organic molecules (including glycoproteins).et al.,ex vivo pig ureter samples. The ureter diameter in the model decreased from approximately 6.0 to 3.0 mm in the proximal segment  and remained almost constant and equal to approximately 2.5 and 2.4 mm in the middle and distal segments, respectively. It then slightly increased to 2.9 mm at the UVJ. It should be noted that previous studies have indicated that there are no significant age and gender differences in ureteric diameter.The ureter model used herein—in both numerical simulations and experiments—was based on the one developed by Clavica in vitro studies using artificial ureter models.In the occluded model, a 7.5 mm long complete occlusion of the ureteric lumen was designed in the proximal region of the ureter, consistently with previous In vitro experiments were subsequently carried out on this model to analyze the accumulation of crystals at regions of interest along the stent with a particular focus on side-holes. The experimental particle-accumulation data were then correlated with the numerical WSS values. Once this correlation was established using the unoccluded model, the developed numerical platform was employed as a predictive tool to evaluate stent performance in the presence of a proximal occlusion of the ureter lumen.As discussed earlier, a primary aim of the study was to evaluate whether there exists a correlation between wall shear stress (WSS) and particle accumulation in a three-dimensional model of the stented ureter. Computational fluid dynamic (CFD) simulations were initially carried out to investigate the flow field within the unoccluded stented ureter, where the stent architecture was reconstructed from a commercially available double-J stent  characterized by a wall thickness of 0.5 mm, a length of 28.5 mm (excluding coils), an internal diameter of 1.5 mm, and a total number of 42 side-holes with an average spacing between side-holes of about 7.5 mm see . In vitret al.x), as shown in CFD simulations were performed to investigate the flow field within the stented ureter, and the computational model was defined in such a way to replicate the experimental conditions. A computer-aided design (CAD) drawing of the ureter model was initially constructed using Autodesk® Inventor Pro 2018 . The fluidic domain was derived by subtracting the stent model from the urinary system model, which was then converted to a format compatible with ANSYS Workbench 18.1 . In order to spatially resolve the wall shear stress (WSS) distribution over the stent surface, the fluidic domain was meshed using tetrahedral volumes . The minimum and maximum mesh element sizes were set to 0.001 and 0.1 mm (edge length), respectively, based on a compromise between computational cost and solution stability see . The inlμm. A comprehensive physico-chemical characterization of the formed crystals was not carried out in the present study but could be the subject of future investigations. The temperature of the solution during the experiment was kept constant at 37 °C, and the pH was equal to 6.8 . Using a peristaltic pump , a constant average flow rate of 1 ml/min was generated, which delivered the artificial urine from a reservoir into the stented ureter model in a closed loop configuration [see ® Hot Plate Stirrer) in order to maintain a constant temperature and prevent crystals sedimentation. Crystal accumulation in the model was captured by taking optical images with a CCD camera  placed above the model. Images were acquired at fixed locations (defined by the location of side-holes) along the ureter model [as shown in The PDMS ureter model shown in tion see . The resIn this study, both CFD simulations and experiments were performed to investigate the correlation between WSS and particle accumulation within a full-scale stented ureter model. Numerical results were analyzed at two different spatial scales: global  and local [i.e., at side-holes of the stent, from hole 1 to hole 42; see supplementary material for numerical values of the maximum velocity magnitude and mean WSS at selected side-holes (Tables S1–S5) and box plots showing the distribution of WSS values over the internal and external surface of the stent .See the


