IntroductionThe Flixene™ vascular graft  has emerged as a new generation synthetic graft, which allows for early needling for haemodialysis. Most studies have shown satisfactory early results. The aim of this study is to report on long-term outcomes of the Flixene graft over eight years in a cohort of patients.MethodsFrom February 2011 to September 2019, 61 patients had 64 arteriovenous graft fistulas (AVGs) by using the Flixene grafts. The median patient age was 67 years; the male to female ratio was 30:31. Diabetes was the reason for the majority of kidney failures (57%). The configuration of the grafts used was mostly upper arm straight AVG. The primary patency rate, secondary patency rate, and surgical complications were assessed.ResultsIn a median follow-up of three years (interquartile range (IQR): 2 - 6), 36 of the AVGs required a fistulogram. Venous side stenosis was the most common cause of high venous pressure or AVG occlusion in 97%. The one-year primary patency rate was 30%. The secondary patency rate was 94.8%, 83.7%, and 77.7% at one, three, and five years, respectively. The longest functional AVG was observed for up to seven years.ConclusionsThis study has shown satisfactory long-term results of the Flixene graft used for hemodialysis. The Flixene graft could be needled within 72 hours without increased complications, which allows the creation of an AVG under an emergency setting to avoid the placement of a central venous catheter (CVC). This strategy should be advocated in future clinical practice. A native arteriovenous fistula (AVF) is the permanent access of choice in patients who require haemodialysis (HD) as it has been associated with a lower risk of infection, less thrombosis, and a better patency rate -4. UnforOver the last decade, new generation vascular grafts have been developed that could be needled earlier within 72 hours after placement, such as the Flixene™ graft  , potentiThe study was conducted by retrospectively analyzing the prospectively collected data in a single-center involving a cohort of patients who required a synthetic vascular graft for the creation of haemodialysis access between February 2011 and September 2019 at Sir Charles Gairdner Hospital, Perth, Australia. The study was approved as a Quality Improvement Project (#QI 16136) by the local Institutional Human Research Ethics Committee of Sir Charles Gairdner Hospital. The indication for using a synthetic vascular graft was that the patients were lacking suitable veins for the creation of a native AVF. The assessment included clinical examination, Doppler ultrasound of the vein mapping, and venography.Surgical techniqueFor an AVG at the Upper Arm (Straight Configuration)A small incision was made at the elbow for brachial artery dissection. A second small incision was made in the axillary area for axillary vein access. A Flixene graft was placed subcutaneously at the upper arm by using a slightly curved tunneler . The venous end of the graft was anastomosed first in an end-to-side fashion to the axillary vein, then the arterial end was anastomosed in the same fashion to the side of the brachial artery by using 6/0 Prolene®. The graft was flushed with heparinised normal saline  prior to the completion of each anastomosis.For the Creation of AVG at the Thigh (Loop Configuration)An incision was made in the groin area parallel to the inguinal ligament and two finger's breadth inferior. The femoral artery and femoral vein were dissected and looped. Firstly, one end of the Flixene graft was anastomosed to the side of the femoral vein, then the graft was tunneled in a loop fashion by a curved tunneler with a small incision made at the apex of the loop at mid-thigh. The other end of the graft was brought to the same groin incision by the curved tunneller meeting the femoral artery and was anastomosed to the side of the femoral artery using 6-0 Prolene. The graft was flushed with heparinised normal saline  prior to the completion of each anastomosis.The blood flow in the AVG was assessed by a handheld Doppler device at the end of the procedure. Overall, two Flixene intraluminal flow guard (IFG) grafts (4-7 mm x 40 cm) and 63 Flixene grafts (6 mm x 40 cm) were used in this cohort. One Flixene IFG graft was replaced by a Flixene graft during the procedure immediately following the completion of anastomoses for concerns of poor flow.Surveillance programAll patients were routinely educated for self-monitoring of AVG function daily. The functional status of the AVG was assessed when on HD. The vascular access nurse specialist collaborated with the dialysis nurses to monitor the AVG through dialysis outcomes and clinical assessment. Monitoring through dialysis outcomes included measurement of flows and pressures, particular attention to the trends in venous pressure, clearances as measured by online KT/V, flow monitoring, and recirculation studies. Clinical assessment included prolonged needle site bleeding time, arm swelling, and changes in thrill and bruit. Any concerns that arose from the dialysis were referred to the vascular access nurse specialist for further assessment and the multidisciplinary team review of the prior imaging. This was followed by a referral to interventional radiology (IR) for fistulogram ± angioplasty as a semi-urgent procedure. All AVG occlusions were primarily referred to IR for endovascular thrombectomy and fistulogram ± angioplasty as an emergency procedure. The surgical team was kept informed of the outcomes. Surgical open thrombectomy was performed only in the cases that failed the IR procedure. Then, the subsequent fistulogram ± angioplasty were routinely performed following open surgical thrombectomy to treat any underlying venous side stenosis.The surveillance program also included the patients who had confirmed stenosis on fistulogram, for which a three-monthly fistulogram ± angioplasty were performed until a proven resolution on updated fistulogram.The primary outcomes were to assess the one-year primary patency rate and the secondary patency rate during follow-up. The primary patency rate is defined as from the time of AVG creation to any first intervention to maintain or restore the function of the AVG or reaching a censored event . The secondary patency rate is defined as from the time of the AVG creation until AVG abandonment or achievement of a censored event  -15. The secondary outcomes were to evaluate surgical complications and long-term graft function. The AVG functional rate is defined as the same as the secondary patency rate in this study. This is due to all the AVGs in this cohort that were used for hemodialysis until abandonment as a result of the unsalvageable condition of occlusion or achieving a censored event . The AVG functional rate and secondary patency rate is presented using Kaplan-Meier statistical method.Data collectionData were extracted from the prospectively collected database included patient characteristics of gender and age, primary kidney disease, dialysis status, the configuration of the AVG, surgical complications, episode of AVG occlusion, the requirement for fistulogram or angioplasty, and the frequency for fistulogram and angioplasty.Statistical analysisData were expressed as number (proportion), mean ± standard deviation for normally distributed continuous variables, and as the median and interquartile range (IQR) for non-normally distributed data. The Kaplan-Meier analysis was conducted for measurement of AVG secondary patency rate and functional rate. R Core Team (2019)  was used for statistical analysis.Sixty-one patients underwent 64 AVG creations by using the Flixene vascular grafts Table . The ageForty-one patients (67%) were using CVC for HD prior to AVG, 14.8% of patients with a history of native AVF failure, 8.2% of patients on peritoneal dialysis for the conversation to HD, 8.2% of patients for second AVG (4.9% of them had their first AVG prior to the study period), and 1.7% patient for the third AVG. Ten patients (16.4%) were pre-dialysis.All AVGs were created successfully, except one Flixene IFG graft, which was replaced by a 6 mm x 40 cm Flixene graft due to poor flow detected by the Doppler device during the procedure. Sixty-three AVGs were in straight configuration at the upper arm with 51 on the left side and 12 on the right side, whereas one AVG was in a loop configuration at the left thigh. All cases were followed up from two months to eight years with a median of three years (IQR: 2 - 6 years). During the follow-up, 19 patients died of other medical comorbidities and six patients received kidney transplants. The primary patency rate was 30% at one year. The secondary patency rates  were 94.8%, 83.7%, and 77.7% at one, three, and five years, respectively . One graft was removed due to severe steal syndrome four weeks after the AVG creation (1.6%). Two AVGs were needled earlier for HD within 72 hours after creation due to a late referral to the surgical team for dialysis access and avoided the use of CVC. During the follow-up period, 36 AVGs required a fistulogram with 23 cases due to elevated venous pressure over 200 mmHg on HD, whereas 13 due to an AVG occlusion. On the fistulogram, 35 cases (97%) had venous side stenosis, while one case (3%) showed arterial side stenosis. The time for the first fistulogram was from one day to 25 months  post-AVG creation. The frequency required for fistulogram ranged from one to 16 times . For the treatment of AVG occlusion, seven cases were managed by IR, whereas six required surgical thrombectomies followed by fistulogram and angioplasty. The first-line treatment for venous stenosis was balloon dilatation. Stent placement was considered if the frequent recurrence (< three months) of stenosis occurred. One case required surgical revision for venous stenosis. Two AVGs required segmental replacement due to a pseudo-aneurysm two years post-AVG creation.Vascular grafts have played an important role in the establishment of HD access as one-third of newly created AVFs failed to mature -2. PatieOver the last decade, new generation vascular grafts have emerged for early cannulation within 72 hours after placement, including AVflo™ , VECTRA® , GORE® ACUSEAL , and Flixene . A recent literature review has shown that early cannulation of these AVGs was satisfactory without an increase in complications and jeopardization of the long-term patency rate , 12, 24.The Flixene vascular graft is a composite trilaminate structure graft that offers exceptional strength and durability allowing early needling. Early studies have reported satisfactory results in comparison with conventional grafts, which were usually cannulated after two to three weeks post-implantation , 24-25.In this cohort, two Flixene grafts were needled earlier within 72 hours without increasing any complications, although the majority of the AVGs commenced use three to four weeks after creation. This was due to a few reasons. Firstly, there was no demand for early use as the most of patients (67%) in this cohort had a CVC line prior to HD. Secondly, dialysis nurses were reluctant to insert the needle into the newly placed graft in the early post-surgery period due to some extent of arm oedema and wound healing. However, the new generation vascular grafts provide an opportunity for early needling in the setting of the emergency for HD. The dialysis nurses should be educated with the information of new generation vascular grafts and encouraged to perform early cannulation to the Flixene graft. This approach could avoid the need for CVC placement or reduce the duration of CVC placement if already in situ. This concept should be advocated in future clinical practice as a strategy to prevent CVC placement and its associated morbidities -12, 24.In this study, it was also observed that the use of the Flixene graft for AVGs achieved satisfactory long-term results. The one, three, and five-year functional rates were 94.8%, 83.7%, and 77.7%, respectively, which are comparable to the native AVF patency rate . From ouIn this study, we had the opportunity to observe the longer durability of the Flixene graft for up to seven years. On the other hand, a couple of Flixene grafts required segmental repair as a result of aneurysmal formation about two years after the graft placement. This could be attributed to repeatedly needling at the same site. Therefore, education for dialysis nurses regarding the needling technique is paramount for preserving the graft. It should be emphasized that the rope ladder technique is preferred for the insertion of the needle. This maneuver could minimize the risk of graft erosion and pseudoaneurysm formation. Understandably, AVG is associated with an increased risk of infection, given the presence of a foreign body. In this cohort, the overall infection rate of the Flixene graft was 4.8%, slightly lower than what was reported in the literature .It is well-known that the common issue of AVGs is the development of venous side stenosis because of venous intimal hyperplasia which requires regular interventional procedures, as observed in this cohort . It has In conclusion, our study has shown that the long-term functional rates and durability of the Flixene graft are satisfactory when used for HD access The Flixene graft is also safe to be needled within 72 hours after implantation, which allows the creation of an AVG under an emergency for HD to avoid the use of CVC. This strategy should be explored in future clinical practice when considering hemodialysis access. We would support the “4R” principle when deciding on HD access to ensure the best practice of patient-centered care.


