In the spring of 1961 a small group of individuals from diverse backgrounds all had one thing in common: They were performing a new technology called extracorporeal circulation (ECC). Less than a decade earlier surgeons and researchers had shown that patients suffering from cardiac disease could be placed on a ‘heart-lung’ machine for a short period of time facilitating intracardiac access for surgical repair. The first formal meeting for individuals practicing and studying ECC, using either ‘heart-lung’ or dialysis machines, was held in 1962. In 1963 the American Society of Extracorporeal Circulation Technicians was formed serving as the first professional society devoted to ECC, and included both perfusionists  and dialysis technicians, who were often nurses. The first formal meeting of AmSECT was held in Chicago at the Sol Fox Lounge in December 14, 1963. In 1967 the Society was formally incorporated with a name change to the American Society of Extra-Corporeal Technology,The Journal of Extra-Corporeal Technology, commonly referred to today as JECT. The goal was to publish the progress and growth of technological knowledge on ECC, and to share the research in a multidisciplinary manner. In the first issue of JECT Jim Wade, the president of AmSECT at the time, wrote in an editorial that the primary goal of both the Society and JECT would be the “communication and the professional exchange of information” by perfusionists, surgeons and administrators [JECT, the goals of the initial formation of the Journal remain similar. During those years there have been many changes to the Journal with perhaps the most obvious being the cover. This year the Journal will undergo its fifth major cover change so it may be fitting to review the history of the front page of this publication.The following year, due to the undaunted efforts of perfusionists and surgeons, a new journal appeared devoted entirely to this emerging field, and was titled strators . While iJECT was Ed Berger a practicing perfusionist from the Charles T. Miller Hospital in St. Paul, Minnesota. Ed was truly a visionary who saw the importance of the scientific pursuit of quality evidence as paramount in supporting methodologies and practices that would improve the conduct of ECC, with the ultimate goal of improving patient care. In his first editorial he established the focus of the Journal as a collaborative effort and stated “by us banding together for the acquiring and sharing of information in order to develop a more perfect technology and more perfect technologists” [The first editor-in-chief of logists” . Indeed,The first cover shows the early juxtaposition of perfusionists and dialysis technicians with images of both professions displayed . In factJournal’s name all in lower case letters. Then in the first issue of Volume 12, published in early 1980, the cover once again was changed to a solid red background and the AmSECT logo changed from the familiar red with to white with black lettering  (Emeritus)Editor-In-Chief (Emeritus)al.stammers@specialtycare.netEmail: Phone: (570) 864-8653


MT) isolated from Sophora flavescens is now utilized as a botanical pesticide in China, in fact, its pesticidal activities are much lower in magnitude than those of commercially agrochemicals. To improve its pesticidal activities, here, the joint pesticidal effects of MT with another alkaloid oxymatrine (OMT) (isolated from S. flavescens) and the monoterpene essential oil 1,8-cineole (CN)  were investigated in the laboratory and greenhouse conditions. Moreover, their toxicological properties were also studied. Against Plutella xylostella, when the mass ratio of MT and OMT was 8/2, good larvicidal activity was obtained; against Tetranychus urticae, when the mass ratio of MT and OMT was 3/7, good acaricidal activity was obtained. Especially when MT and OMT were combined with CN, the significant synergistic effects were observed: against P. xylostella, the co-toxicity coefficient (CTC) of MT/OMT (8/2)/CN was 213; against T. urticae, the CTC of MT/OMT (3/7)/CN was 252. Moreover, the activity changes over time of two detoxification enzymes, carboxylesterase (CarE) and glutathione S-transferase (GST) of P. xylostella treated with MT/OMT (8/2)/CN, were observed. In addition, by scanning electron microscope (SEM), the toxicological study suggested that the acaricidal activity of MT/OMT (3/7)/CN may be related to the damage of the cuticle layer crest of T. urticae.With the increasing development of pest resistances, it is not easy to achieve satisfactory control effects by using only one agrochemical. Additionally, although the alkaloid matrine ( Plutella xylostella Linnaeus  as the major migratory insect pest mainly endangered cruciferous vegetables and caused economic loss in the world of up to US$ 4–5 billion annually . Th. ThMT anMT/OMT (3/7), MT/OMT (3/7)/CN, and spirodiclofen were all prepared at 0.2 mg/mL in 0.1% aq. Tween-80, respectively. Asparagus bean plants were infested with the female adults of T. urticae prior to spraying. Three plants were chosen for one group, and each treatment was three replicates. An airbrush was used to spray 10 mL of the corresponding solution for three replicates. The plants treated with 0.1% aq. Tween-80 alone were used as CK . Their control effects on the 1st, 3rd, and 5th days were calculated in the same way as mentioned above  and MT/OMT (8/2)/CN at 0.4 mg/mL, respectively (treated by acetone as CK). Then, the 30 surviving larvae in the treated group were collected at 12, 24, 36, and 48 h, respectively. They were then snap-frozen in liquid nitrogen and stored at −80 °C for subsequent enzyme activity analysis [According to the above-mentioned leaf-dipping method, 180 robust 3rd instar larvae of analysis .g for 30 min at 4 °C. The homogenous liquid was obtained for glutathione-S-transferase (GST) activity assay when the sample was centrifuged at 8000× g for 10 min at 4 °C.The homogenization treatment (including ten larvae: Weight (g)/Volume (mL) = 1/10) was performed in an ice bath. The homogenous liquid was obtained for carboxylesterase (CarE) activity assay when the sample was centrifuged at 12,000× The absorption values over time of CarE and GST were tested by using CarE (α-naphthyl acetate (α-NA) as a substrate) and GST  assay kits , respectively. Total protein concentration was determined according to the Bradford method  as a standard). The protein content was tested by a BCA protein quantitative assay kit . Finally, the enzymes activity values were obtained according to the absorption value and the protein content. Each treatment was replicated three times .MT/OMT (3/7)/CN) was prepared at 0.894 mg/mL in 0.1 g/L of aq. Tween-80 (0.1 g/L of aq. Tween-80 as CK). Female adult mites with the same physiological status and good growth conditions were selected to cowpea leaves . After 4 h, the leaves were immersed in the above solution for 5 s, the excess solution was absorbed by filter paper, and the leaves were placed in a Petri dish with a moist sponge (three replicates/treatment). Then, Petri dishes were placed in a light incubator . Finally, the dead mites were collected at 24, 48 and 72 h after treatment by the complex for scanning electron microscope (SEM) analysis [The complex , dehydrated with different concentrations of ethanol, and freeze-dried for 3 h. The mites were then placed on a sample table and sprayed with gold under vacuum conditions. The morphology was observed and photographed by S-3400N SEM .p < 0.05). The median lethal concentration (LC50) values were calculated on log-concentration versus probit  regression analysis. The values of r, χ2, df, and P were obtained on regression analysis by IBM SPSS Statistics 20.0 (p < 0.05).Mortality data were corrected with Abbott’s formula and analyzed by a multiple range test using Duncan’s test (MT and OMT) and their binary mixtures at different mass ratios against P. xylostella were tested. As shown in MT and OMT was 8:2, the corresponding 48 h corrected mortality rate (CMR) was 27.9%, which was higher than those of MT (20.9%) and OMT (16.2%). The 48 h CMRs were all 20.9% when the mass ratio of MT/OMT was 7:3 or 5:5. The 48 h CMR was 16.2% when the mass ratio of MT/OMT was 1:9, which was similar to that of OMT. However, when the mass ratio of MT and OMT was 3:7, the corresponding 48 h CMR was decreased to 13.9%, which was lower than those of MT and OMT. Obviously, for MT and OMT against P. xylostella, the best mass ratio of MT and OMT was 8:2. As shown in P. xylostella, 48 h CMRs of 1,8-cineole , MT/OMT , and MT/OMT /CN were 23.3%, 30.2%, 43.1%, and 56.0%, respectively. It demonstrated that when CN (0.4 mg/mL) was combined with MT/OMT, the larvicidal activity of the corresponding complex was significantly improved. Then, 48 h LC50 values of MT, OMT, MT/OMT (8/2), and MT/OMT (8/2)/CN against P. xylostella were further evaluated according to their CMRs at different concentrations , and MT/OMT (8/2)/CN were 0.708 and 0.441 mg/mL, respectively; while 48 h LC50 values of MT and OMT were 0.899 and 1.158 mg/mL, respectively. That is, the larvicidal activity of MT/OMT (8/2)/CN was 2.0–2.6 fold those of MT and OMT. Moreover, the co-toxicity coefficient (CTC) values of MT/OMT (8/2) and MT/OMT (8/2)/CN were 133 and 213, respectively. Clearly, their CTCs were all > 120, so the above two mixtures showed the synergistic effect. Subsequently, the control effects of MT/OMT (8/2) and MT/OMT (8/2)/CN against P. xylostella in the greenhouse at 0.2 mg/mL were tested. As shown in MT/OMT (8/2) and MT/OMT (8/2)/CN against P. xylostella after 5 days were 36.8% and 49.0%, respectively. Symptoms at the fifth day of the cabbage seedling leaves treated with MT/OMT (8/2) and MT/OMT (8/2)/CN against P. xylostella are described in MT/OMT (8/2)- and MT/OMT (8/2)/CN-treated groups, the surfaces of the leaves were smooth and had almost no small puncture; however, there were many small punctures eaten by P. xylostella on the leaves in the CK-treated group. Photographs of control effects of those complexes against P xylostella are shown in First, the insecticidal activities of two compounds  and OMT (18.2%); especially when the