Total Knee replacement (TKR) is performed to relieve pain and restore both the mechanical axis and joint line whenever indicated. Most of prostheses being used for TKR were manufactured using dimensions from Caucasians' measurements. This study documented the dimensions of distal femora of Nigerians and correlated the dimensions with different TKR prostheses.Fifty-six matured femora were measured. Dimensions of distal femora from other regions were retrieved from published articles. The dimensions of TKR prosthesis were extracted from product monographs. Analyses were done with Microsoft excel 2010  and STATA version 13 . Statistical significance was set at p ≤ 0.05The average Mediolateral dimension was 79.3 ± 4.4 mm. The anterioposterior dimensions of the medial and lateral condyles were 63.7 ± 3.6 mm and 64.9 ± 3.3 mm respectively. There were no significant differences between the left and right distal femur. The average aspect ratio calculated was1.23 ± 0.05. There was a mismatch of aspect ratio of the distal femora and those of the prostheses. Equations that can be useful both clinically and in forensic medicine were generated.This study has provided measurements that can be considered when the designing of a suitable femoral component of total knee prosthesis for Nigerians. This study also provided equations that can be used to estimate the dimensions of the medial and the lateral condyles and femoral length from parts of distal femur in forensic medicine.Self-funded Total knee replacement (TKR) surgery has become a worldwide procedure, performed on patients with chronic moderate to severe pain that is refractory to all non-surgical modalities.Most of these prostheses were manufactured using dimensions from Caucasian femora and tibiae osteometric measurements. Physical morphologies differ among racial groups, and so also bony morphologies. Anthropometric and osteometric studies from different racial groups and regions of the world had shown differences in the dimensions of the distal femoral bones around the world. This study was aimed to document the dimensions of distal femura of Nigerians and to compare these dimensions with the documented dimensions from other regions of the world. It also correlated the dimensions of the distal femura with different femoral components of total knee prostheses that are available. Statistical relationships between parts of distal femora were conducted through Pearson's chi square and regression analysis to devise equations that can be used for estimation of different parts of the bone, either for clinical use or in forensic medicine.Ethical approval was obtained from the Lagos University Teaching Hospital Hospitals' Ethics and Research Committee (HREC)-ADM/DCST/HREC/APP/2389 before the commencement of the study.Maximum Femur length (MFL): the distance from the highest border of femoral head to the lowest border of the medial condyle. Femoral Trochanteric length (TL): the distance from the tip of the greater trochanter of femur to the lowest border of the lateral condyle.Medio-lateral dimension (MLD) of the femoral condyles – the widest dimension between the medial and lateral margins of the femoral condyles. Anterior-Posterior Diameter of Medial Condyle (APDMC) - the maximum anterior-posterior dimension of the medial condyle of the distal femur. Transverse Diameter of Medial Condyle (TDMC) - the maximum medial to lateral dimension of the medial condyle of the distal femur.Anterior-Posterior Distance of Lateral Condyle (APDLC) - the maximum anterior-posterior dimension of the lateral condyle of the distal femur.Transverse Diameter of Lateral Condyle (TDLC) - the maximum medial to lateral dimension of the lateral condyle of the distal femur.Intercondylar notch (IN) – the width of intercondylar notchThe dry femoral bones were retrieved and measured within the Department of Anatomy, College of Medicine, University of Lagos. A total of 61 femoral bones were retrieved but five were excluded because they were deformed and distorted. Fifty-six non-sexed, nonpaired, adult dry femoral bones were measured. They were sorted into laterality resulting in 27 right and 29 left femoral bones. Each of the bone was given an alphanumerical identity and labeled. The femoral bones with right laterality were labeled from R1 to R27 while those of the left laterality were labeled L1 to L29. Measurements were done with the osteometric board and digital vernier calipers. All measurements were done twice, and the average of the values was documented and used for the analysis. The dimensions measured were:The aspect ratios of the femora were calculated by dividing the Medio-lateral dimension (MLD) of the femoral condyles with Anterior-Posterior Dimension of Lateral Condyle (APDLC). The dimensions of the femoral components of the total knee prostheses implants that were used in this study were obtained from their product monographs. Analyses were done with Microsoft Excel 2010  and STATA version 13 . Mean and standard deviation were documented. Pearson's Chi square and regression analyses were done for associations. Statistical significance was set at p ≤ 0.05Fifty- six (56) made up of 27 bones with right laterality and 29 bones of left laterality were measured. The average MFL for both sides were 47.9 ±2.1 cm which ranged from 42.1 cm to 51.4cm. The average distal femoral MLD dimension was 79.3 ± 4.4cm (ranged from 65.9cm to 90.4cm) and the APDMC and APDLC were 63.7 ± 3.6cm (ranged from 56.4cm to 71.8cm) and 64.9 ± 3.3cm (ranged from 57.5cm to 71.6cm) respectively. Regression analysis revealed significant statistical relationships between the lengths of the femur and parts that made up the distal femur.  & 5 TherThe scatter plot of the MLD and APDLC of the specimens and the corresponding dimensions of the femoral components of the total knee replacement available were plotted.  The measThe aspect ratios of the distal femora were plotted against the aspect ratios of the femoral prostheses.  there waThe average dimensions of the distal femur in Nigerians were documented in this study. The average distal femoral MLD dimension was 79.3 ± 4.4 mm and the anteroposterior dimensions of the medial APDMC and lateral APDLC condyles were 63.7 ± 3.6 mm and 64.9 ± 3.3 mm respectively. The dimensions of the MLD and APD were also similar to the values documented by Thilak et alThe scatter plot of the MLD and APDLC of the distal femora specimens and the corresponding dimensions of the femoral components of the total knee replacement revealed that measured values of the distal femora were smaller and mostly concentrated within a particular range. The trend lines of the dimensions of the prostheses were steeper compared with the gentle slope of the distal femora. This implies that the increment in dimensions of the femoral prostheses from a lower size to the next available size was larger (both in APDLC and MLD dimensions) than the natural increment observed in the distal femora. This could be a source of mismatch during total knee replacement. It is therefore important for implants manufacturers to review available implants to produce implants that will fit our population.The aspect ratios of the distal femora plotted against the aspect ratios of the femoral prostheses showed no overlapping between the aspect ratios. The aspect ratios of the Caucasians were used to produce these prostheses and may account for the difference. More so, the trend line of the distal femur showed a gentle negative slope. Almost all the trend lines for the aspect ratios of the prostheses were without or almost without a gradient except the trend line of the DuraconR prosthesis with a similar trend like the aspect ratios of the distal femora.The gentle negative slope of the distal femora implied aspect ratios that decreased as the dimensions were getting larger. It must be stated that one of the draw-back of this study was the sample size, which was quite small.The study generated equations that can be used clinically as part of pre-operative planning for total knee replacement or in forensic medicine for identification purposes. Some of the equations includedEquations to estimate the femoral condyles- APDMC = 21.2 + 0.54*MLD- TDMC = 2.45+ 0.27*MLD- APDLC = 23.3 + 0.52*MLD- TDLC = 0.53 + 0.33*MLDThe dimensions of the femoral condyles can be estimated from the mediolateral dimension (MLD). MLD can be measured clinically- APDMC = 29.8 + 0.074*TL- APDLC = 33.2 + 0.069*TLb. Equations to estimate the maximum femoral length (in forensic medicine)- MFL = 32.72 + 0.16*MLD- MFL = 34.3 + 0.22*APDMC- MFL = 29.82 + 0.28*APDLC- MFL = 3.1 + 0.98*TLThe APDMC and APDLC dimensions can also be estimated from TL, which can also be measured clinically, from the tip of the greater trochanter to the lateral knee joint. These equations are:This study has documented average dimensions of the distal femora in Nigerians. It also provided data that can be considered when designing a suitable femoral component of a total knee prosthesis for Nigerians. This study provided equations that can be used to estimate the dimensions of the medial and the lateral condyles and femoral length in forensic medicine.


The SSIIa gene is responsible for the genetic control of gelatinization temperature (GT). The combined effects of BEIIb and SSIIa alleles on the AC, fine structures, and physicochemical properties of starches from 12 rice accessions including 10 recombinant inbred lines (RIL) and their two parents were examined in this study. Under the active BEIIb background, starches with the SSIIa-GC allele showed a higher GT than those with the SSIIa-TT allele, resulting from a lower proportion of A chain and a larger proportion of B1 chains in the amylopectin of SSIIa-GC. However, starch with the BEIIb mutant allele (be2b) in combination with any SSIIa genotype displayed more amylose long chains, higher amylose content, B2 and B3 chains, and molecular order, but smaller relative crystallinity and proportion of amylopectin A and B1 chains than those with BEIIb, leading to a higher GT and lower paste viscosities. These results suggest that BEIIb is more important in determining the structural and physicochemical properties than SSIIa. These results provide additional insights into the structure-function relationship in indica rice rather than that in japonica rice and are useful for breeding rice with