Biomechanical stability assessment of 3 different constructs for proximal fixation of a locking compression plate (LCP) in treating a Worland type C periprosthetic fracture after total shoulder arthroplasty.  27 Worland type C fractures after shoulder arthroplasty in synthetic humeri were treated with 14-hole LCP that is proximally fixed using the following: (1) 1 × 1.5 mm cerclage wires and 2x unicortical-locking screws, (2) 3 × 1.5 mm cerclage wires, or (3) 2x bicortical-locking attachment plates. Torsional stiffness was assessed by applying an internal rotation moment of 5 Nm and then after unloading the specimen, an external rotation moment of 5 Nm at the same rate was applied. Axial stiffness was assessed by applying a 50 N preload, and then applying a cyclic load of 250 N, then increasing the load by 50 N each time, until a maximum axial load of 2500 N was reached or specimen failure occurred.  With regard to internal as well as external rotational stiffness, group 1 showed a mean stiffness of 0.37 Nm/deg and 0.57 Nm/deg, respectively, group 2 had a mean stiffness of 0.51 Nm/deg and 0.39 Nm/deg, respectively, while group 3 had a mean stiffness of 1.34 Nm/deg and 1.31 Nm/deg, respectively. Concerning axial stiffness, group 1 showed an average stiffness of 451.0 N/mm, group 2 had a mean stiffness of 737.5 N/mm, whereas group 3 had a mean stiffness of 715.8 N/mm.  Group 3 displayed a significantly higher torsional stiffness while a comparable axial stiffness to group 2. Periprosthetic fractures occur in approximately 0.6% to 3% of all total shoulder arthroplasties (TSA) . Since pLCP attached to the proximal humerus using 1 × 1.5 mm cerclage wires and 2x locking unicortical screws LCP attached to the proximal humerus using 3 × 1.5 mm cerclage wires LCP attached to the proximal humerus using 2x locking attachment plates allowing bicortical fixation by directing offset locking screws tangentially around either side of the prosthesis stem 27 synthetic humeri with left-sided geometry  were used for this biomechanical study. An anatomical total shoulder prosthesis  was implanted according to the operating instructions of the manufacturer. Each prosthesis was fixed using 40 g of Refobacin Bone Cement . After allowing for the cement to harden, a 14-hole 3.5 mm metaphyseal locking compression plate (LCP)  was positioned along the lateral surface of the humeral shaft. The plate was proximally attached to the synthetic bone using three different fixation constructs. Thereafter, the specimens were divided into three different groups with nine specimens in each group:Following fixation of the LCP onto the proximal segment of the humerus, we performed a 90° transverse osteotomy in the humeral mid-diaphysis 5 cm distal to the end of the prosthesis, simulating a Worland type C periprosthetic fracture and ensuring a stable prosthesis. To focus on the proximal plate fixation and to simulate an unstable fracture situation, only the proximal part of the artificial humerus was used .The biomechanical investigations were carried out on a servo-hydraulic material testing machine  and a custom-made experimental setup. The synthetic humeri were mounted between two universal joints . The disThe specimens were axially loaded with a static force (compression) of 5 N with a loading rate of 0.5 N/s. Afterwards, an internal rotation moment of 5 Nm was applied at a rate of 0.5 Nm/s. After reaching the maximum moment, the specimen was unloaded and an external rotation moment of 5 Nm at the same rate was applied. Finally, the machine adjusted the axial torque back to 0 Nm. The specimens were not damaged during the test. The protocol was carried out in load and torque control. Time, cycles, angle, torque, and force were recorded with a sampling rate of 1 kHz.The axial stiffness investigation was carried out in a cyclic test. Initially, the specimens were loaded with a preload of 50 N. In the first loading stage, a cyclic load with 1 Hz was applied in the sinusoidal form up to a load of 250 N. The lower load in all stages was 0 N, and ten cycles were carried out in each stage. In the following stages, the upper load limit was increased by 50 N each time. Thereafter, the upper load was increased until a maximum axial load of 2,500 N was reached or until the specimen sustained irreversible damage. The protocol was carried out in load and torque control. Time, cycles, angle, torque, and force were recorded with a sampling rate of 1 kHz. Load to failure was assessed by measuring the amount of axial force exerted at the time where the proximal fixation fails.This test was performed to assess the maximum axial load capacity that the LCP could withstand. This test was a pure assessment of the LCP with no additional component attached to it. The plates were fixed in the assembly so that the load being applied onto the plate was identical to the load previously being applied onto both the plate with the humerus fixed to it. In total, three plates were tested with the identical load protocol as for the axial stiffness testing as well as load to failure.U test. The significance level of α = 0.05 was employed.Statistical analysis was performed using SPSS software . The significance of differences between all three groups was tested using the Kruskal–Wallis test. Significant differences between the two groups were assessed using the Mann–Whitney p < 0.001). Furthermore, the difference between the specimens treated with 3 × 1.5 mm cerclage wires and those with the 2x locking attachment plates with bicortical screws were statistically significant (p < 0.001) , while the specimens treated with 3 × 1.5 mm cerclage wires showed an average internal torsional stiffness of 0.51 ± 0.33 Nm/deg, and those treated with 2x locking attachment plates with bicortical screws showed a mean internal torsional stiffness of 1.34 ± 0.16 Nm/deg . The dif< 0.001) .p < 0.001). Furthermore, the difference between the specimens treated with 3 × 1.5 mm cerclage wires and the specimens with the 2x locking attachment plates with bicortical screws were statistically significant (p < 0.001). The difference between the groups treated with 1 × 1.5 mm cerclage wires and 2x locking unicortical screws and 3 × 1.5 mm cerclage wires were statistically significant (p=0.043).After reaching the maximum moment of each specimen in each group and adjusting the axial torque back to 0 Nm, an external torsional stress was applied allowing for the measurement of external torsional stiffness. The mean external torsional stiffness was 0.57 ± 0.20 Nm/deg for the specimens treated with 1 × 1.5 mm cerclage wires and 2x locking unicortical screws, 0.39 ± 0.24 Nm/deg for the group treated with 3 × 1.5 mm cerclage wires, and 1.31 ± 0.24 Nm/deg for the group treated with 2x locking attachment plates with bicortical screws . The difp=0.001).Axial stiffness was assessed by implementing a cyclical axial load to reach a maximum of 2,500 N or until the construct failed after applying an initial axial preload of 50 N. The specimens that were treated with 1 × 1.5 mm cerclage wires and 2x locking unicortical screws showed a mean axial stiffness of 451.0 ± 41.6 N/mm, while the specimens treated with 3 × 1.5 mm cerclage wires showed a mean axial stiffness of 737.5 ± 146.7 N/mm, and the specimens treated with 2x locking attachment plates with bicortical screws showed an average axial stiffness of 715.8 ± 357.7 N/mm . The difWhen assessing the three LCPs without any humerus attached, the mechanical axial load limit of the LCPs was 1,190 N. From this axial load on, the LCPs were irreversibly deformed. When the specimens with the humeri reached this load limit, the LCPs themselves and not the proximal fixation constructs failed. However, the tests were not completed at this point. The loads were increased further until failure of the proximal shoulder implants or the proximal LCP fixation occurred. In the group where the plates were proximally fixed with 1 × 1.5 mm cerclage wires and 2x locking unicortical screws, the LCPs were irreversibly bent in two specimens, the unicortical screws loosened in five specimens, and the attachment between the shoulder prostheses and the servo-hydraulic material testing machine was disrupted in two specimens. In the group with the 3 × 1.5 mm cerclage wires, cerclage wire loosening or rupture occurred in five specimens, the LCPs were irreversibly bent in three specimens, and the distal part of the humerus came into contact with the base of the servo-hydraulic material testing machine in one specimen. In the group with the 2x locking attachment plates with bicortical screws, the LCP was irreversibly bent in eight specimens, and the distal part of the humerus came into contact with the base of the servo-hydraulic material testing machine in one specimen .Given the heterogeneous nature of these fractures, various surgical techniques have been suggested and used to treat these injuries, including cerclage wiring, plating, and interfragmentary screw insertion. There seems to be a general inclination and recommendation leaning towards surgically treating periprosthetic fractures distal to the prosthetic stem when the prosthesis is stable in order to optimize rates of healing, return to function, and painless mobilization. This strategy has been supported by the works of Bonutti and Hawkins and Boyd et al. in their case series of four and seven patients, respectively, with periprosthetic humeral shaft fractures after TSA , 6. HoweMultiple biomechanical studies of periprosthetic proximal femur fracture have mechanically loaded both the proximal and distal bone attachment segments together, with many failures occurring during these tests at the level of the distal fixation –9. Distap < 0.05). This supports the results of Gregory et al. where bicortical screws