mass ratio of MT to OMT was 3:7, the corresponding 72 h CMR was 34.8%. At 0.048 mg/mL, the 72 h CMR of CN against T. urticae was only 11.2%; interestingly, when MT and OMT (mass ratio: 3/7) were dissolved in the solution of CN (CCN= 0.048 mg/mL) in 0.01% aq. Tween-80, and CMTOMT (3/7)/ was 0.5 mg/mL, the corresponding CMR was increased to 38.0%. It may be related to the strong permeability of CN as an essential oil [50 values of MT, OMT, MT/OMT (3/7), and MT/OMT (3/7)/CN against T. urticae were further calculated according to their CMRs at different concentrations  and MT/OMT (3/7)/CN (CCN= 0.048 mg/mL) were 1.405 and 0.894 mg/mL, respectively, while 72 h LC50 values of MT and OMT were 2.014 and 2.377 mg/mL, respectively. That is, the acaricidal activity of MT/OMT (3/7)/CN (CCN= 0.048 mg/mL) was increased to 2.3–2.7 folds when compared with those of MT and OMT. Meanwhile, the CTCs of MT/OMT (3/7) and MT/OMT (3/7)/CN (CCN= 0.048 mg/mL) were 160 and 252, respectively. So, the above two mixtures showed the synergistic effect especially for the combination of MT/OMT (3/7)/CN. On the other hand, the control efficiency of MT/OMT (3/7) and MT/OMT (3/7)/CN (CCN = 0.04 mg/mL in 0.1% aq. Tween-80) at 0.2 mg/mL against T. urticae in the greenhouse was evaluated. As shown in MT/OMT (3/7) and MT/OMT (3/7)/CN after 5 days were 31.3% and 42.3%, respectively. Fifth-day symptoms of asparagus bean seedling leaves treated with MT/OMT (3/7) and MT/OMT (3/7)/CN were described in T. urticae on the seedling leaves in the CK-treated group, whereas in the MT/OMT (3/7)- and MT/OMT (3/7)/CN-treated groups, almost no small white spots were on the seedling leaves. These findings were the same as our previous report [T. urticae are shown in The 48 and 72 h acaricidal results of trations . As shows report . PhotogrP. xylostella, treated with CN, MT/OMT (8/2) and MT/OMT (8/2)/CN at 0.4 mg/mL after 12, 24, 36, and 48 h were depicted in CN-treated group at 12 h (82.1 U/g) was higher than that of the control group (69.4 U/g). It suggested that CN was toxic to P. xylostella and stimulated the detoxification ability of the CarE in P. xylostella at 12 h. At 24 h, the CarE activity value in the MT/OMT (8/2)/CN-treated group (67.7 U/g) was higher than that of the control group (52.4 U/g). It indicated that MT/OMT (8/2)/CN stimulated the detoxification ability of the CarE in P. xylostella at 24 h. After that, the detoxification ability of the CarE in P. xylostella in all treated groups was inhibited. The time for reaching the lowest points of the CarE activities in the treated groups was different. For example, in the CN-treated group, the lowest point of the CarE activity was reached at 36 h, whereas the lowest points of the CarE activity in the MT/OMT (8/2)- and MT/OMT (8/2)/CN-treated group were reached at 48 h.Subsequently, the changes of detoxification enzymes (CarE and GST) activities in MT/OMT (8/2)/CN-treated group at 12 h (3910 nmol/min/g) was higher than that of the control group (2698 nmol/min/g). Similarly, it suggested that the mixture MT/OMT (8/2)/CN stimulated and enhanced the detoxification ability of the GST in P. xylostella at 12 h. At 24 h, the GST activity value in the CN-treated group was slightly higher than that of the control group. Afterwards, the GST activity in the treated groups decreased significantly when compared with that of the control, and it reached the lowest point at 36 h in all treated groups. The GST activity values at 36 h in CN-, MT/OMT (8/2)- and MT/OMT (8/2)/CN-treated groups were 2208, 2591 and 1932 nmol/min/g, respectively, and they were decreased 1.4–1.9 folds of that of the control group (3603 nmol/min/g). Obviously, the GST activity of P. xylostella in the treated groups was largely inhibited at 36 h.As illustrated in T. urticae are flat and have an entire structure, with neat and continuous skin texture; whereas in the MT/OMT (3/7)/CN-treated group, the structure of the cuticles was damaged with visible wrinkles and an uneven arrangement of the inner ridges. The cuticles of wounded mites were thinner, softer, and more asymmetrically bent than those of normal mites, losing their barrier-like function against acaricide penetration. As a result, CN may enhance the penetration ability of this complex on T. urticae [The penetration of insecticide to pest epidermis is the premise of its toxic effect. The cuticle serves as the initial barrier of defense between the body and the outside world, effectively blocking pesticide penetration. The reduction in epidermal penetration can delay the time of pesticide reaching the target site, and pests have sufficient time and faster speed to metabolize the pesticide entering the body . As show urticae ,60.CN was combined with MT and OMT as pesticidal agents: against P. xylostella, the CTC of MT/OMT (8/2)/CN was 213; against T. urticae, the CTC of MT/OMT (3/7)/CN was 252. Furthermore, these mixtures displayed good control efficiency against P. xylostella and T. urticae in the greenhouse. Importantly, the enzymes activity changes over time of CarE and GST in P. xylostella treated with CN, MT/OMT (8/2) and MT/OMT (8/2)/CN were explored. Due to the accumulation of toxicant leading to a decrease in the detoxification ability, the time for reaching the lowest point of the CarE activity in the treated groups was different, whereas the time for reaching the lowest point of the GST activity in the treated groups was the same (at 36 h). Notably, by SEM analysis, the toxicology study suggested that the destruction of the cuticle layer crest of T. urticae by MT/OMT (3/7)/CN may be the main cause of their death. These results will pave the way for the future study of different combinations of MT–OMT–CN and the application of CN as a synergist with other bioactive natural products as pesticidal candidates in crop protection.In summary, the significant synergistic effects were observed when


Now EditorialOffice of Chinese Journal of Lung Cancer has decided to retract it.The editorial office will strengthen checking procedures for the academicpublishing specification to avoid the recurrence of such events in future.Editorial Office of Chinese Journal of Lung CancerJune, 2023


Although uncommon, oxygenator thrombosis in extracorporeal membrane oxygenation (ECMO) devices remains a potential reason for exchanging the circuit, the risk of thrombosis increases with the duration of ECMO therapy and with procoagulant conditions, such as COVID-19 . With a 2 removal (ECCO2R) support in our Intensive Care Unit (ICU) required the injection of rtPA for acute oxygenator thrombosis. Characteristics of the study cohort are reported in Table From March 1, 2020, to March 30, 2022, 3 patients from a population of 99 were treated with either veno-arterial (V-A) and veno-venous (V-V) ECMO or extra-corporeal COThe first patient was on V-V ECMO during the first COVID-19 wave and was fully dependent on the ECMO support because of severe respiratory failure; after 4 days on ECMO, the oxygenator showed rapid signs of clotting  and arterial oxygenation dropped to 55%. Because of no chance of survival considering the time required to have a perfusionist available, the administration of low-dose rtPA  into the drainage section of the oxygenator was decided; within 15 min, the ECMO flow returned to the initial baseline values. Anticoagulation was adjusted to higher targets and no recurrence of oxygenator clotting was observed thereafter. The second patient presented severe respiratory acidemia  with protective lung ventilation requiring ECCO2R; within 3 h from onset of therapy, oxygenator thrombosis occurred in non-working hours and it was decided to administer rtPA to avoid complete circuit and cannula clotting. Drug administration helped to dissolve the clot and result in a normal function of the device. Similar results were observed in the third patient; no hemorrhagic complication was observed within the 5 days following rtPA injection. Heparin was not interrupted during thrombolysis.This is the second report on the use of “off-label” rtPA for oxygenator thrombosis in ECMO patients. In another small cohort, 4 patients were successfully treated with several administrations of rtPA , without


Additionally, heightened braking effects were seen during the acceleration phase of throwing with greater stride knee extension power when pitching with shorter strides . The knowledge gained from this work offers new insight into compensatory stride length adaptation that impacts systemic and throwing arm-specific fatigue to maintain ball velocity, as bilateral ankle joint dynamics can be significantly affected in response to cumulative workload.Altered propulsive and bracing ground reaction forces from lower-body fatigue significantly impact stride length to increase weakness in dynamic elbow stabilizers and risk of medial elbow injury in baseball pitchers. This work investigated altered stride length on three-dimensional ankle joint dynamics to illustrate fatigue-induced changes in ankle motion that can also be impacted by coaching errors. Nineteen pitchers (15 collegiate and 4 high school) were randomized in a crossover design study that encouraged fatigue by throwing two simulated 80-pitch games at ±25% of their desired stride length. An integrated motion-capture system with two force plates and radar gun tracked each throw. Retrospective analysis using pairwise comparisons, including effect size calculations, were undertaken to identify differences in ankle dynamics between stride length conditions for both the drive and stride leg. Longer strides were found to be more effective in drive ankle propulsion and stride-bracing mechanics. Conversely, shorter strides delayed bracing dynamics by demonstrating continued drive ankle plantar flexion moments after stride-foot contact to extend pitchers’ time in propulsion ( Fatigue has been shown to decrease performance in baseball pitchers, where a change in lower-extremity joint power is known to affect throwing arm dynamics that impact the momentum exchange between the throwing arm and trunk, yet can occur with no visible change in ball velocity ,3,4,5,6.In studies featuring adolescent athletes, ball