high amylose content and high resistant starch.Starch branching enzyme IIb (BEIIb) and soluble starch synthase IIa (SSIIa) play important roles in starch biosynthesis in cereals. Deficiency in the BEIIb gene produces the  Waxy (Wx) locus [The emergence of hybrid rice technology has greatly increased the yield potential of rice and solved the problem of food shortages. However, people’s demand for a better life and tastier rice is increasing with the gradual improvement of people’s living standards. Starch is the most important element of rice, accounting for about 90% of the milled rice on a dry weight basis, and its physicochemical properties mainly determine rice cooking and eating quality (CEQ) ,2. Starcx) locus . The synx) locus .Wx and SSIIa is well known and has great effects on the improvement in grain quality since they are responsible for the genetic basis of amylose content (AC) and gelatinization temperature (GT), respectively [GTAT/ AGTTAT) at 5′ splice site of the first intron of Wx pre-mRNA can differentiate indica type aWx (G SNP) and japonica type bWx (T SNP) alleles [Wx gene can be used to classify rice with different AC classes, which can explain >75% of the variation in AC among various rice accessions [SSIIa have been discovered to affect the GT [Natural variations in starch biosynthesizing genes are responsible for the different starch physicochemical properties and CEQ. Although many genes involved in starch biosynthesis have natural variations, the variation in ectively . A G/T s alleles . A simplcessions ,9,10. Twt the GT ,12,13. Tt the GT ,15,16.waxy or glutinous rice with a characteristic of <2% AC is derived from a common mutation of the Wx gene. The knocking out of the Wx gene with physical and chemical mutagenesis, or a new tool such as the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein-9 (Cas9) can easily produce the waxy mutant [amylose extender (ae) mutant is derived from a defect in the BEIIb gene [ae starch is a kind of resistant-starch resource, which can offer a wide array of health benefits to humans [Mutations in the starch biosynthesizing genes may lead to enzyme deficiency and starches with altered structure and functionality. The y mutant . The amyIIb gene ,19 that IIb gene . In addiIIb gene . Therefoo humans .ae and wx/ae double mutant starches display no difference in the chain-length distribution (CLD) of amylopectin and morphology of the starch granule, but wx/ae starch showed a higher pasting temperature and higher peak viscosity. The role of the Wx and SSIIa combination on the amylose fine structure was reported by Wang et al. [Wx SNPs can affect AC but they are unable to alter the CLD of both amylopectin and amylose. Itoh, et al. [SSIIa from an indica rice cultivar to an ae mutant, which led to a higher proportion of amylopectin chains with DP11–18 than those in be2b, and the introduction of Wx from an indica rice cultivar significantly increased AC in the endosperm starch. Ida et al. [ss2a/be2b mutant were significantly higher than those in the be2b single mutant. Although previous studies have reported the effects of allele combinations of different genes on physical and chemical properties and amylopectin chain length distribution (CLD) [SSIIa and BEIIb on the AC, starch fine structure, crystal structure, and functional characteristics of indica rice starch.The combination of alleles of different starch biosynthesizing genes has been reported to change the physicochemical properties and starch fine structures. For example, Kubo et al.  indicateg et al. , who fou, et al.  introduca et al.  found thon (CLD) ,22,24, mBEIIb and SSIIa genotypes. Indica rice recombinant inbred lines (RIL) with different BEIIb and SSIIa genotypes were produced by cross-breeding assisted with a selection of molecular markers. The objective of this study is to characterize the structural and functional properties of the RILs and elucidate the effects of BEIIb and SSIIa genotype combinations on the structure-function relations of starch.In this study, we hypothesize that the starch structure-function relationship would be altered in different combinations of indica rice cultivar Longtefu B (LTFB) with SSIIa allele-TT/BEIIb genotype and an amylose extender (ae) mutant BP577 with SSIIa allele-GC/be2b genotype was crossed to obtain F1. Each F2 breeding line was advanced by the single seed descent method to generate a recombinant inbred line (RIL) population. Molecular markers were applied in the F4 generation to select the recombination of SSIIa and BEIIb alleles. Ten breeding lines (BL01~BL10) were selected and advanced to F7. In this study, a completely randomized design with two replications was conducted at the Zhejiang University farm, Hangzhou, China. Mature seeds were harvested in late September.An Wx allele, the microsatellites (CT)n were amplified with a primer, as