showed significantly higher torsional strength compared to unicortical screws or cerclage wires alone in the treatment of periprosthetic femur fractures [p < 0.05). This result contrasts with the findings of Dennis et al. and Fulkerson et al., which demonstrated that periprosthetic femur fractures treated with unicortical screws displayed a significantly higher torsional stability compared to fractures treated with cerclage wires alone [When comparing torsional stiffness, both internal and external, the group treated with the 2x locking attachment plates with bicortical screws displayed significantly higher stability and stiffness compared with the other two groups . Our findings contrast with the findings of Lenz et al. showed that plates with bicortical screws displayed higher axial stability when compared to cerclage wires in periprosthetic fractures of the femur [When comparing axial stiffness, the groups treated with 2x locking attachment plates and bicortical screws and the group treated with cerclage wires alone displayed comparable axial stiffness, both of which were higher than the group treated with cerclage wires and unicortical screws; however, only the difference between the group with both cerclage wires and screws and the group treated with cerclage wires alone was found to be statistically significant (he femur . We belihe femur .It is worth mentioning that, in our study, the group treated with 2x locking attachment plates and bicortical screws displayed a wider SD range when assessing axial stiffness compared to that of the other two groups. This phenomenon may be due in part to differences in the distance between the humeral transverse osteotomy and the proximal edge of the servo-hydraulic press across specimens, which in turn may be resulted in variable lever arms and a wider standard deviation than expected.in vivo samples. Some complexities in humeral loading were not considered. Loads not acting through the shoulder joint, from muscles and other soft tissues, were, for example, not assessed. The distal segment of the humerus was not considered in the model, precluding failures of this segment in the testing. Moreover, we did not assess the amount of displacement that each fixation method may have displayed before the failure of the setup. Nonetheless, this study provided novel information that was scarcely assessed previously; while biomechanical testing has been previously performed in models of periprosthetic fractures of the femur after total hip arthroplasty, research on periprosthetic humerus fractures after TSA is almost nonexistent. This might be due to the fact that hip arthroplasties have been present since 1925 when the American surgeon Marius Smith-Petersen created the first mold arthroplasty out of glass [The present study has several limitations in addition to the small sample size. Experiments were conducted on synthetic humeri, whose properties differ from those of of glass . Even thof glass , it is oLocking attachment plates with bicortical locking screws provide significantly better torsional stability compared with cerclage wires alone or cerclage wires in combination with locking unicortical screws in the treatment of Worland type C periprosthetic fractures of the humerus after TSA. However, cerclage wires alone provide an economically cheaper alternative while providing comparable axial stability to its more expensive locking plate counterpart. Nonetheless, further biomechanical studies on periprosthetic humerus fractures after TSA are needed to help guide treatment of this increasingly common and challenging injury.


Dietary intake of potato starch could induce a dramatic increase in blood glucose and is positively associated with chronic metabolic diseases . Grape seed proanthocyanidins (GSP) are known to decrease starch digestion by inhibiting digestive enzymes or changing the physicochemical properties of starch. In the present study, GSP were complexed with potato starch to prepare polyphenol–starch complexes. The physiochemical properties and digestibility of complexes were investigated by in vitro digestion model, X-ray diffraction, differential scanning calorimetry, rapid visco analyzer, Fourier transform infrared spectroscopy as well as texture profile analysis. Results indicated that the peak viscosity, breakdown, trough, and setback of the complexes disappeared, replaced by a special continuous increase in paste viscosity. The complexes showed a lower final viscosity and higher thermal stability with the increasing binding amount of GSP. GSP decreased the hardness of the complexes’ gel significantly. FT-IR indicated that GSP might interact with potato starch through noncovalent forces. Additionally, the complexes also showed a higher content of slowly digestible starch and resistant starch than that of the native starch. Thus, we inferred that the addition of GSP could modify the digestibility of potato starch and be an optional way to modify the starch with lower digestion. Solanum tubersum L.) is an important carbohydrate crop that is widely planted around the world [Potato , 1080 and 1020 cm−1 (both due to C–O stretching), 927 cm−1 , 848 cm−1 (CH2 wagging vibration), 761 cm−1 (C–C stretching), 709 cm−1 , and 576 and 526 cm−1  [−1 increased with the increase in GSP, indicating that GSP might strengthen the absorption through H-bonding. We also noticed that in Oladele’s study [−1) in maize starch modified with GPE, which indicated the formation of ether bonds. The formation of covalent bonds (ether bond) might be due to the ionization of starch to form an alkoxide under alkaline condition. Mathew and Abraham [The FT-IR spectra of the native starch and complexes are shown in n starch . The banibration . The othl modes) . Native p < 0.05). However, when the GSP was involved, the parameters of the texture profile showed a decreasing trend with the increasing binding amount of GSP. This indicated that after complexation with GSP, the starch gel could be much softer. The lower final viscosities might lead to lower hardness in gel formation. Cohesiveness implies the strength of the internal bonds. The hydroxyl groups on the lateral side of solubilized polyphenols might interact with starch through hydrogen bonding and prevent the starch chains from combining [The texture profile parameters are shown in ombining . After gNormally, starch is classified as rapidly digestible starch (RDS) and slowly digestible starch (SDS) and resistant starch (RS); RDS and SDS are starches hydrolyzed for 20 min and a further 100 min, RS is the starch not hydrolyzed after 120 min . As show5 U/mL) were bought from Aladdin Chemical Co. Ltd. . Invertase  and α-amylase  were purchased from Sigma-Aldrich Co. Ltd. . The total starch assay kit and the glucose oxidase/peroxidase assay kit were purchased from Megazyme International Co. Ltd. . Other reagents used in the present study were of analytical grade and also purchased from Aladdin Chemical Co. Ltd. .GSP with a proanthocyanidin content over 95% , were generously provided by Tianjin Jian Feng Natural Product R&D Co. Ltd. . Epicatechin standard (SE8100) was purchased from Solarbio Science & Technology Co. Ltd. . Potato starch  and amyloglucosidase  vanillin methanol and 500 μL of 25% (v/v) sulfuric acid methanol. The mixture was placed in a water bath at 30 °C for 15 min, and the absorbance was recorded at 500 nm using a UV-2600 spectrophotometer . The standard curve was made by the epicatechin standard. The binding amount and loading efficiency of GSP can be expressed as Equations (1) and (2), respectively:The GSP that were released in the supernatant obtained in −1 over a scattering range (2θ) of 4–40°. Jade software  was used to analyze the XRD patterns. Relative crystallinity (Xc) was calculated by Equation (3) [X-ray diffractometer  was applied to record XRD patterns of samples as well as GSP at a wavelength of 0.154 nm. A Cu-Kα radiation source (40 mA and 40 kV) was used according to a previous method . XRD pattion (3) .(3)Xc=APasting properties of the samples were characterized by a rapid visco analyzer (RVA) , according to the manufacturer’s instructions. Briefly, 1.5 g of the sample (compensate for 14% moisture basis) was accurately weighed into a canister and mixed with 15 mL distilled water. The samples were held at 50 °C for 1 min, heated to 95 °C for 7.5 min, then held at 95 °C for 5 min before cooling to 50 °C for 7.5 min, and finally held at 50 °C for 2 min. Peak viscosity, trough viscosity, final viscosity, and other parameters including pasting temperature and peak time, breakdown, and setback were recorded.Differential scanning calorimeter  was applied to investigate the thermal properties of the samples . A 3-mic−1 for 32 scans over a wavelength range of 4000 to 400 cm−1. FT-IR Patterns were analyzed with Ominic .Fourier transform infrared spectrophotometer  was used to analyze the short-range ordered structure of the samples according to its introduction. Briefly, the samples were finely ground with KBr and pressed into a pellet. FT-IR patterns were acquired with a resolution ratio of 4 cm−1, test speed of 1.0 mm·s−1, post-test speed of 1.0 mm·s−1, trigger force of 5 g, compression depth of 50%. Hardness, springiness, cohesiveness, chewiness, and resilience were recorded and calculated by Exponent .The pasted samples produced in 2 in beaker flasks and the pH was adjusted to 5.2. The mixture was heated at 90 °C for 20 min with magnetic stirring for the full gelatinization of starch. Then, the fully cooked samples were incubated at 37 °C for 10 min. Three milliliters of enzyme solution (containing 120 U/mL α-amylase and 80 U/mL amyloglucosidase) was transferred to the beaker flasks and initiated the digestion. The beaker flasks were placed at 37 °C for 120 min with an