velocity was reportedly unaffected by intrasubject differences in normalized stride length, and this relationship was also evident among more proficient pitchers ,8,9. GamStride length compensation forms an intuitive starting point to evaluate kinetic chain reactions that may precipitate throwing arm injury risks, and stride length variation effects on multiplane and link-segment biomechanics in baseball pitching remains largely unknown. To our knowledge, no study has investigated bilateral 3D ankle joint mechanics in response to altered stride length that may negatively impact kinetic chain interactions and potentially exacerbate throwing arm stress. Therefore, this study is a continuation of a comprehensive full-body biomechanical investigation, with the purpose of retrospectively analyzing how bilateral ankle mechanics are impacted by stride length differences when throwing fastballs during simulated competitive play. The aim was to demonstrate how changes in stride length evoke compensatory ankle biomechanics, stemming from altered propulsive and bracing ground reaction force profiles. For this investigation, it is hypothesized that closed kinetic chain ankle biomechanics would be affected by changes in stride length. Greater ankle joint dynamics  for both the drive (hind) and stride (lead) ankle are expected to occur earlier in the pitching cycle when adopting longer strides. Drive ankle dorsiflexion is expected to increase with longer strides after stride-foot contact to enact hind ankle braking, as anectodally, toe dragging of the hind foot is common to such deliveries, whereas shorter strides are expected to prolong propulsive drive ankle actions after foot contact and amplify stride-ankle braking dynamics later in the delivery.Secondary analysis was centered on existing data from the original single cohort, with significant changes in temporal, physiological, ground reaction, momentum, and performance indices having been previously reported ,11,12,13Protocols for the subject recruitment, motion capture, and data postprocessing have been previously published ,12,13,14Data collection occurred in the Biomechanics of Human Movement Laboratory at the University at Buffalo and included an integrated biomechanical environment connecting optical tracking with ground reaction force technology in capturing the pitching delivery. Athletes were randomized in being crossed over to throw two simulated 80-pitch games. Subjects delivered the baseball in either an overstride or understride game condition that was set at 25% increased stride length (overstride) and 25% reduced stride (understride) from their desired stride length. Simulated games were held on separate days that were spaced by a 72 h rest window prior to entering a secondary simulated game in the crossover design.A motion-capture system involving eight cameras  synchronized with two floor-embedded force plates  were aligned in series so that drive and stride legs contacted opposing force platforms and provided data that tracked the entire pitching delivery complete with fastball and off-speed pitched baseball velocities. Pitch velocities were recorded using a radar gun and LED display  that was positioned behind a pitching net. Retroreflective markers defined body segments that were captured at 240 Hz through infrared cameras and 960 Hz for force plate data. An LED screen connected to the radar gun provided visual feedback to encourage athletes to deliver fastball pitches with maximum intent.TM, Fabrifoam, Inc., Exton, PA, USA). The elastic wrap was worn securely around the subject’s waist, while ASIS and PSIS markers were attached at the front. The moment of ball release was estimated between the marked ball and throwing hand. For further specifics on anatomical marker placement and subject calibration, please see previous work evaluating stride length impacts on physiological outcomes [After anthropometric measures for height and weight were obtained, retroreflective markers were affixed bilaterally to the whole body, and reflective tape, cut into circles, was secured to the baseball and positioned on the ball in places that were visible to the cameras for the entire arm path that allowed for tracking whole-body motion until the instant of ball release. After static standing and functional hip calibrations, 3D marker trajectories were reconstructed . In total, sixty-three 19 mm and 25 mm retroreflective markers were affixed to body segments for optical 3D tracking, including determining segment inertial properties, subject calibration, and determining hallmark events in the delivery to delineate discrete time points. A hard, sacral thermoplastic shell was tracked optically through three non-collinear markers, which indicated where the pelvis segment was in the 3D space and was positioned over the sacrum using Velcro and elastic overwrap  from flat ground (no pitching mound) into a 6.5′ × 6′ catch net  restricted to a distance of 5.69 m owing to lab dimension constraints rather than from the standard 18.4 m. The pitching delivery was standardized to the stretch delivery  with thrSpatiotemporal parameters were identified from kinematic data and used for desired stride length determination. When the suprapatellar marker of the knee of the stride leg achieved its highest vertical displacement in the windup, it signified peak knee height and was considered the onset of the delivery for kinematic and kinetic analysis. In normalizing bilateral ground reaction forces, body weight was used for both drive and stride leg. Stride-foot contact was determined at the point by which the stride foot landed on the second (opposing) force plate with a vertical ground reaction force that exceeded 5% body weight. Each subject’s desired stride length was calculated as the mean horizontal distance between the drive foot calcaneus at a peak knee height to the stride-foot calcaneus at the stride-foot contact.Thereafter, desired stride length was adjusted ±25% (determined a priori) to reflect overstride and understride throwing conditions and to challenge throwing mechanics. Absolute stride lengths were expressed in meters as well as normalized percentages across all subjects as a proportion of their body height. In previous work, stride length measures were averaged for overstride and understride conditions and equated to 1.40 ± 0.15 and 0.95 ± 0.14 m, respectively, and normalized as relative measures to body height at 0.76 ± 0.07 and 0.52 ± 0.08% body height. As a result, pitchers required minimal adjustments in their throwing mechanics from their mean desired stride length (1.24 ± 0.17 m and 0.67 ± 0.09% body height) . DespiteForce plates embedded in the ground were targeted for subjects to provide constraints for their drive and stride feet so that the desired stride could be visualized and that overstride and understride conditions could be maintained on all throws for each simulated game in the crossover design. For each trial, subjects were acclimated to the respective simulated game stride conditions, and when subjects felt capable of throwing at full effort, the simulated game data collection began. For each recorded pitch, the total delivery time initiated from peak knee height of the lead knee and ended at the point of ball release.Twenty pitches were thrown per inning—16 thrown at maximum effort for fastballs and 4 off-speed pitches thrown at submaximal effort—that were captured over the course of 4 total innings. Between pitches, subjects were allocated approximately 15 s rest timed by a stopwatch and then provided 9 min of rest between innings. Prior to the start of each inning, five warmup throws were provided, which is consistent with competitive baseball standards. The 80th pitch was the last pitch of the simulated play and terminated the data collection. Means from the two highest fastball velocities in the first and fourth inning that were identified from radar data were analyzed. The rationale for involving both the first and last inning’s highest velocity pitches entailed accounting for potential fatigue impacts over the course of simulated games that could be standardized across all participants as it relates to pitch accumulation.As previously reported elsewhere , ball veMER: This is the greatest layback of the throwing arm defined from shoulder axial rotation in the kinematic data, and the point when the greatest negative axial humeral rotation occurred to indicate the humerus achieved furthest position into external shoulder rotation.BR: Upon visual inspection, ball release occurred coincident with peak linear hand velocity that was inspected visually through both the ball and linear kinematic hand trajectory along the leading axis in the capture environment. As a result, peak linear velocity of the hand indicated ball release and formed the endpoint for the pitching cycle.Following data collection, each trial was analyzed kinematically and kinetically through proprietary software used for advanced biomechanical computations . Calculations involved using custom reduction techniques as previously described ,13,16. Bi)(generation phase: from PKH to SFCii) and averaged over the generation, brace-transfer, and acceleration phases over the time-normalized pitching cycle. Ankle profiles were based on Newton–Euler equations of motion ,22,23, wt-tests for hallmark events and phases were undertaken to evaluate ankle dynamic differences between stride length conditions (overstride vs. understride) using SPSS 19 . Statistical significance, predetermined a priori, was set at p ≤ 0.05 for all statistical tests. To illustrate clinical significance for statistically significant findings, Cohen’s d effect size calculations demonstrated standardized mean differences by denoting trivial (<0.2), small (0.2–0.49), moderate (0.5–0.79), and large (>0.8) effects.Paired Triplanar ankle mechanics were found to be significantly different in several pairwise comparisons, with greatest effects seen in the sagittal and frontal plane. Results achieving at least moderate effect size can be seen in accompanying tables A,B, whilp = 0.015, ES = 0.84) at stride-foot contact and during propulsion (generation