described in Bao et al. [The leaves harvested from seedlings of each RIL were used for genomic DNA isolation with the CTAB method of Doyle . The geno et al. .Starch was extracted from rice flour following the methods of Syahariza, Li, and Hasjim . The purX, denoted as wde(logX), obtained from the DRI signal, was converted to the corresponding number distribution Nde(X) by wde(logX) = X2Nde(X) (a relation that holds only for linear polymers) [AP1X and AP2X are the DP value at the peak 1 & 2 of amylopectin, and AMX represents the DP value at the amylose peak, AP2/hAP1h is the ratio of the peak heights of amylopectin, and AMh is the peak height of amylose.The chain-length distributions (CLDs) of the debranched starch were separated with the SEC using an LC20AD system  equipped with three columns   in sequence according to the method described in Zhang et al. . The weiolymers) . XAP1 anX < 10,000 by the whole area, i.e., the whole weight distribution of the debranched starch molecules.The AC was calculated from the SEC weight distributions of debranched starch by dividing the area under the curve (AUC) for 100 < The debranched starches were labeled with the fluorescence 8-amino-1,3,6-pyrenetrisulfonic acid according to the method of Wu et al. . The amyθ from 3° to 40° with a step of 0.05°. The relative crystallinity  was calculated using Origin software  following the method of Zhang, et al. [X-ray diffraction analysis of the starch granules was conducted on an X-ray powder diffractometer , at a voltage of 40 kV and a current of 40 mA. The starches were tightly packed into the glass sample holder, and data were collected over an angular range of 2, et al. .−1 with a resolution of 4 cm−1. The relative absorbances at 1045, 1022, and 995 cm−1, which represent the ordered regions, amorphous regions, and the bonding in the hydrated carbohydrate helices in starch, respectively [−1 ratio represents the degree of order in the starch external region, while the 1022/995 cm−1 ratio represents an index for the ratio of amorphous to carbohydrate structure starch.A Varian 7000 Fourier transform infrared spectrometer equipped with a DTGS detector and an ATR single-reflectance cell containing a germanium crystal (45° incidence angle)  was used to measure the short-range ordered structure of the starch granules . The samectively , were exThree grams of rice flour was weighed in an aluminum can and then mixed with deionized water (25 g). The pasting properties were analyzed using a rapid visco-analyzer (RVA)  according to the method of Bao et al. . The idlo), peak (Tp), conclusion (Tc) temperature, and enthalpy (ΔH) of gelatinization were calculated automatically using the Universal Analysis 2000 program (Version 4.4A) software .The thermal (gelatinization) characteristics were measured by using a DSC 2920 thermal analyzer  equipped with DSC standard and dual sample cells, according to the method of Bao et al. . Rice flp < 0.05). Clustering analysis of the genotypes based on the structural and physicochemical properties was conducted using IBM SPSS Statistics 25.All analyses were carried out in duplicate and reported as mean ± SD. Analysis of variance (ANOVA) was conducted in SAS 9.0  with Tukey’s pairwise comparisons (BEIIb. The PCR products of BEIIb or be2b were digested with MboI restriction endonuclease (BEIIb or be2b (mutant) allele. Two pairs of primers facing each other were used in a PCR reaction to amplify SSIIa GC/TT alleles (SSIIa-GC (GC) and SSIIa-TT (TT) alleles. To confirm whether all rice carried the same Wx allele, the (CT)n microsatellites were amplified, displaying the same allele (amylose extender (ae) mutant, so it has the be2b allele and also harbors the SSIIa–GC allele  was significantly higher than those with the BEIIb allele (26.82%~28.26%). However, there is no significant difference between the SSIIa GC and TT alleles  of 15°, 17°, 18°, and 23° in common with those of common rice and other cereal starches [be2b allele had a C-type pattern with a strong peak at 17° and weak peaks at 5°, 15°, and 23°, which may be derived from incomplete suppression of the expression of the BEIIb [BEIIb mutant were inhibited from elongation due to the action of the SSIIa isoform. It is also suggested that SSIIa forms a heteromeric protein complex with SSI and BEIIb in cereal endosperm to synthesize amylopectin [The XRD patterns of the two parents and 10 breeding lines are shown in starches ,37. The starches ,24,37,40lopectin ,41,42. T−1 region of the FTIR spectra of the two parents and their breeding lines is shown in −1 ratio displayed significant differences between the rice materials, ranging from 0.634 (BL10) to 0.812 (BL01)  . The higity data .o), peak temperature (Tp), conclusion temperature (Tc), and the enthalpy of gelatinization (ΔH) of all the rice starches are presented in BEIIb allele, it is well known