oscillation frequency of 200 rpm. The simulated digestion supernatant (500 μL) was collected at 20 and 120 min, respectively. The supernatant was transferred into 2.5 mL of absolute ethanol immediately to inactivate the enzymes. Then, the solutions were centrifuged at 2000 g for 10 min and 100 μL supernatant was mixed with 500 μL of dual enzyme solution containing amyloglucosidase and invertase  at 37 °C for 10 min to convert all the hydrolyzed starch into glucose [The digestion of potato starch as well as the complexes were performed, according to a previous study . BrieflyThe analysis was performed in triplicate. One-way analysis of variance (ANOVA) in IBM SPSS statistic 23 was used to analyze the data. Tukey’s honestly significant difference were used to separate the means at a significant level of 0.05.GSP has the potential to modify the physicochemical and nutritional properties of potato starch to produce GSP–potato starch complexes. The binding amount indicated a high affinity between GSP and potato starch. The interaction forces between GSP and potato starch might be noncovalent bonding, especially hydrogen bonding, which also retards retrogradation of gels made by complexes. The interaction in complexes could provide a more ordered structure, which implied a better heat and shear stability than the native starch. Furthermore, binding GSP reduced potato starch digestion during starch hydrolysis with a lower content of RDS and higher content of SDS and RS. Complexation between potato starch and GSP might offer opportunities to modify starch with a slowly digestive property. The complexes might be used directly as a bioactive starch or applied in novel functional starchy food processing. Moreover, utilizing the starch–proanthocyanidin complexes to control the release of proanthocyanidins seems promising and needs further study.


Doppler ultrasound is widely used to identify complications following renal transplantation. Evaluation of renal arterial Doppler waveform involves assessment of the peak systolic velocity (PSV), acceleration time or rapidity of the upstroke, and the renal resistive index (RI). The RI measurement is obtained by dividing the difference between PSV and end diastolic velocity by the PSV. The resulting ratio eliminates the inconsistencies commonly encountered in measuring velocities due to inaccurate angle correction and can be considered a measure of vascular impedance. An RI > 0.8 is considered elevated.,In this issue, Moura-Neto et alMultiple studies have shown that a normal RI in the immediate post-operative period is a good predictor for immediate graft function. Moura-Neto et alRI elevation (RI > 0.8) can be seen in various causes of graft dysfunction including, but not limited to, ATN due to ischemia reperfusion injury, rejection, obstruction, and compressionThe role of RI in identifying graft dysfunction has been studied with conflicting results. Most studies suggest that while evaluation of RI has not been able to identify specific causes of early graft dysfunction, elevated RI is significantly correlated with an increased incidence of delayed graft function. Even then, evaluation of RI falls short of effectively predicting the occurrence of graft dysfunction with only a moderate degree of estimated diagnostic accuracyElevated RI not only results from intrarenal factors but may also be secondary to systemic factors, such as heart rate, patient age, hypotension, and aortic stiffness. In a study by Naesens et alIn this study, the authors evaluated the changes of RI over two serial Doppler ultrasound examinations obtained within the first thirty days post-transplant. In current practice, the majority of post-renal transplant patients will only have one post-transplant ultrasound study performed in the immediate post-transplant period. It is unclear if it is feasible or practical to adopt the use of serial Doppler ultrasound examinations in all post-transplant patients. If not suitable for all renal transplant patients, it is necessary to determine which subset of patients would benefit from serial examinations and how often and when the serial studies should be performed. Further studies are also required to evaluate the efficacy of serial ultrasounds in predicting delayed graft function compared to clinical and biochemical markers.1 showed an association between increases in RI on serial Doppler studies in the first few post-operative weeks and delayed graft function due to ischemia-reperfusion injury. Serial evaluations could better identify patients at risk for delayed graft function than RIs measured on a single early post-operative Doppler examination and allow for prompt prevention and intervention.To conclude, the study by Moura-Neto et al


Parent–grandparent co-parenting has become a common mode in Chinese families; however, its correlation with children’s development in the long run remains unclear. Herein, a 10-month follow-up survey was conducted among 253 preschool children and their parents from Chinese parent–grandparent co-parenting families. It aimed to examine the bidirectional longitudinal correlation of children’s effortful control with mother–grandparent and father–grandparent co-parenting relationships, as well as the dissimilarity of the two co-parenting relationships. In addition, the moderating role of maternal parenting self-efficacy in these relationships was also investigated. A cross-lagged model showed that the (1) mother–grandparent co-parenting relationship (T1) positively predicted the father–grandparent co-parenting relationship (T2), (2) dissimilarity of the mother–grandparent and father–grandparent co-parenting relationships (T1) negatively predicted children’s effortful control (T2), and (3) maternal parenting self-efficacy significantly moderated the predictive effect of children’s effortful control on a father–grandparent co-parenting relationship. However, a further simple slope analysis showed that after controlling the father–grandparent co-parenting relationship (T1), the children’s effortful control (T1) did not significantly predict the father–grandparent co-parenting relationship (T2) either in the high or low maternal parenting self-efficacy group. These results indicated that in Chinese parent–grandparent co-parenting families, the father-grandparent co-parenting relationship was influenced by the mother–grandparent co-parenting relationship, and similar mother–grandparent and father–grandparent co-parenting relationships were conducive to the development of the children’s effortful control. Effortful Control is the ability to inhibit a dominant response and/or to activate a subdominant response, to plan, and to detect errors . Ten months later (T2), the parents of 253 children continually participated in the follow-up survey . Attrition was mainly due to the high dropout rate of Chinese children in their final year of preschool. Attrition analysis showed no significant differences in demographic and all study-relevant variables between the longitudinal sample and attrition sample.SD = 0.91, range: 3–6 years). Of the children, 73.7% were the only child in their family, and 79.9% of the children lived in three-generation families with both parents and grandparents. In 132 (52.2%) parent–grandparent co-parenting families, the main grandparent who participated in the child rearing was the parental grandmother. Eighty-four (33.3%) were maternal grandmothers. The number of parental and maternal grandfather was 26 (10.3%) and 11 (4.3%), respectively. The average age of the mothers was 33.95 years old , and 81.8% had a college degree or above. The average age of the fathers was 36.27 years old , and 74.7% had a college degree or above. The average age of the grandparents involved in co-parenting was 60.91 years old , and 94% had a high school degree or less. Furthermore, 53.5% of the participating families had an annual income of RMB 100,000  or above that belonging to the middle- or high-income families according to the The final sample of 253 children whose parents participated in both T1 and T2 surveys was 54.90% male and had a mean age of 4.27 years at T1 , and the follow-up survey was conducted 10 months later in June of the next year (T2). SPSS 21.0 and Mplus 7.4 were used for the subsequent statistical analysis.Cronbach’s α of mother-reported scores of the seven dimensions ranged from 0.63 to 0.85. The Cronbach’s α of father-reported scores of the seven dimensions ranged from 0.67 to 0.85. At T2, the Cronbach’s α of mother-reported scores of the seven dimensions ranged from 0.65 to 0.88. The Cronbach’s α of mother-reported scores of the seven dimensions ranged from 0.62 to 0.87.At T1 and T2, both parents were asked to completed the revised Chinese version of the Parent–Grandparent Co-Parenting Relationships Scale , which wThe average of the total score of each dimension was calculated, with higher scores reflecting a more positive parent–grandparent co-parenting relationship. Dissimilarity of mother–grandparent and father–grandparent co-parenting relationships was represented by the absolute value of the difference between the scores of father–grandparent co-parenting relationship and mother-grandparent co–parenting relationship. Higher scores indicated a greater dissimilarity of mother–grandparent and father–grandparent co-parenting relationships .Cronbach’s α = 0.72 at T1 and 0.83 at T2).At T1 and T2, the mothers completed the 36-item Very Short Form of Children’s Behavior Questionnaire, designed to assess three broad temperament factors  in children aged 3–8 using a seven-point rating scale  . The ChiCronbach’s α of the two dimensions of the scales  were 0.84 and 0.92. The average score of the 31-item scores was calculated, with higher scores indicating higher maternal parenting self-efficacy.At