phase). Drive leg ankle moments differed at SFC and during the brace-transfer and acceleration phase intervals. Plantar flexor moments were significantly higher  with the shorter strides at SFC and brace transfer, whereas ankle dorsiflexor moments observed with longer strides during the brace-transfer and acceleration phases were statistically different from plantar flexor moments experienced with shorter strides . Longer strides presented significantly higher power generated by the drive ankle’s dorsiflexors during the acceleration phase and at BR  whereas shorter stride pitching saw significantly higher power generated by the plantar flexors of the drive ankle during the brace-transfer phase  and power absorption during the acceleration phase .Ankle dorsi and plantar flexor dynamics for the drive and stride leg are depicted, respectively, in p < 0.001, ES = 6.61) with greater angular plantar flexor velocities observed during the acceleration phase with shorter strides . Higher plantar flexor moments throughout the acceleration phase  with coincident positive joint powers at BR  and during the acceleration phase were experienced with shorter strides .As found with the drive ankle, stride leg ankle plantar flexion was statistically higher with a large effect for longer stride lengths throughout the pitching cycle from SFC to BR B. In con(A) Ankle kinematic conventions described by open kinetic chain (OKC) and closed kinetic chain (CKC) movement indicates plantar flexion (−)/dorsiflexion (+) for the drive ankle. Greater plantar flexion and respective angular velocity were indicated by OS during GEN with concentric dorsiflexion power occurring during ACC. Greater concentric plantar flexion during BT and eccentric plantar flexion during ACC was indicated for US.(B) Ankle kinematic conventions describing closed kinetic chain (CKC) movement following SFC (73% and 80% time for respective under- and overstride pitching). Stride ankle plantar flexion (−)/dorsiflexion (+); OS indicated greater plantar flexion from SFC to BR. US had greater plantar flexion velocity, moments and joint power during ACC.p < 0.001, ES = 0.81), with less inversion (moving to eversion) during BT  when compared to shorter strides. Inversion angular velocities were shown to be statistically higher for the drive ankle at the generation phase  with shorter strides. Internal ankle eversion moments were observed for the drive ankle with shorter strides, as longer strides had shown inversion moments at stride-foot contact   ( = 0.24) A. Longer = 1.28) A.p < 0.001, BT ES = 2.18, ACC ES = 16.3) compared to shorter strides. Longer stride pitching revealed greater eversion velocities at BR  and throughout acceleration the phase . After stride-foot contact, mean eversion moments were significantly greater for shorter stride pitching through to BR .For the stride ankle, throwing with longer strides demonstrated greater mean eversion during BT and the acceleration phase (gh to BR B with mogh to BR B. Positigh to BR B. Illust(A) Ankle kinematic conventions described by open kinetic chain (OKC) and closed kinetic chain (CKC) movement indicates inversion (+)/eversion (−) for the drive ankle. OS indicated greater eversion from SFC to BR and greater inversion velocity during ACC. US indicated a greater eversion moments and joint power from SFC to BR.(B) Ankle kinematic conventions describing closed kinetic chain (CKC) movement following SFC (73% and 80% time for respective under and overstride pitching). Stride ankle inversion (+)/eversion (−); OS indicated greater eversion from SFC to BR and greater inversion velocity during ACC. US indicated a greater eversion moments and joint power from SFC to BR.p < 0.001, ES = 0.61). Drive-ankle external rotation velocities were lower during the acceleration phase for longer strides . Shorter strides mediated greater mean absorbance  with the stride ankle in a more internally rotated position  to decelerate the rate of internal rotation during the acceleration phase , whereas shorter strides prolonged propulsive ankle mechanics after foot contact with abrupt stride-ankle braking observed later in the delivery.Altered stride length has been shown to affect propulsive and bracing ground reaction force profiles in both magnitude and timing of peaks in a previous study , where tBoth stride conditions saw maximal drive-ankle plantar flexor moments occurring during 60–65% of the pitching cycle, with peaks close to stride-foot contact observed with the shortened strides as opposed to peaks occurring well before stride-foot contact with the longer strides A. The diFollowing stride-foot contact, the drive ankle experienced greater dorsiflexor concentric joint power, since the foot was plantar flexed to a greater extent during the acceleration phase. Increased dorsiflexor power generation with the foot more plantar flexed later in the delivery may be attributed to greater posterior ground reaction forces acting on the drive foot, potentially to increase friction, as previously reported yet not evident with short-stride pitching . The greThe stride ankle underwent greater dorsiflexion when utilizing the shorter strides, occurring after stride-foot contact and through to maximal external rotation, whereas plantar flexion was maintained throughout the entire pitching cycle with the longer strides and confirmed our secondary hypothesis B. This mEarly in the generation phase, drive-ankle inversion was notably higher with the longer strides, whereas inversion was more pronounced and occurred later in the pitching cycle after foot contact when using the shorter strides. When inverted, the drive ankle with longer strides depicted a greater maximal eversion moment (approximately 35% BW•H) A. The grGreater stride-ankle eversion angles with the longer stride lengths were seen after stride-foot contact. During this period, eversion kinetics were found to be lower with longer strides B, which In closed kinetic chain lower-extremity movement, internal–external ankle rotation is described by transverse tibial rotations relative to either the planted drive or stride foot. Internal tibial rotation about the planted foot increases external ankle rotation, whereas tibial external rotation denotes internal ankle rotation. The greater contribution of external ankle rotation and respective angular velocities, depicted as greater internal tibial rotation during the generation phase, is thought to augment propulsive effort through the ground reaction free moment. Depending on the direction of the free moment, it acts to resist the tendency of the foot to either abduct (toe out) or adduct (toe in) with respect to the ground. One possible advantage is enhanced fictional force by increasing the ability to apply free moments, which occurs early in the pitching cycle before stride-foot contact. With shortened generation times owing to the shorter stride, the lower external foot rotation moments during propulsion combined with minimal external ankle rotation throughout the pitching delivery may further the need to coordinate drive-ankle propulsive effort following stride-foot contact during double support.Greater external rotation stride-ankle moments for shorter strides during acceleration  may be used to distinguish the abrupt deceleration response to slow down closed chain internal ankle rotation . The combination of stride-ankle eversion and internal tibial rotation moments during the acceleration phase may be compensatory for the short stride’s reduced transfer of linear energy to rotation, where such lead ankle biomechanics may enhance angular pelvic and trunk momentum to maintain peak ball velocity .Obvious limitations of this work included the following: (1) research was conducted indoors on flat ground, (2) throwing deliveries occurred at shorter distances, (3) pitching trials did not involve a batter or catcher, and (4) pitchers were not wearing cleats. As a result, these limitations prevent generalizability to live game situations. However, despite these limitations, overhead pitching from flat ground is commonly practiced at all baseball levels, substantiating the importance of this study and making it more representative of pitchers’ training regimens.Among more physically mature pitchers, mound and flat ground throwing deliveries demonstrate similar biomechanical patterns, yet shoulder and elbow loads are augmented with throwing at maximum distance, whereas recent work reports equivalent arm kinetics between conditions ,27.In this work, a simple AB crossover design evaluated exploratory relationships related to ankle dynamics that were influenced by altered stride length. The approach allowed each subject to be their own control, which permitted a lower number of subjects to be studied compared to parallel-group studies. One limitation of this research method was not having comparative data for desired stride length data with the ±25% stride conditions. Desired stride length comparisons increase complexity in this research model, and it is recommended to test this intermediate condition in the future with a more involved research approach at several periods . The addition of desired stride length into a three-way analysis could also give rise to parallel-group research frameworks (ABC) to account for larger samples.In summation, longer strides depicted more advantageous triplanar mechanics in propulsion, whereas shorter strides prolonged propulsion and executed higher bracing dynamics later in the delivery, perhaps to amplify transverse trunk momentum to maintain ball velocity . To protBiomechanical evaluations for baseball pitching should involve examining ankle function dynamically in addition to clinical testing to ensure ground reaction force applications are effective in propulsion and bracing to transfer force from the ground to throwing hand. As a result, this work strongly recommends high-performance teams in baseball to assess stride length critically through 3D optical tracking methods, as compensatory mechanics or ineffective coaching suggestions can be avoided by including ankle motion analysis in their interpretation of efficient throwing mechanics.