that rice accessions with the GC allele had intermediate or high GT, while those with the TT SNP had a low GT [p of around 65 °C , all the rice samples generally had large PV, CPV, SB, CS, and small BD which are characteristic of high amylose rice. Furthermore, these rice samples with the GC allele (BL06 and 07) had larger PV and BD than those with the TT allele. Since SSIIa is generally responsible for the genetic basis of GT, the difference between the RVA profiles might not reflect the allele difference. Among the rice with the be2b allele, all the rice materials had small PV, BD, SB, and CS, showing resistance to heat shearing. This is because high amylose may restrict the swelling of starch during heating. Similarly, rice samples with different GC and TT alleles did not show different RVA profiles , rice materials with different GC/TT were revealed in two subgroups, and the parent LTFB was grouped with the BL08-10, indicating that the group represented the low GT with the TT allele. However, for the be2b-related group, although two or three subgroups could be revealed, they did not follow the GC/TT groups. Instead, parent BP577, BL01, and BL02 formed a subgroup, BL03 and BL05 formed a second subgroup, and BL04 itself formed a third subgroup  and also had a positive correlation with AP1, XAP2, XAM, hAP2/AP1X, fb2, and fb3, but a negative correlation with fa and fb1, suggesting the longer B chains and longer amylose chains led to a higher AC. AC is synthesized by the action of GBSS encoded by the Wx gene. All the rice materials contain the same Wx allele , but had a positive correlation with AP1, XAP2, XAM, hAP2/AP1X, fb3, and −1 ratio, but a positive correlation with the 1022/995 cm−1 ratio , indicating these starches are different from common starches.GT is genetically controlled by SSIIa, whose function is to elongate the short A and B1 chains of amylopectin with DP < 10 to form long B1 chains . To, Tp,−1 ratio . This re content . In thisAP1, XAP2, XAM, hAP2/AP1X, fb2, fb3, The PV, HPV, CPV, SB, and CS had a negative correlation with with fb1 , which iBEIIb and SSIIa alleles. The BEIIb-deficient mutant starches had a higher AC with more amylose long chains, a larger amount of amylopectin long B chains, and a higher degree of molecular order, but a smaller amount of short chains and smaller RC, leading to a higher GT and lower ΔH and pasting viscosities. Therefore, BEIIb is more important in determining the structural and physicochemical properties than SSIIa. The combination of be2b/GC and be2b/TT showed no significant difference, suggesting that the function of SSIIa was not important in the be2b allele background. The resistant starch content was not measured in this study, but the fact that the ae mutant has high resistant-starch content is well known. In the near future, the breeding lines in the background be2b will be used for breeding new rice varieties with high resistant-starch content, which will benefit human health.In conclusion, the structural and physicochemical properties of rice breeding lines changed with different


Of 151 transplanted patients, 34% experienced EAD and had worse 1- and 3-year-survival, compared with those who did not face EAD (NoEAD), 96% and 96% for EAD vs. 89% and 71% for NoEAD, respectively (p = 0.03). None of the donor/graft characteristics was associated with EAD even if the graft was moderately steatotic or fibrotic or from an aged donor. For the first time, this study shows that macrovesicular steatosis >30% might be a warning factor involved in the risk of graft loss or a cause of graft discard after the MP treatment. On the other hand, the MP seems to be useful in reducing the donor and graft weight in the development of EAD.Machine perfusion (MP) has been shown worldwide to offer many advantages in liver transplantation, but it still has some gray areas. The purpose of the study is to evaluate the donor risk factors of grafts, perfused with any MP, that might predict an ineffective MP setting and those would trigger post-transplant early allograft dysfunction (EAD). Data from donors of all MP-perfused grafts at six liver transplant centers have been analyzed, whether implanted or discarded after perfusion. The first endpoint was the negative events after perfusion (NegE), which is the number of grafts discarded plus those that were implanted but lost after the transplant. A risk factor analysis for NegE was performed and marginal grafts for MP were identified. Finally, the risk of EAD was analyzed, considering only implanted grafts. From 2015 to September 2019, 158 grafts were perfused with MP: 151 grafts were implanted and 7 were discarded after the MP phase because they did not reach viability criteria. Of 151, 15 grafts were lost after transplant, so the NegE group consisted of 22 donors. In univariate analysis, the donor risk index >1.7, the presence