T1, the mothers completed the 31-item Parenting Self-efficacy Scale to rate their own parenting self-efficacy on a six-point scale, ranging from 1 (very unconfident) to 6 (very confident) . The ChiCFI) and Tucker–Lewis index (TLI) ≥ 0.90 , root mean square error of approximation (RMSEA) ≤ 0.08 , and standardized root mean square residual (SRMR) ≤ 0.08  framework using Mplus 7.4 . The mod) ≤ 0.08 . Simple rs = 0.44 to 0.68); (2) the mother–grandparent co-parenting relationship was positively correlated with the father–grandparent co-parenting relationship both at T1 and T2 ; (3) both the mother–grandparent co-parenting relationship and father–grandparent co-parenting relationship at T1 were significantly positively correlated with the children’s effortful control at T2 . Conversely, the children’s effortful control at T1 was significantly positively correlated with both the mother–grandparent co-parenting relationship and father–grandparent co-parenting relationship at T2 ; (4) the dissimilarity of the mother–grandparent and father–grandparent co-parenting relationships at T1 was negatively correlated with the children’s effortful control at T2 (r = −0.17), while the correlation between the children’s effortful control at T1 and the dissimilarity of the mother–grandparent and father–grandparent co-parenting relationships at T2 was not significant (r = −0.04); and (5) the maternal parenting self-efficacy at T1 significantly positively correlated with the children’s effortful control, mother–grandparent co-parenting relationship, and father–grandparent co-parenting relationship at T1 and T2 (rs = 0.17 to 0.30).rs = 0.13 to 0.19), so children’s gender and age were used as control variables in the following analysis.Concerning all of the demographic variables, we only found that children’s gender and age were significantly correlated with children’s effortful control at T1 (2(23) = 23.91, CFI = 0.996, TLI = 0.993, RMSEA = 0.02, SRMR = 0.03, Δχ2(1) = 21.87, p < 0.01.After including all the autoregressive paths and concurrent correlations, multiple cross-lagged paths from each variable at T1 to all the other variables at T2 were added in the model see . In addip < 0.01), while no significant predictive effect of the father–grandparent co-parenting relationship at T1 on the mother–grandparent co-parenting relationship at T2 was found; (2) neither the mother–grandparents co-parenting relationship nor father–grandparents co-parenting relationship at T1 was found to significantly predict the children’s effortful control at T2 in the current study. Dissimilarity of the mother–grandparent and father–grandparent co-parenting relationships at T1 significantly negatively predicted the children’s effortful control at T2 , while we did not find that the children’s effortful control at T1 predicted the mother–grandparent co-parenting relationship, father–grandparent co-parenting relationship, or dissimilarity of the mother–grandparent and father–grandparent co-parenting relationships at T2; (3) maternal parenting self-efficacy at T1 significantly positively predicted the children’s effortful control at T2 . In addition, its interaction item with the children’s effortful control at T1 significantly negatively predicted the father–grandparent co-parenting relationship at T2 , which suggested that maternal parenting self-efficacy at T1 moderated the predictive effect of children’s effortful control at T1 on the father–grandparent co-parenting relationship at T2.The path analyses showed that after controlling the children’s gender, age, and stability of variables (themselves) across time, (1) the mother–grandparent co-parenting relationship at T1 significantly positively predicted the father–grandparent co-parenting relationship at T2  in the second step. As shown in Herein, the bidirectional longitudinal relations of children’s effortful control with mother–grandparent and father–grandparent co-parenting relationships, as well as the dissimilarity of the two co-parenting relationships were explored. In addition, the moderating role of maternal parenting self-efficacy in the predictive impact of children’s effortful control on father–grandparent co-parenting relationship was investigated. First, it was found that the mother–grandparent co-parenting relationship played a significantly positive role in predicting the father–grandparent co-parenting relationship; but it was free from the impact of the latter relationship. That is to say, the father had more possibilities to keep a positive co-parenting relationship with the grandparents under the premise of good mother–grandparent co-parenting relationship. Nevertheless, it was hard for the father–grandparent co-parenting relationship to influence the co-parenting relationship of the mother with the grandparents. Such result was consistent with the reality of the Chinese parent–grandparent co-parenting families. As the most principal caregiver of children, the mother actually interacts with grandparents more frequently than the father in the child-raising process. Different parenting concepts make intergenerational co-parenting conflicts inevitable, especially between mother-in-law (grandmother) and daughter-in-law (mother) in context of the Chinese traditional culture . As a reNo significant predictive impact of the mother–grandparent or father–grandparent co-parenting relationships at T1 on the children’s effortful control at T2 was found in the current study. Instead, the investigated children showed a lower level of effortful control at T2 under a greater dissimilarity of the mother–grandparent and father–grandparent co-parenting relationships at T1. As demonstrated by literature, the externalizing co-parenting conflicts may trigger a high-level negative emotional arousal of young children, thus consuming their psychological energy originally used for effortful control and increasing the frequency of children’s emotional and behavioral regulation problems . HoweverNevertheless, in the current study, children’s effortful control at T1 showed no significant predictive effect on mother–grandparent and father–grandparent co-parenting relationship as well as on the dissimilarity of the two co-parenting relationships at T2, which was inconsistent with the assumption mentioned before and the results of previous studies . A possipost hoc power analyses, the Effect Sizes (i.e. represented by f2 in multiple linear regression) for this specific predictive effect in both groups , were also small  on father–grandparent co-parenting relationship (T2) was not statistically significant either in the high or low maternal parenting self-efficacy group. Such result, on one hand, may be attributed to the relatively weak predictive effect of children’s effortful control (T1) on father–grandparent co-parenting relationship (T2) after controlling father–grandparent co-parenting relationship (T1) because of the time stability of the two variables. On the other hand, according to the results of so small , which iThis study has some limitations. First, a 10-month follow-up study may be too short to reveal the significant changes in parent–grandparent co-parenting relationship and children’s effortful control across time because both children’s effortful control and co-parenting relationship were proven to have strong time stability . Longer In conclusion, the current study enriched the existing study results on the topic area of parent–grandparent co-parenting relationship by providing additional data of father–grandparent co-parenting relationship in Chinese parent–grandparent co-parenting families. The study results can be widely implicated in the Chinese family education practice. According to the influence of mothers’ co-parenting relationship with grandparent on fathers’, parents, especially mothers, in parent–grandparent co-parenting, families should not only focus on their own co-parenting relationship with grandparents but also pay attention to their spouse’s co-parenting relationship with grandparents. In this case, parents would communicate more about their attitudes toward grandparents’ parenting behaviors so as to keep their co-parenting relationships with grandparents similar to each other, which would facilitate children’s development of effortful control. In addition, although the current study focused on Chinese parent–grandparent co-parenting families, we introduced more kinds of co-parenting relationships beyond mother–father co-parenting relationship in the topic area of co-parenting, which may inspire future studies across different cultures to think beyond mother–father co-parenting relationship and pay attention to different kinds of co-parenting relationships among multiple child-rearing participants , which may interact with each other and have combined effects on children’s developmental outcomes.The datasets generated for this study are available on request to the corresponding author.This study was carried out in accordance with the recommendations of the Ethics Committee of the School of Psychology, Beijing Normal University. The protocol was approved by the Ethics Committee of the School of Psychology, Beijing Normal University. Written informed consent was obtained from all participants. For all the children who participated in the study, written informed parental consent was obtained from their parents/legal guardians in accordance with the Declaration of Helsinki.XL designed and executed the study, and assisted with the data analyses. SZ analyzed the data and wrote the manuscript. YG wrote part of the introduction and assisted with the data analyses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XL declared a shared affiliation, with no other collaboration, with the authors to the handling Editor at the time of review.