Body surface area of the recipient of ≥1 m2, pretransplantation duration of the recipient on dialysis ≥18 months, hemodialysis prior to transplantation and donor/recipient age difference of ≥25 years were significantly associated with poorer long-term graft survival.Recognizing risk factors that may negatively affect long-term graft survival following pediatric kidney transplantation is a key element in the decision-making process during organ allocation. We retrospectively reassessed all cases of pediatric kidney transplantation performed in our center in the last 20 years with the aim of determining baseline characteristics that could be identified as prognostic risk factors for long-term graft survival. Between 2001 and 2020, a total of 91 kidney transplantations in children under the age of 18 years were undertaken in our center. Early graft failure was observed in six of the 91 patients (7%). The median follow-up of the remaining 85 children was 100 months, and the overall kidney graft survival rates at 5, 10, 15 and 20 years were 85.2%, 71.4%, 46.0% and 30.6%, respectively. Small children with a body surface area of <1 m End-stage renal disease (ESRD), the irreversible damage of the kidneys leading to the necessity of renal replacement therapy, is a major public health issue, and the estimated incidence of ESRD in children across the world was nine per million of the age-related population 4–18 years) in 2008 8 years i. CongeniThe best and most effective renal replacement therapy for children with ESRD is kidney transplantation ,9. Pediap < 0.05 [A retrospective review of all cases of kidney transplantation in recipients aged less than 18 years performed in our center between 2001 and 2020 was conducted. Following the identification of cases using our hospital database, a thorough review of the electronic medical files of the patients was undertaken, and the following baseline characteristics were documented: source of the donor organ (living or deceased), age and sex of the recipients, age and sex of the donor, weight and height of the recipients at transplantation, number of transplantations (primary or retransplantation), pretransplantation duration of dialysis , modality of dialysis performed prior to transplantation , number of HLA mismatches and duration of cold ischemia of the graft. The primary outcome of this study was graft survival, determined as the need to restart dialysis, transplantectomy or retransplantation. Baseline characteristics were compared by chi-squared analysis, and graft survival estimates were analyzed using the Kaplan–Meier method. Continuous covariates were stratified for further analyses. The basis for defining the cutoff values was the population median. Nevertheless, if the distribution of continuous variables gave a clear indication of a biphasic distribution of data values, the cutoff value was modified based on the kernel density distribution in order to reflect the real-life distribution of data values. To determine the independent predictive variable for graft loss, significant factors in the univariate analysis were fitted into a multivariate Cox regression analysis. Statistical analysis was performed using the R statistical computing program  with statistical significance set at p < 0.05 .Between 2001 and 2020, a total of 91 kidney transplantations in children under the age of 18 years were undertaken in our center. Eighty-five of them (93%) originated from a deceased donor (DD), and only six (7%) originated from a living donor (LD). The cause of death of the DD was always brain death, and no DD kidneys following circulatory death (nonheart-beating donors) were accepted. There were thirteen (14%) cases of retransplantation as a consequence of graft failure following transplantation at a younger age.Following transplantation, early graft failure was observed in 6 of the 91 cases (7%). There were two cases of acute graft rejection, two cases of graft thrombosis, one case of sepsis with formation of renal/perirenal abscess, and one case with no graft function following transplantation. Four of the six abovementioned cases were treated with immediate transplantectomy in the days following transplantation. One patient died as a result of a hyperacute rejection episode before removal of the graft. In the case where no graft function was observed, transplantectomy was performed one year later. All cases of early graft failure were transplantations from DD, and only one was a retransplantation.Long-term graft survival was assessed in the remaining 85 children. The median follow-up was 100 months (IQR 53–137 months). During follow-up, loss of function was reported in a total of 30 (35.3%) kidney grafts , and thep = 0.01), age of the donor ≥ 35 years (p = 0.03), donor/recipient age difference of ≥25 years (p = 0.04) (p = 0.02) (p < 0.01) (2 (p < 0.01) (p = 0.014) and a pretransplantation duration of dialysis of ≥18 months  (p = 0.016) and again confirmed the statistical significance of a pretransplantation duration of dialysis of ≥18 months . Because of the small size of our study, fitting all nine parameters  in a multivariate Cox regression model resulted in none of the abovementioned parameters showing statistical significance.A univariate survival analysis revealed a negative statistical significance of the following risk factors: age of the recipient ≥ 12 years ( = 0.04) , a pretr = 0.02) , hemodia < 0.01)  and the  < 0.01) . The sex < 0.01) . A multi= 0.033) . ReplaceKidney transplantation is unequivocally the treatment of choice for ESRD, and epidemiological studies have demonstrated that children who undergo kidney transplantation live on average 30 years longer than those who remain on dialysis . In addiOur study has demonstrated that transplantation of small children is associated with a longer allograft survival time. Lower weight and height and consequently a lower BSA of the child recipient are significantly associated with longer graft survival  ; these cFurthermore, our study confirmed that donor age and donor/recipient age difference play a significant role in long-term graft survival. Specifically, we showed that a donor age ≥ 35 years and a donor/recipient age difference of ≥25 years  were significantly associated with worse long-term kidney graft survival. Accordingly, advanced donor age and high differences in age between donor and recipient have long been associated with poorer outcomes following kidney transplantation ,24,25,26Of the 91 pediatric kidney transplantations that were performed in our center between 2001 and 2020, the number of HLA mismatches exceeded four mismatches in only four cases, with one being a transplantation from a living donor. Similarly, the cold graft ischemia time exceeded 24 h in only three cases. This may explain why the number of HLA mismatches or the duration of cold ischemia time of the kidney graft were not shown to influence long-term graft survival in our study. Despite several studies reporting both factors to be important in influencing long-term graft survival ,33,34, tConcerning pretransplantation duration on dialysis, our study confirms that time on dialysis impacts long-term graft survival. We showed that a duration of ≥18 months on dialysis was significantly associated with poorer long-term graft survival  . In a stp < 0.01 in the univariate analysis, not significant in the multivariate analysis) . In a recent systematic review and meta-analysis of 269,715 adult and pediatric patients, Ngamvichchukorn et al. demonstrated that peritoneal dialysis prior to renal transplantation was significantly associated with a lower risk of overall graft failure and a lower rate of delayed graft function . It is bnalysis) .The main limitations of our study are its small size and the lack of consideration of further recipient- and donor-related baseline data, such as the cause for ESRD, the infection status of the recipient and the donor at transplantation, as well as the BSA of the donor. The small number in some subgroups, such as living donor recipients or transplantations involving a cold-ischemia time of more than 24 h, prohibited a comprehensive investigation of these subgroups. Furthermore, our study does not take into consideration events following transplantation that could very well influence long-term graft survival, such as the immunotherapy schemes used and posttransplant adherence to immunosuppressive medication, delayed graft function, or future infections, as well as the ultimate reason for graft failure. In consideration of the relatively limited number of pediatric kidney transplantations performed in a single center each year, we suggest that longitudinal multicenter studies be conducted to further evaluate other putative risk factors that could influence long-term kidney graft survival.2 , it shows an advantage of peritoneal dialysis prior to kidney transplantation compared to hemodialysis  and it highlights the importance of donor/recipient age differences, as well as the importance of pretransplantation duration on dialysis in long-term kidney graft survival following pediatric kidney transplantation. Our study demonstrates that a donor/recipient age difference of ≥25 years and a pretransplantation duration on dialysis of ≥18 months are significantly associated with poorer long-term kidney graft survival, and both factors should be taken into consideration in the decision-making process during organ allocation.Despite its small size, our study confirms the excellent long-term graft survival rates of small patients with a BSA of <1 m


The HIV Organ Policy Equity (HOPE) Act allows persons living with human immunodeficiency virus (HIV) infection to accept HIV-positive donor organs. Risk of superinfection or viral recombination resulting from the transmission of a genetically distinct donor HIV-1 strain may be a concern. It is unknown if transplantation from acutely infected donors pose additional risk. Analysis of viral quasispecies from donor and recipient samples provides an opportunity to evaluate the transplanted kidney as a viral reservoir and explore the potential for viral superinfection or recombination in the recipient.(Table 1). Single genome amplification of the HIV-1 env gene was performed with viral RNA extracted from recipient urine and plasma and viral DNA from donor kidney cells, recipient PBMC and renal cells. We constructed phylogenetic trees assessed recombination probabilities.A 63-year-old HIV-positive recipient with focal segmental glomerulosclerosis and suppressed viral load (< 20 copies/mL) on antiretroviral therapy (ART) with dolutegravir and lamivudine, received a kidney from an acutely infected deceased HIV-positive donor env sequence was amplified from the recipient’s urine 30 hours post-transplant suggesting transfer of donor virus to recipient. Donor virus became undetectable on subsequent visits while recipient was maintained on ART. Notably, a third virus was detected 1.5 months post-transplant in urine-derived renal tubular epithelial cells and 5 months post-transplant in urine which was genetically distinct from previously detected donor and recipient strains . This third strain did not have genetic features suggestive of recombination. Our findings suggest potential superinfection in the donor or recipient.A new, genetically distinct We documented presence of three unrelated HIV strains within a transplanted recipient and within different viral reservoirs. Donor and recipient recombination did not occur. In acutely infected donors, when the transfer of virus within the kidney occurs, ongoing virologic suppression can be achieved if appropriate ART is maintained. Additional long-term monitoring of viral populations in the recipient is important to fully assess any clinical and virologic implications of this finding.All Authors: No reported disclosures