of hypertension in the medical history, static cold ischemia time, and the moderate or severe macrovesicular steatosis were the significant factors for NegE. Multivariate analysis confirmed that macrosteatosis >30% was an independent risk factor for NegE (odd ratio 5.643,  Extended criteria grafts are increasingly used worldwide , 2 to fiex vivo. The results are positive even with nonstandard grafts  d grafts . The fird grafts . In ItalHowever, the MP is not immune to drawbacks: costs are still high for the most widely used modalities, namely HMP and normothermic MP (NMP). In Italy, a disposable kit for perfusing a graft costs about 8,000–12,000 euros for the Liver Assist Machine . The NMP needs packed red cells, drugs, and nutritional solution . All devData of MP grafts, either by HMP or NMP, were collected from six Italian liver transplant centers. The study period was from the first Italian case in 2015 up to September 2019. Both donation after brain death (DBD) and donation after circulatory death (DCD) were considered.The first endpoint was to identify the risk factors predicting negative events (NegE). NegE were defined as all perfused but discarded livers and implanted grafts lost after LT. Positive events (PosE) included all successfully transplanted perfused grafts .Extended criteria grafts were arbitrarily defined as grafts from donors with at least two of the worst interquartile of pretransplant characteristics if the graft was from DBD, or one of them if from DCD. This way was done to better identify the “very extended” criteria in our specific donor population and not considering “extended” criteria from the literature. MP was used mainly for grafts with some very warning characteristics that could rarely be reported in the literature. Therefore, published definitions of marginal grafts would not have fit for this study of “very marginal” donors. This group of grafts was, therefore, analyzed if significant for NegE with the aim of defining a new concept of “marginal graft” in the MP era.The second endpoint was to analyze the role of MP in terms of post-transplant EAD rates. First, the impact of EAD on patient and graft survival was studied. Finally, donor- and graft-related risk factors of EAD were investigated.National guidelines for liver transplantation were used for organ donation eligibility and graft allocation. All surgeries were performed using a piggyback technique for caval reconstruction.Histology features were reported according to the grade of macro- and microvesicular steatosis and greater or less than 30%. Ishak fibrosis was cited if greater than grade 1.The donor risk index was calculated according to the formula published by Feng et al. . EAD wasex situ, most in hypothermic mode and a few of them in normothermic setting, for at least 2 h until the recipient hepatectomy was completed. For both modalities, the vessel perfusion was used at manually controlled pressure and flow. The arterial circuit was set up at 40 mmHg with pulsatile perfusion pressure, 60 beats for minute with 1,000 ml/min of flow. Portal flow was set up at 6 mmHg continue perfusion pressure with flow up to 2 L/min. University of Wisconsin MP fluid was used for HMP. The grafts were flushed with saline solution or Celsior fluid and implanted after the HMP treatment.For DBD grafts, every center followed its own policy in deciding whether to store the graft in the ice box until the implantation (S-CIT) or to use the MP. For MP grafts, the device was set up at the transplant center after a variable period of S-CIT. All of the grafts were oxygenated ex vivo MP. Only grafts with macrosteatosis ≤30% and Ishak grade ≤1 at biopsy were considered available. National criteria for proceeding with liver retrieval on NRP were ALT <1,000 UI/L (or negative transaminase trend) and negative lactate trend  followed by te trend .Donor and graft characteristics are listed in t test, and variables with skewed distribution with the Mann–Whitney test. Qualitative variables were analyzed with the chi-square or Fischer test, as appropriate. Multivariable logistic regression analysis was performed considering only variables significant to univariate analysis. Odds ratio (OR) and 95% confidence intervals (95% CI) were reported. The Kaplan–Meier modality was used for survival curves, and the log-rank test was used for survival comparison. Results were considered significant if the p-value was <0.05. IBM® SPSS (version 24) was used for statistical analysesThe Kolmogorov–Smirnov test was performed to evaluate the normal distribution of continuous variables. Continuous variables with parametric distribution were compared with the nonpaired sample Student’s From 2015 to September 2019, 158 grafts were retrieved and preserved with an MP. A total of 151 livers were implanted; therefore, seven grafts were not used, with a discard rate of