Journal of Functional Morphology and Kinesiology , which was first released in March 2016, developed greatly in 2020. This journal provides an advanced forum for research studies on functional morphology and kinesiology and the regulatory functions of movement. JFMK meets the growing demand for high-quality, peer-reviewed international journals, supplying the easy access and high publicity of open access, the digital object identifier (DOI), ORCID, and CrossRef to all researchers. We are indexed in the Scopus (Elsevier’s abstract and citation database), DOAJ , Scilit , Google Scholar, World Health Organization, Hinari, FSTA—Food Science and Technology Abstracts (IFIS), and the Norwegian Register for Scientific Journals, Series and Publishers (NSD). Our full texts are archived in CLOCKSS , e-Helvetica , and J-Gate (Informatics India). The In 2020, JFMK was covered by SCImago Journal Rank with a 1.5 Citescore and reached a Q3-quartiles in the following research fields: anatomy, histology, orthopedics and sports medicine, physical therapy, sports therapy, rehabilitation and rheumatology.With great pleasure we announce that in early 2021, we will be indexed by PubMed; we have been recently accepted. We hope we can be included in Web of Science in the near future. JFMK is a member of the Committee on Publication Ethics (COPE). To verify the originality of content submitted to our journals, we still use iThenticate to check submissions against previous publications. MDPI works with Publons to provide reviewers with credit for their work, and MDPI Scitations Alert to provide our authors information on new publications in their research field. The journal publishes articles focusing on molecular, cellular, tissue, system and whole-body responses to a broad range of physical activities. Furthermore, it provides an advanced forum for the analysis of the structure, function, development, and evolution of the cells and tissues of the musculoskeletal system and associated disorders.Journal of Functional Morphology and Kinesiology has continued to grow in functional morphology and kinesiology research dealing with the analysis of the structure, function, development and evolution of cells and tissues of the musculoskeletal system, and the whole body. It is my pleasure to confirm the progress recorded in the last few years [https://www.mdpi.com/journal/jfmk/stats). We are proud to let you know that, thanks to your continuous support, ew years ,2,3, as Journal of Functional Morphology and Kinesiology receives more manuscripts than it is able to publish, and the decision as to which papers are accepted or rejected is a difficult one. The decision is based on several factors, including originality, experimental design, scientific quality, data interpretation, clarity, and English quality, to maintain the high standards of our journal.Indeed, the number of published manuscripts has jumped from 64 in the 2019 volume to 101  in the 2020 volume, with an increase of more than 37% compared with last year. We rejected 39.73% (67 papers) of the contributions to maintain the high standards of our journal. The In 2020, different Special Issues were completed thanks to the huge support of our editors. They include the following: “Role of Exercises in Musculoskeletal Disorders—3rd Edition”, edited by Prof. Dr. Giuseppe Musumeci and Dr. Silvia Ravalli ; “ElectrIn 2020, six distinguished scientists joined the Editorial Board: Prof. Dr. Agata Grazia D’Amico , Prof. Dr. Virginia Tancredi , Dr. Dawson J. Kidgell , Prof. Dr. Jose Antonio , Prof. Dr. Pasqualina Buono , Prof. Dr. Douglas Powell  and Dr. Adam Wells , for a total of 79 editorial board members, seven advisory board members, and the editor-in-chief.Journal of Functional Morphology and Kinesiology will be published in full open access, and in order to provide free access to readers, and to cover the costs of peer review, copyediting, typesetting, long-term archiving, and journal management, an article processing charge (APC) of 1400 CHF (Swiss Francs) will be applied to papers accepted after peer review. All articles published in the In 2021, we shall continue our efforts to improve the journal through further growth and increased visibility. Follow up the planned papers from editorial board members and agreed authors;Contact international conferences recommended by the editor-in-chief or by editorial board members, and try to establish media partnerships with them to make JFMK increasingly well-known among scholars;Communicate with editorial board members regularly and ask for their help and suggestions for journal development;Post high-quality papers through social media  and increase online readership;Reduce the processing time of each submitted manuscript;Try to have publications indexed by the Emerging Sources Citation Index (Web of Science), by EMBASE (Elsevier) and by Web of Science—Clarivate;Try to improve the Citescore in the SCImago Journal Rank in the kinesiology-related sections, such as anatomy, histology, orthopedics and sports medicine, physical therapy, sports therapy, rehabilitation and rheumatology;Try to be reach the First Impact Factor released by Clarivate Analytics;Accomplish, for our authors, the best JFMK paper award and the JFMK travel grant award;Garner, for the sake of journal promotion, support from sponsors for our editors to participate in, and disseminate our journal to, international conferences.In order to achieve this target and lay a strong foundation for publications in 2021, and in our application for indexing, we have made the following plans:From 2021, MDPI will include the accepting Academic Editor’s name on published articles, once they have accepted that manuscript after full peer review. This establishes greater transparency for the readership, demonstrates the care that our Academic Editors take in making decisions, and offers full acknowledgement of the effort put in when making expert judgements about the suitability of a manuscript for publication. We strongly believe that this will also support quality during peer reviews.We hope that you share our enthusiasm for this journal, and we look forward to working with you to make JFMK a leader in its field. Your contributions are vital for the success of this new journal. We look forward to receiving your contributions , and proposals for Special Issues are always welcome.I have personally found this to be quite a challenge, not helped by COVID-19, but more due to the special position that JFMK is trying to negotiate in the highly competitive publishing landscape. I wish you a healthy and prosperous new year, and look forward to continuing to expand the reach and impact of the journal with your help next year.I also take this opportunity to warmly thank, for their confidence, the following: our authors, readers, and reviewers; our editorial advisors; eminent scientists in these fields who, with their experience and important suggestions, guide us in this great enterprise; our excellent editorial board members whose depth of experience covers a very broad spectrum of different disciplines related to the morphology and kinesiology arenas; the managing editor Ms. Molly Lu for her huge support, the publishing manager Dr. Unai Vicario and the other members of the Editorial office who, day after day, thanks to their valuable contributions, ensure the growth of this journal; and, finally, all members of our teams in Basel, Barcelona, Beijing, Belgrade, Romania, Tokyo, and Wuhan, as well as our sponsors.