Eggshell strength is a critical quality factor for consumption eggs as it affects the probability of breakage in practice. In this study, a fast and low-cost methodology for the non-destructive determination of eggshell strength is presented. The method utilized a small steel ball to impact the egg and a microphone to analyse the impact characteristics. Hertz contact theory was applied to relate the measured impact characteristics to the local stiffness of the eggshell. Therefore, a total of 150 eggs were studied on which eight consecutive measurements per egg were taken around the equator at equidistant places. The results showed a strong correlation of 0.93 between the traditional static stiffness measured during quasi-static compression tests and the average stiffness obtained from the new methodology. This paves the way towards fast, low-cost and non-destructive in-line shell strength measurements to reduce the number of cracked eggs reaching the consumer. Modern egg grading is evolving from a labour-intensive process during which each individual egg is handled by humans towards a highly automated process during which up to 180,000 eggs are sorted per hour. In the sorting process, each egg is individually weighed and inspected for the presence of cracks in the shell, for various types of dirt on the shell and for undesired inclusions such as blood and meat spots [The detection of cracks in the eggshell has been researched by many ,3,4,5,6,dynK. The term dynamic was chosen to stress that the shell strength was measured using dynamic measurements, i.e., after being impacted. Coucke [In contrast to the research into the development of crack detection techniques, few studies have aimed at fast techniques to assess the strength of the shell. The main contribution has been made by Coucke , who mod. Coucke  and Couc. Coucke  reported. Coucke  has been. Coucke ,4, who e. Coucke  to a masThe economic importance of shell strength information has been discussed by different researchers ,13,14,15In their work, Mertens et al. ,17 introThe above clearly demonstrates the importance of having available fast, non-destructive and reliable measures of egg quality. Given the lack of methodologies aimed at assessing the shell strength in an online way, the aim of this study was to develop a system that has the potential to provide shell strength information on high-speed grading lines. The proposed system involves the analysis of the dynamic impact of a small steel ball on the eggs’ surface, as outlined in De Ketelaere et al. . This apIn Part I, we outlined the theoretical background for deriving shell strength information using Hertz contact theory and showed that the dynamic impact of a small, steel ball with an egg can be accurately described by this concept. This study aims to bring the theoretical concept into practice using a setup that allows for automatic, high-speed grading.®  data acquisition board connected to a personal computer at a sampling rate of 50 kHz. Next, the digitized signals are analysed using Matlab® software .For impacting the eggs, a commercial Moba crack detector probe  was used . It consFor relating the microphone signal to the actual impact characteristics that form the basis of Hertz contact theory (see Part I), a set of eggs was not only measured with the set-up described above, but also using a laser vibrometer  as a reference. For details, see De Ketelaere et al. . The eggstatK measured under quasi-static compression and the shell thickness—to the Hertz stiffness HK obtained from the experimental set-up described in the above section. The egg mass varied between 42 and 64 g; static stiffness varied between 101 and 193 kN/m and shell thickness between 278 and 432 µm.For investigating the relation between impact speed and contact time from microphone data, a first set of 75 fresh, brown-shelled consumption eggs taken from a local warehouse was used. A second set of 150 eggs taken from another supermarket was used to relate classical shell quality parameters—the static stiffness statK) was measured using a universal tensile and compression test machine . Eggs were placed horizontally between two flat parallel steel plates and compressed at a speed of 10 mm/min. The resolution of the force sensor was 0.001 N. A maximum force of 10 N was exerted. The force and deformation were recorded throughout the test, and used to calculate the static stiffness as the slope of the force-deformation curve in the range 0.98 to 10 N. The measurement was repeated at three equidistant places on the equator of the egg. The average value of the three measurements was used in the statistical analyses. The egg curvature was determined by taking the egg diameter at the equator , measured by sliding callipers up to an accuracy of 1 mm. Additionally, the weight of the egg was measured with up to 0.1 g accuracy.The static stiffness (dynK) of the eggs was also determined based on impulse response measurements. The setup consisted of an impactor rod exciting the egg, and a small microphone recording the vibration of the egg after impact. See De Ketelaere et al. [Alongside the static stiffness, the dynamic stiffness  was extracted from the microphone signal by quantifying the width of the peaks in the signal. Each bouncing of the ball causes such a peak, and the second peak  was used;The contact time n,v1 (m/s) of the second impact (for which the contact time was estimated in the previous point) was estimated by quantifying the time between the first and second impact;The impact speed HK (N/m3/2) was calculated using Equation (1). For details about the Hertz theory, see De Ketelaere et al. [The Hertz stiffness e et al. .c is a constant equalling 3.2145, τ is the contact time (s), and μ is the effective mass of the system (kg):m1 denotes the mass of the impactor  and m2 the egg mass. The maximal normal deflection nmaxδ (m) can also be derived as:maxN (N) was obtained:After those non-destructive measurements on the universal tensile and compression test machine and the 4.For the first set of 75 eggs, those measurements were complemented by measurements taken with the laser vibrometer to have reference values for contact time and impact speed. Since laser measurements are only feasible after demounting the probe, shown in After those measurements, the eggs were broken and the shell thickness was measured at three equidistant locations around the egg equator by means of a micrometre gauge with spherical tips. The resolution of this equipment was 1 µm.2, and the end of the impact was similarly defined as the moment at which the acceleration decreased below this limit value of 50 m/s2. For the microphone signal, the contact time was retrieved in a similar way. The start of the impact was defined as the moment at which the microphone signal exceeded 0.1 V, and the end of the impact was similarly defined as the moment at which the microphone signal went back below this limit value of 0.1 V. De Ketelaere et al.  describeτ from the laser vibrometer and the contact time estimated from the microphone data. As explained above, it was only possible to perform both measurements after demounting the probe in For the initial set of 75 eggs, utilizing data from both the microphone and laser vibrometer allowed for the determination of a correlation between the reference measurement of the contact time In addition to the contact time between impactor and egg, the impact speed is also of importance (see Equation (1)). One way to determine the impact speed of the second impact of the bouncing ball involves the estimation of the flight time between the first and second impact. However, this would require a solution of the coupled differential equations of the impact situation and the magnetic system. Therefore, we followed an alternative approach by exploiting the correlation between the microphone data and the laser vibrometer measurements.nv1 measured by the laser vibrometer and the time of flight Δt (s) of the bouncing ball estimated from the microphone data. This relation is illustrated in n,v1 (m/s) from microphone data using the following formula:For the first set of 75 eggs, a correlation was observed between the impact speed of the second bounce From the above, we can conclude that the impact speed and the contact time between the impactor ball and the egg can be estimated from the microphone data. As these parameters are known to be important factors in relation to shell strength , their pstatK and shell thickness), the second dataset of 150 eggs was used. For each impact with the probe, the impact speed was estimated using Equation (5) and the contact time was estimated as illustrated in statK, the average of the three quasi-static measurements was used.For predicting shell quality parameters (static stiffness statK and the Hertz stiffness HK defined in Equation (1) was investigated  and nmaxδ in Equation (3). The correlation of this linear stiffness with the static stiffness is marginally stronger (r = 0.93) than the relation HK versus statK, confirming the results reported by De Ketelaere et al. [In analogy with De Ketelaere et al. , linear e et al. . It shoum1 << m2) it is only the mass of the smallest body that is important. In the considered case, the impactor ball has a mass of 3 g only, limiting the influence of the actual egg mass. Considering two extreme eggs, one egg of 45 and one of 80 g, plugging these masses into Equation (2) gives effective masses μ of 2.81 and 2.89 g, respectively. Therefore, the impact of the egg mass is, indeed, limited (<3%).De Ketelaere et al.  showed tstatK was also investigated. It can be observed from HK and statK. The sustained high correlation by neglecting the impact speed and the egg mass may be attributed to the relatively high uncertainty in the estimation of the impact speed. This could be seen from Given the limited importance of the impact speed and egg mass, the relation between the contact time and the reference stiffness statK and the shell thickness have been reported, which were confirmed in this experiment (r = 0.86). Hence, a high correlation between the shell thickness and the stiffnesses derived from the impact test was also expected. HK equals 0.88, whereas the correlation between shell thickness and the linear stiffness derived from the impact measurements equals 0.87.In addition to the static stiffness, the thickness of the eggshell is frequently used as an indicator for eggshell strength ,19. StrostatK and the newly defined stiffnesses (HK or its linear version) are high (r > 0.9). In the study of vibration measurements of eggs after impact, Coucke [dynK and statK, all ranging from 0.65 to 0.85. The high correlation found in our study might be mainly due to two different reasons. First, the eight replicates taken for each egg lower the standard error on the measured value per egg. Second, the measurement principles behind the dynamic stiffness and the Hertz-related stiffnesses are completely different. Whereas the dynamic stiffness investigates the vibration behaviour of the egg in response to the impact, the Hertz theory investigates the impact characteristics themselves rather than the response of the egg after being impacted.The correlations between the classical stiffness , Coucke  and De K, Coucke ,20 repordynK, statK and HK. In the diagonal sub-figures, the histograms of the three parameters are shown. The off-diagonal sub-figures show the two-dimensional scatterplots of the considered parameters. The correlation between the Hertz stiffness and the dynamic stiffness dynK is weak (r = 0.39). The correlation between dynK and statK is also low (r = 0.43). These correlations found are considerably lower than those reported by other researchers [dynK to statK.k r = 0.3. The corearchers  pointed HK and its linearized version) and the classical stiffness (statK), it would be interesting to investigate this relation more in depth. This could allow the gaining of more insight into the differences, and whether the HK provides additional information about the shell quality and its relation to breakage in practice. If that would be the case, HK could be used as an additional parameter for breeding companies.As we observed much stronger correlations between the proposed, novel shell strength indices (A low-cost, yet accurate, fast and non-destructive measurement principle was demonstrated yielding accurate eggshell strength information. It uses a small impactor and a microphone as main components. Hertz theory was applied to define the most important impact characteristics. It was first demonstrated that the microphone signal was able to provide an estimate of the contact time between impactor and egg. We also demonstrated that the egg mass itself had only a minor influence thanks to the use of a low-mass impactor. Moreover, the need for an accurate estimation of the impact speed could be avoided by using relatively low impact speeds.The obtained Hertz stiffness was shown to be strongly related to classical shell strength parameters such as static stiffness during quasi-static compression and shell thickness. With the above conclusions about the most dominant impact characteristics, it was also demonstrated that using only the contact time between the impactor and the egg provided an accurate estimate of shell strength. This makes the system attractive from a data processing point of view.Its fast character and suitability for in-line measurements make it a promising tool to include shell strength measurements during egg grading. This would allow packing stations to use weak eggs for breaking or local delivery, while the strongest eggs could be transported longer distances or could be sold as “premium” eggs. Furthermore, the acquisition of massive amounts of shell strength data opens opportunities for automated detection of problems in henhouses, as shell quality is influenced by the health status of the hens.