only 4.4% after MP treatment. After transplant, 15/151 (9.9%) patients lost their grafts . Therefore, the NegE group consisted of 22/158 (13.9%) grafts. The PosE group consisted of 136/158 (86.1%) patients.n = 144, 91.1%).DCD grafts were 53 from Maastricht 3 (33.54%) and 1 from Maastricht 2. Most of the livers were perfused with HMP (The median stay in the intensive care unit (ICU) was 3 days (range = 1–24). Forty-seven (29.7%) donors had deranged transaminases at the time of procurement. The median donor ALT peak value was 49 IU/L , and the median AST peak value was 55 IU/L . Fourteen (8.9%) donors had a peak value of total bilirubin >1.2 mg/dl at the time of procurement, with a median value observed in the whole population of 0.6 mg/dl (range = 0.1–5.0). One hundred forty-three (90.5%) donors had a DRI >1.7 . S-CIT lasted longer than 7 h in 41 cases (25.9%). Notably, one graft had up to 20 h of storage in ice before MP .p = 0.01), and the groups had different distribution among the interquartiles. When donor medical history was available, the rates of diabetes, dyslipidemia, cytomegalovirus IgG, and anti-HBc positivity were not statistically different between the groups. There were significantly more hypertensive donors in NegE ( in NegE .p = 0.03). Median S-CIT was similar in both groups, with a different interquartile distribution (p = 0.01) (p = 0.02)  . Biopsy  = 0.02) .p = 0.023) , only macrosteatosis >30% was found to be a significant variable, with an odds ratio of 5.643  .Separate analysis was performed for the two subgroups of NegE. p-value of 0.01) defined as standard criteria donors (SCDs).Extended criteria donors (ECDs) were defined as all donors with at least of two of the following characteristics: DCD, age > 65 years, BMI > 30, ICU stay >6 days, DRI > 1.9, ALT > 104 UI, AST > 134 UI, bilirubin > 1.2 mg/dl, S-CIT longer than 8 h, and macrosteatosis > 30% . Of the p = 0.03), respectively. A similar difference was noted between grafts survival curves: at 1 and 3 years 93% and 91% for noEAD and 86% and 79% for EAD patients (p = 0.03) . Notably, EAD had a significant impact on patient survival at 1 and 3 years post liver transplant: 96% and 96% for patients who did not develop an EAD (noEAD) vs. 89% and 91% for EAD patients ( = 0.03) .Characteristics of the recipients are shown in This is the first study exploring the possible pretransplant risk factors to discard a liver graft after MP preservation or failure after implantation to help the surgeon determine whether it is worthwhile to set up an MP.p = 0.14). There are no data comparing the sequential of NRP and ex situ preservation vs. NRP alone, but in an evidence-based position paper recently published by the Italian Society of Organ Transplantation (SITO), sequential use in DCD was recommended . Steatotic livers have been often linked to a worse post-transplant outcome, particularly post reperfusion syndrome, EAD, renal injury, and postoperative mortality . As clarified above, NegE includes MP-perfused and discarded plus grafts perfused, implanted, and lost after the transplant. In the literature, graft loss is usually considered as an event to define marginal donors   .EAD had a significant impact on patient and graft survival as shown in This study has several limitations. (A) It is a multicenter study; therefore, it includes an heterogeneous graft population. Each center follows its own criteria regarding standard ice storage and MP, which were not considered in this analysis. In some cases, MP was used to manage logistical issues , rather than to test marginal grafts, and these data were not traceable in the available database. These grafts might have been stored in the ice box. (B) The analysis included both HMP and NMP and this could cause a bias. In any case, so far there is no solid result that clearly endorses the superiority of one of these tools. (C) Most of the recipients have a low MELD score  and this could cause a selection bias for EAD analysis. (D) Finally, the samples of the main endpoint are small, so a larger population study is needed.In conclusion, today MP is an important device to expand the donor pool in the era of very extended marginal grafts. For the first time in the literature, this study showed that a moderate or severe grade of macrosteatosis could be a factor of graft loss post-transplant, even by perfusing these grafts into an MP, or a reason to discard grafts after MP treatment. Macrosteatosis could temper the widespread efficacy of MP, when the rest of the donor bounders have been successfully pushed. Definitely a new donor scoring system is needed in the era of MP grafts.Finally, MP has been useful in combating the well-known donor and graft risk factors for EAD. This potentially life-threatening complication is still reported; therefore, it is necessary to explore what other factors may trigger it.