Hamiltonella defensa differ in their capacity for horizontal transmission. These isolates vary in the protection they provide against parasitoid wasps as well as the costs they inflict on their host, Aphis fabae. We simulated natural horizontal transmission through parasitoid wasps by stabbing aphids with a thin needle and assessed horizontal transmission success of the isolates from one shared donor clone into three different recipient clones. Specifically, we asked whether potentially costly isolates reaching high cell densities in aphid hosts are more readily transmitted through this route. This hypothesis was only partially supported. While transmissibility increased with titre for isolates from two haplotypes, isolates of the H. defensa haplotype 1 were transmitted with greater frequency than isolates of other haplotypes with comparable titres. Thus, it is not sufficient to be merely frequent—endosymbionts might have to evolve specific adaptations to transmit effectively between hosts.Secondary endosymbionts of aphids have an important ecological and evolutionary impact on their host, as they provide resistance to natural enemies but also reduce the host’s lifespan and reproduction. While secondary symbionts of aphids are faithfully transmitted from mother to offspring, they also have some capacity to be transmitted horizontally between aphids. Here we explore whether 11 isolates from 3 haplotypes of the secondary endosymbiont  Buchnera aphidicola, which lives in specialised tissues of their body . The symbiont provides protection against parasitoid wasps, but the strength of this protection varies greatly between different isolates  and had been microinjected with 11 different H. defensa isolates from other A. fabae clones between 2008 and 2012 . The recipient clones were collected in summer 2006 in Europe introduced to it. A fourth H. defensa-free clone, which is visibly distinguishable from all others due to a colour mutation, A08-28H– (Vicia faba) seedlings and under conditions that ensured clonal reproduction .Hemolymph was transferred from each donor to three recipient n Europe  and are n Europe . RecipieA08-28H– , was incH. defensa-infected sublines of clone A06-407 as well as hemolymph from 407H0 as a symbiont-free control. All 36 donor-recipient combinations were replicated 5 times, with each replicate consisting of a batch of 10 stabbed nymphs. Transmission success was determined by testing the offspring of stabbed individuals for the presence of H. defensa. Therefore, we only counted horizontal transmissions as successful if they led to new heritable infections.Subadult aphids (nymphs) of all three recipient clones were stabbed with fine pins contaminated by hemolymph from each of the 11 V. faba seedlings for two generations. Adults used to found the third generation were allowed to reproduce on new plants for 24 h, then they were frozen at −20°C and used to confirm the presence/absence of H. defensa and the correct H. defensa haplotype at the start of the experiment (see section Molecular Methods below).To produce enough aphids for the experiment, five adults of each aphid clone or subline were split off from the laboratory stocks and bred up on To produce the aphids acting as hemolymph donors, 10 adults of the second generation of each subline were placed on a new bean plant, allowed to reproduce for 24 h and then transferred to a next plant. This was repeated over 5 days to produce five consecutive batches of offspring. Offspring were reared for 15 days until they reached adulthood, at which point they were used as donors in the transmission assays.To produce the aphids acting as hemolymph recipients, each of the three recipient clones was reared on 12 separate plants for two generations. We then took six adults from each plant, placed them on a new plant to reproduce for 24 h, before moving them on to another new plant. Again, this was repeated over 5 days to produce five batches of offspring. Offspring were used as recipients in the transmission assays as 3-day old nymphs.2-anesthesia during the procedure. Except for the first day, the donors were frozen after use for later reference. On each day we collected a pool of three donors per subline, which was frozen at −20°C until DNA extraction and subsequent qPCR for estimation of H. defensa titres. A graphical overview of the experimental procedures is provided in Manual transfection of hemolymph from donor to recipient took place over five consecutive days. For every combination of donor and recipient, one 15-day old donor aphid was used to infect 10 three-day-old recipient nymphs. The donor was mounted with double-sided tape on a glass slide and stung with a fine stainless steel needle , which was then inserted briefly into a recipient nymph. This double stabbing procedure was repeated for each of the 10 nymphs. Before use, the needles had been sanitised by soaking in 70% ethanol for 5 min, but they were not cleaned between successive stabs of the same donor-recipient clone combination. Aphids were under COH. defensa via the leaf tissue, we also added two three-day-old nymphs of the H. defensa-free colour-mutant clone A08-28H– as sentinels to each disc. The number of surviving recipients in each disc was scored six days after transfection, when aphids approached adulthood. Either four (batch 1) or three (batches 2–5) survivors were transferred individually to new leaf discs to be reared until their offspring reliably indicated status of infection. Preliminary experiments had established this to be the case at approximately 13-days old, a finding which is corroborated by literature , which contained a broad bean leaf disc (Ø 4 cm) on 1% agar, and were maintained at 21°C and a 16 h photoperiod. To test whether rearing all 10 nymphs on the same leaf disc might allow between-nymph transmission of terature . The twoH. defensa to test whether their stabbed mothers had acquired and passed on the symbiont. The three nymphs from each leaf disc were collected into collection microtubes of the DNeasy 96 Blood & Tissue kit  to which two sterile glass beads were added.Three days after isolating the surviving recipients on separate leaf discs, the number of offspring produced by each survivor was noted (time point t1) and the offspring discarded. After another 4 days, the survivors were moved to new leaf discs and the offspring on the old leaf disc were counted (time point t2) and discarded. After reproducing for 2 days on the new leaf discs, the survivors were removed from the leaf disc and frozen at −20°C. After an additional 5 days, a pool of three offspring of each survivor was harvested for diagnostic PCR for H. defensa, which is only explicable by contamination. To identify its source, we tested all available H0 donors (batches 2–5) and the mothers of all A06-407 recipients for presence/absence of H. defensa. This revealed that the recipient culture was clean, but the donor culture of 407H0 had been contaminated with H. defensa-infected individuals. Four of the twelve H0 donors used in batches 2–5 were found to have carried H. defensa. We discarded their data as well as all data from H0 donors in batch 1, since these donors had not been saved and could therefore not be checked retrospectively.In the second batch, offspring of one A06-407 recipient exposed to hemolymph from donor 407H0 tested positive for H. defensa titres in donor lines and for assessing whether offspring produced by recipient aphids carried H. defensa we used the Qiagen DNeasy 96 Blood & Tissue kit . Extraction success was verified by amplifying part of the 16S rRNA gene of B. aphidicola, the obligate endosymbiont present in all aphids, using specific primers. The presence/absence of H. defensa was also determined by diagnostic PCR with specific primers for the same gene. Primers and cycling conditions are detailed in H. defensa haplotypes, we amplified murE and accD gene fragments , using TaqMan real-time quantitative PCR as described by H. defensa’s dnaK and the aphid’s EF1α gene served as a proxy for H. defensa titre. The 20 μl reactions were run in triplicates on a LightCycler 480 (Roche) and gene copy number was estimated based on a standard curve produced with serial dilutions of a synthetic standard provided by Microsynth AG . For analysis, we used the 2nd Derivative Absolute Quantification approach in LightCycler 480 SW 1.5.1. Two replicates were excluded because we failed to harvest three live adults for DNA extraction (batch 3 of 407H323 and batch 4 of 407H85).The density of Post-hoc tests were performed with multcomp v1.4-13 . We used a generalised linear mixed model (GLMM) with binomial errors, expressing transmission rate as aggregated binomial data juxtaposing number of infected to number of uninfected individuals per treatment and batch. We treated average titre, recipient clone and donor haplotype as fixed effects and included the interactions between recipient and titre and the interaction between recipient and haplotype. Donor identity and batch were treated as random intercepts. We assumed there to be no interaction of titre and haplotype. The data indicates that this is true for haplotype 2 and 3, and it is impossible to determine a reliable estimate of the slope for haplotype 1 since both isolates of this haplotype (H76 and H101) have near-identical transmission rates and titre. To allow the model to converge, we used the Bobyqa optimiser with a set maximum of 200,000 iterations. Uniformity of residuals was confirmed using the package DHARMa  as well as the interaction between recipient and titre and recipient and haplotype were treated as fixed effects, and batch and donor were random intercepts.To analyse survival of stabbed nymphs, we used a linear mixed effects model on the arcsine-square-root-transformed proportion of surviving nymphs. Average titre, recipient clone and donor haplotype  were treated as fixed effects, and batch and donor as random intercepts. In an additional GLMM fecundity of successfully infected aphids was compared. Donor was treated as a fixed effect and batch as a random intercept.The fecundity of survivors during the first week of their adult life was analysed using a GLMM with negative binomial errors. We excluded aphids that had not survived until time point t2 from the data set as their infection status could not be reliably assessed. Recipient clone and infection status  but not between different isolates of H. defensa , and did not depend on titre  or haplotype  of the H. defensa isolate that was transfected. Also non-significant were the effects of the recipient clone , the interaction between titre and recipient  and the interaction between recipient and haplotype . The significant influence of batch on survival was likely a result of improving skill and routine of the experimenter  that were stabbed with hemolymph-contaminated needles, 1,209 (67%) survived the transfection. Survival was different between batches , survivors died before their offspring would reliably indicate H. defensa infection (n = 28), and we discarded recipients stung with 407H0 donors that were not confirmed to be free of contamination with H. defensa (n = 19).A maximum of four (in batch 1) or three (in batches 2–5) nymphs of each treatment that survived the transfection were kept until their offspring would reliably indicate infection with 2 = 36.98, df = 2, p < 0.001), with clone 37H0 producing more offspring . Thus the number of offspring produced in early adult life was similar between aphids that had acquired an infection with H. defensa, those that had rejected an infection and those that were not exposed to infectious hemolymph .On average, survivors produced 40.5 ± 0.5 nymphs during their first week of reproduction. Fecundity in early adult life varied significantly among the three recipient clones  produced H. defensa-positive offspring. These are indeed infections acquired from the stab and not infections