The INFOGEST undigested fractions were subsequently used for in vitro colonic fermentation. Short-chain fatty acids (SCFAs) concentrations, mainly acetate, and propionate were significantly higher in starch-lipid complexes compared to native flour or starch samples. Starch-lipid complexes produced a distinctive microbial composition, which resulted in different gene functions, mainly related to pyruvate, fructose, and mannose metabolism. Using Model-based Integration of Metabolite Observations and Species Abundances 2 (MIMOSA2), SCFA production was predicted and associated with the gut microbiota. These results indicated that incorporating lipids into rice starch promotes SCFA production by modulating the gut microbiota selectively.In this study, two rice varieties (RS4 and GZ93) with different amylose and lipid contents were studied, and their starch was used to prepare starch-palmitic acid complexes. The RS4 samples showed a significantly higher lipid content in their flour, starch, and complex samples compared to GZ93. The static in vitro digestion highlighted that RS4 samples had significantly lower digestibility than the GZ93 samples. The C Therefore, most rice has a high glycemic index (GI). Consuming large amounts of high-GI food could increase the incidence of health issues such as obesity, type II diabetes, and cardiovascular disease2. Consequently, it is imperative to explore viable approaches to slow down the rate of starch digestion in foods, including rice. According to the digestion rate and extent, starches can be divided into three classes: rapidly digestible starch, slowly digestible starch, and resistant starch (RS)3. RS content can be increased by either “in-plant” breeding rice with high content of RS or processing strategies on rice starch such as complexation with lipids.Rice is a staple food for over half of the population in the world. Rice is mainly composed of starch that can be rapidly digested within the human gastrointestinal tract, increasing blood glucose levels4. Lipids such as oil and free fatty acids can be complexed with starch under certain conditions . The hydrophobic chain of lipids can be inserted into the spiral cavity of starch and form a stable structure, which rearranges into undigestible domains5. Several studies showed that the complexation of exogenous lipids and starch leads to decreased starch digestibility4. In the small intestine, the starch-lipid complexes are not digested by amylases, reaching nearly intact the colonic region3. In the lower gastrointestinal tract, the undigestible complex, also called RS type 5 (RSV), is used by the gut microbiota as a substrate4.On the one hand, for “in-plant” breeding, researchers have created wide high RS rice varieties via chemical mutagenesis, radiation mutagenesis, and genetic engineering in recent years. On the other hand, complexation with ligands such as lipids, proteins, and polyphenols can lower the digestion rate of starch6. As a substrate of microbial fermentation, RS significantly influences the gut microbial metabolites, such as increasing the production of short-chain fatty acids (SCFAs), mainly acetate, propionate, and butyrate7. Zhou et al.8 used an in vitro fermentation model to show that complex starch with saturated fatty acids increased SCFAs level during fermentation. An in vivo experiment showed that mice consuming RS diets had higher butyrate concentrations than control diets9. Previous studies showed that dietary RS has potential health benefits, such as reducing the risk of diet-dependent disorders6. RSV intake was reported to reduce postprandial glycemic responses and is promising in preventing metabolic syndromes such as type II diabetes10. However, the colonic fermentation of RSV, especially derived from rice, and its effect on gut health has been barely studied11. Additionally, most studies have examined starch-lipid complexes without taking digestion into account12, exposing the gut microbiota to unreal-life scenarios. Thus, in this study, we comprehensively investigated the in vitro physiological behavior of high-lipid rice RS4, low-lipid rice GZ93, and the palmitic acid-complexed samples, to elucidate the interactions between these samples and the gut microbiota. Samples were first digested in vitro according to the INFOGEST protocol13, and consecutively the INFOGEST undigested fractions were employed as a substrate on fecal batch cultures to investigate gut microbiota functionality. By comparing the physiological effect of naturally high-lipid rice (RS4) and processed high-lipid rice  with low-lipid rice GZ93, we aim to reveal the role of high-lipid rice modulating the gut microbiota.RS intake modulates the gut microbiota, and the effects of various types of RSs on the composition of microbial communities are differentFirstly, we measured the total starch, protein, and lipid content of the samples Fig. . No sign15. Therefore, this study used the most abundant fatty acid in rice oil  to complex with starch.Before palmitic acid, commercial rice oil (with triglyceride >80%) was used to complex with RS4 and GZ93 starch to mimic natural starch-endogenous lipid complex. However, the complex was hardly obtained due to the relatively large molecular structure of triglyceride, which served as a large steric hindrance that made it difficult for triglyceride to enter the cavity of starch for complex formationTp 97 °C) was attributed to the melting of amylose-lipids complexes, which was also observed by Zhang et al. (2019)1. The melting peak of the starch-fatty acid complex usually appeared at around 94–121 °C14. The Tp of amylose-lipid complex in RN was 110 °C16. After complexation with palmitic acid, RPA had two typical endothermic peaks. The first sharp peak (Tp 61 °C) represented the melting of palmitic acid. The second peak was irregular and predicted to be a merged peak of double helical amylose crystallites and amylose-palmitic acid complex16, while the latter contributed to the most ΔH of the peak with Tp at about 105 °C. For this peak, ΔH in RPA and RN was 12.1 and 0.4 J/g, respectively, indicating the different amounts of the amylose-palmitic acid complex. Similar results were observed in GZ93 samples. While the lower ΔH (3.7 J/g) of GPA, compared to 12.1 J/g of RPA, may be due to the lower amylose content containing lipid ligands in GZ93.To have more indications of the structure of the starch-lipids complex, the thermal properties of samples were measured using DSC Fig. . RS disp13. As shown in Fig. ∞ than GN and GS, while RPA had a 24.7% and 18.5% lower C∞ than RN and RS  was slightly lower than RS (67.3%), maybe because the endogenous lipid and protein in RS4 flour inhibited starch digestion. Consistent with our results, previous studies also observed decreased starch digestibility after complexation with palmitic acid17.To verify the influence of endogenous or exogenous starch-lipid complex on rice starch digestion, this study applied INFOGEST protocol to measure the digestion properties of different samples RS Fig. , indicat17. Since RS4 had a higher content of endogenous amylose than GZ933, RPA had more amylose complexed with lipids than GPA. These results suggested that complexation with lipids decreased the digestibility of starch.After in vitro digestion, the starch content in the INFOGEST undigested fractions is shown in Supplementary Table 11. SCFAs have several physiological functions, such as mediating colon pH to inhibit the growth of certain pathogenic bacteria, supplying energy to gut microbiota and intestinal epithelial cells, and sustaining the barrier function of the intestinal mucosa19. The higher content of SCFAs produced from high-lipid rice RS4 samples compared to GZ93 samples can potentially improve gut health.SCFAs are the main end-products of starch fermentation and (bio)indicators of the activity and composition of gut microbiotaThe in vitro digested materials  were fermented and slurries were collected at 4 h, 24 h, and 48 h to measure SCFAs concentrations. The 3D PCA , which may be due to the higher content of RS in RS4 rice Fig. . MoreoveAfter 48 h fermentation, each sample had similar SCFA production to that after 24 h fermentation, indicating that microorganisms fully utilized the remaining starch after 24 h fermentation Fig. . Notably11 reported that the production of SCFAs reached the maximum after 12 h fermentation. The total content of acetic and propionic acids was about 76% of all SCFAs after 24 h fermentation. These conclusions are consistent with our results, except that acetic acid was the dominant product in the research of Qin et al.11. Zhou et al.8 claimed that the anaerobe could not utilize saturated fatty acids in the colon. However, it could boost fermentation after complexing with high-amylose maize starch, resulting in more acetic acid, propionic acid, and total SCFA production than high-amylose maize starch alone or debranched starch. The propionic acid and butyric acid production pathways are conserved and substrate-specific, while the production pathways of acetic acid are widely distributed among bacterial groups20. Duncan et al.21 reported that reduced intake of carbohydrates decreased the proportion of butyric acid to total SCFA in feces. Starch is an essential substrate for lactate production, and butyrate is the main product of lactate fermentation by human intestinal microbiota22. When the carbon substrate is limited, gut microbiota may decarboxylate the intermediate product succinate to propionic acid, which regenerates CO2 to convert C3 acid to C4 acid (succinate pathway)22. The proportion of propionic has been considered beneficial in preventing obesity and type II diabetes23. Thus, the higher percentage of propionic acid produced by RSV fermentation may benefit human health.Qin et al.22. The difference in SCFA production may be caused by microbial diversity. To study the effects of RSV on the gut microbiota composition and the cross-linking between SCFAs and microorganisms, the microbial communities of samples after 48 h fermentation were researched by 16S rRNA gene sequencing. The α-diversity indexes were calculated to comprehensively determine the richness and evenness of the community  association suggesting that propionate was microbiome-governed29. This negative but strong association can occur for several reasons. For example, the AGORA model used only one propionate synthesis gene, ‘PPAt2’, and one degradation gene, ‘PPAt2i’, distributed across 28 different taxa  scores with SCFAs measurements across GPA, RPA, GN, RN, and RS. We used a regression model to assess whether CMP scores significantly predict SCFA levels, then identified specific taxa that explain variation in each SCFA on the MIMOSA2 websiteate Fig. . The maprt Table . Moreoveles Fig. , which ples Fig. . FlavoniThe present study, which mimicked human digestion and colonic fermentation in vitro, showed that RSV obtained by complexing rice starch with palmitic acid decreased the in vitro digestibility of starch. When the INFOGEST undigested fraction was exposed to the gut microbiota, certain microbial taxa were boosted changing the specific functions to degrade the matrix. During the microbial fermentation, the palmitic acid complexed sample increased SCFA production compared to native flour or starch samples, and SCFA concentrations were strongly associated with specific taxa.RS4 samples produced higher concentrations of acetic and propionic acids, indicating a better fermentation effect of the high-lipid rice. These results suggest