acquired secondarily from other nymphs feeding on the same leaf disc, because none of the 177 sentinel aphids we tested were positive for H. defensa. Horizontal transmission success varied between different H. defensa isolates and batches, which was reflected in a highly significant random effect of H. defensa isolate and a significant effect of batch  = 39.86, p < 0.001] but not between haplotypes   was likely too short to detect the reproductive costs imposed by H. defensa, which are typically late-acting costs, mediated by curtailed lifespan rather than a reduced daily fecundity (Previous studies have shown that infection with some oduction . One reaecundity . We woulH. defensa isolate determine its horizontal transmission success. This casts doubt on our initial assumption that costly isolates providing limited benefit to the host are maintained through an increased disposition for horizontal transmission, since two strongly protective and nearly avirulent isolates also showed frequent horizontal transmission. Further studies will be required to understand why such seemingly omnipotent isolates do not dominate in aphid populations.In conclusion, our experimental simulation of a known horizontal transmission route showed that both titre and haplotype of a https://doi.org/10.5061/dryad.s4mw6m95n.The data has been provided to Dryad Digital Repository and can be accessed at HK collected the data and wrote the first draft of the manuscript. Both authors designed the study, contributed to data analysis, contributed to and commented subsequent versions, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It was noted that the EL molecules in the graft had very high mobility in the hydrated state. The EL–SF grafts were implanted into the abdominal aorta of rats to evaluate their patency and remodeling ability. No adverse reactions, such as bleeding at the time of implantation or disconnection of the sutured ends, were observed in the implanted grafts, and all were patent at the time of extraction. In addition, vascular endothelial cells were present on the graft's luminal surface 2 weeks after implantation. Therefore, we conclude that EL–SF artificial vascular grafts may be useful where small-diameter grafts are required.Globally, increasing mortality from cardiovascular disease has become a problem in recent years. Vascular replacement has been used as a treatment for these diseases, but with blood vessels <6 mm in diameter, existing vascular grafts made of synthetic polymers can be occluded by thrombus formation or intimal hyperplasia. Therefore, the development of new artificial vascular grafts is desirable. In this study, we developed an elastin (EL)–silk fibroin (SF) double-raschel knitted vascular graft 1.5 mm in diameter. Water-soluble EL was prepared from insoluble EL by hydrolysis with oxalic acid. Compared to SF, EL was less likely to adhere to platelets, while vascular endothelial cells were three times more likely to adhere. SF artificial blood vessels densely packed with porous EL were fabricated, and these prevented the leakage of blood from the graft during implantation, while the migration of cells after implantation was promoted. Several kinds of  Atherosclerosis and heart disease caused by dietary and lifestyle changes account for a significant proportion of morbidity and mortality worldwide. Vascular replacement surgery is a common procedure for such vascular diseases, and bypass surgery for revascularization is in great demand for such patients  is a natural protein produced by the silkworm and is well-known as an excellent textile material  is an important component of the extracellular matrix of vessels. It provides elasticity in arteries, lungs, and skin , 13C cross-polarization/magic angle spinning (13C CP/MAS) NMR, and 13C dipolar decoupled-magic angle spinning (13C DD/MAS) NMR   in the dry and hydrated states  vascular grafts.In this study, the water-soluble EL, as prepared by Miyamoto et al., was shown to improve cell adhesion and demonstrated anti-thrombogenic properties compared with SF in For this study, we used water-soluble EL  . The soluble EL-A sample was prepared by fractionation based on the elastic modulus and aggregation temperature of soluble EL, prepared from the insoluble EL of porcine aortas, by treatment with 0.25 M oxalic acid  to the SF at a concentration of 10% (w/v) and then boiling at 70°C for 1 h. The solution was then filtered to remove residual solid components and dialyzed with a cellulose dialysis membrane against distilled water at 4°C for 3 days to produce the aqueous solution of SF.The aqueous solution of SF was prepared as described in our previous papers . The number of cells was counted using a fluorescence microscope BIOREVO BZ-9000 with BZ-H1C software . Three points were selected on each plate, and the number of cells was determined and averaged. The cultivations were performed three times with HUVEC.Human umbilical vein endothelial cells  were used for the cell adhesion experiment with SF and EL- coated 24-well plates . The PRP on EL and SF- coated 24-well plates was incubated at 37°C for 3 h. After light cleaning with PBS, the EL was fixed with glutaraldehyde for 1 h at room temperature. After washing with PBS and drying, the number of platelets at three points was counted on the images with a scanning electron microscope (SEM)  and averaged. The experiments were performed three times with PRP.The 1.5 mm diameter double-raschel knitted silk tubes with high elongation and contraction properties were prepared with silk fibers containing reduced SS. We used a computer-controlled double-raschel knitting machine  , when compressed to 25% of the diameter, was calculated using the analysis software TRAPEZIUM. Ten dry, short, ring-shaped specimens with an axial length of 10 mm were prepared for the determinations, conducting four parallel tests, and the values were averaged.The circumferential tensile strengths of the SF graft, SF-coated SF graft, and EL–SF graft were determined using an EZ-graph manufactured by SIMAZU, Kyoto, Japan  1H decoupling during acquisition, 2,176 data points, 8 k scans, and 4 s recycle delay at room temperature. Details of the NMR experimental conditions for the 13C DD/MAS NMR experiments have been described earlier , a refocusing delay of 1JCH/6 or 1JCH/3. The 13C chemical shifts were calibrated externally through the methylene peak of adamantane observed at 28.8 ppm with respect to external tetramethylsilane (TMS) at 0 ppm . The rats were managed and cared for in accordance with the standards established by TUAT and described in its “Guide for the Care and Use of Laboratory Animals. EL–SF grafts were implanted in the abdominal aorta of 12 rats under a stereoscope   shows the pictures of HUVEC attachment to SF and EL-coated 24-well plates. The number of HUVECs was greater for the EL-coated plate than for the SF-coated plate, 1684 ± 180, and 449 ± 54, respectively, A: right). Thus, EL exhibited a three-fold higher attachment rate of HUBECs. B: left) shows SEM images of platelet adhesion on SF and EL-coated 24-well plates. The attachment rate of platelets was three-fold higher for SF, compared to EL, B: right). The platelet adhesion count for EL and SF-coated 24-well plates was 76 ± 6 and 210 ± 22, respectively. In summary, EL had a higher attachment rate for HUVECs and a lower attachment rate for platelets than SF. Therefore, EL has promising anti-thrombosis properties that may be beneficial for future applications of small-diameter grafts.The SEM images are shown in 13C solid-state NMR at the molecular level. 2) was lower than the SF-coated SF graft (0.012 N/mm2), as shown in A further examination of the EL–SF grafts was performed by determining the physical properties and characterization by several kinds of 13C r-INEPT, (B) 13C DD/MAS, and (C) 13C CP/MAS NMR spectra of EL–SF grafts in the hydrated state, and (D) shows the 13C CP/MAS spectrum of the graft in the dry state. The expanded region (0–80 ppm) of these spectra are also shown in 13C chemical shifts and assignments are listed in 13C r-INEPT spectrum, 13C nuclei with fast motion in the hydrated EL–SF graft  and Phe  residues (13C CP/MAS NMR spectra, 13C chemical shifts of the peaks, the 13C DD/MAS NMR spectrum could be assigned as shown in 13C CP/MAS NMR spectrum of the hydrated graft, indicating the high mobility of these residues. The presence of the Pro residues in the primary structure is thought to be a source of higher hydration of the pro-containing protein fibers such as spider silk fiber and elastin fiber (Asakura et al., As shown in residues . The carresidues . HoweverMost of the peaks from EL could be seen in the dry state along with the peaks from the SF fibers, as shown in After implanting the EL–SF grafts , all ratHE staining was used to investigate how the implanted graft changed after implantation. At 2 weeks, there was no cell infiltration into the grafts because the EL sponge had not decomposed . Since Ein vivo, and its shape and properties can be controlled by various processing methods (Horan et al., We developed a small-diameter artificial vascular graft using SF (Enomoto et al., Elastin is found in the extracellular matrix of the intima of blood vessels. It accounts for 50% of the dry weight of arteries, and its properties include endothelial cell compatibility, blood compatibility, and elasticity. It is also attracting attention as an artificial vascular graft combined with other biomaterials (Wilson et al., In this study, we used water-soluble EL prepared from insoluble native elastin by hydrolysis with oxalic acid to improve SF vascular grafts. The beneficial characteristics of EL, i.e., higher attachment of endothelial cells and lower attachment of platelets, were still maintained compared with SF. Thus, we conducted an experiment to develop an EL–SF artificial vascular graft with high biocompatibility to promote endothelialization, antithrombogenicity, and elasticity. Since thrombus formation is the primary cause of occlusion in <6 mm diameter grafts (Begovac et al., 13C solid-state NMR spectra of the EL–SF graft in the dry and hydrated states enabled the characterization of the local structure and dynamics of EL and SF molecules at the molecular level. The hydration of the SF chains of the EL–SF graft is suppressed because of the crosslinking with EL molecules and their creation of a hydrophobic environment around the SF chains. On the other hand, the EL molecules in the EL–SF graft are highly mobile in the hydrated state, especially for the Pro and Leu residues. Thus, the EL properties effectively minimize platelet adhesion and promote endothelial migration in the EL–SF graft.The various After implantation, patency was confirmed in all EL–SF grafts. No signs such as hematuria, diarrhea, inflammation, tail disintegration, or leg immobility were observed in the rats. The EL molecules on the SF fibers should be antithrombotic, as EL is first exposed to blood when flow resumed (Fernández-Colino et al., The use of a crosslinking agent for glutaraldehyde was not a major problem in this study because it was implanted in rats. For humans, however, it is important to use low-inflammatory elastin (Wise et al., 13C solid-state NMR studies indicated that the EL molecules in the graft have very high mobility in the hydrated state. This graft showed excellent biocompatibility in vitro, and excellent operability and remodeling ability in vivo.In this study, we developed an EL–SF 1.5 mm diameter double-raschel knitted vascular graft. The SF graft was densely packed with porous EL, which prevented blood leakage from the graft during implantation. The migration of cells after implantation was promoted. The The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Tokyo University of Agriculture and Technology.TA and YA designed the study. TT, YA, and AN performed experiment. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.