that high-lipid rice, whether naturally or through processing strategies, may potentially have more health effects than low-lipid rice. Further in vitro and in vivo studies are needed to investigate the mechanism of how the starch-lipid complex interacts with the gut microbiota and intestinal epithelial cells.2PO4, NaCl, MgCl2·(H2O)6, CaCl2·(H2O)2, and pure ethanol were purchased from VWR International B.V. (the Netherlands). KH2PO4, NaCl, (NH4)2CO3, NaOH, and HCl, as well as the yeast extract, peptone, mucine, and L-cysteine HCl were purchased from Sigma-Aldrich Chemie B.V. (the Netherlands). All the chemicals used are of analytical and chromatographic grade.Pepsin (P6887), α-amylase (A3176), pancreatin , amyloglucosidase (10113), and palmitic acid were purchased from Sigma-Aldrich . KCl, KH3. Briefly, dry grains of R7954 were treated by 300 Gy 60-Cobalt gamma rays’ irradiation. Mutants with different endosperm lipid traits were selected from the M2 generation and verified in the subsequent M3–6 generations. The highly advanced generation mutants with stable endosperm lipid contents were chosen for the current study.This study used two rice varieties: RS4 (apparent amylose content 44.7%) and GZ93 (apparent amylose content 16.4%). They were derived from gamma irradiation (300 Gy) of R7954 as previously describedThe two varieties were grown in Hainan , China in 2019. The two varieties were harvested, dried, and stored at 4 °C in darkness. Before analysis, grains were dehulled using a paddy husker . Then the outer layer  of brown rice was removed using a rice whitener machine  to get white rice. White rice was milled using a cryogenic grinder  and sieved through 90–160 µm mesh to obtain the rice flour.35 with modifications. Briefly, 6 g of rice were weighed into a 50 mL centrifuge tube, then 30 mL of 0.2% sodium hydroxide solution and a glass bead were added. The tubes were kept at room temperature with shaking for 3 h. Then, the mixture was centrifuged at 4000 g for 20 min. After centrifuge, the supernatant was discarded and the upper layer of the sediment was carefully removed. Then, the sediment was washed with distilled water for three times and the upper layer was removed each time after washing. During the last washing, the pH value of starch suspension was adjusted to 7 and the final sediment was freeze-dried. The starch was milled by a ball miller and sieved through 125 µm mesh for further analysis.Starch was isolated based on the method reported by Lumdubwong & Seib1.Total starch content was measured using the Total Starch Assay Kit (AA/AMG) . Protein content was measured by Dumas  using a protein-to-nitrogen conversion factor of 6.25. D-Methionine was used as a standard. Lipids content was measured using the method reported by Zhang et al.17 with modifications. Briefly, 0.5 g starch was mixed with 7.5 mL of water in a 50 mL centrifuge tube, then 0.05 g palmitic acid was added. The mixture was vortexed for 2 min for full dispersion. Then the mixture was incubated in boiling water for 30 min, immediately shaking every 5 min. After cooking, the tubes were cooled down at room temperature for 1 h to allow the retrogradation of starch and the formation of the complex.The starch-palmitic acid complex was produced using the method reported by Soong et al.Rice flour and rice starch samples were cooked in the same way without palmitic acid added. The prepared samples were immediately used for digestion, or freeze dried for thermal property measurement.3. The prepared samples were freeze-dried and milled. For each sample, 2.5 mg was weighed in an aluminum DSC pan, then 7.5 µL of deionized water was added. After hermetically sealed, the pans were equilibrated at room temperature for 12 h. Then the sample pans were scanned at a heating rate of 4 °C/min from 30 to 130 °C with a hermetically sealed empty pan as a reference. The onset temperature (To), peak gelatinization temperature (Tp), and heat of gelatinization (ΔH) were determined using the analysis tool available in the Universal Analysis software .The thermal properties of the samples were measured with a differential scanning calorimeter DSC Q200 , according to Shen et al.13 with modifications. Briefly, the freshly cooked and prepared samples as described in the part of the preparation of starch-palmitic acid complex were immediately digested through a simulated oral phase, simulated gastric phase and simulated intestinal phase (SIP) with specific conditions . During the SIP, 100 μL of the samples were taken out from each tube at 0, 10, 20, 30, 45, 60, 90, 120, 150, and 180 min and mixed with 0.4 mL of ethanol to stop the digestion. Then the samples taken out at each time point were centrifuged at 4000 g for 15 min, and the supernatant was collected for glucose measurement.The in vitro digestion was performed using the standard protocol reported by Brodkorb et al.g for 15 min, the supernatant was collected for glucose measurement using GOPOD reagent . The digested amount of starch was calculated by the glucose quantity with a conversion factor of 0.9. The starch digestion data were fitted to a first-order model as previous described by Goñi, Garcia-Alonso, & Saura-Calixto (1997)36:t, C0 and C∞ are the concentrations of the percentage of digested starch at time t, 0 and infinite time and k is a pseudo-first order rate constant. Box Lucas model as the nonlinear curve fit model in OriginPro 9  was used for estimating k and C∞ value.The supernatant (100 µL) was combined with 0.5 mL of amyloglucosidase solution (27.16 U/mL) and incubated at 37 °C for 1 h. The tubes were then boiled for 5 min to inactivate the enzyme. After centrifugation at 4000 After digestion, the tubes were immediately centrifuged at 4000 g for 30 min at 4 °C. The supernatant was discarded and the sediment was stored at −20 °C for fermentation. The starch content in the remaining was measured using the Total Starch Assay Kit (AA/AMG) .37. Donors were based in the Netherlands, and fecal samples were collected on different days to perform the biological replicates. Fecal samples were quickly collected in a sterile container, in which an anaerobic atmosphere generation bag was added just before fecal collection, to obtain an anaerobic environment and stored at 4 °C before use. The subsequent process was completed within 2 h. All used equipment such as gloves, collection tubes, and swabs are sterilized to minimize the risk of contamination. Before fermentation, phosphate buffer for fecal inoculum , fermentation medium , and milli-Q water were sterilized by autoclave. Before use, the phosphate buffer was added with sodium dithionite (15 mg/L). The fresh feces (40.0 g) was combined with 200 mL anaerobic phosphate buffer and prepared by a Stomacher 400 circulator  for 10 min at speed 300. The fecal suspension was centrifuged at 500 g for 2 min, and the supernatant was collected as the final fecal suspension. The digestion sediment was dispersed with 10 mL sterilized milli-Q water and added to penicillin bottles. The same amount of milli-Q water with or without palmitic acid was used as a control and blank sample. Then 21.5 mL medium and 3.5 mL fresh fecal sample was added into the penicillin bottles and covered by rubber caps. The bottles were flushed with nitrogen for 30 min for anaerobic conditions and then incubated at 37 °C on a rotating shaker (300 rpm). At each time point , 1.0 mL sample was taken out from the bottles with a sterilized syringe. All samples were stored at −20 °C for further analysis.In vitro batch fermentation was performed using the method reported by Guo et al.Briefly, donated fresh feces were collected from two healthy adults  who declared that they did not smoke and had not consumed antibiotics for 6 months. Healthy volunteers gave written consent for a single fecal donation, and their anonymity was granted. According to the Medical Ethical Advisory Committee of Wageningen University (METC-WU) guidelines, this research did not need ethical approval.g (4 °C) for 5 min. The SCFAs content was measured using the method reported by Guo et al37.. For 500 µL supernatant, 250 µL internal standard  was added for better SCFA separation and quantification. The samples were then injected into gas chromatography , equipped with a Restek Stabilwax column 30 m × 0.32 mm × 0.5 µm (T Max 240 °C)  and a flame-ionization detector . The injection volume was 1.0 μL. The carrier gas was nitrogen, and the rate was 10 mL/min. The temperature profile starts at 100 °C, then increases at 10.8 °C/min to 180 °C, and is kept at 180 °C for 2 min. Then, it increased at 50.0 °C/min until 240 °C and kept at 240 °C for 2 min. The standards of acetic, propionic, butyric, valeric, iso-butyric and iso-valeric acids in concentrations of 0.01–0.45 mg/mL were prepared for identification and quantification.The samples collected at each time point were centrifuged at 9000 38. Select Auto-scaling (mean-centered and divided by the standard deviation of each variable) and Log transformation (based 10) for data normalization. The 3D Principal Component Analysis (PCA) plots and heatmaps were generated, and Correlation Analysis and ANOVA Simultaneous Component Analysis were processed.The concentrations of acetic acid, propionic acid and butyric acid at 4 h, 24 h and 48 h were calculated and subjected to Time-series + one-factor analysis using MetaboAnalyst 5.0 online platformBacterial genomic DNA extraction from chosen samples after 48 h fermentation was performed using the Qiasymphony SP automated nucleic acid purification system . The extraction property and concentration were tested. 16S ribosomal RNA sequencing of the V3−V4 region was performed by PCR, using the forward primers (5’-CCTACGGGNGGCWGCAG-3’) and the reverse primer (5’-GACTACHVGGGTATCTAATCC-3’). Sequencing library was generated and quality was assessed by Quant-iT PicoGreen . Then the library was sequenced on Illumina MiSeq Platform , which generated about 300 bp double-end reads with over 50,000 readings per sample and base mass was greater than 30.39. Alpha diversity indexes  were obtained using QIIME2 to indicate the gut microbiota composition in samples. The difference in microbial communities among samples was characterized by β-diversity calculated by principal coordinate analysis (PCoA) with weighted UniFrac analysis. The composition of each sample at the phylum level and the sequence number difference at the genus level were analyzed by alignment with Greengenes database. Bacterial metagenomics functions were predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) on the 16S rRNA gene abundance data. Heatmap was performed to show diversity of gut microbiota composition and difference in predictive function among samples based on the most abundant ASVs (the relative content >1%) by R package. Integration of microbiome and metabolomics data was performed using Model-based Integration of Metabolite Observations and Species Abundances 2 (MIMOSA2), freely available at http://elbo-spice.cs.tau.ac.il/shiny/MIMOSA2shiny/29.The 16S ribosomal RNA sequences were uploaded to NCBI as PRJNA1002494. Sequences were analyzed using Quantitative Insights into Microbial Ecology2 (QIIME2) software. DADA2 method generated feature sequences based on amplicon sequence variants (ASVs)p < 0.05) were performed for statistical analysis by SPSS 26 .All tests were carried out in triplicates. One-way ANOVA and Duncan factorial scheme (significance level at Further information on research design is available in the Supplemental MaterialReporting Summary